Luring the vector: A systematic review of sand fly attractants

Abstract

Sand flies are vectors of Leishmania spp. parasites, responsible for causing leishmaniasis in humans and animals. Effective control of sand fly populations is essential to interrupt pathogen transmission, yet conventional insecticide-spraying methods have shown limited and often unsustainable impact. As part of Integrated Vector Management (IVM) systems, attractant-based strategies offer a promising complementary approach by luring the vector for improved surveillance and control. Understanding the sensory cues that drive sand fly behavior is essential for the development of effective attract-and-kill or monitoring tools. However, the application of attractants in sand fly control remains underutilized, partially due to fragmented and inconsistent evidence across studies. To address this gap, we conducted a systematic review, according to the PRISMA guidelines, to summarize current knowledge on sand fly attractants and evaluate their potential role within IVM frameworks. Articles published up to the end of 2024, were retrieved from four databases. The search strategy was adapted to the PEO (Population, Exposure, Outcome) framework, with tailored search queries designed for each database in order to identify relevant field and laboratory studies. The 100 included studies were assessed using a customized tool and classified into five categories: “visual cues”, “olfactory cues”, “combined cues”, “attractive toxic sugar bates (ATSB)”, and a “special category”. To the best of our knowledge, this is the first systematic approach to comprehensively and systematically summarize existing knowledge regarding sand fly attractants.

Graphical abstract

Highlights

• •Sand fly attraction reviewed across, visual, olfactory, and combined stimuli.
• •LED lights and volatiles emerged as the most tested, effective attractants.
• •Lack of standardized methodology and reporting protocols leads to knowledge gaps.
• •Attractant-based traps show potential within IVM for low-resource endemic settings.
• •More research needed on species-specific cues and life stage behaviors.

1. Introduction

Vector-borne diseases (VBDs) represent a major economic and public health burden worldwide, accounting for nearly 20% of all infectious diseases with a high death toll each year (WHO, 2024). Among the VBDs, leishmaniasis is caused by more than 20 species of Leishmania parasites and transmitted by phlebotomine sand flies. The disease remains a major public health burden in resource-limited, endemic regions, affecting both local populations and deployed military personnel (Wasserberg et al., 2014). However, its relevance is extended beyond traditionally endemic tropical and subtropical areas, particularly in the context of international travel, migration, climate change, and ongoing humanitarian crises (Kim et al., 2025).

Τo date, there are no available preventive vaccines for human leishmaniasis, and the therapeutic treatment relies mainly on chemotherapy, which is accompanied by several adverse side effects, high cost, and poor patient adherence (Moafi et al., 2019; Dinc, 2022; Mikery et al., 2022). Current sand fly control strategies include personal protection, pathogen reservoir host management, environmental vector management strategies, and chemical interventions such as indoor residual spraying. However, the long-term feasibility of some of these strategies is limited by factors such as the evolution of insecticide resistance, environmental concerns, and inconsistent efficacy, particularly in rural settings where implementation and monitoring are more challenging. Non-chemical approaches may also face barriers, including logistical constraints, limited community participation, and the need for ongoing maintenance to remain effective over time (Balaska et al., 2021). Recognizing the shortcomings of relying on “silver bullet” solutions or single mode-of-action chemical pesticides, the World Health Organization endorsed the Integrated Vector Management (IVM) principle (WHO, 2022). IVM is a multiple-hurdle strategy that combines locally customized, evidence-based methods ranging from traditional tools to innovative, semiochemical-based approaches like attract-and-kill and mass trapping, to achieve more sustainable and effective vector control (Chanda et al., 2017).

Our objective in the present paper was to collect, collate and synthesize the available relevant data from both field and laboratory studies on the cues that attract sand flies, in the context of a systematic review. A cohesive summary of the existing knowledge in the field was produced, aiming to provide insights into the potential factors driving the attraction mechanisms of these vectors, highlight research gaps, guide future research and ultimately support the development of more efficient surveillance and control methods.

2. Materials and methods

A detailed protocol was developed and approved by all the authors prior to the systematic review. The protocol followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) standard reporting guidelines (Supplementary file 1) (Page et al., 2021) and outlined the key steps to ensure methodological rigor and transparency. These steps included: (i) scoping (formulation of the focused question and preliminary research on the topic); (ii) research planning (search strategy, defining databases and eligibility criteria); (iii) conducting the research; (iv) screening and assessing the studies; (v) extracting and evaluating the data; and (vi) presenting (interpreting) the obtained data.

Furthermore, to facilitate comprehension, a summary of key terms used in this systematic review is presented in Table 1.

Table 1.

Glossary.

Term	Definition
Kairomone	A substance produced or emitted as an activity result of an organism that attracts or influences in a positive way the behavior of another species. It provides an adaptive value to the organism receiving it.
Light intensity	Brightness or power of light emitted from a source per unit area.
Light wavelength	Distance between two consecutive corresponding points on a wave, such as two crests or two troughs. It is a fundamental property of light waves and is measured in units of length, commonly nanometers (nm) for visible light.
Pheromone	A chemical substance, usually a glandular secretion, which is used in communication within a species, such that one individual releases the material as a signal and another responds after sensing it.
Semiochemicals	Chemical substances that transmit signals between organisms, influencing their behavior or physiology. Τhey include both pheromones and allelochemicals (allomones, kairomones, synomones).
Thigmotropic response	A mechanosensory response due to contact with a surface.
Volatile organic compounds (VOCs)	Organic (carbon-containing) compounds that have a high vapor pressure, causing them to evaporate readily into the atmosphere.

2.1. Design of the search strategy

The search strategy was designed to identify relevant studies across multiple databases to address the focused question: “Which cues are attractant and what is their effectiveness to lure sand flies in both laboratory and field studies for the monitoring and/or control of their populations?“. PubMed, ScienceDirect, Scopus, and Web of Science were systematically searched for articles published up to 24th December 2024. No temporal restrictions were set, based on the earliest date of publication. The search terms were tailored to align with the PEO framework (Population, Exposure, Outcome), and the specific search strings used for each database are provided in Supplementary file 2: Table S1. For Science Direct specifically, three different search string combinations were used to accommodate the database’s word limit restrictions. The results obtained were combined, and repetitions were removed. Comparative terms were not included in the search strings in an effort to ensure inclusivity across various research methodologies. This approach was chosen to capture a wide range of relevant literature without bias towards a predefined set of comparative measures. Comparisons between treatments and controls were considered during study assessment and throughout data extraction and synthesis. Boolean operators (AND, OR) as well as filters regarding the type of article, language, and text availability were applied, depending on the functionality of each database, to refine the results. In an effort to locate additional fitting studies, manual search was also carried out by going through the reference lists of the included studies as well as pertinent review articles.

2.2. Definition of inclusion/exclusion criteria

Eligibility criteria were established to identify studies meeting the objectives of the systematic review. Studies were included if they: (i) were written in English; (ii) were available in full text or the full text could be retrieved through personal contact with the authors; (iii) were published in peer-reviewed journals; (iv) were original research articles reporting primary data including laboratory, semi-field or field experiments; and (v) reported the results of studies related to specific cues, i.e. lights, colors, chemical substances, pheromones, kairomones, animals etc. and attractancy of sand flies and/or attractant efficacy of the used traps. Short communications and scientific notes were included if they provided sufficient information regarding the experimental design, a clear description of the treatments and control groups studied, statistical analysis and assessment of the results. Articles were excluded if: (i) they were duplicates; (ii) only abstracts were available; (iii) they were conference papers, operational notes, expert opinions or reviews; (iv) they were written in languages other than English; (v) the research was performed on vectors other than sand flies; (vi) they compared different trap types with the same attractant, as mechanical differences in how insects are retained introduce variability that impacts the final trap efficiency; (vii) the aim of the research was to compare different brands of the same trap type; (viii) no statistical analysis comparing sand fly attraction between treatment (cue) and control groups, or no sufficient quantitative data (e.g. means, standard deviations, raw counts) from which statistical analysis can be conducted were provided; and (ix) critical aspects of the experimental design, such as control groups and implemented treatments, were not provided or were poorly described.

2.3. Study selection process and data extraction

The retrieved articles were stored in Mendeley Reference Manager software (Elsevier, Mendeley Ltd., Amsterdam, The Netherlands) for organization purposes, and two independent reviewers (P.T. and A.G.) screened titles and abstracts to assess their relevance. Full-text articles were then evaluated for inclusion based on the predefined criteria. Any discrepancies were resolved through discussion until a consensus was reached, or through consultation with a third reviewer (A.C.). Details extracted from each study included the first author’s name, publication year, article title, objective of the study, setting of the study (e.g. laboratory, field) and geographical location, sand fly species, type of the studied attractant, descriptions of treatments and experimental controls, main results, and key findings. For sand fly species of the American taxa, we report the nomenclature as provided in the original publications and, in parentheses, include the updated classification according to the taxonomy proposed by Galati (2018). The abbreviations of genera follow the system proposed by Marcondes (2007). Outcomes of interest included the number of sand flies attracted to specific cues or traps, attraction rates or percentages, behavioral responses (e.g. activation, landing, oviposition or host-seeking behavior), and comparisons between different attractants or experimental controls. Due to significant variations in methodologies, experimental designs and outcome measures across the studies, the results were synthesized narratively without meta-analysis.

2.4. Risk of bias assessment

In an effort to identify potential sources of bias in the selected articles, we developed a customized questionnaire (Supplementary file 2: Table S2) based on the JBI Critical Appraisal Tool for randomized controlled trials (Tufanaru et al., 2020). This questionnaire was designed as a tool to systematically evaluate the risk of bias across diverse experimental designs and protocols, which is a characteristic challenge in entomological research. The questionnaire aimed to identify possible sources of bias specific to the study context, such as treatment descriptions, confounding variables or unaccounted experimental factors, measurement reliability etc. A categorical response format with four predefined options: “Yes”, “No”, “Unclear”, and “Not applicable” was utilized. These options were designed to provide a framework for evaluating each article, with “Yes” indicating the criterion was met, “No” indicating it was not met, “Unclear” reflecting insufficient information, and “Not applicable” denoting that the criterion was irrelevant to the study design. The bias assessment was primarily performed by one reviewer (P.T.) using the developed questionnaire. In cases of uncertainty or ambiguity, the opinion of a second reviewer (A.C.) was sought to ensure accuracy and consistency.

3. Results and discussion

3.1. Studies selection and characteristics

The systematic literature research results are depicted in the flow diagram (Fig. 1). More than 3000 publications were initially identified through the electronic databases, and 22 additional records were manually selected. After the screening process, following the PRISMA methodology, and the application of the inclusion/exclusion criteria, a total of 100 studies were considered eligible and thus included in the review. As there were no limitations based on publication date, the oldest included study was published in 1980. To date, most studies (62% of the total) were published since 2010, while the steady increase in publication output regarding sand fly attractants is reflected by the ascending trendline in the distribution chart of the included studies (Fig. 2A). In order to highlight the most frequently occurring terms among the studies, a qualitative visual tool was created by entering the keywords, MeSH terms, or the whole title of the study (when the previous were not available) into an online word cloud generator (https://www.freewordcloudgenerator.com). The proportional frequency of the 100 most used terms (out of 374 possible terms in total) is depicted in Fig. 2B. The displayed font size of each term is indicative of the frequency of occurrence. The most common term was “sand flies” (43 mentions), followed by “leishmaniasis” (31 mentions), and “phlebotomus” (27 mentions). Interestingly “leishmaniasis” was not included in the search strings, but its mention count emphasizes the importance and relevance of this term in the reviewed studies.

Fig. 1.

Flow diagram of the studies selection process according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Note: The filters used are listed in Supplementary Table S1.

Fig. 2.

A Distribution of studies according to publication period (n = 100). B Word cloud of the 100 most mentioned keywords and terms in the included studies. The displayed font size of each term is indicative of the frequency of occurrence. C Number of studies per type of tested cue and setting of the experiment. Note: Two studies were placed in two categories each; therefore, 102 studies are summarized. D World map (created with mapchart.net) showing the geographical distribution of the field studies (n = 54). Note: One study was conducted in two countries; therefore, 55 study locations are listed.

Specific characteristics of the 100 eligible studies in terms of tested cues and experimental settings are given in Fig. 2C. Based on the tested cue type criterion, the life stage of the tested sand flies, and/or the stated aim of each study, 5 broad categories were defined, namely: (i) visual cues (n = 18): including studies on trap lights (n = 14), and trap colors (n = 4); (ii) olfactory cues (n = 51): consisting of diverse stimuli, including carbon dioxide (CO₂) sources (n = 4), plants (n = 4), animals used as baits (n = 6), animals infected with Leishmania (n = 6), several pure chemical substances, isolated host odors, fruit etc. (n = 19), and sex/aggregation pheromones (n = 12); (iii) combined cues: the 8 studies that fall into this category, address combinations of visual and olfactory or other types of cues (e.g. temperature, humidity); (iv) attractive toxic sugar baits (ATSB) (n = 4) which combines olfactory attractants and phagostimulants; and (v) special category: where 2 studies are dedicated to the responses of immature stages (eggs, larvae) of sand flies to attractant cues and 19 studies investigate the effects of cues on oviposition. The first four categories were grouped together as they all refer to studies on adult sand flies of both sexes. Oviposition was treated as a separate category due to its focus on a specific behavior unique to female sand flies, while immature stages were also categorized separately, given the limited number of studies and their developmental focus. Overall, 49 studies were conducted in the field, 46 studies took place under laboratory conditions, and 5 studies described both laboratory and field applications. It should be noted that two studies (Kline et al., 2011; Machado et al., 2022) have been added in two categories each (visual/combined and visual/olfactory, respectively), as their methodologies encompassed elements of both categories.

Finally, among the countries and regions where the 54 field applications were conducted (Fig. 2D), Brazil holds the lead (n = 22), followed by the Jordan Valley region (n = 9), Kenya (n = 4), and Iran (n = 3). It is worth mentioning that in the study of Moncaz et al. (2013), the experimental sites were in Ethiopia and in a region near the Dead Sea Basin (here cited as Jordan Valley region), so the total number of study locations in Fig. 2D is 55. The studies are almost evenly distributed between Old World (n = 26) and New World (n = 27) regions. However, within the New World, Brazil clearly dominates, accounting for 22 out of 27 studies. This highlights a particularly strong research focus on sand fly species and vector-related dynamics in Brazil, likely reflecting the country’s high burden of vector-borne diseases such as leishmaniasis.

3.2. Critical appraisal of the studies

The risk of bias was assessed using the evaluations of the 100 included studies to 12 predefined methodological quality domains (Q1-Q12). The distribution of the categorical responses across these domains is presented in Fig. 3. The goal was to identify the sources of bias, so each domain was evaluated separately. Most of the domains (Q1-Q4 and Q7-Q9) are characterized as low-risk, since the majority of the studies (> 90%) had a “Yes” response. These domains address the clarity of study objectives and contextual details (Q1-Q2), the adequacy of treatment and experimental control descriptions (Q3-Q4), and the validity and precision of outcome measurement and reporting (Q7-Q9). For specimens excluded from the analysis (Q11) – such as those with inadequate identification – and for any missing data, appropriate information on their handling was provided where relevant. However, in most cases, this assessment domain was not applicable (“N/A” 87%) due to the experimental designs followed and the nature of the studies.

Fig. 3.

Distribution of risk of bias assessment responses across the 12 methodological domains used to evaluate the included studies.

Moderate or high biases of studies included the identification (Q5: “Yes” 59%, “No” 22%, “Unclear”, 19%) and control (Q6: “Yes” 60%, “No” 17%, “Unclear” 23%) of confounding/unaccounted experimental factors, and the discussion about potential limitations in each study (Q12: “Yes” 50%, “No” 38%, “Unclear” 12%). This weak or omitted description might be related to “common knowledge assumption” by the researchers-authors, inexperience or oversight, and/or space limitations due to journal guidelines in some cases (especially for older publications). Either way, lack of information on these factors may raise readers’ concerns about internal validity, or lead to low reproducibility of the experiment and biased effect estimates as unaccounted confounders may have influenced the observed behaviors. Furthermore, inconsistent assessment of the practical significance of the results (Q10: “Yes” 77%, “No” 12%, “Unclear” 11%) suggests a gap in their applied interpretation. Statistical significance of an observation without clearly articulated implications for actual application in real conditions undermines the utility of research findings to the design of effective vector control strategies.

3.3. Visual cues

Most sand fly species are crepuscular or nocturnal and exhibit positive phototaxis towards artificial light. Although there is little information on the vision and photosensation of sand flies, there are data from electroretinogram studies indicating that Lutzomyia longipalpis has two main light absorption peaks, one in the ultraviolet (340 nm) and another in the blue-green-yellow range (520 nm for females and 546 nm for males), with minimal sensitivity in the near-infrared (670 nm) (Mellor et al., 1996). The perception of ultraviolet (UV) and blue-green light is crucial to facilitate the biological needs that are essential for sand flies’ survival and reproduction under low light conditions, in which sand flies are typically active (de Felipe et al., 2023; de Freitas Milagres et al., 2024).

In particular, sensitivity to UV and blue-green wavelengths has been linked to wide-field motion detection and visual navigation by contrasting the UV-rich sky against the less reflective ground. This ability is especially beneficial at dawn or dusk, when UV radiation remains relatively abundant and at twilight (before dusk) when blue-shifted spectral conditions prevail (Mellor and Hamilton, 2003). Such spectral sensitivity allows sand flies to detect landmarks, identify suitable resting or feeding sites, and potentially recognize mates. The importance of visual cues in sand flies’ behavior is well-established, as light traps are the most common method of surveillance in entomological research (Mellor and Hamilton, 2003; Cohnstaedt et al., 2008; Silva et al., 2024).

Since the 1960s, Centers for Disease Control and Prevention (CDC) light traps and, more recently, several CDC-type traps, which are modified configurations of the original, are widely used as a reliable benchmark tool for the field trapping of biting dipterans, including sand flies (Silva et al., 2024). The common visual attractant of CDC traps is an incandescent light bulb, emitting light mostly in the infrared range (> 700 nm) of the spectrum. Over the last 20 years, as lighting technology has evolved, different colors of light-emitting diodes (LEDs) have become a point of research to better understand the color-attraction of sand flies and boost the collection efficiency of light traps. LEDs offer a plethora of advantages over traditional incandescent bulbs. These include high energy efficiency with minimal heat emission, extended operational lifespan, compact design, greater durability, low maintenance requirements, and the ability to customize the light color output, as LEDs can emit specific colors at narrow (±3 nm) or broad spectra of wavelengths (±250 nm) without the use of filters. The aforementioned features make them particularly well-suited for portable field traps (Cohnstaedt et al., 2008; Silva et al., 2015, 2016; Gaglio et al., 2018).

The included studies concerning visual cues of sand flies are summarized in Supplementary file 3: Table S3. Among the early research attempts, aiming to study the light-induced attraction response of sand flies to specific lights, was the one conducted by Mellor and Hamilton (2003). They tested the attraction response of male and female Lu. longipalpis to seven monochromatic beams of light from 350 nm (ultraviolet) to 670 nm (deep red). Two-choice experiments were conducted in three separate sets where each tested wavelength was presented at intensities higher than, equal to, or lower than the 400 nm control. Attraction was evaluated based on the percentage of sand flies that left the release chamber and landed within a specific area (6-cm diameter circle) centered on each light source. Sand flies were exposed to the lights for 7 min, and their landings were recorded with a camera. The results indicate that overall, the ultraviolet and the blue-green-yellow regions of light were more attractive to this species, unlike blue-violet (400 nm), and orange-red (600–670 nm), which were the least attractive. Sand flies appeared capable of discriminating wavelengths regardless of intensity. This behavior could be interpreted as evidence of true color vision (Mellor and Hamilton, 2003). Furthermore, the responses with respect to a wide range of intensities (from 8.4852 × 10¹⁶ up to 15.089 × 10¹⁸ T photons s⁻¹cm⁻²) were assessed using the same experimental protocol for three wavelengths (350, 490, and 520 nm). The response percentage, indicating attraction, increased with rising light intensity for 490 nm and 520 nm (almost parallel graphical curves between these wavelengths). Α difference was observed for the 350 nm wavelength, showing a higher response percentage up to a point (sharper slope in the graph) and a slight decrease at 4.7716 T photons s⁻¹cm⁻², but finally reaching the same levels of attraction response as the other two tested wavelengths. These results suggest that more than one type of photoreceptors may be involved in flying and landing behaviors. However, further research is needed to fully understand the role of light in sand flies’ behavioral regulation (Mellor and Hamilton, 2003). It should be mentioned that 400 nm was set as the control, as Lu. longipalpis eyes have shown minimum sensitivity to this wavelength based on the electroretinograms from their previous study (Mellor et al., 1996).

The color discrimination ability was also confirmed in a field study in Brazil, where, blue (470 nm) and green (520 nm) LED traps, deployed at three different luminous intensities per color, were compared. During each sampling night, three traps of each color, set to distinct intensities, were positioned simultaneously but at separate locations 100 m apart. Trap positions were rotated nightly to reduce site bias. The results demonstrated that sand fly captures (mostly Lu. longipalpis) generally increased with light intensity, and while blue LEDs tended to attract more sand flies overall; a significant difference was only observed against the lowest intensity of green LEDs (10,000 mcd) (Lima-Neto et al., 2018). These findings highlight the importance of both color and intensity in sand fly attraction and support their ability to distinguish wavelengths independently of light intensity.

Varying results have been reported in terms of species-specific attraction to LED colors. Field studies conducted in Brazil at various environments have revealed that New World species (i.e. Nyssomyia whitmani, Lu. longipalpis, Evandromyia (Aldamyia) walkeri) are mainly attracted to green LED (520 nm) (Silva et al., 2015, 2016) or blue LED (470 nm) (Lima-Neto et al., 2018; Silva et al., 2020). Even though the differences may not be statistically significant in some cases, blue and green spectra are generally attractive to New World sand fly species. On the other hand, Old World species suggest a different pattern of phototactic behavior. In a field study carried out in a rural, arid agricultural environment in southern Egypt, significantly more Phlebotomus papatasi were collected with red LED traps than with incandescent light, blue or green LEDs (Hoel et al., 2007). Similarly, red LED appears to be more attractive to Sergentomyia minuta sand flies, as reported in a study conducted in an animal shelter in Spain (de Felipe et al., 2023). It should be noted that results from the same study showed that besides red LEDs, Se. minuta males were also significantly attracted by green LED, while total phlebotomine and Phlebotomus perniciosus captures showed no preference for any specific light source.

The findings regarding the attraction towards the red LEDs were unexpected, especially given that there are no studies on sand fly photoreceptor pigments, and therefore assumptions rely on what is known from other Diptera (Drosophila models), most of which possess three types of photoreceptors (R1-6, R7, and R8) and generally lack red-sensitive photoreceptors (White, 1985). Therefore, the observed attraction towards red light in the study by Hoel et al. (2007) was attributed by the authors to either the higher intensity of the red LED compared to the others tested (although red and green LED intensities were almost similar), or to the reflected light (due to the use of metal rain shields) of this specific LED. Another explanation could be the interpretation of red color as black or a shade of gray, like in the case of other insects (Simuliidae) that are attracted to dark colors or to the perceived contrast of the color which is crucial for the visual attraction of Aedes aegypti that do not have red-sensitive rhodopsin in their rhabdomeres (Burkett and Butler, 2005; de Felipe et al., 2023).

Field studies in both Neotropical and Mediterranean regions have also confirmed the attraction of sand flies to UV light. Jeraldo et al. (2012) found that a 4 W UV fluorescent black light attracted significantly more Lu. longipalpis in an urban environment in Brazil than the incandescent control light. Old World species, such as Ph. perniciosus and Se. minuta, also showed a preference for UV LED (at 395 nm) light sources when used alone (Gaglio et al., 2018) or in combination with a colored LED (de Freitas Milagres et al., 2024). As previously mentioned, UV detection plays an important role in both navigation and identification of plants (de Freitas Milagres et al., 2024). Despite evidence highlighting species-specific preferences for various LED colors and for UV, some studies have shown that incandescent bulbs can be equally attractive (da Silva et al., 2022; de Souza et al., 2024; Silva et al., 2024). It should be noted, though, that differences in UV source type (LED or fluorescent lamp) could influence sand fly responses due to variations in wavelength emission and intensity.

In addition to species-specific patterns, other parameters, such as sex and physiological stages also affect the attraction preference of sand flies to different light sources. In the study by Mellor and Hamilton (2003), a trend of female attraction towards blue-green light (490 nm) was observed at low intensities, while green-yellow light (546 nm) was more attractive to males. In a recent laboratory study, Machado et al. (2022) assessed more nuanced behavioral responses. They evaluated the activation (insects leaving the release cage in response to a stimulus) of colony-maintained Nyssomyia neivai sand flies to incandescent light, assessing differences across different female physiological stages, including unfed, gravid, and blood-fed female individuals. No activation was recorded for blood-fed females, while nearly 50% of unfed and gravid females were activated. Overall, the results from Mellor and Hamilton (2003) and Machado et al. (2022) imply sex- and life-stage-specific responses to visual cues and therefore are valuable for the development and optimization of trapping technologies tailored for specific physiological stages.

Beyond wavelength and intensity, studies have also investigated how trap colors, as visual cues, can affect sand fly behavior and attraction. For instance, Elnaiem et al. (2020) showed that the bright colors (white, yellow, transparent plastic sheets) of sticky traps caught more Phlebotomus orientalis than black and red traps, at different lunar phases in a field study conducted in Sudan. Kline et al. (2011) evaluated different CDC trap colors (black or white catch bags combined with black or white lids, or without a lid) for capturing Ph. papatasi and found that all-black traps captured the highest numbers, particularly males, whereas all-white traps were the least effective. In Brazil, black Shannon traps attracted more than 90% of total captures, with Lutzomyia almerioi as the predominant species (Galati et al., 2001). Conversely, in another Brazilian field study, white Shannon traps were more attractive to Psychodopygus carrerai carrerai, Nyssomyia shawi, and Psychodopygus davisi, although the difference was statistically significant only for the latter two species (Brilhante et al., 2017). The findings suggest again that sand fly responses to trap colors (regardless of the trap type) are species-specific.

Given the diversity of findings across studies regarding visual cues, it is essential to evaluate the factors that influence light trap efficiency and consider the broader implications for trap design and vector surveillance. The lack of consistency in the methodological approach, coupled with the diverse ecological settings where studies take place, contributes to the variability in observed behaviors and responses to visual cues (de Souza et al., 2024). Moreover, the light trap efficiency might be affected by biotic or abiotic factors such as the presence/absence of hosts, weather conditions, positioning and placement of the trap, ambient illumination from natural or artificial sources, etc. (Silva et al., 2016; Lima-Neto et al., 2018; de Felipe et al., 2023).

In addition to these confounding variables, wavelength and light intensity remain key determinants of phototactic behavior in sand flies, as evidenced by both laboratory and field experiments previously analyzed. Frequently, studies use commercially available LEDs and do not control for the intensity of light, therefore, direct comparisons of collection rates with different light colors in most of the studies above are not always consistent, as some LEDs are 10–1000 times brighter than other colors in the study. The spectra of the LEDs are not well characterized either and most studies simply report the lambda max or the most intense wavelength and not the entire spectra of the LED which may overlap sensitive areas in the sand fly vision making that LED attractive despite not being the optimal wavelength.

Current evidence underlines the critical influence of both the spectral quality (wavelength) and quantity (intensity) of light on sand fly attraction. To further elucidate the mechanisms driving these responses and uncover interspecific differences in photosensory capability, future research should aim to investigate the structure and function of visual receptors in additional sand fly species beyond Lu. longipalpis, particularly those with differing ecological and behavioral traits. Expanding our understanding of the photosensory biology of sand flies will provide crucial insight into their sensory ecology and contribute to a more complete picture of their biology. Such knowledge is foundational for interpreting species-specific light responses and achieving broader consensus across experimental approaches. Ultimately, this could guide more tailored experimental designs, improve the comparability of results across studies, and support the development of species- and stage-specific surveillance that reflect the ecological and sensory realities of the target vector populations.

3.4. Olfactory cues

The present section analyzes the findings of sand fly attraction studies, categorized by the type of tested olfactory cue. Further details on the individual studies are available in Supplementary file 3: Table S4.

3.4.1. Carbon dioxide (CO₂) sources

Carbon dioxide (CO₂) is a ubiquitous chemical cue that plays a vital role in the biology of numerous insect species. It serves as a critical long-range activator and attractant in the foraging and host-seeking behavior of many haematophagous dipterans, including sand flies (Guerenstein and Hildebrand, 2008; Tchouassi et al., 2024a). Results from the study of Pinto et al. (2001) conducted in an agricultural environment in Brazil indicate an approximately linear relationship between the responses of Lutzomyia intermedia and Lutzomyia whitmani (now classified as Nyssomyia intermedia and Nyssomyia whitmani, respectively) to increasing CO₂ doses (0.08–0.55% CO₂). This pattern aligns with findings with other haematophagous insects and may explain the proportional attraction of sand flies to host size (Pinto et al., 2001). In the same experiment, Lu. (Ny.) intermedia (and, to a lesser extent, Lu. (Ny.) whitmani) males seemed to be more responsive to CO₂ than females, indicating sex-specific and species-specific differences in olfactory sensitivity and attraction.

Several carbon dioxide sources have been employed in CDC light traps and enhanced the specificity and efficiency of the vector sampling process. The most commonly used forms integrated with the traps are compressed CO₂ (in cylinders or tanks) and dry ice (solid CO₂). Despite their efficiency, both forms pose practical challenges, particularly in remote or resource-limited settings such as parts of Africa and/or Asia, where logistical constraints (supply availability, transportation, storage, and disposal) can limit their feasibility for routine field use (Hoel et al., 2011; Benante et al., 2019). As a result, alternative methods for CO₂ generation have been explored to overcome these limitations.

Hoel et al. (2011) reported that traps with the prototype FASTGAS (FG) CO₂ generator system, based on water and food-grade chemicals, were equally attractive with the traps using compressed CO₂ (releasing 250 ml/min) and 1.5 kg of dry ice (renewed each day during the experiment). Similarly, Benante et al. (2019) tested three CO₂ generator system prototypes (TDA, CUBE, and Moustiq-Air Med-e-Cell) to evaluate their efficiency compared to dry ice when used to bait CDC light traps for mosquito surveillance in Thailand. The prototypes, each with a different CO₂ generation mechanism, were tuned to release CO₂ at a rate between 250 and 400 ml/min, a range considered effective for attracting anthropophilic vector species. The TDA prototype generated CO₂ via an acid-base reaction using sodium bicarbonate and malic acid. The Moustiq-Air Med-e-Cell (MEC) system used an electrochemical process for CO₂ stripping from oxalic acid, while the CUBE prototype produced CO₂ through combustion of liquid fuels like diesel or JP-8 using a compact Micro-Furnace.

Following the initial evaluation, the best-performing system (TDA-baited trap) was selected for further field trials targeting sand flies in Greece. In these follow-up trials, the TDA-baited trap was tested alongside two other configurations: a CDC light trap used alone and another paired with dry ice as the CO₂ source. The TDA-baited trap collected effectively high numbers of all sand fly species present at the study sites. In the case of Phlebotomus tobbi, no significant difference in trap catches was observed between dry ice and TDA-baited traps, while the prevalent species (Phlebotomus perfiliewi) was collected in greater numbers in the dry ice-baited traps. Overall, the TDA system performed well, offering a practical, field alternative for CO₂-based sand fly surveillance when dry ice is not available.

In addition to its use in suction traps, CO₂ has also been employed as a bait in sticky traps to increase sand fly capture rates. Moncaz et al. (2013) used large horizontal white polypropylene boards coated with castor or sesame oil. They evaluated near the Jordan Valley region the effect of CO₂ released from dry ice or from sucrose fermentation when used as bait on these sticky traps. The performance of CDC light traps baited with the same CO₂ sources was also assessed. The majority of the caught sand flies (94%) were Ph. papatasi, and both sexes were attracted to CO₂ irrespective of the trap type. More specifically, unbaited sticky traps captured very few Ph. papatasi individuals (mean number ∼2.4 sand flies/trap/night), while when baited with CO₂, the trap catch increased significantly to 100 and 250 sand flies/trap/night with sucrose fermentation and dry ice, respectively. A similar pattern was observed with CDC light traps, where dry ice-baited traps captured on average 97 sand flies/trap/night, compared to 33 sand flies/trap/night in traps baited with CO₂ derived from fermentation. In the same study, experiments using sticky traps baited with CO₂ produced from fermentation were also conducted in Ethiopia. The predominant species captured was Ph. orientalis, accounting for 70.3% of the total catch. Although overall trap catches were low, CO₂ significantly increased the number of females captured in the sticky traps. It must be noted that the efficiency of dry ice as bait outperformed the fermentation-derived CO₂ in terms of overall attraction, likely due to its substantially higher rate of CO₂ emission. Nevertheless, since CO₂ production can be achieved through fermentation with the use of low-cost and readily available ingredients (water, table sugar, and baker’s yeast), this method can be a practical alternative for both light or sticky traps in settings where access to dry ice is limited, or its use is logistically challenging (Moncaz et al., 2013).

The development and evaluation of such alternative CO₂ sources remain an active area of research, focusing on identifying solutions that balance efficacy, affordability, and operational feasibility across diverse ecological and logistical contexts. Future field studies should focus on better controlling and monitoring CO₂ release rates and duration of use. Furthermore, contamination of the solutions by bacteria can greatly compromise fermentation; therefore, additional advances are needed in this field to meet the consistency of controlled release from a CO₂ source.

3.4.2. Plants

Both sand fly sexes rely on plant sugar meals to obtain the energy needed for all important functions of their life-cycle, such as survival, flying, host-seeking, and mating. These sugar meals are typically derived from nectar, honeydew, and phloem sap. Therefore, the composition of local plant communities can have a significant effect on the spatial and temporal dynamics of sand fly populations, especially considering that some plants may even impair sand fly survival or leishmaniasis transmission competence. Despite the importance of plant meals, visual and chemical cues that attract sand flies to specific plant species remain largely unexplored (Abbasi et al., 2018; Hassaballa et al., 2021).

Scarce research studies, conducted in the greater Jordan Valley area, investigated the attraction of Ph. papatasi to several local plants (Schlein and Yuval, 1987; Schlein and Jacobson, 2008; Müller et al., 2011). According to the data obtained, plants with high respiration rates during the night tend to attract more sand flies. This trait is particularly advantageous for these crepuscular and nocturnal insects that forage under low-light conditions, as CO₂ emission reflects indirectly sugar availability. Moreover, higher sugar concentrations in plant tissues increased the feeding rates of sand flies. However, sugar concentration does not appear to play a significant role in initial attraction but rather influences the intensity of feeding once contact is made (Schlein and Jacobson, 2008). Interestingly, Ph. papatasi were attracted to specific plants when tested in the field but did not feed on them in laboratory experiments, indicating that sand flies are attracted to plants not only for sugar meals but also for shelter and breeding (Schlein and Yuval, 1987). Furthermore, some evidence shows differentiations in cue preferences between the sexes (Müller et al., 2011; Hassaballa et al., 2021). The limitation of these studies is that although some plant species were identified as attractants, the specific compounds that act as cues and are responsible for this effect were not determined. In follow-up research that tested individual volatile organic compounds (previously identified from preferred plants), all compounds elicited a dose-dependent positive response in sand flies. Among those tested, linalool oxide emerged as the most effective attractant (Hassaballa et al., 2021). These findings highlight the potential of leveraging specific plant-derived volatiles in developing novel sand fly control/monitoring strategies yet underscore the need for further research to fully characterize the chemical ecology underlying plant-sand fly interactions.

3.4.3. Animals as baits

Female sand flies, which require blood meals to develop their eggs, are not only important vectors of leishmaniasis, but are also a biting nuisance, even when they are not actively transmitting pathogens (Kasili et al., 2009). In order to design and apply control methods for reducing biting pressure in endemic regions, but also understand the bionomics of leishmaniasis that has a direct influence on pathogen dispersal, it is critical to identify the preferred hosts and potential reservoirs of Leishmania parasites (Gebresilassie et al., 2015). Blood-meal analysis techniques (molecular and serological methods) as well as animal bait bioassays have been used in an effort to assess host preferences (Gebresilassie et al., 2015; Yousefi et al., 2023). Most sand fly species are considered opportunistic feeders, which means that host abundance and accessibility in the field could bias the interpretation of host preference based on blood-meal analysis. In this context, experiments with specific animal baits, provide a controlled, reproducible, and ecologically meaningful understanding of sand fly host-seeking behavior and preferences.

The fragmented information on sand fly host preferences makes comparisons across studies challenging, primarily due to the limited number of investigations, variability in trapping methods, and differences in experimental design. However, some tendencies can be identified. For example, Ph. orientalis were more attracted to and fed on larger animals (cows and donkeys versus chicken, dog, goat, sheep, and humans) (Gebresilassie et al., 2015). Similarly, Phlebotomus guggisbergi females showed a clear preference for larger animals (goats and sheep versus hyrax, hamster, crested rat, dog, rabbit, and cat) in a field study conducted in Kenya (Johnson et al., 1993). Larger animals provide a bigger surface area for landing and feeding and produce more body heat and CO₂, which are critical cues for locating hosts. In another field experiment, goat- and dry ice-baited traps were equally attractive to Phlebotomus duboscqi (Kasili et al., 2009). Analysis of the data revealed a positive relationship between bait weight and sand fly attraction; the goat was the largest and heaviest animal tested. Interestingly, when the goat’s attractancy data were excluded, no difference was detected among the other baits (hamster, Nile grass rat, gerbil, and chicken), despite their varying weights. This result supports the notion that insects identify and navigate towards a host through a combination of visual, thermal, tactile and olfactory cues (Lehane, 2005). This point is further supported by the findings of Christensen and Herrer (1980), who studied the attraction of the endemic species in Panama to a wide variety of hosts. Their results suggest species-specific preferences, with a clear attraction of Lutzomyia vespertilionis (Dampfomyia vespertilionis) to bats and a preference of Lutzomyia panamensis (Psychodopygus panamensis), Lutzomyia olmeca bicolor (Bichromomyia olmeca bicolor) and Lutzomyia sanguinaria to other mammals (specifically rodents). Despite being among the smallest of the used baits, the aforementioned animals attracted the highest number of sand flies.

In the frame of Integrated Vector Management (IVM), understanding sand fly host preferences is essential for targeting interventions more effectively. By identifying the primary hosts that attract sand flies, IVM programmes can prioritize control measures around these hosts, whether through selective use of repellents, treatment of domestic animals, or environmental management. Additionally, knowing which wild mammals act as reservoirs can guide wildlife-focused interventions, contributing to the broader goal of reducing disease transmission (Balaska et al., 2021).

3.4.4. Animals infected with Leishmania

Accumulating evidence suggests that parasites can manipulate the behavior and physiology of hosts and vectors to facilitate their transmission (O’Shea et al., 2002). This parasite-host-vector relationship has prompted interest in whether Leishmania-infected animals attract more sand flies. Although only a limited number of studies have addressed this hypothesis, it was shown that infection with Leishmania infantum, the causative agent of visceral and cutaneous leishmaniasis, can increase host attractiveness to sand flies compared to uninfected animals. These studies have tested whether the parasite could affect the emitted odors from both natural parasite reservoirs, such as dogs (Chelbi et al., 2021; Staniek and Hamilton, 2021), and experimental animal models (Nevatte et al., 2017). The primary vector species examined have been Lu. longipalpis, although one study investigated the attraction of Ph. perniciosus and Phlebotomus perfiliewi (Chelbi et al., 2021). Notably, this latter study assessed attraction under both laboratory and field conditions, providing a broader ecological perspective. Results on the preference of male and female sand flies for infected hosts have been inconsistent. While some studies report greater attraction of females (Nevatte et al., 2017; Staniek and Hamilton, 2021), others have observed stronger responses in males (Magalhães-Junior et al., 2019). These inconsistencies may stem from differences in experimental approaches, including the use of live animals, odors collected from hair or whole bodies, or specific volatile compounds previously identified as infection biomarkers in dogs. Moreover, a variety of setups and behavioral assays have been employed, such as Y-tube olfactometers, wind tunnels, and two-choice bioassays, each of which may influence the observed outcomes. Regardless of sex, i.e. if the increased attraction involves male or female sand flies, any observed preference is epidemiologically relevant. Female sand flies are the vectors responsible for Leishmania spp. transmission, while males help sustain insect population growth and may contribute in some species to female attraction (to either infected or uninfected hosts) through their aggregation and pheromone production (Staniek and Hamilton, 2021).

In contrast to findings with L. infantum, two studies investigating cutaneous leishmaniasis parasites, namely Leishmania braziliensis and Leishmania amazonensis, using mice and hamsters as hosts, found no significant differences in sand flies’ attraction between infected and healthy animals (da Rocha Silva et al., 2019, 2024). Specifically, neither Ny. neivai nor Lu. longipalpis (both colonized species) showed a distinct preference for infected over uninfected animals or blood meal intake. Additionally, no difference in the volatile organic compound profiles between infected and uninfected hosts was detected.

There are still many aspects that need to be explored to fully understand the mechanisms and implications of parasite-induced changes in host attractiveness. Future studies should aim to identify the specific odor compounds involved in the attraction, and the effect of competing odors from the environment. Additionally, the role of parasite load and sex-specific vector responses should be assessed across different host species, sand fly vectors, and Leishmania parasites (ideally under natural or semi-natural conditions). This way, the broader applicability of these mechanisms in diverse epidemiological contexts could be unravelled, and their potential for developing targeted vector surveillance and control tools will be more accurately assessed.

3.4.5. Chemical compounds, kairomones, and diverse sources of olfactory cues

Significant aspects of the sand flies’ behavior are driven by chemical cues, which serve as essential signals in their ecological interactions. Chemicals that mediate behaviors, including host-seeking, foraging, mating, and oviposition, allow sand flies to locate blood or sugar sources, potential mates, and suitable breeding sites (Tchouassi et al., 2024a). Mating and oviposition will be analyzed separately in the following sections (3.4.6 and 3.7.1, respectively) as they rely on distinct and specific chemical signals and behaviors.

Beyond the use of specific baits (vertebrates or plants) to assess sand fly preferences, odors obtained from human hands or other vertebrate hosts (hamsters, fox) have been reported as attractants and/or activators (Hamilton and Ramsoondar, 1994; Oshaghi et al., 1994; Dougherty et al., 1999; Rebollar -Tellez et al., 1999; Bray and Hamilton, 2007). Indicatively, the response of sand flies to human emanations, as evaluated by the number of contacts (landings) on handled Petri dishes, was 130, compared to 70 contacts for the control group (Hamilton and Ramsoondar, 1994). A study on specific volatile organic compounds from human leg hair showed that phenylacetaldehyde, sulcatone, and icosane resulted in activation (defined as the initial flight response up to one-third of the distance toward the odor source), and attraction (closer approach of the odor source) effects on wild-captured species (75.4% Lu. (Ny.) intermedia) in wind tunnel experiments (Tavares et al., 2018). As far as odors from other vertebrates are concerned, traps baited with entrained volatiles from a hamster caught 64.4% of the sand flies, whereas the unbaited control trap caught only 15.6% (Oshaghi et al., 1994). In addition, Dougherty et al. (1999) revealed through bioassays and electrophysiological tests that female Lu. longipalpis detect fox gland odors via distinct receptor neurons in the ascoid sensillum. Sixteen identified compounds, including ketones, alcohols, aldehydes, and carboxylic acids, evoked specific, class-related neural responses. Each chemical type triggered a distinct neuron, with different signal strengths and interaction effects. This suggests that sand flies have sophisticated olfactory discrimination abilities.

Special attention has been given to specific compounds such as 1-octen-3-ol (octenol), a common alcohol found in mammalian breath and sweat, which has demonstrated consistent effectiveness in attracting several haematophagous flies. Interestingly, responses to octenol vary widely among sand fly species, ranging from strong attraction to neutrality or repellency, indicating that its effectiveness as a lure depends on the species and ecological context (Pinto et al., 2011). In general, New World species seem to present a consistent dose-dependent attraction response to octenol, with responses to the neat compound reaching up to 80% (Magalhães-Junior et al., 2014; Machado et al., 2015). It is worth mentioning that even the two isomers of octenol induce different responses in certain sand fly species, with the R-form generally acting as a more potent kairomone (Tchouassi et al., 2024b).

Alongside octenol, other alcohols and commercial synthetic lures have been tested to improve trapping methods and better mimic natural host odors (Pinto et al., 2012; Ortiz et al., 2020; Machado et al., 2015, 2022). However, their effectiveness varied considerably. For instance, while pentanol, hexanol, heptanol, octanol, nonanol, and decanol elicited significant activation and, to a lesser extent, attraction responses in Ny. neivai females, often in a dose-dependent manner, propanol and butanol did not differ significantly from the control. Notably, hexanol and octanol emerged as the most effective attractants, even surpassing octenol at lower concentrations. A mixture of highly responsive alcohols (heptanol, octanol, and nonanol at 1:1:1 ratio) showed enhanced activation (93%) but only moderate attraction (63%), suggesting that increased activation does not necessarily translate to higher attraction (Machado et al., 2015). Additionally, commercial lures like BG-Lure demonstrated inconsistent species-specific responses, effectively increasing Ny. whitmani catches in the field, but not attracting other species such as Nyssomyia antunesi or Pintomyia nevesi in the field or Ny. neivai in wind tunnel bioassays (Pinto et al., 2012; Ortiz et al., 2020).

A few studies have explored more complex and less defined odor sources, such as cow manure, several fruits, blood, and urine from livestock species, and monofloral kinds of honey as a proxy for natural floral nectar, which serves as a sugar source for sand flies in the wild (Schlein et al., 1989; Junnila et al., 2011; Wasserberg et al., 2014; Mong'are et al., 2015; Yousefi et al., 2020). Overall, these odor sources elicited species- and sex-specific responses, likely reflecting differences in feeding habits, reproductive status, and ecological niche across sand fly populations.

Despite significant progress, current knowledge on the olfactory cues mediating sand fly behavior remains incohesive and species-specific, often limited to isolated compounds or host contexts. A more integrated approach that would link electrophysiological, behavioral, and ecological data, is essential to fully understand the sensory mechanisms underlying host-seeking, mating, and sugar-feeding behaviors.

3.4.6. Pheromones

Pheromones are chemical signals released by an individual that influence the behavior or physiology of other members of the same species. Their use as attractants to lure insects into traps or enhance the efficiency of insecticide spraying has become a standard approach in agricultural pest management (Chelbi et al., 2011). This concept is being also explored in the context of vector control, especially for medically important insects such as sand flies.

Research has focused on the identification and functional characterization of pheromones in sand fly species, with particular attention to Lu. longipalpis, the main vector of visceral leishmaniasis in Latin America. Lutzomyia longipalpis is recognized as a species complex, with ongoing debate regarding how to delineate its members. A distinguishing feature among these sibling species is the specific sex-aggregation pheromone produced by males (Bell et al., 2018). The pheromone composition varies among members of the complex, suggesting a role in reproductive isolation and species divergence (Bray et al., 2009; Magalhães-Junior et al., 2019). In early publications, these pheromones were broadly categorized based on their chemical structure as “diterpenoid-like” and “farnesene-like”, prior to the formal identification and naming of specific compounds. To date, up to four distinct pheromone types have been identified among Lu. longipalpis populations from several Brazilian states and other South American countries.

In nature, male Lu. longipalpis commonly form aggregations (leks) on or above host animals, which attract females for mating. These leks are driven by male-produced terpene pheromones, synthesized in abdominal glandular tissue from isoprene units, which, in combination with host odors, attract both males and females and are crucial to the species’ mating behavior. Supporting this field observation, laboratory experiments have shown that an extract from male tergal glands (diterpenoid-like pheromone, C₂₀H₃₂) can elicit strong and rapid attractancy of Brazilian (Sobral, Ceará State) Lu. longipalpis females to live hosts, such as hamsters, even at distances reaching almost 2.5 m (Morton and Ward, 1989). Hamilton et al. (1994) analyzed tergal gland extracts from Lu. longipalpis males collected in Jacobina, Brazil (Bahia State) and identified a major nonpolar hydrocarbon peak as the primary sex pheromone component. Behavioral bioassays showed that this compound elicited strong attraction in virgin females, though the response was slightly lower than to the full extract. When combined with a minor component, the response matched that of the whole extract, indicating a synergistic effect. In contrast, the minor component alone and other fractions produced little or no attraction, similar to solvent controls. Similar behaviors by females of the same species, originating from Colombia, were observed when conspecific male pheromone extract was used in combination with several artificial host factors. The Melgar (Colombia) flies studied in this work produce a farnesane-like pheromone, which has a shorter carbon chain compared to the diterpenoid-like pheromone produced by other Lu. longipalpis populations (Nigam and Ward, 1991). Furthermore, Spiegel et al. (2005) provided evidence that the male-produced pheromone (1S,3S,7R)-3-methyl-α-himachalene (natural or synthetic) of Lu. longipalpis from Jacobina (Brazil), not only attracts females but also serves as an aggregation stimulus for males. Through electrophysiological recordings and behavioral assays, they demonstrated that receptor cells in the antennae of both sexes respond specifically to this pheromone isomer and that both males and females fly upwind toward synthetic pheromone sources or gland extracts in wind tunnel experiments. Kelly and Dye (1997) have investigated the aggregation dynamics of Lu. longipalpis and the factors that affect them. They found that males migrate to aggregation sites earlier than females and often return to the same location on subsequent nights, indicating a kind of spatial memory. Male immigration rates increased with both host and sand fly abundance, while female immigration depended primarily on the number of sand flies already present. Females tended to leave aggregations more quickly than males, likely driven by the need to blood-feed and find a resting site, while males remained longer, possibly influenced by pheromones.

Subsequent studies have demonstrated the practical potential of leveraging male-produced sex pheromones for vector control. Bray et al. (2009, 2010, 2014) developed and tested synthetic compounds of the pheromone (±)-9-methylgermacrene-B, showing its effectiveness in attracting both male and female Lu. longipalpis under field conditions, even in insecticide-treated sites. These lures, when optimized for release rate and longevity, could remain effective for up to 12 weeks and significantly enhance the impact of residual spraying by concentrating sand fly populations at treated locations. Additionally, host odor enhanced the attractiveness of the pheromone, particularly for females. Using pheromone dispensers greatly increased trap catches, whether mechanical (e.g. CDC traps) or passive (e.g. sticky traps). Later studies (Bell et al., 2018; González et al., 2020) revealed that attraction is dependent on pheromone (9-methyl-germacrene-B) quantity rather than the proximity of lures, making clustered deployment feasible and that sand flies can detect and respond to the pheromone from distances up to 30 m, particularly in the presence of host odors. These findings suggest that integrating synthetic pheromones into control programmes can enhance sand fly suppression and potentially reduce the transmission of visceral leishmaniasis, at least for this sand fly species.

While the pheromone-mediated behavior of Lu. longipalpis has been studied and leveraged in vector control research, there is comparatively limited literature on Old World vectors of leishmaniasis. Notably, among the studies reviewed here, only two specifically address pheromone-related phenomena in Ph. papatasi, focusing on the existence of sex- and/or aggregation-related chemical cues and their potential role in mediating attraction between individuals. Early findings of Schlein et al. (1984) suggest that there is a pheromone produced by females that induces attraction and stimulates feeding of other conspecific females, as evidenced by a three-fold increase in the number of feeding females when membranes previously used by engorged females were reapplied. Extracts of different body parts of both males and females have been tested in laboratory experiments as potential sources of the aggregation pheromone. Female palps and mouthparts exhibited pheromonal activity, with particular attention given to large vacuolated cells located in the third- and fourth palpal segments and in Newstead’s sensilla, suggesting these structures as the likely sites of pheromone production. The response was shown to be olfactory and optimal at around 28 °C, while higher temperatures eliminated the effect.

Contradictory results were found in a separate study. Chelbi et al. (2011) provided evidence for a male-produced sex pheromone in Ph. papatasi. Using both laboratory and field-based approaches, they demonstrated that young females (≤ 3 days old) were attracted to headspace volatiles from small groups (5 individuals) of males or mixed male-female groups (5 individuals per sex), but not to females alone. In contrast, males showed no attraction to either sex or to mixed groups. Interestingly, larger groupings of males or mixed flies were found to be repellent under laboratory conditions, suggesting a dose-dependent or concentration-sensitive response in female attraction as measured with a Y-tube olfactometer bioassay. The response in Ph. papatasi was chemically driven, as visual and auditory stimuli were eliminated due to experimental design. In the same work, field trials using CDC light traps baited with small mixed-sex groups captured significantly more females than unbaited controls, while traps with larger groups caught fewer flies overall (though the proportion of females among the total catch remained higher). These results imply that female Ph. papatasi are highly sensitive to male pheromone concentration and may prefer to approach low-density male aggregations, a pattern markedly different from the lekking behavior observed in Lu. longipalpis. Further research is essential to identify the specific pheromonal chemical cue(s), if any, in the Old World sand fly species and also to evaluate any potential application to targeted vector control tools.

3.5. Combined cues

In the previous sections, most studies have analyzed the responses to the tested parameters (visual or olfactory cues) individually. However, sand flies likely respond to complex combinations of these signals in natural settings. Investigating how multiple sensory modalities interact offers a more ecologically valid perspective and can reveal synergistic effects that may not be apparent in single-cue experiments. This section includes studies that examine the behavioral responses of sand flies to combinations of sensory cues, including olfactory, visual, thermal, and humidity-related stimuli (Supplementary file 3: Table S5). From the simple addition of CO₂ sources (Chaniotis, 1983), host odors (Teodoro et al., 2007), or the use of chemical attractants (da Silva et al., 2019; Mikery et al., 2022) in light traps, to more complex trap configurations incorporating several colored LEDs, chemicals, heat, and humidity (Mann et al., 2009; Kline et al., 2011; Müller et al., 2015), combined sensory cues have consistently resulted in significantly higher sand fly captures compared to controls.

For instance, adding a controlled CO₂ source (dry ice in a box) to light traps increased significantly sand fly captures (especially females) compared to light alone. Most importantly, the species composition did not differ between control and treatment, highlighting the wide attractiveness of CO₂ across multiple sand fly species (Chaniotis, 1983). Likewise, in a study conducted in a peridomicile area, the presence of hens combined with electric light (particularly at an intensity of 3 W) increased significantly the number of Ny. whitmani sand flies captured. Light alone or hens alone attracted fewer sand flies, indicating a synergistic effect of visual and host cues (Teodoro et al., 2007). The electric aspirator collected more sand flies than the Falcão trap under these conditions, suggesting device efficiency also plays an important role in the final outcome (Teodoro et al., 2007). The combined effect of olfactory and visual stimuli likely results from how different sensory cues operate over varying distances. Host-related odors such as CO₂ act as long-range attractants and can guide sand flies from several meters away, while visual stimuli, such as light, are generally effective at shorter distances. Light disrupts insect’s ability to orient in the environment and, as a result, guides it toward the light source in the trap (Müller et al., 2015). Mann et al. (2009) noticed a species-specific response of Lutzomyia (Psathyromyia) shannoni and Lutzomyia (Micropygomyia) vexator to combinations of colored LEDs and odor attractants (CO₂, octenol, and hexenol), but in general, multi-modal traps were more effective than single-cue traps. Female Ph. papatasi also showed increasing capture numbers when parameters mimicking hosts (heat, moisture, contrasting or dark colors) were combined in the traps (Kline et al., 2011; Müller et al., 2015).

Collectively, these findings underline the importance of considering cue interactions rather than treating each modality in isolation. Such an approach is particularly valuable for the design of integrated vector management tools, which may benefit from the strategic combination of sensory stimuli to increase specificity and efficacy.

3.6. Attractive toxic sugar baits (ATSBs)

Attractive Toxic Sugar Baits (ATSBs) are an “attract and kill” strategy that exploits the natural sugar-feeding behavior of sand flies and other insects by combining phagostimulant sugars with oral toxins and attractants (Balaska et al., 2021). The four studies included in this review were conducted in the Jordan Valley region, Morocco, and Iran and demonstrated significant reductions in sand fly densities, often exceeding 80%. More specifically, ATSBs comprising sugar sources and attractants like juice of over-ripe nectarines, wine, and brown sugar or commercial baits were used in formulations with insecticides such as boric acid, spinosad, and dinotefuran (Schlein and Müller, 2010; Müller and Schlein, 2011; Qualls et al., 2015; Saghafipour et al., 2017). The applied methods investigated in the studies included spraying vegetation, treated barrier fences, and bait stations, with spraying vegetation typically showing the highest efficacy (Supplementary file 3: Table S6). The study sites varied from arid desert environments with sparse vegetation to agricultural and rural village settings, encompassing both sugar-rich habitats dominated by fruiting cacti and sugar-poor landscapes with limited floral resources, indicating the applicability of the method across different ecological settings. For example, ATSB treatments using dinotefuran, whether applied to vegetation or delivered via bait stations, proved effective in both sugar-rich and sugar-poor environments, sustaining reduced sand fly populations for up to five weeks post-application. However, the observed decline in sand fly populations was more delayed in the sugar-rich sites. While the ATSB approach seems to be promising, care should be given on ecological safety and the potential impact on non-target species (Kapaldo et al., 2018).

3.7. Special category

3.7.1. Oviposition

Gravid females are considered the most epidemiologically important physiological stage, as they have taken at least one blood meal and may have acquired pathogens. Additionally, ovipositing females contribute to the amplification of the sand fly population by laying eggs. Vector control tools targeting these females could significantly improve the efficacy of IVM programmes by reducing the potential for population growth while minimizing the risk of pathogen transmission (Wasserberg and Rowton, 2011; Marayati et al., 2015).

In the absence of parental care, as is typical for most insects, female sand flies rely heavily on the selection of suitable oviposition sites to maximize the survival and reproductive success of their progeny (Kowacich et al., 2020). The oviposition behavior is determined by several factors, including environmental conditions, i.e. season, temperature, relative humidity, photoperiod (Schlein et al., 1990), and various cues that have a direct impact on the oviposition site selection and oviposition rate. Gravid females locate places to lay their eggs by using a combination of different sensory systems, like receptors on the antennae, tarsi, and mouthparts (Rama et al., 2014a; Shymanovich et al., 2018). Sand flies typically lay their eggs in terrestrial microhabitats rich in organic material, where they also develop through four larval stages before pupating and finally emerging as adults (Shymanovich et al., 2018).

Studies have begun to unravel the importance of environmental cues in oviposition site selection and oviposition stimulation. The reviewed studies on oviposition, grouped by sensory cue type are summarized in Supplementary file 3: Table S7. The role of conspecific eggs (whole or extracts) as oviposition stimulators has been confirmed in laboratory experiments for several sand fly species, including Lu. longipalpis, Phlebotomus dubosqui, Ph. papatasi, Phlebotomus argentipes, Sergentomyia schwetzi and Sergentomyia ingrami, although for the latter the effect was marginally not statistically significant (Elnaiem and Ward, 1991; Dougherty et al., 1992, 1994; Basimike, 1997; Rama et al., 2014a; Kowacich et al., 2020). It was found that the stimulation substance of the Lu. longipalpis eggs was secreted onto them by the accessory glands of the sand flies (Dougherty et al., 1992), and it was later identified as dodecanoic acid (Dougherty and Hamilton, 1997). Elnaiem and Ward (1991) also investigated the relations between oviposition rate of Lu. longipalpis and age or number of the eggs used as stimulators. They concluded that the oviposition rate was not affected by the age (days) of the stimulators, but the oviposition response was positive when more than 80 eggs were included per rearing pot. Responses with 20 or 40 eggs were not different from controls, while oviposition rates increased between 80 and 160 eggs but showed no further change when the number of eggs was raised to 320. The dose-dependent response was also confirmed with recordings from the electrophysiological responses of a single sensillum from the antennae of gravid Lu. longipalpis females, when the synthetic pheromone (dodecanoic acid) was used at doses up to 1000 μg (Dougherty and Hamilton, 1997). Kowacich et al. (2020) used Ph. papatasi to study separately the two stages of oviposition response, i.e. attraction to oviposition site and stimulation of oviposition, and the effect of egg or dodecanoic acid dose. They found that at low doses (0.01–0.1 μg) of dodecanoic acid there was strong attraction and oviposition stimulation, while higher doses resulted in neutrality or repellence/deterrence. The relationship between the site attraction and the number of eggs as baits followed a bell-like shape. In contrast, oviposition results showed an opposite shape curve (U-like curve) with the increasing number of eggs, indicating a qualitatively different response. These opposing patterns with respect to conspecific egg numbers suggest that attraction and oviposition may be regulated by distinct semiochemicals with differing dose-response dynamics.

In addition to conspecific eggs, other sand fly stages and body parts have been tested as potential oviposition attractants/stimulants, including larvae, pupae, adults, accessory glands, and bodies without glands (Dougherty et al., 1992; Basimike, 1997; Kowacich et al., 2020). All stages and body parts elicited an attraction/stimulation response, except for bodies without glands (Dougherty et al., 1992). In the case of the early immature stages (eggs, first-instar larvae), whole bodies, or glands, the positive response is attributed to the presence of dodecanoic acid. A single published work was retrieved, concerning the effects of several conspecific developmental stages (eggs, first- and fourth-instar larvae, pupae, male and female adults) on the number of laid eggs by Se. ingrami, Ph. duboscqi and Se. shwetzi (Basimike et al., 1997). In this study, gravid females were exposed to filter papers smeared with crushed eggs, first- and fourth-instar larvae, pupae, and adult males and females, which were placed separately in vials serving as potential oviposition sites. Each test material was paired with an unbaited control, and oviposition responses were evaluated after seven days by counting the number of eggs laid in each oviposition site. Interestingly, the results showed species-specific patterns, with Se. shwetzi showing significant positive responses to all stages, while Ph. duboscqi exhibited strong responses to eggs, pupae, and adults, but not to larvae. Sergentomyia ingrami showed significantly increased egg-laying in response to fourth-instar larvae, pupae, and adult stages, but not to eggs (marginally) or first-instar larvae. Furthermore, interspecific tests revealed that Se. ingrami responded significantly to adult female Ph. duboscqi. In all cases, the oviposition responses to conspecific and interspecific test materials were positive, even when statistical significance was not detected, supporting the idea that oviposition in sand flies is mediated by cues elicited from almost all developmental stages. It is also plausible that, besides dodecanoic acid, these responses were influenced by the release of other chemicals from the excreta and/or bacteria of the smeared sand flies (Kowacich et al., 2020). To sum up, gravid sand flies are likely to prefer recently colonized oviposition sites (presence of first-instar larvae and/or eggs), indicating suitable conditions and high offspring survival potential, but possibly tend to avoid sites with more developed stages, indicating potential competition and resource depletion. Attention has also been given to the role of larval food sources and other organic materials commonly found in natural breeding microhabitats. Organic materials (rabbit and chicken feces, cow manure, rabbit chow, hay) or their extracts, as well as chemical compounds identified from fecal extracts (2-methyl-2-butanol and hexanal), but also saprophytic and gut bacteria, have been shown to enhance both attraction and stimulation in various sand fly species, i.e. Lu. longipalpis, Ph. argentipes, and Ph. papatasi (Elnaiem and Ward, 1992a; Dougherty et al., 1993, 1995; Peterkova-Koci et al., 2012; Rama et al., 2014b; Marayati et al., 2015; Faw et al., 2021; Kakumanu et al., 2021). These substrates not only provide essential nutrients for the developing larvae but also emit volatile compounds that may serve as olfactory cues influencing oviposition behavior. Studies have shown that gravid sand flies are able to detect and respond to these chemical cues, suggesting that the presence of suitable larval food plays an important role in guiding oviposition site selection and oviposition rate.

It must be noted though, that oviposition substrates not only elicit semiochemicals but also stimulate thigmotropic responses (Dougherty et al., 1994). Limited studies have shown that tactile cues from substrates also affect sand fly oviposition behavior. Irregular surfaces that mimic natural breeding sites, such as crevices, were preferred for oviposition by both Lu. longipalpis and Lutzomyia (Migonemyia) migonei gravid females compared to flat surfaces (Elnaiem and Ward, 1992b; Nieves et al., 1997). The size of the oviposition pots did not seem to play a significant role.

Regarding the effect of visual cues on oviposition behavior, a single laboratory study was retrieved. The obtained results suggest that gravid Ph. papatasi females are attracted to features that resemble terrestrial microhabitats (dark round objects, i.e. black jars, possibly resembling openings of rodent burrows), while other female stages or males do not seem to be influenced by them (Shymanovich et al., 2018). Moreover, the researchers were able to record that, as expected, under complete darkness, sand flies were attracted by the olfactory cue (larval frass) regardless of the color of the oviposition site. On the contrary, as the light level increased, visual cues prevailed, and sand flies preferred the darker sites. It must be highlighted, though, that this shift took place under specific laboratory conditions, where sand flies could clearly distinguish the black oviposition jars from a brighter background. In nature, oviposition occurs during crepuscular periods. Therefore, visual and olfactory response systems to cues are most likely interrelated and complement each other. Shymanowich et al. (2018) suggested that vision might be used to orient and navigate sand flies from a long-to medium distance under dim light conditions, but olfactory stimulation determines the suitability of a site for oviposition. However, this remains a hypothesis based on controlled experimental conditions, and it is unclear how well these findings translate into more complex natural environments. Building on the experimental findings from controlled laboratory and semi-field studies, observational field investigations provide additional ecological context. For example, Vivero et al. (2015) investigated the breeding sites of sand flies in rural and urban areas of Colombia. They identified immature sand fly stages in soil samples and associated them with specific vegetation and physicochemical soil properties. Natural breeding sites of phlebotomine sand flies were found to vary across habitats. Tree species and soil characteristics, including organic matter, pH, and moisture, played a decisive role in larval survival, highlighting the strong influence of microhabitat features on sand fly development. Although observational, these findings shed light on habitat features that influence sand fly oviposition, linking laboratory results to conditions encountered in natural environments. It is apparent that oviposition site selection and oviposition stimulation involve the integration of chemosensory, visual, and thigmotropic cues, as articulated by Dougherty et al. (1993), who proposed that gravid females are initially attracted by the physical and chemical properties of the substrate and are later stimulated to oviposit by conspecific egg pheromones in close proximity.

A better understanding of oviposition behavior will not only facilitate the development of targeted control strategies but will also contribute to overcoming key challenges in sand fly research. In particular, it may help address the high mortality of gravid females during oviposition in laboratory conditions, which hampers colony establishment, biological studies, and experimental investigations of Leishmania and arbovirus transmission.

3.7.2. Immature stages

The terrestrial breeding nature of sand fly immature stages, in combination with their extremely small size, poses significant challenges to their detection and control under field conditions. As a consequence, the research focus has shifted to adults, resulting in a notable lack of effective detection and control strategies targeting the immature life stages.

A recent effort to gain a better understanding of the early stages of sand fly development, has studied the influence of environmental factors, such as organic matter and the presence of conspecific eggs, on egg hatching regulation. Nguyen et al. (2021) found that Ph. papatasi eggs were stimulated to hatch in the presence of an aqueous extract of larval rearing substrate when the eggs were dispersed. On the contrary, a hatching inhibition tendency was observed when the eggs were accumulated (placed in clusters). These findings imply, for the first time, that there is a regulation mechanism that modulates the emergence of larvae in response to environmental cues that indicate the suitability of the habitat for subsequent development and survival. Clarifying these mechanisms could offer valuable insights into population dynamics, which are critical for epidemiological studies.

A proof-of-concept study on the chemotaxis of sand fly larvae was recently published by Tsikolia et al. (2024). In their study, they used Ph. papatasi larvae in a series of novel bioassays to investigate their response to gustatory and olfactory cues from the larval food. According to the results, the larvae were able to distinguish food sources and showed a clear food preference, within a few hours of exposure in a two-choice bioassay. In addition, they seem to be able to detect and respond from a distance to olfactory cues from their preferred feeding sources. The identification of the specific chemicals or mixtures that trigger the larval response could be further valorized in designing targeted larval surveillance tools in the frame of an attract-and-kill strategy.

3.8. Limitations

This review has certain limitations that should be acknowledged. The literature search was restricted to publications in English. However, given that most peer-reviewed research in this area is published in English by a concentrated group of specialized institutions and researchers, we consider the risk of omitting significant findings minimal. Another limitation is the exclusion of environmental and ecological factors that may modulate attraction, such as moonlight/starlight, ambient humidity, wind conditions, and temperature gradients, factors known to influence sand fly activity but not systematically reported or controlled across studies.

4. Conclusions

This systematic review identified, retrieved and synthesized the available research on the attraction of sand flies to various stimuli, including visual and olfactory cues, and their combinations, with a focus on critical behaviors for sand fly survival (such as host-seeking, sugar-feeding, oviposition site selection). The vast majority of knowledge involves adult sand flies, with limited but promising new research efforts targeting the chemical ecology of immature stages. Significant progress has been made, especially since 2010, in identifying potential attractants (and combinations thereof) and understanding their effects on sand fly behavior under laboratory and field conditions. Studies, however, vary considerably in focus and methodology, leading to a fragmented picture of the topic and thus substantial knowledge gaps remain. With a view toward practical applications, integrating these findings into IVM strategies, particularly through the development of attract-and-kill methods, represents a promising avenue for sustainable sand fly control. However, the true potential of attractants will only be realized through a comprehensive and coordinated approach that addresses existing limitations, including variability in efficacy, environmental context, and species-specific responses. Future research should aim for greater standardization in experimental design and reporting, consider the integration of environmental variables, and expand investigations to lesser-studied sand fly species and behaviors. Additionally, a deeper understanding of the biochemical or molecular mechanisms influencing responsiveness to attractants along with the multi-modal cues driving sand fly attraction, across their life stages, will be crucial for developing effective, species-specific surveillance tools and control interventions.

CRediT authorship contribution statement

Panagiota Tsafrakidou: Conceptualization, Investigation, Data curation, Writing – original draft, Writing – review & editing. Arsen Gkektsian: Investigation, Data curation, Writing – original draft. Michael Miaoulis: Investigation, Data curation, Writing – original draft. Lee W. Cohnstaedt: Writing – original draft, Writing – review & editing. Alexandra Chaskopoulou: Conceptualization, Writing – original draft, Writing – review & editing, Supervision, Resources, Project administration.

Ethical approval

Not applicable.

Statement on the use of AI-assisted technologies

During the preparation of this paper, the authors used ChatGPT in order to improve data extraction, language and readability. After using this tool, the authors reviewed and edited the content as needed. The authors take full responsibility for the content of the published article.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

All data analyzed in this study are included in this published article and its supplementary files.