Exploiting nectar and blood feeding cues and phagostimulants to optimise Attractive Targeted Sugar Baits against a sand fly vector of leishmaniasis

Daniele Pereira Castro; Fernando Ariel Genta; Matthew E. Rogers

doi:10.1371/journal.pntd.0013888

Abstract

Background

Leishmaniasis presents a major public health problem for a large number of countries requiring effective integrated management of the vector, sand flies, for sustained control. Such strategies need to be economically and environmentally sustainable and adaptable to the behaviour of local vectors. One such tool is Attractive Targeted Sugar Baits (ATSB) that exploit the necessity of sand flies to acquire sugars between bloodmeals. Here we explored the kinetics and cues for sugar and blood feeding to improve the efficacy of ATSBs against sand flies.

Methods

A fluorescent assay was developed to quantify sugarmeals to assess the feeding efficiency of colony-reared female Lutzomyia longipalpis sand flies.

Results

Sand flies showed a range of preferences for different sugars presented on cotton wool and could be manipulated to deposit them into the crop and/or midgut. We found that the combination of 10% sucrose and 10% fructose allowed flies to obtain the largest sugarmeals taken to the crop. Sugarmeals were taken to both the crop and midgut when it contained 200 mM bovine serum albumin (BSA) as a source of protein and 1 mM adenosine triphosphate (ATP) as a phagostimulant. Using this combination, the efficacy of the ingested insecticide fipronil was significantly increased; reducing the 50% lethal concentration from 584 µM to 1.65 µM in a sugarmeal that promoted the simultaneous uptake of the insecticide into the midgut as well as the crop.

Conclusions

In this study we highlight the potential of understanding the cues used by vectors to sugar feed and blood feed. By incorporating blood feeding phagostimulants, such as BSA and ATP, in ATSB we vastly improve their killing efficiency against sand flies. This demonstrates a new approach to target these disease vectors.

Author summary

Many insect vectors of disease use sugars, such as nectar, as fuel to enable them to obtain blood, that in turn sustains parasite transmission. In most blood feeding Diptera sugars are diverted to the crop where it is stored and released into the midgut for energy whereas blood is taken directly to the midgut where it is digested to provide nutrients for egg development. For sand flies the cues required to switch between sugar and blood feeding programmes are unknown but if they were they could be exploited to improve Attractive Targeted Sugar Baits (ATSB) as a means of poisoning sand flies when they sugar feed. In this study we find that the sand fly, Lutzomyia longipalpis, uses a combination of physical cues (biting/piercing vs sucking) and meal composition to allow them to efficiently feed on nectar and blood. We show that they sense protein and adenosine triphosphate (ATP) to direct blood to the midgut and we demonstrate that this can be turned to our advantage to improve the lethality of ATSB. We show that inclusion of bovine serum albumin as a source of protein and the phagostimulant ATP can trick sand flies to blood feed on sugar and direct the insecticide fipronil to the midgut where it was 355-fold more potent than in sugar alone. These results show the potential of phagostimulants to improve the efficacy and selectivity of ATSB towards blood feeding arthropods, including sand flies, and highlights the value of understanding the feeding programmes of disease vectors in general.

Citation: Castro DP, Genta FA, Rogers ME (2025) Exploiting nectar and blood feeding cues and phagostimulants to optimise Attractive Targeted Sugar Baits against a sand fly vector of leishmaniasis. PLoS Negl Trop Dis 19(12): e0013888. https://doi.org/10.1371/journal.pntd.0013888

Editor: Adly M.M. Abd-Alla, International Atomic Energy Agency, AUSTRIA

Received: July 2, 2025; Accepted: December 22, 2025; Published: December 26, 2025

Copyright: © 2025 Castro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The original data is provided as a supplementary information file.

Funding: DPC received a salary and was funded by a CAPES - SENIOR VISITING PROFESSOR CAPES-PRINT - 88887.878914/2023-00. FAG received a salary and was funded by the National Council for Scientific and Technological Development – CNPq - Produtividade em Pesquisa - 312305/2022-2 and FAPERJ – Cientista do Nosso Estado – E-26/200.454/2023. MER received a salary and was funded by the Biotechnology and Biological Sciences Research Council (David Phillips Fellowship awarded to MER, BB/H022406/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Attractive Targeted Sugar Baits (ATSB) have been shown to effectively control Anopheles vectors by providing an ‘attract-and-kill’ approach [1]. They take advantage of the necessity of mosquitoes and other dipteran vectors to feed on sugars for daily survival. It consists of a bait that is attractive to the target vector species, combined with an oral toxin and sugar to stimulate feeding. The toxin is then diverted in to the midgut to exert its effect when the vector requires the sugars for energy to aid their survival, fecundity, flight ability and host-seeking activity. Reducing the vector density is the primary aim of ATSB [2]. With the development of insecticide resistance in malaria vectors and increased outdoor transmission, primary control methods such as insecticide-treated nets (ITN) and indoor residual spraying (IRS) are becoming less effective [3–5]. Therefore, supplementary vector control tools like ATSBs are urgently required. However, to be effective any ATSB toxin needs to access the midgut as quickly as possible and in sufficient amounts to kill the insect.

Female sand flies are the principal vector of leishmaniasis, a group of neglected tropical diseases afflicting people in 99 countries globally with at least 700,000 new cases reported annually [6]. Sand flies acquire and transmit Leishmania parasites when they bloodfeed. Between bloodmeals sand flies require sugars, obtaining them from various sources, including, flower nectaries, plant sap, fallen fruits and honeydew from other sap-sucking insects [7–9]. Sand flies use these sugars to fuel their metabolically demanding lifestyle and flight. Sand flies are small (1–3 mm) but can rapidly engorge on blood and take large meals 2–3 times their body size in 1–2 minutes to obtain sufficient protein to produce 30–50 eggs [10,11]. Blood is diverted through the stomodeal valve into the midgut where it is concentrated, surrounded with a peritrophic matrix and digested [12]. Less is known about the sugar feeding habit of these vectors (due to their cryptic nature and distribution across a wide diversity of ecotypes) but they are expected to take small, intermittent nectar meals that are initially deposited into the crop and delivered to the midgut to provide energy as required [13,14]. Therefore, sand flies have distinct blood and nectar feeding programmes allowing them to direct these resources into different organs to be used for different metabolic functions.

Although not known, these programmes are expected to be controlled by sensory input from the meals, cues from the host and the physical act of biting. In this study we sought to begin to uncover these cues by using a fluorescent dye to quantify the volume of sugars imbibed by female Lu. longipalpis sand flies and its diversion to the crop and midgut. This allowed us to show that these sand flies mostly use physical cues to activate their bloodfeeding programme but blood-derived phagostimulants could override them to direct sugars into the midgut. Field trials of ATSB have shown to reduce local sand fly abundance in arid ecotypes [15–17]. Here we show that phagostimulants can be exploited to further improve the efficacy of ATSB towards sand flies.

Materials and methods

Reagents

All reagents were sourced from Sigma Aldrich, UK, unless stated otherwise.

Sand flies

The Lutzomyia longipalpis colony of sand flies (originating from Jacobina, Bahia State, Brazil) was maintained at the London School of Hygiene and Tropical Medicine as previously described [18]. Flies were kept at 26°C and 75% ± 5% relative humidity in a daily light:dark cycle of 6:18 hours. The colony was maintained on fresh human blood and the larvae reared on an autoclaved mixture of compost, daphnia, mouse diet and fine sand. In all experiments unfed female flies 2–3 days post-emergence were used.

Sugar meal determination assay

Sand flies were exposed to cotton wool pads soaked in various sugar solutions or water containing a fluorescent dye to determine the feeding proportion, the volume of sugar imbibed and the deposition of sugars into the crop and midgut. Female sand flies were transferred to a 15 cm³ netting cage and allowed to acclimatise for 2 hours at 26°C in a humidified (70%) 25 L clear plastic bag. During a feeding experiment, the starved sand flies (3 days post-emergence, deprived access to sugar or water) were supplied with a 6 cm diameter (0.7 g) cotton wool pad (makeup removal pad) placed on top of each cage and soaked with 6 mL 10% sucrose and 10% fructose (w/v, 1:1) dyed with 0.002% (w/v) fluorescein and placed in darkness. After 2 or 4 hours the sugar-soaked pads were removed. Different phagostimulants, 1 mM adenosine triphosphate (ATP), 200 mg/mL bovine serum albumin (BSA), and different salts, 140 mM sodium chloride (NaCl) and 25 mM sodium bicarbonate (NaHCO₃), were added to the fluorescein-dyed 10% sucrose-10% fructose solution either singularly or in combination to determine the best sugarmeal components for midgut engorgement.

Crop and midgut collection and processing

Sand flies were knocked down on ice for 5 mins then washed in a weak detergent solution to remove setae from the body. Washed sand flies were transferred to a dissecting microscope and the entire alimentary tract (crop, midgut, hindgut and Malpighian tubules) was removed as one unit in PBS. To do this the head was removed first using the bevelled edge of a 0.1 mL insulin syringe and the 3^rd segment of the abdomen was partially cut. Using the needle the whole alimentary tract was drawn out of the carcass in one careful motion by pulling on the separated tip of the abdomen. The hindgut and Malpighian tubules were trimmed away from the midgut and the crop separated from the midgut. Working quickly, the crop and midgut were pipetted with 5 µL of the surrounding PBS and transferred into separate 1.5 mL Eppendorf tubes containing 95 µL PBS and placed on ice. Samples were frozen at -40°C until use.

To measure the fluorescence dissected crops and midguts were freeze-thawed three times by exposing them to hand-hot water for 2 min followed by vortexing for 10 s and dry ice for 2 min. Samples were centrifuged at 2000 rpm for 1 min to pellet any debris before transferring 15 µL to 85 µL PBS in wells of black 96 well flat-bottomed microtitre plates (Nunc). Fluorescence was measured using a Spectramax M3 plate reader (Molecular Devices) set at 485 nm excitation and 520 nm emission with 15 s shaking before reading.

Crop volume scoring

The sand fly crop was scored for their relative volume. Immediately after dissection the crop was recorded as either empty (presenting as a thin translucent diverticulum, joining the alimentary canal just anterior to the stomodeal valve), partially or fully fed with sugar.

Artificial membrane and cotton wool feeds

Different meals (10% sucrose-10% fructose, blood 50% in PBS and 50% blood in fluorescein-dyed 10% sucrose-10% fructose solution) were offered to unfed females either through chicken skin, an artificial membrane of parafilm in a Hemotek apparatus or cotton wool in the presence of the human sensory cues CO₂ (by blowing twice for 10 seconds into the cage immediately after adding the meal) and heat (37°C) for 2 hours.

Sugar insecticide assay

Thirty to forty female unfed sand flies (2–3 days post-emergence) were transferred to 455 mL carboard soup containers (Vegware), with a 115 mm diameter aperture, covered securely with a fine mesh and allowed to acclimatise for 2 hours at 26°C in humidified (70%) 5 L boxes. During the toxin-feeding experiment, the unfed sand flies were supplied with a cotton wool pad soaked with 6 mL 10% sucrose-10% fructose (w/v, 1:1) with or without 200 mg/ml BSA + 1 mM ATP and varying concentrations of fipronil (1–1000 µM) (RSPCA, UK) or water for 30 min at 26°C. Sand flies were monitored every 20 min for the first 140 min, followed by a 24 hour endpoint check. Feeding and survival were determined. The number of dead sand flies per total number was used for assessing survival and the median lethal concentration (LC50) was obtained through variable slope nonlinear regression using a Hill equation in GraphPad Prism 10.6.0.

Statistical analyses

Sample size calculation was carried out with the following criteria: (i) two or more independent groups with a continuous endpoint (volume of fluorescent sugar solution); (ii) detect differences between means with an anticipated mean ± standard deviation (s.d.) volume of 99 ± 97 nL and 20 ± 28 nL of 10% sucrose-10% fructose in 2 hour fed control crops and midguts (determined from a pilot experiment) and a 100% increase in the test; (iii) significance level alpha (chance of a Type I error – i.e., including false positives) of 0.05; beta (chance of a Type II error – i.e., including false negatives) of 0.20; (iv) study power (1-b) of 0.80; (v) sampling ratio (N_control/N_test) of 1 and (vi) minimum effect of interest of 200 nL and 40 nL in crops and midguts, respectively. This retuned a sample size of 6 flies per group [19]. The number of sand flies per cage was between 20–40 and assays were repeated 2–3 times depending on insect availability. Our sample size was in line with similar studies [11].

As the data was not normally distributed (assessed by a Shapiro–Wilk normality test), one-way analysis of variance (ANOVA) was used to evaluate the differences among experimental groups to compare a single factor across multiple groups. Post hoc multiple comparisons were conducted using the Dunn’s test to identify specific group differences. Results are presented as mean ± s.d., and statistical significance was defined as p < 0.05. All analyses were performed using GraphPad Prism software, version 10.6.0. Data for this study can be found in the supplementary information (S1 Data).

Results

Sand fly sugarmeal intake and fate

Sand flies, like other dipteran vectors, require frequent sugarmeals to maintain their activity which ATSBs exploit as a method for control. To understand the process of sugar uptake we determined the volume of sugar ingested into the Lu. longipalpis crop or deposited into the midgut using fluorescein serially diluted and homogenised with an unfed midgut and crop, followed by freeze-thawing. This allowed us to construct a calibration curve and determine that the average crop volume was 12 ± 6 nL (s.d.) after 24 h exposure to our standard rearing concentration of 50% sucrose (1.46 M) (S1 Fig). By comparison, the midgut had relative little sugar in it (1.8 ± 2.75 nL), which is consistent with the selective diversion of sugars into the crop. To find an optimal sugar meal for female Lu. longipalpis we tested a range of sucrose concentrations and found that 10% resulted in the largest crop volumes (35 ± 18 nL), and the highest proportion of sand flies that fed (31/40, 78%) (Fig 1A). Next, we tested a panel of mono- and di-saccharides commonly found in nectar [20] at the standardised concentration of 292 mM. Individually, sucrose resulted in the highest volume imbibed and stored in the crop. In combination with fructose, sand fly crops contained 3- and 4-fold more sugar than either sucrose or fructose alone (Fig 1B), yet the midgut remained relatively devoid of sugar.

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Fig 1. Intake and fate of nectar sugars in female Lutzomyia longipalpis sand flies.

Unfed sand flies were exposed to different nectar sugars (S = sucrose, F = fructose, M = maltose, Glc = glucose, Gal = galactose) dyed with fluorescein. (A and B) Volume of sugarmeals in the crop of sand flies after 4 hours exposure to increasing concentrations of sucrose (A) or different sugars and sugar combinations (10% final concentration) (B). (C) Volume of 10% sucrose-10% fructose meal in crop (CR) and midgut (MG) over 24 hours after exposure to sugars for 4 hours. Data pooled from 3 independent replicate experiments, n = 10–35/group. Bars represent the mean ± s.d. Asterisks indicate values that are statistically significant (**P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001) using a one-way ANOVA with Dunn’s multiple comparison test.

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To further understand the kinetics of sugar intake and deposition into the midgut we next measured crop and midgut sugar volumes in sand flies exposed to fluorescent meals of 10% sucrose-fructose (292 mM) for 4 hours. Flies were allowed to feed on this sugarmeal and sampled 2, 4, 6, 8 and 24 hours later (Fig 1C). We found that Lu. longipalpis rapidly filled their crop within the 4-hour exposure period and retained the sugar meal for the following 6 hours, during which the midgut contained only minimal amounts of sugar. Thereafter, the sugar volume in the crop gradually decreased, and a small but consistent amount became detectable in the midgut, presumably to be metabolized for energy. These findings suggest that, under experimental conditions, sugar ingestion by Lu. longipalpis occurs rapidly, is temporarily stored in the crop, and is gradually released into the midgut throughout the day.

Sand flies have separate blood and nectar feeding programmes, but they are not mutually exclusive

Mosquitoes have the ability to discriminate between nectar sugars and blood using specialised neurons within the stylet that pierces the skin [21]. This may allow them to feed regularly on nectar whilst maintaining their appetite for blood. In contrast, the control of the nectar and blood feeding programmes of sand flies is unknown. To see if blood feeding and nectar feeding are separate processes in sand flies, we presented blood and fluorescein-dyed 10% sucrose-fructose to unfed females either through chicken skin, an artificial membrane of parafilm or cotton wool in the presence of the human sensory cues CO₂ and heat. The proportion of flies that took these meals into their crop or midgut and the relative amount (none, partial, full) was recorded after 2 hours (Fig 2; S2 Fig). As expected, sand flies fed readily on blood though chicken skin in the presence of CO₂ and heat (100% fed with 87% obtaining full meals in their midgut) and obtain sugars from cotton wool irrespective of the presence of sensory cues (73% fed with 45% obtaining full meals in their crop). A similar result was found for sand flies feeding on blood through parafilm although the overall proportion that fed was much less compared to skin (27% fed with 100% obtaining full meals in their midgut). Flies exposed to blood alone did not take any to the crop, irrespective of the mode of delivery or presence of sensory cues. In contrast, sand flies did not take any blood to the midgut from cotton wool in the presence of CO₂ and heat but could feed on sugar through skin or an artificial membrane (via skin: 33% fed with 15% obtaining full meals in their crop; via parafilm: 5% fed with none obtaining full meals in their crops). These sugarmeals were found exclusively in the midgut.

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Fig 2. Sugar and blood ingestion through different modes of feeding.

Sand flies were fed 10% sucrose-10% fructose, 50% blood in PBS, or 50% blood in 10% sucrose-10% fructose solution through cotton wool, chicken skin or parafilm membrane for 2 hours. (A and B) Proportion of flies with sugar or blood in the crop (CR) and midgut (MG) and the relative amount (empty, partial meal, full meal). (C-E) Example images of midgut and crop from sand flies fed sugar (C) or blood + sugar (D&E) through cotton wool. Scale bar = 500 µm. Data pooled from 2 independent replicate experiments, n = 30-36/group.

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A mixture of blood and sugars resulted in blood being present in the midgut and crop when sand flies fed via cotton wool, skin, or parafilm (via cotton wool: 53% fed, 100% in the midgut and 25% in the crop; via chicken skin: 100% fed, 100% in the midgut; via parafilm, 34% fed, 100% in the midgut) in the presence of heat and CO₂. These results highlight the combined effect that the feeding mode (skin penetration, nectar sucking) and meal composition can exert on the feeding programme and destination of the meal. Further, they indicate that although sand flies use different blood and nectar feeding programmes in nature, they are not mutually exclusive and can be potentially manipulated to deliver products to the crop and midgut simultaneously.

Phagostimulants found in blood divert sugar into the sand fly midgut

From the results of Fig 2 it appears that sand flies can ingest sugar into the midgut but not ingest blood into the crop unless it is offered in combination with sugar. Previous work with Aedes aegypti has shown that it is possible to get these mosquitoes to engorge on an artificial meal of serum proteins or protein-free saline only if the phagostimulant adenosine triphosphate (ATP) was co-present with other plasma components, such as sodium chloride (NaCl) and sodium bicarbonate (NaHCO₃) [22,23]. This indicates that mosquitoes sense a combination of chemical cues present in blood and nectar to enable them to select an appropriate feeding programme. To see if female sand flies do something similar we tested whether the ingestion of sucrose-fructose into the crop and midgut could be influenced by the presence of the phagostimulants NaCl and NaHCO₃, bovine serum albumin (BSA) or ATP; either singularly or in combination. Fig 3A shows that after a two hour exposure to sucrose-fructose the amount taken to the crop can be substantially increased through the inclusion of ATP + NaCl, NaHCO₃, with a smaller increase shown for flies fed on sugars plus BSA + NaCl, NaHCO₃. In all groups the meal volumes in midguts are much lower than crop volumes but between groups the midguts of flies fed sugars with BSA + ATP + NaCl,NaHCO₃ had more sugar solution diverted to the midgut (17% vs 5% of the average sugarmeal). Twenty four hours later, Fig 3B, crop volumes were lower in the majority of groups and most of the midgut volumes remain unchanged with the notable exception of sand flies fed sugars with BSA + ATP, with or without NaCl and NaHCO₃. In these flies the average midgut volume increased 16-fold, demonstrating that sand flies could be manipulated to take large sugarmeals and divert them to the midgut.

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Fig 3. Role of bloodmeal-associated phagostimulants on sugar ingestion and deposition.

Volume of meals in crop (CR) and midgut (MG) of female sand flies fed 10% sucrose-10% fructose (SF) with or without NaCl + NaHCO₃ (salts), BSA or ATP after 2 hours exposure (A) and 24 hours later (B). Data pooled from 2 independent replicate experiments, n = 10–32/group. Bars represent the mean ± s.d. Asterisks indicate values that are statistically significant (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001) one-way ANOVA with Dunn’s multiple comparison test.

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Inclusion of phagostimulants in ATSB result in more effective killing of sand flies

Lastly, we tested the potential of mimicking a bloodmeal in an ATSB format for its ability to kill sand flies. Sand flies fed on 10% sucrose-10% fructose with or without the insecticide fipronil (100 µM) showed little difference in the amount ingested and diverted to the crop or midgut (Figs 4A and 4B). However, sand flies fed sugars and insecticide with BSA + ATP took larger meals into the crop and diverted significantly more to the midgut (Fig 4C shows an example image). Over a wide range of concentrations, fipronil in a sugarmeal containing phagostimulants was up to 355 times more potent than in sucrose-fructose alone, with a LC50 of 1.65 µM within by 24 hours of exposure compared to a LC50 of 584 µM for fipronil in sugars alone (Fig 4D and 4E, S3 Fig). This is 8- and 6-fold lower than the topical LC50 and the acute oral LC50 of fipronil to honey bees, respectively [24,25]. The lethality of fipronil in sugar meals with phagostimulants was enhanced and could induce significant mortality in sand flies as early as 60–80 mins post-exposure (Fig 4D). Most concentrations of fipronil tested with phagostimulants (5–1000 µM) showed close to 100% lethality after 24 hours compared to 50% mortality of the group fed sugars and 500–1000 µM insecticide without phagostimulants. Collectively, these results show that the use of phagostimulants to obtain larger sugarmeals that are directed to the midgut substantially improves the lethality of the ATSB towards Lu. longipalpis and presents the opportunity to lower the concentration of insecticides or toxicants to reduce non-target effects.

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Fig 4. Mock ATSB with phagostimulants against sand flies.

Sand flies were exposed to 10% sucrose-10% fructose (SF) with or without phagostimulants (ATP + BSA) or the insecticide fipronil (100 µM). (A) Proportion of crops with a meal of sugar and the relative amount, n = 50 flies/group. (B) Volume of sugar in crop (CR) and midgut (MG), n = 21-27 flies/group. (C) Example image of sand fly with a sugarmeal in crop and midgut after feeding on phagostimulants. Scale bar = 500 µm. (D) Kaplan-Meier survival curve of sand flies fed with increasing concentrations of fipronil, n = 30-68 sand flies/group. (E) Concentration-response curve of fipronil in the presence or absence of phagostimulants in the sugarmeal after 24 hours, n = 40-59 sand flies/group. Data pooled from 2 independent replicate experiments. Bars (B) or symbols (E) represent the mean ± s.d. Asterisks indicate values that are statistically significant (**P ≤ 0.01, ****P ≤ 0.0001) using a one-way ANOVA with Dunn’s multiple comparison test.

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Discussion

Sugar feeding is an essential activity of many dipteran vectors of disease, required to to locate bloodmeals and egg-laying sites in order to use the proteins and lipids from blood to generate large batches of eggs. Thus, sugar feeding holds great potential for exploitation in the control of disease vectors, serving as a versatile delivery method for toxicants, insecticides, antiparasitic compounds, anti-feeding agents, or microbes engineered to express anti-pathogen proteins or peptides - a strategy known as paratransgenesis [26]. However, Attractive Targeted Sugar Baits (ATSB) currently rely on sugars as the sole phagostimulant. This presents a problem as many vector species take small, intermittent sugarmeals from a variety of sources, requiring multiple visits to the bait to achieve toxicity.

In recent years there has been a drive to understand the feeding behaviour of mosquitoes and this has identified a number of phagostimulants that show promise in improving ATSBs [21,27]. A limited number of sand fly studies using ATSBs, either as bait stations or directly sprayed on to vegetation have shown promising reduction of sand fly numbers, ranging from 60%-96% [15–17]. In this study we sought to understand the sugar and blood feeding response of sand flies to improve the ingestion of sugar and its diversion to the midgut – the site of action for many insecticides and toxicants used in ATSBs. Adult sand flies of both sexes feed routinely in sugar sources as nectar, honeydew, or fruit juices, and only females require a blood meal from a vertebrate host for egg development [28–30]. In laboratory or field conditions, sand fly sugar meals are mimicked artificially using sugar baits (e.g. cotton wool embedded in a sugar solution). Importantly, sugars are used by Leishmania in the female sand fly midgut: (i) as a stimulus for the complete development of the parasite to an infectious metacyclic stage: (ii) to establish chemotactic sugar gradients that allow Leishmania to colonise the anterior midgut and stomodeal valve for transmission and (iii) as an energy source to survive in the sand fly [12, 31] and references therein]. This emphasizes the potential value of ATSBs, since they exploit a behaviour that is essential both for sand fly survival and parasite transmission.

To compete in the field with the vector’s natural sugar sources, the quality of the attractant is a crucial component of the ATSB design. Equally important but rarely considered is the quality of the meal itself - to promote as much toxin or insecticide to be taken during a single feed. This may be a missed opportunity as, currently, no ATSB has been designed to get a vector to engorge on a sugarmeal and directly target the insect’s midgut.

To see if this was possible for sand flies we used a robust fluorometric assay to sensitively measure the quantity of a sugar in the crop and midgut of the neotropical leishmaniasis vector, Lu. longipalpis; to assess the effect of different sugars baits on meal size and how quickly it can access the midgut. We show that the combination of sucrose and fructose provided the best sugar source, resulting in the largest crop meals within hours of exposure to the sugar source. Interestingly, glucose, the only sugar found in both blood and nectar, was infrequently taken into the crop. This suggests that for sand flies nectar and blood feeding cues are largely distinct but show a degree of overlap that may be sensed, in the context of other cues, to efficiently switch between these two feeding modes. In seminal work by Hosoi and Galun the meal components for the dengue vector Ae. aegypti to engorge were determined and, importantly, were shown to work without blood [32–34]. They demonstrated that blood feeding could be uncoupled from its value as a protein-source by showing that ATP induced engorgement only when co-presented with plasma components such as NaCl and NaHCO₃. Later, Jove et al. (2020) elegantly demonstrated that female Ae. aegypti use specialist neurons in the stylet (the mouthparts that pierces the skin and cannulate blood vessels) to discriminate nectar and blood and are insensitive to the nectar sugars sucrose and fructose [21]. They are activated by glucose only in combination with other bloodmeal cues, including NaCl, NaHCO₃, ATP and serum proteins. Interestingly, only half of the neurons were activated in response to bloodmeal cues indicating that others are important but are as yet unknown.

In a simple, experiment, we show that female sand flies ingest blood only when presented through a membrane (of skin or parafilm) and not through cotton wool, unlike sugars that could be consumed from cotton wool or through a membrane. However, a mixture of nectar sugars and blood on cotton wool resulted in a significant proportion of flies with blood in their crop and midgut. This indicates that the destination of a meal in sand flies can be manipulated according to its constituents and that cues in blood have the potential to direct a sugarmeal to the midgut. In mosquitoes nectar is taken to the crop, whereas blood bypasses the crop and is routed to the midgut, where it is digested. Mosquitoes will readily engorge on blood through a membrane provided with host-sensory cues such as heat and CO₂, however, if the bloodmeal is replaced with sugars the mosquitoes will reject this meal if these sensory cues are present [35]. This indicates that cues in the odour and the taste of a meal allow these vectors to flexibly chose which feeding programme to employ. To this end, Zhilin et al. (2022) found that activation of ATP-sensitive potassium channels in the salivary glands misdirected blood into the crop [36]. Collectively, this offers the premise to explore phagostimulants, such as ATP, as a means to improve ATSB function.

In contrast, little is known of the mechanisms underlying sugar and blood feeding in sand flies, however, what we do know is quite revealing. Physical cues appear to be important and how the sand flies use their mouthparts. Meals derived from plant surfaces, such as honeydew from aphids or flower nectar, were observed to be diverted to the crop, whereas meals taken directly from plants through piercing, as seen in blood feeding, are largely directed to the midgut [13]. Using dyed sugarmeals, Tang and Ward showed that female and male Lu. longipalpis sand flies interrupted during feeding had traces of sugar in the midgut, but that the rest of the meal was diverted into the crop [37], something that was also seen in this study. Next, they investigated the ultrastructure of the stomodeal valve (an invagination of the midgut with an underlying ring of muscle that allows the midgut to retain ingested blood) finding basiconic sensilla on the inner side of the oesophagus at the junction with the stomodeal valve [38]. They proposed that these sensilla may control the movement of the valve after contact with fluids entering the midgut and may be responsible for diverting the sugarmeal to the crop by closure of the valve only after a small volume of sugar has passed through it. Supporting this, we could detect the fluorescent sugar meal in the midgut of Lu. longipalpis, albeit at low levels, as early as 2 hours after exposure to a sucrose-fructose meal.

All blood-sucking insects use a range of phagostimulants to discriminate sugar and blood that could potentially allow them to obtain large reserves of sugar without affecting their appetite for blood. Therefore, we tested the effect of a range of blood feeding cues to encourage female sand flies to ingest large sugarmeals rapidly into their crops and divert it directly to the midgut – i.e., trick them to blood feed on the sugar bait. A mixture of four components of blood - BSA, ATP, NaCl and NaHCO₃ - were tested. We found that ATP could induce sand flies to take large sugarmeals into their crop, with the largest (548 nL) found in the combination with NaCl and NaHCO₃. After 24 hours the flies with the largest sugarmeals remaining in the crop were those presented ATP in the sugar solution (in combination with any of the other cues), however, in these flies there was little or no sugar diverted to the midgut. By far the largest diversion of sugar occurred after 24 hours in sand flies fed with a combination of ATP and BSA - capable of introducing 709 nL into the midgut. This compares well with the volume of blood that a fully engorged female Lu. longipalpis can take (1 µL [10]) and the mean bloodmeal volume ingested by a number of other phlebotomine sand fly species (0.47 to 1.01 µL [11]). Confirming this result, a follow up experiment found that the combination of BSA and ATP led to the largest proportion of female sand flies with full crops; larger amounts of sugar taken into the crop and diverted to the midgut and dramatically more rapid killing of sand flies using the example insecticide fipronil, commonly used in ATSB (Fig 4).

By introducing cues that promote rapid midgut engorgement on sugars there is scope to improve ATSB to control vector-borne diseases. In our mock ATSB we show the potential of combining phagostimulants with the sugar bait and how they can be improved to be more lethal more quickly against sand flies and, potentially, other disease vectors.

Therefore, these findings may be leveraged to improve ATSBs currently in use but only if it can be shown to work at scale and in regions of endemicity. For sand flies, ATSB have been shown to be effective in arid ecotypes, where nectar sources are either scare or absent [15–17]. To be able to compete with other sugar sources in plant-rich habitats, overall bait efficacy could be further improved by incorporating host kairomones or sand fly-derived pheromones to increase bait attractiveness and enhance competition with natural sugar sources. A good candidate for Lu. longipalpis could be the terpene-based synthetic male sex/aggregation pheromone that has been shown to attract male and female sand flies up to 30 m in peridomestic settings across Brazil [39]. This attractant has been shown to be an efficient lure and can reduce the number of indoor sand flies and the incidence of canine leishmaniasis when used in conjunction with pyrethroid insecticides sprayed on chicken coups [40,41], Terpenes have been found in other sand fly species [42], suggesting that the correct combination of phagostimulant ATSB and pheromone lure could be modified to target specific sand fly species. However, the specific chemical composition of the sex/aggregation pheromone can differ between populations, sometimes allowing for different “chemotypes” within the same species complex, such as Lu. longipalpis [42], and it has yet to be established how such pheromone-based attractants would affect sugarfeeding. A more immediate limitation of the current phagostimulant ATSB is the instability of ATP to above room temperatures. Non-hydrolysable ATP analogues are available, such as adenylyl imidodiphosphate and adenylyl methylene diphosphate [43] and may offer better thermostability but they remain to be tested as a phagostimulant for vectors other than mosquitoes.

Although not tested in this study, lowering the dose of toxicant or insecticide may also substantially reduce their off-target effects on insect species that also nectar feed, such as pollinators and has the potential to improve the effects of ATSB targeting the Leishmania parasite [28,44]. With this in mind, careful combination of blood feeding cues may also improve the selectivity of a ATSB towards blood-sucking arthropods but more needs to be known about them and how vectors other than mosquitoes sense and switch between nectar and blood feeding programmes. Sand flies belong to a subgroup of disease vectors that feed upon a pool of blood by lacerating the upper dermal capillary loops in the skin. Pool feeding may generate unique cues that mosquitoes may not encounter, such as skin constituents or wound-associated cytokines, chemokines and volatiles [45], so it would be prudent to explore these in greater detail to expand the range of vectors of this emerging control tool.

Supporting information

S1 Fig. Calibration curve for quantitation of sugarmeal volume in a sand fly crop or midgut.

A serial dilution of 50% sucrose dyed with fluorescein was made with a crop and midgut from a single unfed female sand fly. For each dilution, sugar, dye and dissected organs were homogenised before reading the fluorescence (485 nm excitation and 520 nm emission). Values represent the average of 4 replicates per dilution.

https://doi.org/10.1371/journal.pntd.0013888.s001

(TIF)

S2 Fig. Representative images of sugarfed crops from sand flies.

Flies were fed on 10% sucrose-10% fructose solution dyed with 0.002% fluorescein through cotton wool. (A) empty crop, (B) partial sugarmeal or (C) full sugarmeal. Crop (CR), midgut (MG). Scale bar = 500 µm.

https://doi.org/10.1371/journal.pntd.0013888.s002

(TIF)

S3 Fig. Mock ATSB against sand flies, without phagostimulants.

Kaplan-Meier plot of survival of sand flies fed with increasing concentrations of fipronil. Sand flies were exposed to 10% sucrose-10% fructose with or without the insecticide fipronil (100–1000 µM). Data pooled from 3 independent experiments, n = 39–59/group.

https://doi.org/10.1371/journal.pntd.0013888.s003

(TIF)

S1 Data. Supporting data for study.

https://doi.org/10.1371/journal.pntd.0013888.s004

(XLSX)

Acknowledgments

We would like to thank Ms Shahida Begum for assistance with sand fly colony rearing at LSHTM.

Financial disclosure statement

DPC was funded by a CAPES - SENIOR VISITING PROFESSOR CAPES-PRINT - 88887.878914/2023-00. FAG was funded by CNPq – Produtividade em Pesquisa - 312305/2022–2 and FAPERJ – Cientista do Nosso Estado – E-26/200.454/2023. MER was supported by the BBSRC (David Phillips Fellowship awarded to MER, BB/H022406/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Figures

Abstract

Background

Methods

Results

Conclusions

Author summary

Introduction

Materials and methods

Reagents

Sand flies

Sugar meal determination assay

Crop and midgut collection and processing

Crop volume scoring

Artificial membrane and cotton wool feeds

Sugar insecticide assay

Statistical analyses

Results

Sand fly sugarmeal intake and fate

Sand flies have separate blood and nectar feeding programmes, but they are not mutually exclusive

Phagostimulants found in blood divert sugar into the sand fly midgut

Inclusion of phagostimulants in ATSB result in more effective killing of sand flies

Discussion

Supporting information

S1 Fig. Calibration curve for quantitation of sugarmeal volume in a sand fly crop or midgut.

S2 Fig. Representative images of sugarfed crops from sand flies.

S3 Fig. Mock ATSB against sand flies, without phagostimulants.

S1 Data. Supporting data for study.

Acknowledgments

Financial disclosure statement

References