Can floral nectars reduce transmission of Leishmania?

BACKGROUND: Insect-vectored Leishmania are responsible for loss of more disability-adjusted life years than any parasite besides malaria. Elucidation of the environmental factors that affect parasite transmission by vectors is essential to develop sustainable methods of parasite control that do not have off-target effects on beneficial insects or environmental health. Many phytochemicals that inhibit growth of sand fly-vectored Leishmania-which have been exhaustively studied in the search for phytochemical-based drugs-are abundant in nectars, which provide sugar-based meals to infected sand flies. PRINCIPLE
FINDINGS: In a quantitative meta-analysis, we compare inhibitory phytochemical concentrations for Leishmania to concentrations present in floral nectar and pollen. We show that nectar concentrations of several flowering plant species exceed those that inhibit growth of Leishmania cell cultures, suggesting an unexplored, landscape ecology-based approach to reduce Leishmania transmission. SIGNIFICANCE: If nectar compounds are as effective against parasites in the sand fly gut as predicted from experiments in vitro, strategic planting of antiparasitic phytochemical-rich floral resources or phytochemically enriched baits could reduce Leishmania loads in vectors. Such interventions could provide an environmentally friendly complement to existing means of disease control.

Introduction

Plant secondary metabolites have a long history of use against human disease and provide the basis for both traditional medicines and many modern drugs [1], including treatments for neglected tropical diseases [2]. The sand fly-vectored Leishmania parasites are estimated to cause disease in >2 million humans each year, with 10% of the world’s population at risk, and have a greater health burden (as measured by loss of disability-adjusted life years) than any human parasite besides malaria [3]. These infections include an estimated >0.2M cases of visceral leishmaniasis, which, if untreated, results in >90% patient mortality [3,4]. Due to their clinical significance, Leishmania spp. have been studied intensively in a search for affordable and effective treatments for human infections [5], including exhaustive testing of plant extracts and their components against both mammal- and insect-associated parasite life stages [2,6]. These studies have suggested new treatments for trypanosomatid-associated infections of humans [7] and related parasites of beneficial insects [8,9]. As in humans, antimicrobial phytochemicals can enhance resistance to infection in plants themselves [10] and in other plant-consuming animals, including insects [11].

The diets of blood-feeding, disease-vectoring insects such as sand flies and mosquitoes include sugar-containing plant tissues as well as blood [12]. Sugar sources differentially affect not only vector survival, but also the development of parasitic Plasmodium falciparum malaria in Anopheles mosquitoes [13] and Leishmania major in sand flies (Phlebotomus papatasi) [14], with effects mediated by secondary metabolites [15,16]. Sand flies feed on plant sugars between acquisition and transmission of Leishmania to humans and other mammals [17], as demonstrated by caging flies with dye-infused branches, spectrophotometric detection of sugars or plant cell walls in the gut, and molecular analysis of field-collected flies showing the presence of plant DNA [18-20]. The importance of dietary sugars is evident from their effects on fly longevity. Flies survive less than a week under sugar source-poor desert conditions [21] and less than 2 weeks when reared on comparatively sugar-poor branches [22], but more than 7 weeks on 20% sucrose solution [22]. The abundance of sugar meal-inducible glucosidases expressed by the sand fly and by its Leishmania parasites provide additional evidence of mutual adaptation to an omnivorous lifestyle that exploits diverse plant sugars as food sources [23-25], and that sugar sources could be manipulated to control vectors and their parasites [16,22].

Although sand flies may acquire sugar meals from plant sap, fruit, or aphid- or cicada-derived honeydew [26], floral nectar appears to be a preferred food source, as evidenced by the attractiveness of flowering bushes and branches (relative to those soiled with honeydew) in a desert oasis [12]. The small (<1 μL) meal sizes of sand flies [23] would make the concentrated sugars found in nectar a profitable foraging resource, in spite of the small volumes available at each flower, explaining the general attractiveness of flowering plant food sources to sand flies and related dipterans [27]. The size of sugar meals is, however, impressive on a mass-specific basis—increasing the mass of females by >30% over 48 h [28]—consistent with the strong effects of meal chemistry on gut-dwelling Leishmania.

The role of nectar chemistry in insect disease ecology has recently been highlighted by work on infections of pollinators. Floral nectar and pollen, their constituent secondary metabolites, and the composition of flowering plant communities can ameliorate trypanosomatid growth and infection in bumble bees [8,9,29-31]. Both nectar and pollen—which may mix with and influence the chemistry of nectar at flowers [32]—contain diverse secondary metabolites that shape plant-pollinator ecology and plant-microbe ecology [33-37]. Flavonoids are one class of antimicrobial and antileishmanial compounds [38,39] that are ubiquitous in both nectar and pollen, with concentrations in pollen often exceeding 1% of total dry matter [40,41]. This suggests that consumption of secondary metabolite-rich nectars could mitigate Leishmania transmission by reducing infection intensity in nectar-feeding sand fly vectors [12], pointing to a new strategy for drug- and insecticide-free disease control. However, despite appreciation for the clinical antileishmanial potential of plant metabolites [2], growing recognition of the role of plant metabolites—including those in nectar and pollen—in insect infection, and the critical role of plant sugars in sand fly diets, there has been surprisingly little investigation into the potential for antileishmanial phytochemicals in the diets of sand flies to mitigate Leishmania transmission [14,15].

To assess the potential for floral resource-associated phytochemicals to reduce vector-borne infection, we compared phytochemical concentrations previously shown to inhibit Leishmania to concentrations previously found in floral nectar and pollen. Our synthesis of prior work on Leishmania phytochemical sensitivity with nectar and pollen secondary chemistry shows that many floral nectars contain antileishmanial compounds at concentrations sufficient to inhibit parasite growth. These findings suggest an unexplored, landscape ecology-based approach to reduce transmission of widespread and virulent Leishmania infections. If phytochemical concentrations that inhibit Leishmania in vitro are equally effective in the sand fly gut, incorporation of antiparasitic nectar sources into landscapes and domestic settings could simultaneously benefit pollinator and public health.

Methods

We compared the flavonoid concentrations found in a previous survey of methanolic extracts from 26 floral nectars and 28 pollens [40,42] with previously published results from in vitro screening of various Leishmania spp. (Table A in S1 Text). We focused on flavonoids because these compounds were the most consistently present class of compounds across both nectar and pollen [40] and—particularly in the case of quercetin—some of the most potent and selective compounds against Leishmania [39,43,44]. To prevent overestimation of inhibitory potential that could result from including flavonoids of lesser or unknown antiparasitic activity, we further distinguished between total flavonoid concentrations and those with a kaempferol, quercetin, apigenin, or luteolin aglycone, each of which has well-documented antileishmanial effects [39,45,46] (Table A in S1 Text).

We analyzed micromolar concentrations to enable pooling across compounds with different parent flavonoids and glycosides. Flavonoid glycosides—including those of quercetin and kaempferol—can be less potent against Leishmania than are their parent aglycones [39], which can more easily cross cell membranes [47]. However, we included flavonoid glycosides because these compounds are hydrolyzed by intestinal glucosidases—a variety of which are found in sand flies [28]—to their corresponding aglycones [48,49]. These glucosidases have been shown to form antileishmanial aglycones from glycosylated coumarins in intestinal extracts [16]. We focus our discussion on nectar because sand flies, like other Diptera, do not have chewing mouthparts that would enable direct consumption of pollen and other solid foods [20]. However, incidental presence of pollen in nectar can dramatically increase the nectar’s concentrations of amino acids [50], with ecologically relevant effects on nectar-feeding insects [32]. Pollen could similarly affect nectar’s phytochemical profile and antimicrobial effects. For example, presumably pollen-derived cinnamic acid-spermidine conjugates were found in nectar of two species in our previous survey—Digitalis purpurea and Helianthus annuus [40]. In H. annuus, nectar concentrations averaged 1.7% of pollen concentrations, despite exclusion of large insects that contribute to such "contamination" [50] for 24 h prior to sampling. We therefore also discuss pollen concentrations that exceed the Leishmania IC50 estimates by >100-fold, on the grounds that the much (235-fold [40]) higher flavonoid concentrations found in pollen could meaningfully alter the antiparasitic activity of nectar, even when pollen accounts for <1% of nectar volume.

Results

We compiled 18 Leishmania IC50 estimates for 4 flavonoids—quercetin (n = 8), kaempferol (n = 4), apigenin, and luteolin (n = 3 each) that have been relatively well studied for effects on Leishmania spp. cell cultures (Table A in S1 Text). Most (11 of 18) of the assays used the promastigote (i.e., insect-associated) life stage; the remainder used either intracellular (n = 4) or axenic (n = 3) amastigotes (Table A in S1 Text). These Leishmania IC50 estimates were then compared to the flavonoid concentrations found in a previous survey of secondary metabolites of nectar and pollen [40].

Flavonoids were found in the nectar of 21 of 26 species (81%) and in pollen of 26 of 28 species (93%), accounting for 30% of the total phytochemical content in nectar and 41% in pollen [40]. Total flavonoid concentrations exceeded 100 μM in 8 of 26 nectars (31%, median concentration 30.9 μM, IQR 4.24–127 μM; median 61.4 μM after exclusion of the five species without nectar flavonoids) and exceeded 104 μM in 18 of 28 pollens (64%, median 1.36 ∙ 104 μM, IQR 4.31 ∙ 103 to 2.30 ∙ 104 μM) (Fig 1). Glycosides of quercetin (found in 9 of 26 nectars and 14 of 28 pollens) and kaempferol (5 of 26 nectars and 19 of 28 pollens) were most common [40].

Fig 1

Published Leishmania IC50 estimates for selected flavonoids (A) relative to concentrations of the corresponding compounds in nectar and pollen (B).

Shapes in panel (A) correspond to the Leishmania stage tested. Boxplots in panel (B) show medians and interquartile ranges for concentrations of quercetin, kaempferol, apigenin and luteolin derivatives (red boxes) and total flavonoids (blue boxes). Points show median concentrations (pooled across individual samples) by species. Text annotations denote species with >100 μM of the selected flavonoids in nectar (Brassica napus, Dicentra eximia, Helianthus annuus, and Thymus vulgaris). Literature references for Leishmania IC50 estimates are given in Table A in S1 Text.

Compounds with a parent aglycone of quercetin, kaempferol, apigenin, or luteolin accounted for 62% of flavonoid compounds and 54% of molar concentrations in nectar, and 72% of compounds and 75% of molar concentrations in pollen. In nectar, median concentration of this subset of compounds across all species (20.3 μM, IQR 9.37–58.9 μM) was remarkably close to the 23.1 μM median IC50 for Leishmania (based on 18 references (Table A in S1 Text)). Concentrations exceeded 100 μM (i.e., more than the highest Leishmania IC50 for any of the parent compounds) in nectar from 4 of 26 species (Dicentra eximia, Brassica napus, Helianthus annuus, and Thymus vulgaris). In pollen, median concentrations exceeded 104 μM (i.e., >100-fold the greatest Leishmania IC50) in pollen from 12 of 28 species, including two species (Lythrum salicaria (1.21 ∙ 105) and Solidago canadensis (1.19 ∙ 105)) with concentrations >105 μM—over three orders of magnitude above the greatest Leishmania IC50 (Fig 1).

Antileishmanial compounds in nectar were not limited to flavonoids. Seven nectars contained chlorogenic acid, with a median concentration (51.2 μM) similar to the IC50 for L. donovani promastigotes (54 μM [51]) and 100-fold greater than the IC50 for L. amazonensis promastigotes (0.5 μM [52]). The species with the highest median concentration of chlorogenic acid (Dicentra eximia, 184 μM) also had the highest concentration of the selected flavonoids (Fig 1). Nectar concentrations of two additional species (Penstemon digitalis, 134 μM) and Rhododendron prinophyllum (56.7 μM) also exceeded the L. donovani promastigote IC50 (Fig 2). Chlorogenic acid was also found in seven pollens at up to 3760 μM (Persea americana), with a median concentration (1227 μM) over 20-fold greater than that found in nectar and over three orders of magnitude above the L. amazonensis promastigote IC50 [52] (Fig 2).

Fig 2

Concentrations of chlorogenic acid in nectar and pollen in comparison with inhibitory concentrations for Leishmania.

Points represent median concentrations from species with detectable chlorogenic acid (sampled in [40]). Horizontal lines show published IC50 values [51,52]. Sampled plant species (labeled by genus) were Dicentra eximia, Penstemon digitalis, Rhododendron prinophyllum, Malus domestica, Vaccinium corymbosum, Cucurbita pepo, Persea americana, and Geranium maculatum. For Vaccinium, "cult" refers to cultivars and "wild" refers to wild plants.

Nectar of one species (Thymus vulgaris) contained the caffeic acid-dihydroxyphenyl lactic acid ester rosmarinic acid. Median concentration (165 μM, IQR 87.7–206 μM) was 10-fold greater than the IC50 for L. donovani promastigotes (16.3 μM [51])—against which rosmarinic acid and apigenin were the most selective of the compounds evaluated—over 30-fold greater than the 4.8 μM IC50 for L. amazonensis amastigotes [52], and over 200-fold greater than the 0.7 μM reported for L. amazonensis promastigotes [52] (Fig 3). Nectar of T. vulgaris is also notable for its high thymol content (26.1 μg mL-1 [53]), which exceeds six of the eight IC50 values reported for Leishmania promastigotes (Table A in S1 Text, [54,55]).

Fig 3

Concentrations of rosmarinic acid in Thymus vulgaris nectar in comparison with inhibitory concentrations for Leishmania.

Points represent individual nectar samples (from [40]). Horizontal lines show published IC50 values [51,52].

Discussion

Our synthesis of a previous survey on the quantitative phytochemical composition of nectar and pollen with the extensive body of research on phytochemical-mediated inhibition of Leishmania in vitro reveals the potential for floral resources to ameliorate vector-mediated transmission of Leishmania. The most common compounds in nectar and pollen—flavonoids and their glycosides—have shown strong inhibitory effects against Leishmania [39,44,56]. Our findings indicate that a subset of the floral nectars analyzed to date—including the common garden herb Thymus vulgaris (thyme) and the widespread crop species Helianthus annuus (cultivated sunflower) contain bioactive flavonoids at concentrations that inhibit growth of diverse Leishmania in vitro. Incidentally, both plant species have also been shown to mitigate transmission and infectivity of bumble bee trypanosomatids, including in field mesocosms and landscape surveys [30,31]. Further investigation of the effects of specific nectar and other sugar sources on sand fly infection is needed. However, deliberate encouragement of these and other plants with trypanosomatid-inhibiting chemistry could reduce infection in disease-vectoring, hematophagous insects as well.

Our data suggest that around 20% of nectars contain flavonoids at strongly antiparasitic concentrations, although this number likely varies by region and season. Our analysis was focused on bee-pollinated species in the Northeast United States, where sand flies are absent, and therefore contained few of the specific plant species naturally used by sand flies in Leishmania-endemic regions. However, sand flies have been observed to prefer cultivated gardens (to which such plants could be introduced) over endemic vegetation [20], and have been associated with plants in the same taxonomic families as those represented by the species analyzed here. For example, Brassica napus (Brassicaceae) nectar was among the highest in flavonoids; flowers of another member of this family (Sinapis alba) elicited feeding by Phlebotomus papatasi [57]. Nectar of Impatiens capensis (Balsaminaceae) had flavonoid concentrations (20.9 μM, all from strongly antileishmanial compounds) close to the median of the nectars examined (20.3 μM) and the median Leishmania IC50 (23.1 μM); branches of the congener I. balsamina were fed upon by Lutzomyia youngi in Colombian coffee plantations [58]. On the other hand, nectar flavonoid concentrations were considerably lower (1.6 μM total) in Trifolium pratense, the only Fabaceae species tested; plants of this family have been strongly associated with sand flies in field sampling [59] and DNA metabarcoding studies [60,61]. Flavonoid concentrations were also low (<1 μM) in Cucurbita pepo (Cucurbitaceae) and undetectable in Catalpa speciosa (Bignoniaceae), two other plant families associated with sand flies [20]. Based on these results, the amounts of antileishmanial flavonoids ingested by flies could vary substantially in different landscapes.

Besides floral nectar, other known sugar sources may also possess flavonoid-mediated antileishmanial activity. Flavonoid concentrations of sand fly-attracting fruits [26] appear similar to those found in nectar. Combined quercetin and luteolin contents ranged from unquantifiable in honeydew melon to 22.8 μM in nectarine, 33.1 and 39.7 μM in red and white guava, and 53.6, 84.1, and 91.1 μM in white, black, and red grapes respectively [62]. Honeydew from sapsucking insects such as aphids [12] likely also contains types and quantities of flavonoids similar to those found in floral nectar, based on the similar flavonoid profiles of honey from these two sources [63]. Further experiments are needed to assess the chemistry of local, fly-attracting, sugar-providing plant species and their effects on insect host and parasite mortality, as demonstrated for lectin-rich plant sugar sources in Israel [14,15,22]. Given that sand fly feeding on branches [19], flowers [12], and fruits [26] tends to be highly selective on a few local species, the scope of such research is likely achievable.

We predict that our analysis—which accounts only for direct effects of a few compounds on parasites as estimated from in vitro studies—provides a conservative estimate of the effects of plant compounds on disease transmission. First, we focused on a limited subset of nectar components whose effects on Leishmania have been thoroughly studied, ignoring the effects of co-occurring chemicals that could also affect parasites (e.g., other flavonoids, lectins, and alkaloids), whether present in the ingested sugar source or formed during sand fly digestion (e.g., deglycosylation of coumarins or cyanogenic glycosides to compounds that reduce parasitic infection [16,64]). Second, these direct effects could be amplified by host-mediated reductions in levels of parasites due to phytochemical ingestion. For example, nectar-derived flavonoids stimulated immune gene expression in honey bees [65]; similar flavonoid-induced immune stimulation could enhance parasite clearance in flies. In addition, besides their effects on protozoa specifically, flavonoids are generally antimicrobial [38], and could inhibit growth of midgut bacteria that facilitate Leishmania infection [66]. It would be of interest to contrast the effects of similar flavonoid concentrations taken directly from plant tissues—which are delivered to the sand fly midgut—versus those from surface sugars (e.g., nectar and honeydew), which are first stored in the crop [57]. The gradual release of nectar and honeydew from the crop to into the anterior midgut [57] could limit the exposure of midgut-dwelling parasites to phytochemicals from these sources.

The effect of sugar-containing meals from plant sources was a long-overlooked component of sand fly ecology that proved crucial in Leishmania transmission [17]. However, feeding of sand flies on several plant taxa causes marked parasite mortality—up to 88% in the case of castor bean (Ricinus communis) [14], the lectins of which agglutinate a variety of insect trypanosomatids [67]—paralleling the strong effects of plant sugar sources on malaria infection in mosquitoes [13]. Although flowering plant nectar sources might at first glance appear to be a liability for Leishmania transmission due to the food they provide to sand flies, sugar starvation in fact results in greater vector infection intensity and natural selection for flies with lesser parasite resistance [68,69]. This finding is consistent with the preponderance of Leishmania hotspots in arid regions, where plant sugar sources are scarce [68,69]. High parasite loads also alter sand fly feeding on mammals in ways that promote transmission to new hosts [70]. These lines of evidence suggest that despite their role as vector food sources, phytochemical-rich floral nectar sources could have a net transmission-reducing effect.

Feeding of sand flies on floral nectar may also result in incidental pollen exposure that, due to pollen’s high flavonoid concentrations, has strong effects on Leishmania in the fly gut. Such incidental exposure was suggested by the high prevalence of Pinaceae DNA associated with sand flies at sites apparently lacking such plants [20]. This association was postulated to reflect exposure of flies to windblown pollen, which could also account for at least some of the DNA from Cannabis sativa—another wind-pollinated species not visibly present [20]. Introduction of pollen to nectaries by bees can increase nectar amino acid concentrations by an order of magnitude, and potentially introduce antiparasitic compounds from con- and heterospecific pollens as well [50]. Given that flavonoid concentrations in pollen are 200-fold higher than those in nectar [40], incidental ingestion of even small amounts of pollen could substantively inhibit proliferation of parasites and the transmission potential of their vectors. In H. annuus, pollen-associated spermidines occurred at concentrations >1% of those in pollen even when pollinators were excluded [40]. In our meta-analysis, eight of the 28 pollens previously surveyed contained flavonoids at concentrations that exceeded 100-fold the maximum inhibitory concentration reported for Leishmania (Fig 2). This suggests that as little as 1% incidental addition of pollen to nectaries might be sufficient for Leishmania inhibition, even for nectars that lack antileishmanial flavonoids initially.

Whether antiparasitic compounds are present in secreted nectars or due to incidental introduction of pollen, nectars rich in phytochemicals are promising candidates for ecological mitigation of Leishmania transmission. Parasites of this genus appear both sensitive to flavonoids and, given the parasite’s establishment in the midgut and forward migration in the alimentary canal [17], directly exposed to ingested compounds before appreciable metabolism of these compounds—by hosts or microbiota in the abdominal midgut—can occur. The limited intestinal absorption of ingested flavonoids [49], hydrolysis of glycosides found in plants to their more potent aglycones in the intestine [28,49], and likelihood of direct contact between parasites in the anterior midgut and ingested phytochemicals all indicate the potential for flavonoid-rich nectars to reduce Leishmania infection in sand flies. However, empirical testing of these compounds in sand fly diets is necessary to confirm their efficacy in the insect vector and model the effects of sugar sources on parasite infection, vector longevity, and disease transmission, as was recently done for malaria [13]. In addition, the broader ecological effects of floral compounds—whose effects are unlikely to be limited to sand flies alone—must be considered before implementation of interventions, particularly those that involve introduction of non-endemic plant species.

Conclusions

The global toll of Leishmania infection and the difficulties of eradicating its sand fly vectors and non-human reservoirs demand the development of new, environmentally compatible strategies to reduce parasite transmission [71]. Our synthesis of existing data shows that sugar-seeking sand flies are attracted to floral resources, and that floral nectars contain antileishmanial phytochemicals at concentrations that inhibit replication of parasite cell cultures. The extent to which floral resources influence Leishmania epidemiology will depend on the contribution of nectar to sand fly diets and the extent to which in vitro inhibitory effects are realized in the guts of infected flies. If the effects of nectar on insect infection are commensurate with predictions based on nectar phytochemistry, reduction of transmission via supply of antiparasitic nectar sources in local landscapes—or phytochemical-based, transmission-blocking baits [16]–could positively influence public health. Such interventions could reduce reliance on drug treatments that may be costly, inaccessible, or potentially hazardous [3] while simultaneously supporting populations of beneficial insects and their resistance to insect-specific trypanosomatid infections [29,30]. The fields of insect ecology and medicinal chemistry for insect-vectored parasites have thus far developed more in parallel than in concert. Integrating knowledge of medicinal plant chemistry and plant-mediated tritrophic interactions that affect parasites in disease-vectoring insects holds promise for environmentally friendly control of trypanosomatid threats to global health.

Supplementary Table A, references, and metadata.

(PDF)

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Zipped folder with data spreadsheets for Leishmania inhibitory concentrations (leishmania_ic50) and nectar and pollen flavonoid concentrations (nectar.pollen.flavonoids).

(ZIP)

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6 Dec 2021

Dear Mr. Palmer-Young,

Thank you very much for submitting your manuscript "Can floral nectars reduce transmission of Leishmania?" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: As this is a meta-analysis their is no testable hypothesis. So the other questions are irrelevant

Reviewer #2: This section methods is fine for me except some points to address. Please find below my comments

1- Line 101-102 (Ref 27), could you explain how flavonoid glycosides are less effective than aglycones?

Reviewer #3: The study design was clear, however it is not strong enough support the hypothesis raised in this study.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: As this is a compilation of other scientists' research there was no analysis plan to begin with.

The results are clearly stated.

Figures:

Figure 1 A: There are no values even close to 1,000 (1e+03), so why have a graph that goes to 1e+05 (100,000)? B: Is very busy and hard to read – can it be simplified?

Figures 2 & 3. N.B. fig. 2 uses 1e + scale, while fig 3 uses 0.1 to 100. The second is so much easier to read.

Table

Table includes data on amastigotes, a stage in the life cycle of the parasite that is very transitory in the sand fly.

Reviewer #2: Good, but is the concentration of floral resources that you use the same concentration that could Phlobotomus ingest in the wild?

Reviewer #3: Concentrations evaluated here are based only in vitro exposition of the parasite to compounds, ignoring their effect during Leishmania-sand fly interactions, i.e., ignoring the behavior and physiology of the insect host, in which is essential to the potential for the vector control.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusions are adequate, but the limitations are (i.e. lack of field data) not mentioned.

No evidence is presented that sand flies actually feed on the various plants mentioned.

In the field of Public Health the authors wrote that their work could lead to an environmentally friendly control of trypanosomatid threats to global health.

Reviewer #2: Good

Reviewer #3: The compilation done here would be extremely helpful as a screening process to test experimentally these compounds in the insect vector. Such conclusion would be more restrictive, but certainly more accurate to the data presented.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Lines 13 and 53: Insect-vectored Leishmania are the second-most debilitating of human parasites worldwide. The actual quote is “Among parasitic infections, this disease is responsible for the highest number of disability adjusted life years (a measure of health burden) after malaria.” (Lymphatic Filariasis is surely more debilitating than cutaneous leishmaniasis caused by Leishmania major). While the authors repeat several times this health burden, it is somewhat over empathized.

Line 54 The CDC numbers: For cutaneous leishmaniasis, estimates of the number of new cases per year have ranged from approximately 700,000 to 1.2 million or more. For visceral leishmaniasis, the estimated number of new cases per year may have decreased to <100,000. WHO numbers are less, so perhaps “reported” should be cautiously used.

Line 66: “floral nectar appears to be a preferred food source” Perhaps it should be noted that this study was carried out in a lush irrigated farming village surrounded by desert.

Line 137 and Discussion: Is there any evidence that sand flies feed actually on for example (Dicentra eximia, Brassica napus, Helianthus annuus, or Thymus vulgaris)? Examples of mesocosm would augment the meta-analysis. A real world example of this would be “DNA barcode for the identification of the sand fly Lutzomyia longipalpis plant feeding preferences in a tropical urban environment”: Leonardo H G de M Lima et al Sci Rep, 2016 Jul 20;6: 29742 or if the authors comb the literature they can find Suaeda asphaltica as a sand fly food source or Cameron’s work in South America on bean plants as actual sand fly food or Alexander B and Usma MC, study in Colombian coffee fields.

Could it be that the compounds the authors are promoting would make better pharmaceutical products?

While the MS is partly speculative in nature, the inclusion of some of the references add little to overall question.

Other references that may interest the authors for their discussion:

Lectins and toxins in the plant diet of Phlebotomus papatasi (Diptera: Psychodidae) can kill Leishmania major promastigotes in the sandfly and in culture. Jacobson RL, Schlein Y. Ann Trop Med Parasitol. 1999 Jun;93 (4):351-6.

Sand fly feeding on noxious plants: a potential method for the control of leishmaniasis Y. Schlein, R L Jacobson, G C Müller Am J Trop Med Hyg. 2001 Oct; 65(4):300-3

While the MS is partly speculative in nature, the inclusion of some of the references adds little to overall question?

Reviewer #2: Good

Reviewer #3: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The MS reviews the research of others on the in vitro screening of various extracts of nectar and pollen on Leishmania species and discuss the use of secondary metabolites in the control of the infections in the sand fly vector.

The inclusion of known plant food sources would improve the MS

Reviewer #2: Phlebotomus take the nectar as nutrition. The effects of components in nectar are various. This study focuses on floral nectars and Leishmania transmission. The conclusion is that planting phytochemical-rich floral resources or phytochemically enriched baits could reduce Leishmania loads in vectors, providing an environmentally friendly complement to existing means of disease control. With these findings, there is a great hope to come over some diseases as Leishmaniasis without chemical solution, using natural environmental resources.

The manuscript how it is standing now, is well written, clear and shows good results to fight against the Leishmania. However, to improve the manuscript some points raised below need to be more detailed.

1- Line 117-118 (Ref 13), in this paper they have shown that some plant species reduce the transmission of malaria and other plants maintain the transmission, so, could you consider this in the discussion?

2- In your references 2, 12, 17, 23 there is no DOI as well as in all your references in Supporting information 1, please add these details

3- Some species scientific names in your references are not italicized, you should correct that too

Reviewer #3: The information discussed in this study is certainly interesting, but it is not being consistent with the broader potential speculated by authors. I suggest authors either support the study with some experimental tests of nectar compounds on insects or restrict claims to avoid the speculative character.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

24 Jan 2022

Submitted filename: leishmania_hypothesis_responses_v1.1.pdf

Click here for additional data file.

11 Feb 2022

Dear Palmer-Young,

Thank you very much for submitting your manuscript "Can floral nectars reduce transmission of Leishmania?" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Although the modifications in the text have improved the manuscript, there are still some alterations that deserve attention by the authors.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Claudia Ida Brodskyn

Associate Editor

PLOS Neglected Tropical Diseases

Alvaro Acosta-Serrano

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Although the modifications inthe text have improved the manuscript, there are still some alterations that deserve attention by the authors.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: Good, very clear to me now

Reviewer #3: As previously mentioned the study is clear and presents a satisfactory design.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: Good

Reviewer #3: Data presented previously was clear and modifications have not big effect in the current manuscript.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: Good

Reviewer #3: In my view, the conclusion of this work still remain very broad considering the data based only in vitro tests to discuss a bigger question, a more restrictive statement would be more plausible.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: Good

Reviewer #3: I acknowledge all changes performed by the authors in the text, it improved considerably the manuscript, however I still would recommend some "major revision" in this work.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have used the remarks of the referees to refine their MS. It has been improved.

Reviewer #2: Good from my point of view

Reviewer #3: Despite all modifications performed, the hypothesis raised in this work still consider a large knowledge in nectar compounds and their effect in the parasite in vitro, it has poor information related to the vector interaction, in which is essential considering to the potential claimed in this manuscript.

I still recommend authors discuss more about the physiology and behaviour of the insect that could justify the hypothesis. Even simple details regarding the insect could make all the difference and should be not selectively ignored. For instance, if the nectar amount taken by the insect would enough to affect the parasite considering the in vitro results presented here?

Please check here some papers that might help :

Ferreira, T.N., Pita-Pereira, D., Costa, S.G. et al. Transmission blocking sugar baits for the control of Leishmania development inside sand flies using environmentally friendly beta-glycosides and their aglycones. Parasites Vectors 11, 614 (2018). https://doi.org/10.1186/s13071-018-3122-z

Ferreira ME, de Arias AR, Yaluff G, de Bilbao NV, Nakayama H, Torres S, et al. Antileishmanial activity of furoquinolines and coumarins from Helietta apiculata. Phytomedicine. 2010;17:375–8.

Gontijo NF, Melo MN, Riani EB, Aleida-Silva S, Mares-Guia ML. Glycosidases in Leishmania and their importance for Leishmania in phlebotomine sand flies with special reference to purification and characterization of a sucrase. Exp Parasitol. 1996;83:117–24.

Jacobson RL, Schlein Y, Eisenberger CL. The biological function of sand fly and Leishmania glycosidases. Med Microbiol Immunol. 2001;190:51–5

Dillon RJ, EEl K. Carbohydrate digestion in sand flies: α-glucosidase activity in the midgut of Phlebotomus langeroni. Comp Biochem Physiol. 1997;116B:35–40.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

19 Feb 2022

Submitted filename: leishmania_hypothesis_responses_v2.3.pdf

Click here for additional data file.

9 Mar 2022

Dear Palmer-Young,

Thank you very much for submitting your manuscript "Can floral nectars reduce transmission of Leishmania?" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Dear authors,

The manuscript is improved a lot, but two reviewers pointed out some minor issues that deserve your attention.

Please, answer these points and send the manuscript back to be analyzed again.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Claudia Ida Brodskyn

Associate Editor

PLOS Neglected Tropical Diseases

Alvaro Acosta-Serrano

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Dear all,

The manuscript is improved a lot, but two reviewers pointed out some issues that deserve your attention.

Please, answer these points and send the manuscript back to be analyzed agains.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #2: Good for me

Reviewer #3: Methods are clear and well described by the authors

Reviewer #4: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #2: Good

Reviewer #3: Result analysis is consistent with the study design and clearly explained.

Reviewer #4: see below

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #2: Good

Reviewer #3: Lines: 289-292

I would like just to point that some technical interspecies issues may need to be addressed here when extrapolating these results. It is unlikely that the phytochemistry from these floral compounds would be specific to sand flies. The ecological concerns would be similar to using synthetic compounds.

Reviewer #4: see below

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #2: Good

Reviewer #3: Accept

Reviewer #4: see below

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #2: Good for me now, but they need to consider changing Phlebotomus langeroni in italic in reference 23 and the figure 1 in supporting information section is blurred

Reviewer #3: I acknowledge all modifications performed in the manuscript. The text is more consistent with the data analysed.

Reviewer #4: Revised version of the manuscript is significantly improved as helpful comments of reviewers were accepted. In my opinion, just few relatively minor changes are required:

Authors are aware that the sugar meal is directed first to crop (see lines 237-240). However, I am missing important information that only small droplets of sugar meal are released from the crop to the anterior (thoracic) midgut where the sugar meal is relatively quickly digested by glycosidases. This mechanism of sugar digestion may significantly decrease the effect of anti-leishmania compounds in the sugar meal. It is necessary to mention this fact either in Discussion or Conclusions.

The above mentioned fact should be also reflected by more cautious deductions in the text, for example „would be“ should be replaced by „might be“ on line 267.

Line 274: the proportion of microbiota in sand fly hindgut is negligible and unimportant in comparison to microbiota present in abdominal midgut of sand flies. Therefore, „hindgut microbiota“ should be replaced by „microbiota in abdominal midgut“.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

11 Mar 2022

Submitted filename: leishmania_hypothesis_respones_v3.1.pdf

Click here for additional data file.

29 Mar 2022

Dear Palmer-Young,

We are pleased to inform you that your manuscript 'Can floral nectars reduce transmission of Leishmania?' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Claudia Ida Brodskyn

Associate Editor

PLOS Neglected Tropical Diseases

Alvaro Acosta-Serrano

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

After three rounds of revision, the manuscript can be accepted for publication.\\Best regards

Cláudia Brodskyn

16 Apr 2022

Dear Palmer-Young,

We are delighted to inform you that your manuscript, "Can floral nectars reduce transmission of Leishmania?," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases