Species-Specific Color Preferences During Foraging in Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus Across Varying Light Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

line 168 - are you saying that the mosquitoes were allowed to forage for 44 -48 hours? Please make clear. 

---

However, your study lacks critical ecological context and nuance that must be addressed.  You keep mentioning 'foraging' but you have really assayed is likely water foraging or sugar feeding (by way of detecting water) or even (if its 48 hours of assaying) that they happened to take a likely water drink because they were resting against the colored sugar meal anyways.

Therefore in the abstract and conclusions, and possibly intro It needs to be addressed, specifically what you think they are foraging for . . . and why you think that.

you do hint at refuge seeking - looking at picture of experimental arena, the e.g. green dot may be the only dark surface in there and that is a common cue for mosquitoes to seek refuge in.  Were the day biters more likely to seek darker colors? That would support this.

Much previous work exists you could possibly cite or do your own experiments where they just assayed refugee color selection itself to try and resolve this what was the cause and what was the effect issue .

Do you think some sugar sources are 'supposed to ' a certain color ? (a flower red, a stem green for example ? Could this play a role?

finally, can you address more how you see this information being used?  You are certainly not trying to call this host seeking .

Author Response

We thank the reviewer for the careful reading of the manuscript and for the constructive and insightful comments, which have helped to improve the clarity and interpretation of our study. Below, we address each comment in detail.

line 168 - are you saying that the mosquitoes were allowed to forage for 44 -48 hours? Please make clear. 
->  Yes, mosquitoes were allowed to forage freely on the colored sugar-water solutions during this period. We have clarified that mosquitoes were maintained in cages with continuous access to the dyed fructose solution for 44–48 hours, during which they could feed or remain unfed. After this exposure period, mosquitoes were aspirated and examined under a stereomicroscope to assess abdominal coloration and determine sex.

-> l170-178: “Mosquitoes were maintained in cages with continuous access to the colored fructose solution for 44–48 hours, during which they could voluntarily feed or remain unfed. After this exposure period, mosquitoes were aspirated and visually examined under a stereomicroscope, including determination of their sex. Following ingestion, the mosquitoes’ abdomens displayed coloration corresponding to the consumed dye. Based on abdominal coloration, mosquitoes were categorized into the following groups: single colors (red, blue, green, or black), mixed colors (mosquitoes that consumed both colors), and uncolored (unfed or water-fed) mosquitoes. Mosquitoes that died during the ex-periment were excluded from the analysis.”

However, your study lacks critical ecological context and nuance that must be addressed.  You keep mentioning 'foraging' but you have really assayed is likely water foraging or sugar feeding (by way of detecting water) or even (if its 48 hours of assaying) that they happened to take a likely water drink because they were resting against the colored sugar meal anyways.
Therefore in the abstract and conclusions, and possibly intro It needs to be addressed, specifically what you think they are foraging for . . . and why you think that.
you do hint at refuge seeking - looking at picture of experimental arena, the e.g. green dot may be the only dark surface in there and that is a common cue for mosquitoes to seek refuge in.  Were the day biters more likely to seek darker colors? That would support this.

-> We agree that our experimental design does not allow us to definitively distinguish whether mosquitoes were primarily seeking water or sugar, and we therefore clarify that the assay captures voluntary contact with, and ingestion from, liquid resources. However, we consider it unlikely that the observed abdominal staining merely reflects incidental intake during resting. In our cages, mosquitoes typically rest on the vertical cage walls rather than on the cage floor where the colored substrates were placed. If the colored pads had primarily served as resting sites, we would have expected prolonged aggregation on specific colors, which was not observed. Instead, stained mosquitoes were commonly observed dispersed on the cage walls during the assay, with only a few individuals present on the feeding substrates at any given time. Importantly, mosquitoes had access to an additional water source without sugar or ink, and we observed engorged but non-stained (clear) abdomens, indicating that water intake without dye ingestion occurred. Taken together, while we cannot conclusively attribute the behavior exclusively to sugar feeding, these observations argue against a purely resting-driven or incidental explanation and are consistent with intentional ingestion from the dyed sugar solution.

-> l30-32: “This study investigates the color preferences of Aedes aegypti, Aedes albopictus, and Culex quin-quefasciatus during foraging, using an ink-based staining method to assess sugar-feeding behav-ior under varying light intensities.”

-> l83-84: “This method allows us to track the mosquitoes' color preferences in the context of nectar- and sugar-feeding behaviour.”

-> l 370-377: “In this context, it is important to note that the present experiment does not allow a definitive separation between active sugar–water foraging and color-dependent resting with incidental ingestion. However, the observed behavioral patterns are not consistent with classical resting or refuge selection. At the observation time points at the beginning and at the end of the experiments, mosquitoes were predominantly located on the vertical cage walls, and no pronounced aggregation on any colored substrate was observed (not quantified in this study), in contrast to patterns reported in some resting-focused studies [23,24]. Only few individuals were in contact with the feeding pads at these time points.”

-> l471: “Our findings reveal that mosquito color preferences observed in the context of sugar feeding are finely tuned to species- and sex-specific visual ecology and ambient light conditions:…”

Much previous work exists you could possibly cite or do your own experiments where they just assayed refugee color selection itself to try and resolve this what was the cause and what was the effect issue .

-> We agree that experiments explicitly isolating refuge or resting-site color selection would be valuable to further disentangle cause and effect. In our study, however, we did not observe a strong aggregation on any color, neither at the beginning nor at the end of the experiments; at any given time, only a few individuals were typically present on the feeding substrates, while most mosquitoes were resting on the cage walls. This pattern contrasts with classic refuge-selection assays, where large numbers of mosquitoes aggregate on preferred dark surfaces. Nevertheless, we acknowledge that we cannot definitively exclude the possibility that some color contacts occurred opportunistically during resting rather than as part of resource-directed behavior. This limitation has now been explicitly addressed and discussed in the discussion section, where we differentiate between resource-associated color effects and potential refuge-related explanations, and outline how future color-only assays could help resolve this question.

-> l 370-377: In this context, it is important to note that the present experiment does not allow a definitive separation between active sugar–water foraging and color-dependent resting with incidental ingestion. However, the observed behavioral patterns are not consistent with classical resting or refuge selection. At the observation time points at the beginning and at the end of the experiments, mosquitoes were predominantly located on the vertical cage walls, and no pronounced aggregation on any colored substrate was observed (not quantified in this study), in contrast to patterns reported in some resting-focused studies [23,24]. Only few individuals were in contact with the feeding pads at these time points.”

Do you think some sugar sources are 'supposed to ' a certain color ? (a flower red, a stem green for example ? Could this play a role?

-> Yes, color could plausibly act as a visual cue indicating sugar source quality or accessibility. In natural settings, accessible sugar is often associated with flowers, fruits, or extrafloral nectaries, which provide conspicuous visual signals, whereas green vegetative tissues typically do not reliably indicate a sugar reward. It is therefore conceivable that mosquitoes use color and contrast as a coarse heuristic to prioritize potential sugar sources, via innate biases and/or learned associations between visual cues and reward. This interpretation is consistent with studies using rewarding artificial flower mimics, where Aedes aegypti showed faster responses, higher visitation, and increased sugar feeding on colored “flowers” compared with white controls, and also exhibited color-dependent resting on those floral mimics (e.g., Dieng et al., 2018). In our study, we did not test natural plant structures, but our results support the idea that color cues can modulate contact and ingestion in a sugar-feeding context. We have incorporated this perspective as a brief, clearly framed hypothesis in the discussion.

-> l410-415: “From an ecological perspective, these patterns are consistent with resource-oriented behavior, as accessible plant sugars in natural environments are associated with visually conspicuous structures such as flowers or fruits rather than green foliage, which may rep-resent less accessible or less rewarding sugar sources [26,27]. Dedicated follow-up experiments, explicitly separating color-only resting assays from liquid-feeding contexts, will be required to disentangle resting-site selection from resource-associated color cues.”


finally, can you address more how you see this information being used?  You are certainly not trying to call this host seeking .

-> Yes, we agree, and we would like to clarify that the behaviors described in this study are not interpreted as host-seeking. The experiments exclusively involved fructose-based sugar solutions and therefore address nectar- and sugar-feeding behavior. We view the practical use of this information primarily in the context of attractive toxic sugar baits (ATSBs), where success depends on bait detection, landing, and voluntary sugar feeding rather than host attraction. In this framework, species- and light-dependent color preferences could be used to optimize bait visibility and feeding probability (e.g., through color choice of bait substrates or bait stations) without altering bait composition or toxicant dose. This applied perspective has been clarified in the discussion.

-> l457-468: “Taken together, these species- and context-dependent differences in color-driven behavior indicate that visual preferences emerge from the interaction between sensory physiology, light environment, and sugar-feeding ecology rather than representing fixed traits. Beyond their relevance for understanding nectar- and sugar-seeking behavior, the color preferences identified here may therefore also find practical application. In addition to serving as simple and effective markers for feeding assays in the laboratory, they could be exploited to improve applied control strategies such as attractive toxic sugar baits (ATSBs)[28] . Specifically, adapting bait color to target species and ambient light conditions—using dark, low-reflectance substrates for crepuscular mosquitoes or red stimuli to enhance attraction of diurnal Aedes species under moderate illumination—may increase bait detectability and feeding probability without modifying bait composition or toxicant dose, thereby enhancing the efficiency of sugar-based mosquito control.

Please find the changes in the attached manuscript highlighted in yellow.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General and Formatting

• Do italicize all scientific names (e.g., Aedes and Culex species) consistently throughout the manuscript. These terms can write as Aedes spp. or species of Aedes/Culex
• Consider using Cx. quinquefasciatus instead of Culex pipiens quinquefasciatus, as this nomenclature is widely accepted also
• Do standardize light intensity units and formatting throughout the manuscript (e.g., use lx instead lux and “1,600” rather than “1.600”)

 

Abstract

• Page 1, line 32: consider revise light intensity values to (0, 130, and 1,600 lx)

 

Introduction

• At first mention, consider introducing species in full: Culex (Cx.) quinquefasciatus, Aedes (Ae.) albopictus, and Aedes aegypti, and use the abbreviated forms (Cx. quinquefasciatus, Ae. albopictus, and Ae. aegypti) thereafter to avoid repetition

 

Methodology

• Page 3, line 135: Please add the estimated age and physiological status (host-seeking or blood-fed) of the mosquitoes used.
• Page 3, line 136: Could the authors explain why mosquitoes up to seven days old were used? Compared with 2–3-day-old mosquitoes, older mosquitoes may be more inclined to blood seeking activity
• Page 3, line 136: Were mosquitoes released at a 1:1 male-to-female ratio in each reps?
• Page 4, lines 145–148: Could the authors clarify why, rather than following previous studies that tested all color combinations together, the color combinations were tested separately?
• Page 4, lines 145–148: Were the color positions within a petri dish changed to reduce any positional bias?
• Page 4, line 167: Could Figure 1 be revised to add other colour combinations as well?

 

Results

• Page 5, line 189: Minor formatting issue
• Page 6, line 217: Did any mosquitoes die during the experiment, and if so, were they included in the analysis?
• Page 6, line 217: A fairly large number of mosquitoes were unstained, could this be due to dye dilution from the water provided or differences in metabolism? Were any control mosquitoes used to check this?
• Figure 4: Do italicize scientific names.

 

Discussion

• The findings appear particularly relevant to attractive toxic sugar bait (ATSB) studies. Do the authors agree, given that the colored dye may better represent flower-associated and host-seeking cues rather than oviposition trap colors? Including a brief ATSB-related discussion could help strengthen the applied relevance

 

Supplementary Material

• Supplementary File 3: Spelling error for Cx. quinquefasciatus

Author Response

We thank the reviewer for the careful reading of the manuscript and for the constructive and insightful comments, which have helped to improve the clarity and interpretation of our study. Below, we address each comment in detail.

General and Formatting

Do italicize all scientific names (e.g., Aedes and Culex species) consistently throughout the manuscript. These terms can write as Aedes spp. or species of Aedes/Culex

Consider using Cx. quinquefasciatus instead of Culex pipiens quinquefasciatus, as this nomenclature is widely accepted also

Do standardize light intensity units and formatting throughout the manuscript (e.g., use lx instead lux and “1,600” rather than “1.600”)

 

• corrected

 

Abstract

Page 1, line 32: consider revise light intensity values to (0, 130, and 1,600 lx)

 

• corrected

 

 

Introduction

At first mention, consider introducing species in full: Culex (Cx.) quinquefasciatus, Aedes (Ae.) albopictus, and Aedes aegypti, and use the abbreviated forms (Cx. quinquefasciatus, Ae. albopictus, and Ae. aegypti) thereafter to avoid repetition

• corrected

 

 

Methodology

Page 3, line 135: Please add the estimated age and physiological status (host-seeking or blood-fed) of the mosquitoes used.

• added

 

Page 3, line 136: Could the authors explain why mosquitoes up to seven days old were used? Compared with 2–3-day-old mosquitoes, older mosquitoes may be more inclined to blood seeking activity

• The age range of mosquitoes was relatively broad; however, ages were randomly distributed among experimental groups. As a result, this is expected to increase variability rather than cause systematic bias. An age-dependent analysis would indeed be of interest; however, based on the data generated in this study, we expect only minor behavioral differences between very young mosquitoes and those up to seven days old within the experimental setting used (sugar feeding and dye uptake).

 

Page 3, line 136: Were mosquitoes released at a 1:1 male-to-female ratio in each reps?

• Prior to the experiments, mosquito numbers were estimated; consequently, the final group sizes and sex ratios were random. Variation in sex ratios may have introduced additional variability due to sex-specific differences in physiology and behavior; however, as sex ratios were random, no systematic bias is expected
• L138-139: “Prior to the experiments, mosquito numbers were estimated, resulting in some variation in group sizes and sex ratios”

 

Page 4, lines 145–148: Could the authors clarify why, rather than following previous studies that tested all color combinations together, the color combinations were tested separately?

• In our initial study using Culex pipiens molestus, all four color solutions were tested simultaneously. However, the subsequent analysis proved challenging, as many mosquitoes ingested multiple colors in varying amounts, making it difficult to reliably distinguish between single-color uptake and mixed-color patterns. Consequently, this experiment was primarily used as an exploratory approach to identify the most promising color combinations. In the present study, we deliberately did not include this experimental setup, as the limited resolution of the resulting data reduced analytical accuracy. Instead, we focused on pairwise color combinations identified in the preliminary study, which allowed for clearer interpretation and more robust comparisons.
• L144-146: “However, unlike the referenced study, a comprehensive screening involving all four colors simultaneously was not conducted, as this approach previously resulted in ambiguous mixed-color uptake that limited analytical resolution.”

 

Page 4, lines 145–148: Were the color positions within a petri dish changed to reduce any positional bias?

• No, the positions of the colored sugar solutions within the Petri dishes were standardized and remained unchanged across experiments. All treatments were therefore conducted under identical spatial conditions to ensure comparability between replicates.
• 156-162: “The position of the petri dishes and pads within the cages was standardized such that identical colors were oriented toward the same cage corner, thereby avoiding potential biases related to flight distance, cage geometry, or visual field and colored pad position and to minimize positional effects, particularly given the predominantly wall-resting behavior of mosquitoes (Fig 1). Additionally, the uncolored water source was always in equal distance to both petri dishes.

 

Page 4, line 167: Could Figure 1 be revised to add other colour combinations as well?

• done

 

Results

Page 5, line 189: Minor formatting issue

• corrected

 

Page 6, line 217: Did any mosquitoes die during the experiment, and if so, were they included in the analysis?

• Information added
• L 177-178: “Mosquitoes that died during the experiment were excluded from the analysis.”

 

Page 6, line 217: A fairly large number of mosquitoes were unstained, could this be due to dye dilution from the water provided or differences in metabolism? Were any control mosquitoes used to check this?

• We thank the reviewer for this comment and have clarified this point in the manuscript. We assume that the relatively high number of unstained mosquitoes can be attributed to multiple factors. First, the applied light intensities themselves may have influenced sugar-feeding behavior. Second, mosquitoes were not starved prior to the experiments, which may have reduced immediate sugar uptake. In addition, dye excretion occurs at variable rates; we observed that ingested dye was excreted after approximately two days, although this process was not quantified in detail. For these reasons, and to avoid increased variability due to dye excretion and the emergence of multicolored abdomens, the experimental duration was limited to a maximum of 48 hours. Extending the feeding period would likely have increased variability and the risk of false-negative classifications. We further note that color intensity of the abdomen varied depending on the amount of colored sugar solution ingested and potential additional intake of uncolored water. Consequently, abdominal coloration was not assessed based on intensity but scored categorically as either colored or uncolored. This approach may have led to rare false-negative classifications in individuals that ingested only very small amounts of dye. However, all scoring was performed by a single observer, which likely minimized inter-observer variability and ensured consistent classification of borderline cases across experiments. No separate control mosquitoes were included for dye metabolism; instead, internal consistency was maintained through standardized scoring and experimental duration.
• L432: Further studies are needed to better understand the attractiveness of ink for albopictus, whether it is particularly attractive or repellent and to investigate its digestion and excretion dynamics in greater detail.”

Figure 4: Do italicize scientific names.

• corrected

 

 

Discussion

The findings appear particularly relevant to attractive toxic sugar bait (ATSB) studies. Do the authors agree, given that the colored dye may better represent flower-associated and host-seeking cues rather than oviposition trap colors? Including a brief ATSB-related discussion could help strengthen the applied relevance

• We agree with the reviewer that our findings are particularly relevant to attractive toxic sugar bait (ATSB) studies. The dye used in this study was applied to fructose-based sugar solutions and therefore represents cues associated with nectar- and sugar-feeding behavior, rather than oviposition-related stimuli. As such, the observed color preferences are more appropriately interpreted in the context of sugar source detection and feeding, which is the behavioral foundation of ATSB strategies. We have now added a brief discussion highlighting how species- and light-dependent color preferences could be exploited to improve ATSB visibility and feeding probability
• l436-447: Taken together, these species- and context-dependent differences in color-driven behavior indicate that visual preferences emerge from the interaction between sensory physiology, light environment, and sugar-feeding ecology rather than representing fixed traits. Beyond their relevance for understanding nectar- and sugar-seeking behavior, the color preferences identified here may therefore also find practical application. In addition to serving as simple and effective markers for feeding assays in the laboratory, they could be exploited to improve applied control strategies such as attractive toxic sugar baits (ATSBs). Specifically, adapting bait color to target species and ambient light conditions—using dark, low-reflectance substrates for crepuscular mosquitoes or red stimuli to enhance attraction of diurnal Aedes species under moderate illumination—may increase bait detectability and feeding probability without modifying bait composition or toxicant dose, thereby enhancing the efficiency of sugar-based mosquito control.

 

 

Supplementary Material

Supplementary File 3: Spelling error for Cx. quinquefasciatus

• Corrected

Please find the changes in the attached manuscript highlighted in yellow.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript “Species-Specific Color Preferences During Foraging in Aedes aegypti, Aedes albopictus, and Culex pipiens quinquefasciatus across Varying Light Conditions” presents a research study demonstrating that three major arboviral vectors exhibit differences in food-source color preferences that are influenced by their wild feeding behaviors (diurnal and nocturnal).

The manuscript is well written and clearly discussed. I have no major concerns, and the findings of this work are relevant to the field of vector behavior.

Author Response

We thank the reviewer for the time and effort invested in evaluating the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

thank you for addressing my comments.

Author Response

We also sincerely thank you for your careful and detailed re-evaluation of our revised manuscript.