Phlebotomine sand flies are vectors of human leishmaniases, important neglected tropical diseases. In this study, we investigated diel patterns of oviposition behavior, effects of visual cues on oviposition-site selection, and whether these affect the attraction of gravid Phlebotomus papatasi (Scopoli), the vector of old-world cutaneous leishmaniasis, to olfactory cues from oviposition sites. To evaluate these questions, we conducted a series of experiments using attraction and oviposition assays within free-flight test chambers containing gravid females entrained under a 14: 10 hrs light: dark photoperiod. By replacing sticky-screens or moist filter papers every three hours, we showed that oviposition site search occurs mainly in the latest part of the night whereas peak oviposition occurs during the early part of the night. Behavioral responses to olfactory oviposition cues are regulated by time-of-day and can be disrupted by transient exposure to a constant darkness photoperiod. Gravid females, but not any other stage, age, or sex, were attracted to dark, round oviposition jars, possibly resembling rodent burrow openings. This visual attraction disappeared in the absence of an illumination source. Egg deposition rate was not affected by jar color. Olfactory cues had the strongest effect when the visual cues were minimal. Our study showed, for the first time, that visual cues in the form of oviposition-site color, lighting level, and photoperiod are important in guiding the oviposition behavior of phlebotomine sand flies. Furthermore, such visual cues could modify the flies’ sensitivity to olfactory oviposition cues. Our results suggest that chemosensory and visual cues are complementary, with visual cues used to orient gravid females towards oviposition sites, possibly at long- to medium-ranges during crepuscular periods, while olfactory cues are used to approach the burrow in darkness and assess its suitability at close-range. Implications to sand fly control are discussed.
Phlebotomine sand flies (Diptera: Psychodidae) transmit protozoan parasites (Leishmania spp.), as well as pathogenic bacteria (Bartonella bacilliformis) and viruses [1–3]. Most significant are the human leishmaniases that, following malaria and dengue, are the most pervasive vector-borne diseases. Leishamaniases are found worldwide in warm tropical, semi-arid, and arid environments. The population at risk is estimated at approximately 350 million people with annual incidence estimated at 1–1. 5 million cases of cutaneous leishmaniasis, 500,000 cases of visceral leishmaniasis, and an estimated Disability-Adjusted Life Year (DALY) of 2. 3 million [4,5]. Leishmaniasis is distributed in the poorest regions of the world, and considered a neglected disease in terms of under-reporting, under-funding of research, and inadequate health care [6–8]. With no vaccine to protect against the etiologic agent, reduction of exposure to sand fly bites using insecticides is the most effective prevention measure [6,9, 10]. This strategy is seriously constrained, however, by the evolution of insecticide resistance, which renders residual sprays less effective [10,11]. Also, given the limited knowledge of sand fly breeding sites [12,13], source reduction using biolarvicides has not been an effective approach [10,14]. Hence, a more focused, targeted, and efficient control method is urgently needed [15]. An alternative approach to delivery of the insecticide to the vector is to bring the vector to the insecticide using attractants [16–18]. Oviposition-site attractants can provide the basis for a novel control and surveillance approach targeting gravid females that are typically responsible for pathogen transmission and population amplification. Oviposition traps have been used for the control of mosquitoes [19–23], but no such tool yet exists for the control of sand flies. Unlike other biting Diptera, sand flies develop in terrestrial rather than aquatic microhabitats [1,3]. Eggs are typically laid in soil rich in organic material on which the larvae feed and develop through four instars before pupation and adult emergence [1,3, 24,25]. For both new- and old-world sand fly species, olfactory cues originating from organic matter of various sources have been identified as important sources of oviposition attractants and stimulants [26–29], and some of these cues were shown to be produced by microbes [30,31]. Conspecific eggs are also attractive to sand flies [32–34], with dodecanoic acid identified as an oviposition stimulant for Lutzomyia longipalpis (Lutz and Neiva) [35]. Insects orient to oviposition sites using multi-modal sensory systems. Chemosensory cues play a central role as oviposition attractants and as lures in oviposition traps, especially for nocturnal insects [18,20,21]. However, visual cues may also play an important role, especially in diurnal and crepuscular insects [19]. For example, Aedes albopictus (Skuse) mosquitoes prefer to lay eggs in black plastic cups rather than in white, blue, or orange cups, or cups with black-and-white contrasting patterns [36], and Aedes triseriatus (Say) and Anopheles spp. visually prefer to oviposit in dark water [37,38]. Although sand flies were shown to respond to visual cues [39–44], nothing is known regarding their visual assessment of oviposition sites. Many sand fly species, including our study species Phlebotomus papatasi (Scopoli), are tightly associated with animal burrows within which they find shelter, blood-feed, lay eggs and where coprophagic larvae feed on decaying fecal matter [13,45]. Therefore, it is reasonable to hypothesize that gravid females would be visually attracted to dark round objects resembling burrow openings. The timing of trap deployment may also affect the efficacy of oviposition traps because different species prefer to lay eggs at different times. The circadian oviposition behavior of mosquitoes has been relatively well studied, but not so for sand flies. For example, Ae. aegypti, a day-time biter, consistently laid eggs in the afternoon irrespective of ambient temperatures, and when maintained on a 12: 12 hrs photoperiod it laid eggs just before light-out [46]. Given the nocturnal or crepuscular nature of sand flies [3,47,48] it is often assumed that they oviposit at night [49,50]. Indeed, gravid females of Ph. papatasi, which typically breed in rodent burrows, were observed to exit the burrows in the early part of the night, suggesting that oviposition-site search activity may start early in the night [51]. In contrast, the majority of Phlebotomus orientalis (Parrot) gravid females were caught after midnight, suggesting that oviposition-related activity occurs mostly in the later part of the night [52]. However, both studies relied on indirect evidence based on locomotor activity, and to date nothing is known about the timing of oviposition-related activities in sand flies. An important observation with mosquitoes was their integration of sensory inputs from olfactory cues and visual cues [53–55], and that it may vary during the diel cycle [56–59]. With sand flies, evidence for such interactions is scant. By studying circadian clock gene expression of Lu. longipalpis, researchers observed an evening peak that anticipated the lights-off cue [60]. With Ph. papatasi, adding a humidity source to a CDC trap increased capture only for white or clear traps but not for black traps [40]. To our knowledge, no studies with either mosquitoes or sand flies have documented such interactions in the context of oviposition behavior. In this work, we used Ph. papatasi, a vector of Leishmania major, the etiological agent of zoonotic cutaneous leishmaniasis (ZCL) in the Middle East [3,61]. We investigated its diel pattern of attraction to oviposition sites and timing of egg deposition, the effects of visual cues on oviposition-site selection and evaluated whether visual cues and photoperiod affect the attraction of gravid females to known olfactory oviposition cues. Specifically, our study focused on the following four aims: (1) Evaluate if time-of-day affects: (A) the timing of oviposition-site search activity, (B) timing of egg deposition, and (C) attraction to known olfactory cues; (2) Evaluate if transient changes in the photoperiod affect the attraction of sand flies to known olfactory oviposition cues; (3) Evaluate if visual cues, in the form of oviposition site color and illumination level, affect oviposition-site selection; and (4) Evaluate if visual cues affect the attraction of sand flies to known olfactory oviposition cues.
As part of the sand fly colony maintenance, sand fly blood-feeding on ICR white mice was conducted at the SoBran research facility, Gateway University Research Park in North Carolina under SoBran IACUC Protocol number UNC-002-2016 dated 12/12/2016. This protocol was approved by the SoBran Gateway Animal Care and Use Committee; Chaired by Stephanie Strahan. This institution is USDA-registered and PHS-assured, therefore, SoBran’s animal care and use standards adhere to the Animal Welfare Act (USC 7, Sections 2131–2159,1966, as amended) and Animal Welfare Regulations (9 CFR, Chapter 1, Parts 1–4); the PHS Policy on Humane Care and Use of Laboratory Animals USHHS, NIH, 2015); and The Guide for the Care and Use of Laboratory Animals (NRC, 8th edition), among others. In experiment #1, we evaluated the change in the number of flies caught on a sticky screen trap or the number of eggs laid on a filter paper over a diel cycle. We analyzed these longitudinal count data using a random-intercept negative-binomial regression model, with cage as the clustering factor [62]. We used paired Student’s t-test to compare the effect of treatment versus control within a cage. In order to compare the relative attraction of sand flies for the treatment, among treatment levels (e. g. , illumination level, time window, jar color) we calculated the proportion of flies caught in the treatment jar compared with total flies trapped in the two jars combined (hereafter, preference). Values above 0. 5 indicated preference for the treatment and values below 0. 5 indicated aversion to it. Given that “preference” is a proportion, we analyzed these data using weighted logistic regression, with number of flies making a behavioral choice used as the weighting factor [63]. Analyses were conducted using Stata (StataCorp. , College Station, TX).
In this experiment, sand flies that were reared under a 14: 10 light: dark photoperiod were tested for their olfactory preference to a known oviposition attractant (larval frass) under different photoperiod conditions: the 14: 10 photoperiod to which they were entrained or constant darkness. Flies exposed to the entrainment photoperiod exhibited stronger attraction to the rearing medium compared with flies switched transiently to constant dark conditions with an average preference rate of 60. 1 ± 3% and 49. 0 ± 4%, respectively (t = 2. 15, df = 59, P = 0. 03) (Table 1). On average, under the standard 14: 10 photoperiod, significantly more flies were trapped in the frass-baited jar than in the sand only control (4. 52 ± 0. 3 versus 3. 06 ± 0. 3 flies, respectively; paired-t = 3. 32, df = 30, P = 0. 001), whereas in assays conducted under constant dark conditions (0: 24 light: dark) no significant difference was found between the two jar types (4. 1 ± 0. 5 versus 3. 7 ± 0. 3, respectively; paired-t = 0. 612, df = 29, P = 0. 27). This pattern was consistent in the two rooms in which the assays were conducted (Table 1). Also, no significant difference was found with respect to the response rates, with an average response rate of 45. 3 ± 3% and 46. 3 ± 3% under these respective conditions (t = 0. 238, df = 59, P = 0. 81). Here too, results were consistent in the two rooms in which the experiment was conducted (Table 1). We conducted two-choice assays, using free-flight cages containing a pair of sticky traps of the same color with one jar containing larval frass (olfactory cues) and the other jar serving as control (moist sand only). Under complete darkness, gravid females exhibited clear olfactory attraction to the treatment jar irrespective of jar color (Fig 4). As light level increased to low-level (a 120-fold increase in brightness), olfactory attraction to the frass-containing jars decreased and became non-significant for all jar colors (Fig 4). This decrease was particularly steep and statistically significant for the black jars where attraction disappeared altogether (Fig 4). For other jar colors, preference showed a non-significant decreasing trend. No significant difference was found among jar colors (deviance = 2. 89, df = 2, P = 0. 23). With further increase in luminance (dim lighting level, 2-fold brighter than low-light), olfactory attraction to frass-baited jars remained low and non-significant for both black and white jars. For transparent jars, however, preference for the frass-baited jars increased and became significantly greater than for the black jars (paired-t = 2. 73, P = 0. 014) (Fig 4).
Essentially, nothing was known about the timing of oviposition-related behaviors in sand flies. With sand flies being predominantly nocturnal or crepuscular [3,47,48], it was assumed that they oviposit during the dark phase [49,50]. However, the questions as to when they search for oviposition sites and when they actually deposit their eggs have remained unanswered. With specially-designed exit-entrance traps, Yuval and Schlein (1986) observed that gravid Ph. papatasi females constituted a large fraction of flies exiting rodent burrows in the early part of the night, suggesting that oviposition-site search activity is more likely to occur early in the night [51]. In contrast, our results show a clear peak in activity related to oviposition-site search activity in the last three hrs of the scotophase that seems to also spill-over into the dawn and early hours of the morning. Furthermore, our results indicate that this time-period (the last three hours of the scotophase) is also when olfactory attraction to known oviposition attractants peaks. Obviously, our experiments should be repeated using field-caught or freshly produced lab-reared flies under field conditions. However, assuming our findings extend to natural sand fly populations, these results suggest circadian regulation of oviposition site-search behavior that affects the sand fly’s activity level and possibly sensory responsiveness to olfactory and visual oviposition cues. Why oviposition site-search activity is delayed until the later part of the night is yet to be determined. It is possible that, initially, gravid females need to search for a sugar-meal in order to replenish their energy reserves before switching to an oviposition site-search mode. This speculation is consistent with an observation by Yuval and Schlein (1986) showing that a high proportion of females entering burrows at the later part of the night were sugar fed, compared with those exiting burrows in the earlier part of the night [51]. With respect to timing of egg-laying, we observed a distinct peak of oviposition during the first three hours of the scotophase (which also included 1 hr of dusk) and lower oviposition rates before or after. These results highlight the adaptive value of circadian organization, which enables the coordination of metabolic processes and behaviors, including the expression of sensory receptors, relative to local entrainment conditions. The circadian organization of Ph. papatasi gravid females may be as follows: gravid females initially search for a sugar-meal during the night, then they switch to actively seeking a suitable oviposition site (often a rodent burrow) late in the night, they shelter within the burrow during the day, and then, possibly triggered by declining lights, other environmental changes such as relative humidity, or activity of the rodent host, they initiate egg-deposition at the beginning of the following night. Such a pattern is consistent with the behavior of many nocturnal mosquito species that tend to oviposit during dusk and early evening [19]. It is possible that a resting period within the burrow, before oviposition, enables females to complete egg maturation, mate, or even take supplementary blood meals from the resting host within the burrow. We cannot, however, discount the possibility that gravid females might also be laying eggs during the day within the dark burrow, and this possibility will be evaluated experimentally in the future. Attraction to a known olfactory substrate (larval frass) was shown to vary along the diel cycle with few flies attracted during the photophase or early in the scotophase, a slight increase towards midnight, and many flies attracted to frass during the last three hours of the night. Furthermore, our results indicate that the level of olfactory attraction of gravid sand flies to an attractive substrate is affected by the photoperiod regime to which they are exposed. Specifically, sand flies entrained under a natural photoperiod (14: 10 hrs light: dark, including 1 crepuscular hr at the start and end of the photophase) exhibited greater attraction to frass under these conditions than when switched to constant darkness (0: 24 hrs) for the 6-hr assay. On the other hand, activity levels, as expressed in terms of the flies’ response rate, did not differ between the two photoperiod regimes (Table 1). These results suggest that visual cues associated with the dawn crepuscular period and early photophase may affect the flies’ oviposition site-selection behavior by modifying their sensory responses to volatile olfactory cues. We validated experimentally that preference for the black jars was due to visual cues and not due to olfactory attraction to potential volatile components of the black paper, because flies did not differentiate between jars containing concealed black or white papers. Gravid females clearly preferred black jars over transparent (clear) jars or white jars, but they failed to differentiate between white and clear jars. To further explore the role of visual cues, we performed the black-versus-clear-jar choice experiment at three illumination levels: complete darkness, low light (simulating dark moonless night conditions), and dim light (simulating moon-lit night conditions). In the absence of any light, jars of different colors should not be visually differentiable, and indeed, sand flies did not exhibit a preference for black or clear jars. In contrast, in low and dim light gravid females clearly preferred the black jar over the clear jar. Likewise, gravid females preferred the black jar over the white jar in dim light, but showed no preference between clear and white jars. We also discovered that the effect of the visual cue pertains only to the oviposition site-search phase but not to the actual egg-laying phase. When provided with the choice of laying eggs on a wet black versus a wet white paper no difference in egg numbers was found. This observation is consistent with previous studies on both old- and new-world sand flies indicating that oviposition stimulation is triggered by chemosensory cues of fecal or conspecific origin [26,27,29–31]. Most striking was the fact that attraction to black jars over white jars was only exhibited by mature gravid blood-fed females. In contrast, non-blood-fed mature females, 74% of which were autogenous, did not exhibit such visual attraction (although a slight trend in that direction was suggested). This suggests that the level of female gravidity (% gravid) and fecundity (per-capita rate of egg production), which were shown to be higher in blood-fed females, are associated with the sensory acuteness of these females to visual oviposition cues. Visual attraction of adult males to black jars could be expected to mirror that of gravid females if mate-seeking males would await incoming gravid females in burrow entrances. The non-significant attraction trend we observed might be consistent with such a trend, but more study on that is obviously required. We also observed that activity level of young flies of both genders was considerably lower than that of mature flies, suggesting that activity of young flies may be stimulated by other cues such as those indicating presence of sugar- or blood-meal that are more relevant to flies at this stage [17,51]. Overall, this stage-specificity of responses to visual cues suggests that the black jars are perceived by gravid females as a visual oviposition cue and not as a generic cue. This result somewhat differs from findings that host-seeking Ph. papatasi flies were attracted to CDC traps where the collection cup was wrapped in black paper [40]. Attraction of gravid flies to black round jars that might be perceived as rodent burrow openings is consistent with our knowledge of the biology of Ph. papatasi that in arid areas are highly dependent on rodent-host burrows (typically Psammoys obesus or other Meriones spp.) as suitable oviposition and shelter sites [64,65]. The slight, non-significant affinity of sand flies of both genders and age/stages to black jars suggests a certain generality of this cue. Yet, the strong attraction of gravid females to a dark and round object suggests that they have evolved to become highly attuned to such a visual cue, which would facilitate the detection of sparsely distributed oviposition sites in arid environments. In complete darkness, gravid females exhibited significant olfactory attraction to used larval frass, a known source of volatile oviposition attractants [26,27], irrespective of oviposition jar color. As the light level increased, however, the degree of attraction to the frass-containing jar decreased or even disappeared altogether as visual cues became more apparent. This effect was particularly evident with black jars, which, as described above, provided the strongest visual cue to gravid females. Our results suggest that although visual and olfactory cues are integrated in the central nervous system to drive behavior, different sensory modalities take precedence during the orientation sequence. Such effects have been observed with mosquitoes [19]. In the confines of the relatively small, dimly lit, arena under our experimental conditions, visual cues appear to prevail over olfactory cues. Under natural conditions, however, we suspect that a cryptic dark burrow entrance on the surface of a relatively poorly lit ground might not be as apparent as a jar with a black vertical silhouette against a light-colored background, as used in our assays. We further suspect that chemosensory and visual cues are complementary, with visual cues used to orient gravid females towards potential oviposition sites at long-to-medium ranges during the crepuscular periods, while olfactory cues are used to approach the burrow in darkness and assess its suitability at close-range. Direct evaluation of the complementary effect of these cues would require a separate factorial-design experiment, including manipulation of the visual apparency (signal-to-noise) of the burrow entrance. Results of this study add important novel insights for our understanding of the basic biology of Phlebotomine sand flies and have important implications for management of sand fly populations. First, in the context of the development of an oviposition trap, it is clearly necessary to take visual cues into account. Such a trap should be black and round resembling a rodent burrow entrance. Optimizing trap shape and design is an important next step, and of particular importance are its vertical aspect and apparency relative to the ground terrain. Then, visual cues should be complemented with chemosensory cues that include olfactory stimuli to guide the flies to the burrow and contact and gustatory cues to arrest females and stimulate oviposition. Our results show that visual cues might transcend olfactory cues, but this might not be the case under natural light and landscape conditions. Our findings that oviposition site search activity occurs in the late scotophase would suggest that traps should be activated in the field during the second part of the night and early morning. This would require that traps either be deployed in the later part of the night or that the olfactory lure is operational throughout the night. Nevertheless, these results require confirmation in the field with traps or rodent-burrow monitors under various natural-light conditions. Finally, results of this study contribute three important features and conditions for optimizing bioassays for further studies of sand fly oviposition site-selection. First, when screening olfactory attractants, visual cues should be minimized and probably transparent jars would serve this purpose best. Second, attraction bioassays with gravid sand flies should be implemented in the later part of the scotophase under minimal light, conditions under which chemosensory information takes precedence over visual cues. Finally, flies should be reared and maintained using a natural photoperiod representative of summer conditions to optimize the sensitivity of bioassays. These recommendations are already implemented in current studies in our lab.
Sand flies are vectors of human leishmaniases, an important neglected tropical disease. An alternative approach to the conventional delivery of an insecticide to the vector is to bring the vector to the insecticide using oviposition (egg-laying) -site attractants. Olfactory cues originating from organic matter have been identified as important sources of oviposition attractants. However, nothing is known regarding visual assessment of oviposition sites by sand flies. Also, little is understood about diel egg-laying patterns of sand flies. Finally, it is not known if visual cues and time-of-day may affect their sensitivity to olfactory cues. In this study, we investigated these questions in a series of lab experiments using free-flight cage arenas, with Phlebotomus papatasi (vector of cutaneous leishmaniasis). We showed that peak oviposition-site search and sensitivity to olfactory cues occurs mainly in the latest part of the night whereas peak oviposition occurs during the early part of the night. We demonstrated that only gravid females, but not any other stage or sex, were attracted to dark, round oviposition jars resembling burrow openings. Finally, we showed that sensitivity to olfactory cues is reduced in the presence of strong visual cues and in the absence of natural diel photoperiod.
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Diel periodicity and visual cues guide oviposition behavior in Phlebotomus papatasi, vector of old-world cutaneous leishmaniasis