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UNIVERSIDAD DE ZARAGOZA FACULTAD DE VETERINARIA Departamento de Patología Animal “Fototropismo en dípteros del género Phlebotomus ” Proyecto de Fin de Grado en Biotecnología dirigido por Javier Lucientes y Ronald Vladimir Oropeza José Herrera Russert Curso 2013/2014 Index ABBREVIATIONS 1. Abstract ........................................................................................................................ 1 2. Introduction ................................................................................................................. 3 2.1 Main characteristics of sandflies .............................................................................. 3 2.2 General aspects of leishmaniosis ............................................................................. 4 2.2.1 Introduction .................................................................................................................. 4 2.2.2 Biological cycle of Leishmania, mecanism of pathogeny and clinical profile ............ 4 2.2.3 Diagnosis and treatment ............................................................................................... 5 2.3 Control and prevention of leishmaniosis ................................................................. 6 2.3.1 The use of light traps in sandfly control ....................................................................... 6 3. Aims .............................................................................................................................. 7 4. Materials and methods ................................................................................................ 8 4.1 Species used ............................................................................................................. 8 4.2 Rearing of Phlebotomus spp .................................................................................... 8 4.2.1 Preparation of larval substrate ...................................................................................... 8 4.2.2 Obtention of live material ............................................................................................ 8 4.2.3 Colony establishment ................................................................................................... 9 4.2.4 Colony maintenance ..................................................................................................... 9 4.3 Study of the phototropism of P. papatasi .............................................................. 10 4.3.1 Design of the experimental enclosure ........................................................................ 10 4.3.2 Phototropism tests ...................................................................................................... 11 4.3.3 Analysis of obtained data ........................................................................................... 12 5. Results ......................................................................................................................... 13 5.1 Results for the first experimental enclosure ........................................................... 13 5.2 Results for the second experimental enclosure ...................................................... 13 5.2.1 Young males and young females in same-sex groups ................................................ 13 5.2.2 Young males and Young females in combined groups .............................................. 16 6. Discussion ................................................................................................................... 18 6.1 Influence of the experimental enclosure ................................................................ 18 6.2 Light as the only stimulus ...................................................................................... 18 6.3 Effect of the diodes ................................................................................................ 19 6.4 Differences in the response of males and females ................................................. 20 7. Conclusions ................................................................................................................ 21 8. Bibliography ............................................................................................................... 22 9. Annex of tables ........................................................................................................... 24 ABBREVIATIONS ANOVA: analysis of variance CDC: Center for disease control cm: centimeter g: gram LED: light-emitting diode l: liter L1-3: larval instars 1 through 3 mg: milligram mm: millimeter NJLT: New Jersey light trap PCR: polymerase chain reaction spp: multiple species Th: T helper (lymphocyte) V: Volt W: Watt ºC: degrees Celsius 1. Abstract 1 Phlebotomus papatasi (Diptera: Psychodidae) es el flebotomo vector de de la leishmaniasis cutánea zoonótica causada por Leishmania major (Trypanosomatidae: Kinetoplastida), una enfermedad parasitaria causada por un protozoo prevalente en la mayoría de regiones mediterráneas, siendo considerada en muchos casos un problema de salud grave tanto animal como humano. En este trabajo se estudió el tropismo positivo de individuos adultos de esta especie, confinados en un recinto experimental tubular desarrollado durante el transcurso del mismo, hacia la luz emitida por dispositivos LED (diodo emisor de luz) de tres colores principales, el verde, rojo y azul, además de varias pruebas adicionales con luces violeta y amarilla. Se empleó la luz ultravioleta procedente de trampas CDC como control positivo y un control negativo en ausencia de luz para todas las pruebas. Los individuos proceden de una colonia de flebotominos (P. perniciosus y P. papatasi) ubicada en el departamento de patología clínica veterinaria de la universidad de Zaragoza. Los resultados indican que existe una atracción elevada hacia la luz ultravioleta en los casos estudiados, mientras que las luces led no generan una atracción muy significativa. Específicamente la luz verde dio lugar a un grado menor de atracción en los casos estudiados. Tanto en las pruebas con machos aislados como con una combinación de machos y hembras, los machos resultaron ser más atraídos por las luces que las hembras. Estos resultados podrían resultar de utilidad en el diseño de nuevas trampas para el control o monitoreo de flebotominos y en el desarrollo de sistemas de iluminación menos peligrosos para animales domésticos. 1. Abstract 2 Phlebotomus papatasi (Diptera: Psychodidae) is a sandfly that acts as a vector of the zoonotic cutaneous leishmaniasis caused by Leishmania major (Trypanosomatidae: Kinetoplasmida), a parasitic disease caused by a protozoan that is prevalent in most Mediterranean regions, and that is considered a serious threat to the health of both humans and livestock. In this paper the attraction towards light emitting diodes (LEDs) experienced by adult individuals of the aforementioned species was tested within a tubular experimental circuit constructed specifically to this end. The experiments focused on three main colors; green, red and blue, and additionally yellow and purple lights were also tested for. Ultraviolet lighting from CDC insect traps was used as the positive control, and every experiment was also accompanied by a lightless negative control. All sandfly specimens used were from a phlebotomine colony (that features both P. papatasi and P. perniciosus) located in the department of clinical pathology of the University of Saragossa. The results indicate that there exists a strong attraction towards ultraviolet light in the experimental conditions, whereas LED lighting does not generate a strong tropism. Green light specifically appeared to show the lowest degree of attraction. Males were more attracted to the lights than females in all the tests, both in a same sex group and combined with females. These results could prove useful in the design of new insect traps for pest control or phlebotomine population monitoring, as well as in the development of safer lighting systems for livestock. 2. Introduction 3 2.1 Main characteristics of sandflies Sandflies (Phlebotominae) are a subfamily of insects belonging to the order Diptera and specifically to the family Psychodidae. Amongst this group, only two genera function as vectors of leishmaniosis, Lutzomyia in the new world, and Phlebotomus, which is the focus of this paper, in Europe, Africa and Asia [1]. In Spain, the genus is represented by 13 species but only those belonging to the subgenus Larrossius are capable of transmiting the disease [2]. This paper has its focus centered upon the species P. perniciosus and P. papatasi, the most important vectors in the Spanish interior [3]. These dipterans may be distinguished by the naked eye thanks to their small size of between 2 and 3 mm, very long legs, two lanceolate wings that are held open in a V-like shape when at rest, and a color that varies between light brown and beige. They are nocturnal insects, with a peak in their activity that starts at dusk and continues until midnight. As for their habitat, sandflies are to be found in natural environments such as animal nests and at the foot of trees and bushes, but also in man-made areas like sewers, gardens, farming land, etc [2]. The full life cycle of the sandfly lasts about 45 days in optimal conditions. The animal completes 4 developmental phases: egg, larva, pupa and an imaginal or adult stage. It is not possible to give more precise time periods for each stage because their length varies significantly depending on air temperature and humidity [1]. Ideal growth conditions include a temperature of 17-30ºC, the exact value depending on the species, and a humidity level of 80%. [2, 4]. A lower temperature would increase the duration of the cycle, whereas temperatures above 40ºC and low humidity levels can kill the sandflies [2, 5]. In laboratory conditions, the eggs take 7-10 days to hatch. The larvae, worm-like in appearance, take about 3 weeks to pupate and go through 3 molts that separate the 4 larval stages, termed as instars, and referred to as LI-IV. The adult sandflies finally emerge from the pupae after about 10 days, with males emerging earlier than females [1]. Nourishment depends on the stage of the insect. Larvae have chewing mouth parts and feed off organic matter. Adults bear cutting-sucking mouth parts that allow them to cut through skin and sever capillaries to let the blood flow out, which they then directly ingest [2]. However, the adult insects also feed on the nectar of flowers, ripe fruits and other plant sources of sugar, as well as aphids. The ingestion of blood is a needed requirement for the female to be able to develop eggs. Only the females have sufficiently developed mouth parts to be able to cut through skin, so that they are the sole vectors of leishmaniasis. On occasion blood-filled males may be observed, but in these cases it is assumed that they have obtained it from an already open wound [1]. Throughout their life females may bite between 3 and 5 times, although most of them only bite once in their life time. The number of eggs laid is directly correlated to the quantity of blood ingested, and may reach 200. Most species, among them those mentioned in this paper are also gonotrophic, meaning they lay their eggs after each blood ingestion[1, 2]. 2. Introduction 4 2.2 General aspects of leishmaniosis 2.2.1 Introduction Leishmaniasis in human medicine, or leishmaniosis in a veterinarian context, refers to a disease caused by all the tripanosomatidean protozoa belonging to the genus Leishmania. It is a vector disease that is always transmited by the bite of a sandfly [6]. It may be zoonotic, when the reservoir is wild animals or livestock, or anthroponotic when the reservoir is human [1]. The first depictions of human leishmaniasis trace back to the year 650 b.C. in Babylon [7]. Currently 88 countries are affected, especially those located in tropical and subtropical areas, and it is associated with about 70000 deaths every year [8]. The clinical manifestations can be more or less severe, giving name to three distinct types of leishmaniasis: cutaneous, mucocutaneous and visceral [6]. Thirty species are known within the genus Leishmania, out of which about twenty are known to be pathogenic to humans [9]. In Europe, and more specifically in the Mediterranean region, L. infantum is the main causative agent of the disease, which causes cutaneous and visceral forms in humans as well as in dogs, its other main reservoir [10]. 2.2.2 Biological cycle of Leishmania, mecanism of pathogeny and clinical profile Leishmania presents two stages, the stage present depending on the host: in sandflies it is present as a promastigote, the elongated extracellular flagellated form, and in its vertebrate hosts it is found as an amastigote, oval in shape, much smaller and located within the phagolisosomes of mononuclear phagocytic cells [11]. The cycle begins when a female sandfly feeds on the blood of a mammal with amastigoteinfected macrophages. These amastigotes turn into promastigotes in the sandflies’ intestine and quickly rise in number as they traverse a series of non-infective middle stages. On reaching the stage of metacyclic promastigotes, the protozoans are already infective and extend a phlagellum that allows them the mobility to migrate to the sandflies’ proboscis. During the sandflies’ next blood ingestion the promastigotes are regurgitated and penetrate the wound caused during the bite. In the vertebrate host they are engulfed by macrophages and other phagocytes, then transform into amastigotes and are included into a parasitophorous vacuole that constitutes the phagolysosome. There the protozoan reproduces without being detected until it causes its host cell to lysate. Once free in the blood vessels it can again be phagocyted by other cells, completing the cycle [11]. In dogs the mechanism of patogenicity adjusts to the following description. When the promastigotes are inoculated into the mammalian host a local inflammatory response is triggered that attracts neutrophils, eosinophils, natural killer cells, and later on macrophages. The antigens carried by Leishmania are phagocyted by dendritic cells or are freely transported by way of the lymphatic ganglia, where they are presented to activate T lymphocytes. This initially triggers a Th2 type response that leads to the release of various cytokines. Depending on the cytokines released the Th1 or the Th2 type response will prevail, which in turn 2. Introduction 5 determines whether the individual is susceptible or resistant to the disease, respectively. In the Th1 response, activated Th1 lymphocytes release cytotoxic NO that causes the death of the parasite, and at the same time the proliferation of B lymphocytes is activated so that immunoglobulins are secreted that encourage resistance to intracellular microorganisms, and the destruction of infected macrophages occurs through Fas-dependent apoptosis. In the Th2 response however, the parasite causes a polyclonal stimulation of B lymphocytes that causes an excess in immunoglobulins. The high concentration of circulating immune complexes that results is the direct cause of the pathologies related to this disease [12]. The clinical profile of leishmaniasis in humans, as well as in dogs, depends on a variety of factors that determine the immune response: parasite genotype, size of the inoculum, zone of inoculation, number of bites received, the sandflies’ saliva, certain genetic traits of the host, the existence of concomitant infections, etc. This originates three types of leishmaniasis. In the first one, the cutaneous type, the parasites remain isolated in the macrophages of the area that was bitten as a result of a strong cellular immune response. This causes the appearance of erythematose papules in the site of the bite that grow and ulcerate, generating scabs of dry exudate that may heal by themselves leaving behind hypopigmented scars. In the mucocutaneous form, the parasites spread towards the mucous membrane and can potentially destroy and disfigure it, especially the nasal and buccal mucosa. Finally, in its visceral form, the parasites spread into inner organs as a result of a failed cellular response and cause fever, weight loss, hepatosplenomegaly, glomerulonephritis, anemia and kidney failure, among other alterations [13]. It should be noted that specifically canine leishmaniasis is frequently both cutaneous and visceral [14]. 2.2.3 Diagnosis and treatment Diagnosis, in humans as well as in animals, can be achieved through microscopy, examining the presence of the parasite in dyed blood films from splenic biopsy, bone marrow and lymph nodes, in skin extracts and tissue biopsies. If the infection is of a low degree, parasite detection should be accomplished by isolating it in vitro or in vivo or with a polymerase chain reaction (PCR). Since the differences between species are few, any isolated Leishmania must be identified by molecular, biochemical and immunological methods [15]. The treatment depends on the location, the type of Leishmania and the seriousness of the disease. The first line of treatment consists in the administration of pentavalent antimony derivates: N-metilglucamine antimoniate, generally in Europe, and sodic estibogluconate, normally in America. More and more resistances to these drugs are appearing, however. Noteworthy second line pharmaceuticals include Amphotericin-b, that can cure visceral leishmaniasis in spite of its toxicity and, more recently, Miltefosine, a new drug that has proven itself safe and effective in the short term, but also has teratogenic properties. In addition to these, levamisol and the Calmette and Guerin bacillus are used as coadjuvants. Despite these medical approaches, the death rate among patients suffering from visceral leishmaniasis remains high even with a proper treatment [13]. 2. Introduction 6 2.3 Control and prevention of leishmaniosis No effective vaccine against leishmaniasis exists currently [15]. For this reason, control measures for the disease focus on the early detection and treatment of cases, and on the elimination of vectors and reservoirs. The sacrifice of seropositive dogs is an option hardly considered because of ethical and social reasons. Despite this, a study was conducted in Brazil where this prophylactic measure was taken, and it was observed that a reduction in seropositive dogs does not correlate with a decrease in the prevalence of human leishmaniasis [16]. On the other hand, the use of insecticide-impregnated collars for dogs has been suggested. Experimental results indicate that this could significantly lower infection in humans and dogs, but more studies on the subject are still needed [17]. Treatment of infected animals has also been attempted as a control method. However, in a study conducted in Italy where all asymptomatic and oligosymptomatic dogs were treated and symptomatic ones were suppressed, it was concluded that prevalence of the disease did not vary much either [18]. Finally, fighting off the vectors has been tried, spraying the walls of houses and animal nests such as rabbit holes and termite mounds with insecticidal agents. However, more studies of this kind are required, and also a better knowledge of the feeding, resting, and reproductive habits of the various sandfly species so as to characterize them before insecticide implementation. There is also evidence that insecticide-resistant sandfly populations are starting to arise [17]. 2.3.1 The use of light traps in sandfly control Within the context of leishmaniasis control programmes, it is important to have methods of vector monitoring. One of the most frequently used is the capture of sandflies with CDC light traps [1] the history of which will be briefly outlined. The first prototypes of light traps were the New Jersey traps during the 1920s [19]. The NJLT uses a 25 W incandescent light bulb as a means to attract numerous mosquito species, and it is still in ample use as an element in mosquito population assessment. Later on, the need for smaller and more portable traps led to the development of CDC type traps [20], which use an incandescent lamp powered by a 6 V battery and are very often supplemented with a CO2 emitting agent. In the last 70 years, investigators have tried a wide variety of traps that incorporate artificial light of different colors, intensity and/or frequencies in an attempt to improve the effectiveness of the present capture systems [21, 22, 23]. The color of the trap itself (reflected light) and the color of the lamp are among the most studied stimuli in the attempt to increase the efficacy of traps for the capture of mosquitoes, sandflies, and other biting Diptera [22]. 5. Results 13 5.1 Results for the first experimental enclosure The first experiments that were performed used the first described enclosure, which consisted in three cages linked by tubes. Several repeats were carried out with the incandescent light bulb and with the ultraviolet light, yielding a negative response from the sandflies, which all remained in the first cage, with isolated individuals reaching the tubes or the intermediate cage on occasion. The vast majority of the specimens tended to perch in the upper corners of the cage, where they remained oblivious to the possible attraction exerted by the light. The groups that were tested for were comprised of about fifteen individuals each and without making any sex distinctions, since these tests were performed in the initial phase where a valid enclosure model was still being sought for. Since this first model was clearly inadequate, it was discarded and a second model was constructed. Because the sandflies appear to congregate and get stuck in any crevices the space may have, such as the corners of the cages, it was clear that the second model would have to take the form of a smooth cylinder or a funnel. 5.2 Results for the second experimental enclosure Initial testing with this enclosure showed that it was clearly adequate, as opposed to the first model, because in this case a response by the sandflies was clearly recorded and all specimens were equally under the influence of the light, there being no corners or darker areas where they could preferentially congregate. The results shown here refer to groups of young adult individuals, as a few tests were carried out using newly hatched adults and even a few representatives of P. perniciosus, but these did not make out the bulk of the experiments and the data were deemed quantitatively insufficient to be worth treating further. 5.2.1 Young males and young females in same-sex groups Young individuals would be of ages comprised between 3 and 5 days after emergence. This is a representative age for tropism studies, since young, as yet unfed individuals disperse at a higher rate [25]. Experiments carried out using same-sex groups formed by ten individuals each yielded the plot shown in figure 5.1. The data used for this graph belonged to six different repeats for each individual color in the cases of red, green and blue, including one negative control for each one of these (see tables A1 and A2). Data for white, yellow and purple were not as abundant, originating from only three repeats. The mean of these groups, each composed by three tubes, was obtained and plotted. 5. Results 14 The results point towards similarities between the control and the main colors studied. Among the colors that received a more comprehensive treatment, red, while not being overly attractive does seem to stand out for males while green showed the lowest levels of attraction. Yellow and white seemed to be more attractive to males, but more experiments should be conducted to prove this. Differences between the sexes were outlined comparing overall performance of males as compared to females, as can be seen in figure 5.2. Figure 5.1 Male or female response to individual colors: Results for males and females are expressed in percentages of attraction and non-attraction related to each individual group. Red line depicts female tendencies for each color and blue line depicts those of the male. There was a clear bias in the performance of males and females in isolation from each other, with males showing a much higher tendency to move towards the light than females. The negative control already showed a slight imbalance with more males than females reaching the end of the tube though, so these results may not necessarily indicate that the males are drawn to the light. The discrepancy was however further increased by the presence of the light. Current effect: F(6, 120)=11,760, p=,00000 Effective hypothesis decomposition Vertical bars denote 0,95 confidence intervals MALES FEMALES RED Var2: ATTRACTION NO ATTRACTION -2 -1 0 1 2 3 4 5 6 7 % GREEN Var2: ATTRACTION NO ATTRACTION BLUE Var2: ATTRACTION NO ATTRACTION WHITE Var2: ATTRACTION NO ATTRACTION YELLOW Var2: ATTRACTION NO ATTRACTION PURPLE Var2: ATTRACTION NO ATTRACTION CONTROL Var2: ATTRACTION NO ATTRACTION 5. Results 15 Figure 5.2 Male or female response to all colors: Results for males and females are expressed in percentages of attraction (blue) and non-attraction (red). Response to ultraviolet light was also tested in these same conditions, and gave the results summarized in figure 5.3. Data for this type of light stemmed from three repeats like the ones described above (see tables A5 and A6). This was sufficient to demonstrate a clear tropism towards this kind of light in the conditions imposed by the experiment, with males still showing higher values than females Figure 5.3 Male or female response to ultraviolet light: Both males and females are shown to develop an increased attraction, in blue, and a lower attraction, in red, to ultraviolet light as compared to the control. Data are expressed as percentages of attraction in each repeat. Current effect: F(1, 20)=186,94, p=,00000 Effective hypothesis decomposition Vertical bars denote 0,95 confidence intervals ATTRACTION NO ATTRACTION MALES FEMALES Phlebotomus papatasi -0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 % Current effect: F(1, 8)=1,5000, p=,25551 Effective hypothesis decomposition Vertical bars denote 0,95 confidence intervals ATTRACTION NO ATTRACTION ULTRAVIOLET Var1: MALES FEMALES -2 -1 0 1 2 3 4 5 6 7 % CONTROL Var1: MALES FEMALES 5. Results 16 5.2.2 Young males and young females in combined groups Combined groups composed of males and females were also tested. In this case four repeats of three tubes and an accompanying negative control each were tested (see tables A3 and A4). Group composition included 5 males and 5 females for each tube. Results are shown in figure 5.4. Red again stood out in comparison to blue and green, albeit with rather modest results. Specifically males seemed to be attracted the most to this type of light. Green showed the most negative results. The combination of males and females did not seem to exert a noticeable influence over their response to the light. Figure 5.4 Male and female response to individual colors: The colors tested were red, green and blue. Percentages of attraction and non-attraction are shown for males in blue and for females in red. Male and female behavior was also summarized in figure 5.5, which shows results analogous to those from figure 5.2. The same concept applies for the tests run with ultraviolet light (see tables A5 and A6), figure 5.6, where the results clearly resemble those from figure 5.3. In conclusion, the presence or absence of members of the opposite sex does not seem to influence phototrophic behavior in experimental conditions. Current effect: F(3, 36)=6,2634, p=,00156 Effective hypothesis decomposition Vertical bars denote 0,95 confidence intervals MALES FEMALES RED Var2: ATTRACTION NO ATTRACTION -2 0 2 4 6 8 10 12 % GREEN Var2: ATTRACTION NO ATTRACTION BLUE Var2: ATTRACTION NO ATTRACTION CONTROL Var2: ATTRACTION NO ATTRACTION 5. Results 17 Figure 5.5 Male or female response to colors: Attraction, in blue, and non-attraction, in red, are shown as percentages for each repeat. Lights included here are red, green and blue as per figure 5.4. Figure 5.6 Male or female response to ultraviolet light: Attraction, in blue, and nonattraction, in red, are shown as percentages for each repeat for ultraviolet light and its corresponding negative controls. Current effect: F(1, 12)=183,49, p=,00000 Effective hypothesis decomposition Vertical bars denote 0,95 confidence intervals ATTRACTION NO ATTRACTION MALES FEMALES Phlebotomus papatasi -1 0 1 2 3 4 5 6 7 8 9 10 11 % Current effect: F(1, 8)=1,2800, p=,29067 Effective hypothesis decomposition Vertical bars denote 0,95 confidence intervals ATTRACTION NO ATTRACTION ULTRAVIOLET Var1: MALES FEMALES -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 % CONTROL Var1: MALES FEMALES 6. Discussion 18 6.1 Influence of the experimental enclosure It was confirmed that a member of the phlebotomines, P. papatasi, does not show an elevated attraction towards light emitting diodes, to which it was exposed in groups of between 5 and 20 colony-bred individuals in the above experiments. Even upon exposure to the ultraviolet lamp, attraction was not unanimous with a significant number of specimens, especially females, staying away from it. This last result is partially attributable to the design of the experimental enclosure where the insects were located during the tests, as was shown by their behavior in the initial three cage system, and the fact that much higher levels of attraction were achieved keeping an undetermined but high number of insects inside the glass cylinder of the aspirator, a space with a 1 cm diameter. Differences between the aspirator and the experimental enclosure which could help explain this change are available space, number of individuals (a lot higher in the aspirator and hence a higher density), and the sex ratio, unknown in the aspirator but specified for the experiments. 6.2 Light as the only stimulus Studies performed by Müller and Hogsette show that, in field conditions, which necessarily differ from those found in the laboratory, light is not the determining stimulus in the tropism of the P. papatasi captured near the Dead Sea [26]. In one of these studies, traps without attractive stimuli were presented in addition to different combinations of CO2, light, attractive colors, temperature, humidity and chemical attractants. CO2 turned out to be the main attractant, followed by ultraviolet light, which has also exerted an undeniable effect on the animals in our experiment, with the effect of the other stimuli being lower. It was also discovered that there exists a synergistic effect between these stimuli. Many other studies referred to Phlebotomus and other genera also imply that attraction towards ultraviolet light is the highest, followed by incandescent light [26], but the presence of animal odors in the form of octanol, CO2, caproic acid or the presence of avian livestock [27], is a more powerful factor in itself than light, synergistic in the cases studied, and probably necessary to achieve an elevated positive tropism, which could add to explain why the levels of attraction in our tests were far from maximal. This also concurs with the experiments conducted by Kirstein and Faiman [28], where the traps that were not equipped with chemical lighting actually yielded a higher number of representatives of the genus as compared to CO2 traps additionally equipped with lights. 6. Discussion 19 6.3 Effect of the diodes Light emitting diodes only began to be employed in light traps for insects a few years ago [29], and thus far most of the work has been focused on the capture of Culicidae. Studies that used traps equipped with diodes of various colors (blue, green, yellow, orange, red and infrared) have proven useful for the capture of several species of Anopheles, Culex, Culiseta, Ochlerotatus and Psorophora, with the most effective colors being blue and green, which equaled incandescent light in effectiveness measured as the number of mosquitoes lured in one night. Burkett also proved that these culicids are more readily attracted to reflected light than to emitted light, a point that has not been covered in this study, but that also holds true for P. perniciosus [30]. The differences in the effect exerted by the different LED colors in our study were not very high, but there was a sustained tendency towards a lower attraction for green diodes. In a study conducted by Hoel and Butler in Egypt, CDC traps were modified for their use with red, green and blue diodes, resulting in high numbers of captured P. papatasi [30]. In this case the red lights attracted more than twice as many specimens as the incandescent controls and more than four times those lured by the blue and green lights. These results would suggest that red light could be an adequate substitute for conventional traps, with a higher and more specific yield and also a reduced energy budget since the light comes from diodes. Although in our study, much more limited in space and subjected to the conditions imposed by the experiment, such a high difference between the performance of individual colors was not noted, there was a distinctly lower attraction towards green lights as compared to red or blue for the young adults of this species, but the effect of the red light remains small nevertheless, with the only clearly positive response being exerted by the ultraviolet light, a fact that is already well known and covered in studies for many kinds of dipterans, and the basis of how CDC traps and others work. Even though light emitting diodes have been successfully deployed to capture culicids, our result suggests that the same doesn’t necessarily hold true for P. papatasi. For the same reason, lighting systems that employed diodes instead of incandescent light bulbs, perhaps with the exception of red light in view of the field results from Hoel and Butler and the slight inclination of our own specimens in comparison to blue or green, could be more adequate in a situation of risk of Leishmania transmission to humans and livestock. 6. Discussion 20 6.4 Differences in the response of males and females The clearest bias in this study was originated by differences in the response of males and females. Males generated a clearly higher number of positive results in all the conditions that were tested for. There are differences in the distances covered by sandflies, mainly related to their sex, physiological status and type of habitat. Generally it is young females that travel the greatest distances in search of a source of blood, while males lack such a need and disperse more slowly as a result [25]. However, it has also been observed that, in adults of P. perniciosus that enter homes right after sunset, the males are the first ones to appear, followed by the females after an interval of approximately an hour and a half [31]. These observations illustrate that there are differences in movement and dispersal in field conditions, explained mainly by the need for blood of females. The relation of these differences in dispersal to phototropism is hard to establish, and it doesn’t seem to carry much validity in the conditions established in our experiments with the diodes due to the restrictions in space and time available to them. The differences in dispersion capacity have been tentatively explained through reproductive behavior, whereby males would tend towards aggregation on domestic animals, though not sucking blood and they would be the ones exerting attraction on the females by means of secreted pheromones. In this way, the males would be the ones to find a suitable location where they would form “mating swarms” and contribute to the approach of females [31]. This behavior was observed in our case during the times of feeding females, where males also perched in high numbers on the hamster. This effect of males over females has been used to optimize light traps, with the conclusion that small groups of males used as bait in these traps increase the number of captured females in comparison to baitless traps [32]. However, the explanation of the differential results found for males and females in this paper is not clearly feasible due to the restrictions imposed by the space where the experiments took place and the time they lasted, and it will suffice to say that there has been a clear bias in the behavior of the sexes, not necessarily related to the light since it also happened to an extent for the negative controls. 7. Conclusions 21 From the data obtained in this paper, the following conclusions may be drawn: 1. The rearing of Phlebotomus spp in laboratory conditions is a viable process and a useful one to obtain live material for behavior studies. 2. The data obtained in the experiments suggest that Phlebotomus sandflies do not experience a strong attraction towards the monochromatic light emitted by LED devices, in clear contrast with a strong attraction towards ultraviolet light. This idea could find its importance in the development of lighting systems that are less prone to attract potential vectors of Leishmania, consequently safer for domestic animals. 3. There don’t seem to be very significant differences between the different light colors, with a slightly lower response to green light. In all the cases males are the more mobile specimens and more readily drawn to the light, which has its implications since females are the sole vector of the disease. A partir de los datos obtenidos en este trabajo pueden extraerse las siguientes conclusiones: 1. El mantenimiento de colonias de Phlebotomus es un proceso viable en el laboratorio y útil para disponer de material vivo para realizar estudios de comportamiento. 2. Los datos obtenidos en los experimentos con luz sugieren que los Phlebotomus no experimentan una atracción muy intensa por la luz monocromática emitida por los dispositivos LED, en contraposición con una atracción muy intensa por la luz ultravioleta. Esta idea podría encontrar su importancia en el desarrollo de sistemas de iluminación menos propensos a la atracción de potenciales vectores de Leishmania, consecuentemente más seguros para animales domésticos. 3. No parece haber grandes diferencias entre los distintos colores de luz, con una respuesta ligeramente más baja a la luz verde. 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