The scarcity of information on the immature stages of sand flies and their preferred breeding sites has resulted in the focus of vectorial control on the adult stage using residual insecticide house-spraying. This strategy, along with the treatment of human cases and the euthanasia of infected dogs, has proven inefficient and visceral leishmaniasis continues to expand in Brazil. Identifying the breeding sites of sand flies is essential to the understanding of the vector' s population dynamic and could be used to develop novel control strategies. In the present study, an intensive search for the breeding sites of Lutzomyia longipalpis was conducted in urban and peri-urban areas of two municipalities, Promissão and Dracena, which are endemic for visceral leishmaniasis in São Paulo State, Brazil. During an exploratory period, a total of 962 soil emergence traps were used to investigate possible peridomiciliary breeding site microhabitats such as: leaf litter under tree, chicken sheds, other animal sheds and uncovered debris. A total of 160 sand flies were collected and 148 (92. 5%) were L. longipalpis. In Promissão the proportion of chicken sheds positive was significantly higher than in leaf litter under trees. Chicken shed microhabitats presented the highest density of L. longipalpis in both municipalities: 17. 29 and 5. 71 individuals per square meter sampled in Promissão and Dracena respectively. A contagious spatial distribution pattern of L. longipalpis was identified in the emergence traps located in the chicken sheds. The results indicate that chicken sheds are the preferential breeding site for L. longipalpis in the present study areas. Thus, control measures targeting the immature stages in chicken sheds could have a great effect on reducing the number of adult flies and consequently the transmission rate of Leishmania (Leishmania) infantum chagasi.
Members of the Lutzomyia longipalpis (Lutz & Neiva) species complex are the main vector of Leishmania (Leishmania) infantum chagasi (Cunha & Chagas), the causative agent of the visceral leishmaniasis (VL) in Brazil and Latin America [1]–[3]. Its recent introduction and adaptation to domiciliary habitats in the urban and peri-urban areas of municipalities in every region of Brazil, including São Paulo state, has resulted in higher incidences of human and/or canine visceral leishmaniasis [4]–[8]. From 1995 to 2010, Brazil recorded 53,633 new cases of the disease, with an annual mean of 3,352 new cases [9]. From 1999 to 2011, São Paulo state recorded 1,927 new cases of the disease, with 169 deaths [10]. In Brazil, the VL control program focuses on the treatment of human cases, the euthanasia of seropositive infected dogs and insect vector control [5], [11], [12]. Since this disease is zoonotic, treatment of human cases does not affect transmission. The incidence of human infections seems to be associated with the number of infected dogs and vectors [13]–[15]. Culling dogs is unpopular and its impact on the reduction of the incidence of human VL is contradictory [12]–[14], [16]–[18]. Vectorial control is focused on the sand fly' s adult stage through residual house-spraying insecticide [1], [5], [11], [12], [19]. Insecticide application is generally accepted by residents in affected areas, is more practical and, in theory, more effective than the other methods at controlling transmission [13]. However, due to the irregularity and low coverage of application, possible repellent action of insecticide, high cost when used on a large scale and its short residual effect this method is considered inefficient in the control of VL [12], [14], [19], [20]. In Brazil, the high abundance of L. longipalpis in conjunction with domestic animals, particularly dogs, which act as the amplification hosts for L. (L). i. chagasi, means that transmission occurs within relatively small areas represented by peridomiciles in rural and urban areas [19], [21]–[23]. In addition, dispersion studies have shown that the movements of L. longipalpis individuals are spatially focal [24], [25]. Such observations, coupled with the high frequency of males and females of different physiological age (empty, engorged and gravid) present in collections carried out in urban areas of several municipalities of São Paulo state (unpublished), indicate that the complete life cycle of L. longipalpis occurs in peridomicile habitats of these areas. Nevertheless, finding the preferred breeding microhabitat of the L. longipalpis and other sand flies species is a difficult task [26]–[28]. It is generally assumed that the immature stages of sand flies develop in shaded and moist terrestrial microhabitats, rich in organic nutrients, such as in or near leaf litter, bases of trees, animal burrows, animal sheds, and rock crevices [27], [29]–[32]. Nevertheless, there is no precise information on the preferred breeding sites of these important vectors. Among the techniques used to identify the natural breeding sites of sand flies, soil emergence traps are the preferred indirect method employed [26], [27], [29], [30], [33]–[37]. In this study, a new soil emergence trap design was used to identify the natural breeding sites of L. longipalpis in urban and peri-urban areas of two municipalities identified as endemic for VL in São Paulo state. A more detailed knowledge of the immature stages and their preferential breeding sites is essential to understand the vector' s population dynamics and could be used to develop novel control strategies.
The municipality of Promissão (21°32′S 49°51′W) and Dracena (21°28′S 51°31′W) are located in the Western region of São Paulo state, Brazil (Fig. 1). Promissão has a total area of 787 km2 and approximately 37,570 inhabitants. Dracena has a total area of 489 km2 and approximately 41,000 inhabitants. According to Köeppen' s climate classification, these areas are identified as Aw - Tropical wet and dry [38]. Lutzomyia longipalpis was first detected in Promissão and Dracena in 1999 and 2003, respectively. At the time of the present study, canine and human transmissions have been established in both municipalities. The houses in the urban and peri-urban areas of these municipalities usually have non-paved peridomicile with bushes and trees (e. g. fruit trees), and domestic animals shelters for dogs and chickens are common. In both municipalities, of the 20 blocks where the study was performed, chickens and dogs were present in approximately 10% and 30% of the dwellings respectively. Two emergence trap designs were used in this study. The first one was that described by Casanova (2001) and a new one (described below) was developed by the same author (Fig. 2A–2D) through modifications in the polyvinyl chloride (PVC) pipe tube trap described by Ferro et al. (1997) [26], [35]. The emergence traps were 20 cm tall, with varying diameters of 10. 16,20. 32,25. 40 and 44. 60 cm, sampling 0. 008,0. 032,0. 051 and 0. 156 m2 of substrate respectively (the diameter reduced where necessary due to site conditions). The bottom edges of the PVC tubes were serrated to allow insertion at least 5 cm into the soil (Fig. 2A) of different microhabitats. After fixing it to the ground, the inner surface of the superior part of the tube was totally covered with a 4 cm wide adhesive paper (Fly – Catcher “The Stable”, Silva – made in Sweden), which was attached to the tube wall using paper clips (Fig. 2B). To prevent sand flies escaping, the top of the tube was covered with voile (a fine-mesh, fabric gauze) fixed with rubber bands (Fig. 2C). This way, the sand flies which emerge and fly inside the PVC tube end up landing on the adhesive paper, which works as a sticky trap. Finally, to protect the trap from the sun and rain, a small tent was placed over them, taking care to leave approximately 5 cm clearance between the trap and the tent walls (Fig. 2D). Two series of continuous soil emergence trap collections were carried out from March 2005 to February 2006 and from September 2006 to January 2007 in the urban and periurban area of the municipalities. The first period was exploratory and its main objective was to identify possible L. longipalpis breeding sites and the fauna associated with them. The second period aimed to evaluate the spatial distribution pattern of immature stages in the two principal microhabitats positive to L. longipalpis in Promissão, during the first study period. Chi-square analysis, used to compare the proportion of positive microhabitats for L. longipalpis in each municipality, and Pearson correlation coefficient, calculated to examine the relationship between the number of L. longipalpis collected in emergence and CDC traps, were carried out using BioEstat (version 5. 0; Mamirauá/CNPq, Belém, PA, Brazil). To evaluate the spatial distribution pattern of the immature stage, the variance-to-mean ratio (s2/), with chi-square statistic test to determining significantly departure from randomness χ2 = SS/ where s2 is the variance and is the sampling mean and SS is the sum of squares) and the Morisita' s Index (Id = nΣ x2−N/N (N−1), with chi-square statistic test to determine significant departure from randomness χ2 = (nΣ x2/N) −N, where n = number of samples; N is the total number of individuals collected, and Σ x2 is the squares of the number of individuals per sample, summed over all samples) were used [42]. In these indexes, values equal the unity indicate a random disposition, values smaller than the unity show a regular or uniform distribution, and values significantly higher than one indicate an aggregated or contagious disposition. The frequency of sand flies in CDC traps was obtained by month using William' s geometric mean [43].
The new design of emergence trap used in the present study is easy to construct, very straightforward to install and allows for longer periods between visits to the traps to remove sand flies (every 20 days, approximately). Clearly, the chicken shed was the microhabitat that contributed the most to the high positivity of investigated dwellings, for the high proportion of positivity of emergence traps and to the increased number of L. longipalpis collected in the municipality of Promissão. In Dracena, chicken sheds were also the most productive microhabitat for L. longipalpis, however with a much lower number of positive emergence traps and sand flies than found in Promissão. Probably, this was due to both the smaller number of chicken shed investigated (about 50% smaller) and sampled area in the chicken shed microhabitat (about three times smaller) than that in Promissão. Compared to the leaf litter under tree microhabitat, the chicken shed and uncovered debris areas are much smaller, and the population boundaries are readily apparent. This facilitates sampling of the population and estimating its size, particularly if the population shows contagious spatial distribution. In this sense, although in the present study the leaf litter under tree microhabitat has contributed only 5. 40% of the total L. longipalpis collected, one should not ignore the possibility of a high production of L. longipalpis in this microhabitat, because this species is by far the most common in the peridomiciles of the studied areas. However, the differences in microhabitat size were most likely not the reason for the great difference observed between the positivity and productivity of these microhabitats. The chicken sheds are probably more favourable breeding sites because they are important blood feeding and resting sites for females of L. longipalpis and the abundance of faeces can provide a source of organic material for larval food. One advantage of employing emergence traps to detect sand fly breeding sites is that they allow estimates of population densities from the observed productivity of breeding sites, expressed in adults/area/time [29], [45]. Considering the period of investigation, the number of the traps and the sampled area, the estimated production of L. longipalpis in the chicken shed microhabitat was by far the highest among all the researched microhabitats in the present study. For instance, the density of 17. 29 sand flies per m2 or 41. 17 sand flies per 100 m2 per day estimate for the total of chicken shed microhabitat in Promissão is a much higher value than found for L. longipalpis and other species in Neotropical region. In emergence traps set near pigsty microhabitats Ferro et al. (1997) estimated a density of 4. 96 L. longipalpis per m2 sampled [26]. Considering other sand fly species, Rutledge & Ellenwood (1975) in Panama, and Arias & Freitas (1982), Casanova (2001) and Alencar (2007) in Brazil, estimated from the total of sand flies species captured in the litter of the forest floor, a production of 24. 4,4. 1,24. 0 and 5. 8 sand flies per 100 m2 per day, respectively [29], [33], [35], [36]. Notably, the 34 L. longipalpis collected in one emergence trap with 20. 32 cm of diameter (0. 032 m2), set in a chicken shed in Promissão, corresponded to a density of 1,062. 5 sand flies per m2. Besides that, the high density of 50. 7 sand flies per m2 obtained in chicken shed microhabitats during August 2005 in Promissão allowed to estimate a production of 121. 7 L. longipalpis per 100 m2 per day. This high potential production of L. longipalpis agrees with the high abundance of adults in CDC traps set in the peridomicile. The sexual ratio pro males may be a result of a possible greater male vagility after emergence facilitating their contact with the adhesive paper. Some of the male flies collected in the emergence traps still showed partial or no rotation of their genitalia. However, there is still the necessity to evaluate the efficiency of the traps of different dimensions to collect the emergence population inside them. This can be tested by releasing a known number of pupae or newly emerged adults into the traps, preferably at the central point [45]. Although the sampling of the microhabitats in the first period of study had been done with an uneven number of traps of different sizes, the intensive sampling, the concentration of a great number of L. longipalpis in few traps and the great number of traps with few or no flies seemed to be a strong indication of its contagious spatial distribution pattern. During the second study period, designed to identify where the concentrations occurred, and how great and how frequent they were, the indices used also showed a contagious distribution pattern. However, the abundance of L. longipalpis was low and new long term experiments, with greater number of traps and an exact positioning in the grid are necessary to allow the use of more accurate methods. These results suggest that females lay their eggs in clutches in restricted microhabitats, as indicated by Ferro et al. (1997) [26]. Probably the female sand fly in this, and other, species has the ability to detect food, shade, humidity and physical-chemical soil constitution, which are generally graded in a habitat. Some studies have shown that female sand flies locate an appropriate site by orienting towards semiochemical oviposition attractant components of eggs and animal faeces [46]. Contagious spatial distribution is the pattern commonly reported for insects in natural environments [45] and has already been suggested for sand flies species in forest floor microhabitats [29]. The contagious spatial distribution pattern can be responsible for the commonly reported difficulty in finding immature forms in soil samples or adult forms in emergence traps [27], [28]. The associated fauna may offer important information on species which might act as a sand fly breeding site indicator. Detecting larvae from a Diptera predator group in the same chicken shed sample where L. longipalpis were found is interesting from the biological control perspective. Collecting E. cortelezzii, E. lenti, E. termithophila and N. neivai in chicken shed together with L. longipalpis also points to the importance that this environment can have in the ecoepidemiology of cutaneous leishmaniasis. The high abundance of L. longipalpis detected in the CDC traps fixed in peridomicile associated with animal pens, especially chicken sheds, has been frequently observed in several other rural and urban areas of Brazil [21], [47]–[49]. The lack of correlation between the number of adult sand flies collected in CDC traps and the number of immature stages in soil samples, or adults in emergence traps, is not uncommon [26], [50], [51]. This could be due to an accumulation of multiple generations of flies in the CDC trap, in addition to the fact that adults are attracted to the host from an area considerable larger than the emergence microhabitats that are sampled. Probably the combined effects of rainfall, air temperature and evapotranspiration, which determine the soil water balance, influence the quality of the breeding site microhabitats and consequently determine the adult sand fly population fluctuations [26], [29], [48], [52], [53]. In the present study, moderate water deficit periods seem to be favorable for population increase and periods of high water surplus seem to negatively affect the immature and adult populations of L. longipalpis. For Promissão, the rising curve for adults from October on, which led to a peak in December, may have been influenced by moderated water surplus periods between October and December, which only happened in this municipality and not in Dracena. On the other hand, longer periods of intense draught, like the one which happened more evidently in Dracena (water balance deficit from March to December), may be detrimental to the immature stages. The same has been observed with L. longipalpis and other sand fly species in urban and rural areas of São Paulo state [48], [53]. Both the high proportion of positive sites and high density of individuals in emergence traps indicate that the chicken shed microhabitat is the preferential breeding site of L. longipalpis in the study areas. In this sense, a new item – act as breeding sites to L. longipalpis - can be added to list of Alexander et al (2002) as factor associated to chicken sheds that increases the risk of transmission of L. (L.) i. chagasi to humans in Brazil [22]. Raising chickens is relatively common and both culturally and socio-economically important in urban, peri-urban and rural areas of the municipalities in all regions of Brazil. Its importance in the ecoepidemiology of zoonotic visceral involves some type of balance between zooprophylaxis, maintenance of sand fly populations and attraction of reservoir hosts [22]. Spatial analysis studies to detect areas at increased risk for visceral leishmaniasis such as those developed by Cerbino-Neto et al (2009), could evaluate the presence of chicken in the neighbourhood as an environmental factor associated with incidence of the disease [54]. It would be of interest to evaluate the efficiency of vector control interventions, such as environmental change and insecticide (chemical or biological), in chicken shed microhabitats, with the aim of reducing the adult population. The abundance of the female population size is a critical parameter of vectorial capacity [53], [55]. If the chicken shed, as a preferential breeding site, has an important role in determining the population abundance of adult forms of L. longipalpis, control strategies aimed at immature stage population in this microhabitat could be cheap, practical, and effective at reducing the populational abundance of females and consequently, the transmission rate. Furthermore, the efficacy of residual insecticide and others potential control strategies against adult forms - i. e. impregnated dog-collar [17], [56], zooprophylaxis [22] synthetic sand fly pheromone in conjunction whit insecticide [47], [57] – could be improved through of the control of immature stages of L. longipalpis population in chicken shed microhabitats.
Sand flies are the vectors of a number of pathogens that infect man, especially cutaneous and visceral leishmaniasis. The control of sand flies through residual house-spraying insecticide has proven inefficient in preventing the spread of these diseases in several areas of the globe. Sand flies have a life cycle including eggs, larvae and pupae that are found in the ground, and winged adults. We developed a soil emergence trap that allowed us to identify chicken sheds as the preferred breeding sites for L. longipalpis that is the vector of visceral leishmaniasis in urban areas of municipalities in South-eastern region of Brazil. This finding opens up the possibility of controlling the immature stages which could complement the usual control strategies that focus on the adult fly.
Larval Breeding Sites of Lutzomyia longipalpis (Diptera: Psychodidae) in Visceral Leishmaniasis Endemic Urban Areas in Southeastern Brazil