Few microhabitats have been previously identified as natural breeding places for phlebotomine sand flies so far, and little is known about the influence of climate variables in their density. The present study was conducted in a dry region with a semiarid climate, where visceral leishmaniasis occurs in humans and dogs. The occurrence of breeding places in specific microhabitats was investigated in soil samples collected from five houses, which were also the location used for sampling of adults. All the microhabitats sampled by our study were identified as natural breeding places due to the occurrence of immature forms of sand flies. On a weekly basis, the number of adult sand flies captured was positively correlated with the mean temperature from preceding weeks. These results, in addition to promoting an advance in the knowledge of sand flies biology, may furnish a tool for optimizing the control of the sand flies, by indicating the most suitable periods and microhabitats for the application of insecticides.
Despite the medical importance of leishmaniasis, little is known about the natural breeding places of its vectors. Most previous attempts to identify the preferred microhabitats for the oviposition of sand flies in the Neotropical region have produced disappointing yields, resulting in a small number of positive soil samples and immature forms [
As observed by Newstead [
Dry climate regions experience more pronounced climatic variations than regions with humid climates typical of rain forests, so the density of vectors may exhibit different characteristics as well. Studies conducted in rain forest regions (e.g., Hanson [
The knowledge about natural breeding places for sand flies and vector density may represent useful information for directing efforts at biological control, leading to reduce the density of the vectors and consequently control the incidence of disease [
The Cavunge district (12.3°S; 39.3°W) is located in a semiarid region of Bahia state (Figure
Location of Cavunge district and positive microhabitats (a) Typical scenario from Cavunge: trees with exposed roots along with exposed rocks (b) soil accumulated between exposed roots of a
During the first period of this study, from June 2007 through July 2008, soil samples at low-light places with apparent decomposed organic matter, were collected from areas immediately adjacent to the five residences selected by our study group. These samples were gathered with a regular spoon and stored in cylindrical receptacles with a capacity of 1 L. Microhabitats from which positive samples were collected were then chosen for more intensive sampling during the subsequent study period.
During the second period of our study, from August 2008 through July 2010, approximately 64 soil samples were monthly collected at the same five residences and stored in similar receptacles. According to the results from the first year of study, the soil was sampled in the following microhabitats: cavities in rocks; areas between the roots of a
During the entire period of study, three different techniques: direct-observation, flotation-sieving, and flotation technique were utilized to identify the presence of sand flies in the soil samples.
The direct-observation technique was used to examine all soil samples until sixty days after their arrival at the insectarium. This technique consisted by daily observation of the receptacles containing the soil samples in two different ways: by naked eye, so to find adult sand flies through the translucent cover of the 1L receptacles, and with a stereomicroscope, aiming to find immature forms in the soil. To count the number of sand flies in a soil sample, we included immature forms that reached the adult stage, immature forms that did not reach the adult stage but were similar to other larval stages (larvae and pupae) present in our laboratory colony of
After the period corresponding to the direct observation technique, all soil samples were submitted to the flotation-sieving technique or the flotation technique.
The flotation-sieving technique, as described by Hanson [
Due to the retention of immature forms on the sieves used to process the soil samples from the first part of the study, and consequently loss of information about the real quantity of immature forms in the soil samples, the sieves were replaced by flotation for the second part of the study. With the flotation technique, a saturated salt solution was simply added to the soil samples and allowed the immature forms to float.
Following floatation-sieving and flotation, the immature forms suspended by a high density solution, were collected with a brush and disposed separately in 1L capacity cylindrical receptacles, remaining there for 60 days. These vessels were one-third filled with plaster covered by a ration composed by a mixture of rabbit feces, commercial hamster ration and small quantity of soil from the original substrate. The 2 inch hole in the bottom of these containers allowed keeping the plaster constantly humid due to the directly contact with a moist paper towel.
The adult sand flies gathered from direct observation, flotation-sieving and flotation techniques were stored in covered glass slides with mounting media and identified at species level based on the identification key proposed by Young and Duncan [
During each month of the second part of the study, one day before the soil sampling, two HP light traps, one inside the residence, and another in the adjacencies, were placed in every one of the same five residences used for soil sampling. The light traps were operated from 6 pm to 6 am, period also used by other authors due to the known nocturnal activity of several sand flies species [
Climate data were obtained by the National Institute of Meteorology (INMET) from the Feira de Santana meteorological station, placed at about 40 km of distance from Cavunge district. Daily means from temperature, and relative humidity were provided directly from the INMET, which calculated these data through the arithmetic means from records of maximum and minimum temperature or relative humidity at 9 am, 3 pm and 9 pm, respectively. Otherwise, daily rainfall was measured by a single record of millimeters of rain in a day.
Daily rainfall and daily averages for temperature, and relative humidity were used to obtain the averages of these variables on a weekly basis. Weekly averages of rainfall, temperature and relative humidity were calculated through the arithmetic means of daily rainfall, mean temperature and mean relative humidity, respectively, at one up to three weeks before the soil or adult sampling days. Thus, the density of immature or adult sand flies was correlated by statistical analyses with the preceding weekly averages from the climate variables.
From Minitab software, multiple linear regression analyzes were performed for the yield of sand flies in the soil or adult sampling in each month and the average of climate variables at one up to three weeks before soil or adult sampling days.
During the first period of this study, 234 soil samples were collected from five residences in the Cavunge district. Nine of these samples were identified as positives by direct-observation or flotation-sieving technique in the microhabitats and included rock cavities (2), between the roots of
During the second period of this study, a total of 1,523 soil samples were collected from different microhabitats (Table
Positive microhabitats and sand flies yield.
Microhabitat | Collected samples | Positive samples | Sand flies |
---|---|---|---|
Chicken feces | 87 | 1 | 1 |
Fallen tree trunk | 55 | 5 | 14 |
House interior | 105 | 1 | 1 |
Rock cavities | 521 | 21 | 24 |
Tree roots | 371 | 9 | 11 |
Water tank | 384 | 12 | 13 |
Total | 1,523 | 49 | 64 |
A total of 2,002 sand flies were collected with light traps during the second period of the study. Species in addition to those found in the soil samples were identified as follows: 9
Species of sand flies identified following sampling of adults.
Species | Inside houses | Adjacent to houses | Total | ||
M | F | M | F | ||
2 | 1 | 4 | 2 | 9 | |
2 | 0 | 0 | 0 | 2 | |
16 | 36 | 6 | 24 | 82 | |
181 | 685 | 237 | 804 | 1,907 | |
2 | 0 | 0 | 0 | 2 | |
Total | 203 | 722 | 247 | 830 | 2,002 |
The yield of adult sand flies captured by HP light traps and the average temperature from the preceding week were associated at a moderated strength of association (Table
Multiple-regression analysis among climate variables and sand flies yield from sampling of adults.
Variable | R² | R² (adjusted) | |
---|---|---|---|
Average temperature | |||
1 week | 43.6% | 35.1% | 0.016 |
2 weeks | 0.134 | ||
3 weeks | 0.964 | ||
Average rainfall (mm) | |||
1 week | 10.0% | 0.0% | 0.216 |
2 weeks | 0.664 | ||
3 weeks | 0.747 | ||
Average rainfall (days) | |||
1 week | 14.4% | 1.5% | 0.543 |
2 weeks | 0.470 | ||
3 weeks | 0.626 | ||
Average humidity | |||
1 week | 16.4% | 3.9% | 0.500 |
2 weeks | 0.652 | ||
3 weeks | 0.910 |
Multiple-regression analysis among climate variables and sand flies yield from soil sampling.
Variable | R² | R²(adjusted) | |
---|---|---|---|
Average temperature | 9.5% | 0.0% | |
1 week | 0.740 | ||
2 weeks | 0.825 | ||
3 weeks | 0.895 | ||
Average rainfall (mm) | 1.4% | 0.0% | |
1 week | 0.903 | ||
2 weeks | 0.938 | ||
3 weeks | 0.845 | ||
Average rainfall (days) | 9.7% | 0.0% | |
1 week | 0.579 | ||
2 weeks | 0.253 | ||
3 weeks | 0.640 | ||
Average humidity | 13.5% | 0.5% | |
1 week | 0.535 | ||
2 weeks | 0.422 | ||
3 weeks | 0.626 |
Natural breeding places for phlebotomine sand flies, as the most likely places for the development of immature forms, may be represented by different microhabitats in different regions. In our study at Cavunge, a district with semiarid climate and Caatinga vegetation, positive soil samples were collected from a fallen trunk and between trees roots, as found in previous studies conducted at rain forests [
Rock cavities in exposed rocks and cracks around a water tank, two of the most positive microhabitats, exhibit characteristics previously identified by Deane and M. P. Deane [
As expected in a region affected by visceral leishmaniasis, most of the sand flies from the soil samples or captured by the light traps were identified as
Analysis of the relationships between sand fly density and climate variables resulted in moderated strength association between adult sand flies captured and the average temperature from the preceding weeks. Such relationship was not observed by previous studies of associations between sand flies density and climate variables, which identified the absence of climatological influence in sand flies density [
The analyses among climate data and sand flies density made by our study suffered limitations, because data gathering was not made
The sand flies natural breeding places, as well as climate variables associated with vectors density, despite their potential to assist the biological control, are poorly understood so far. Our study points to several microhabitats that may serve as natural breeding places for phlebotomine sand flies in a semiarid region and also elucidate that temperature decreasing may be a predictor for the increasing of vector density in a dry climate region. Altogether, our results may furnish a tool for optimizing the control of the sand flies, and consequently, the anthropic and zoonotic cases of visceral leishmaniasis.
The authors are grateful to the fellowships provided from Fundação de Amparo à Pesquisa do. This work was supported by grants from CNPq, program PAPES V (FIOCRUZ/CNPq) and FAPESB. Bruno Sangiorgi was supported by a fellowship from FAPESB. Aldina Barral is a senior investigator from CNPq.