Bionomics of phlebotomine sand flies from three villages in Bihar, India

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1 S106 Journal of Vector Ecology March 2011 Bionomics of phlebotomine sand flies from three villages in Bihar, India David Poché 1, Rajesh Garlapati 2, Kate Ingenloff 1, Jennifer Remmers 1, and Richard Poché 1* 1 Genesis Laboratories, Inc., P.O. Box 1195, Wellington, CO 80549, U.S.A. 2 Boring Canal Road, Patna, India ABSTRACT: This study examined the spatial distribution and seasonal fluctuations of population densities of phlebotomine sand flies and was designed to obtain baseline data on the population trends of Phlebotomus argentipes, P. papatasi, and Sergentomyia spp. in a visceral leishmaniasis endemic area of Bihar, India. Beginning on 28 October 2009 and through 20 October 2010, 63 CDC light traps were evenly distributed in human homes, cattle sheds, combined dwellings, chicken coops, and adjacent vegetation areas in three villages in the Saran District of Bihar State. Sand fly collections were made on a weekly basis, sorted, and identified according to species, sex, and feeding status of the two genera. The daily temperatures and relative humidity ranges were collected in a representative human home, cattle shed, and combined dwelling in each of the three study villages. Village census surveys were conducted in the three study villages in February 2010, acquiring human population data, structural composition data, and livestock census information, and documenting the history of visceral leishmaniasis within each household. A total of 52,653 sand flies was trapped and identified over 3,276 trap-nights. Peaks in abundance were observed in November 2009, March and April, June through August. Of the sand flies trapped, 72.1% were P. argentipes, 27.1% Sergentomyia spp., and 0.8% P. papatasi. Distribution of the sand fly captures included 30.6%, 26.7%, 18.6%, 12.1%, and 12.0% from vegetation, combined dwellings, cattle sheds, housing, and poultry houses, respectively. Journal of Vector Ecology 36 (Supplement 1): S106-S Keyword Index: Visceral leishmaniasis, sand fly, Phlebotomus, India, vector. INTRODUCTION Leishmaniasis is second only to malaria as a vectorborne disease of humans. Some 20 species of Leishmania are transmitted by approximately 90 proven phlebotomine vectors, infecting people in 88 countries, 72 of which are developing nations. Visceral leishmaniasis (VL) is the most severe clinical form of this disease and is usually fatal if untreated (Alvar et al. 2006). It is estimated that 500,000 new cases of visceral leishmaniasis are contracted annually, causing an estimated 59,000 deaths globally. Nearly 90% of all VL cases occur in low socioeconomic peridomestic areas of Bangladesh, Brazil, India, Nepal, and Sudan (Desjeux 2002, Desjeux 2004). In India, the majority of cases occur in 28 of 37 districts of Bihar state, where approximately 100,000 new cases of VL are reported annually (Desjeux 2001, Bern et al. 2005, Ranjan et al. 2005, Singh et al. 2006, Dey et al. 2007). VL is largely considered a rural disease, often correlated with malnutrition, poor sanitary conditions, and other factors associated with poverty. The majority of individuals that contract VL are involved in agriculture in some capacity (Thakur 2000, Ranjan 2005). Studies also indicate an increased risk for urbanized areas as livestock populations increase (Desjeux 2001, 2002). In India, the known vector of Leishmania donovani is the sand fly, Phlebotomus argentipes Annandale and Brunetti, and humans are believed to be the only or primary reservoir. However, adult P. argentipes preferentially feed on cattle, and the proximity of cattle is a risk factor for the development of VL in humans (Mukhopadhyay and Chakravarty 1987, Thakur et al. 2004, Barnett et al. 2005, Palit et al. 2005). Knowledge of phlebotomine population dynamics in India is limited and most research encompasses only the vector species, P. argentipes. In those studies, conflicting data exists regarding the seasonal abundance of P. argentipes. Several studies conducted suggested a single annual peak of P. argentipes populations between April and November while other research claims that two peaks occur a large population peak in the pre-monsoon season followed by a second smaller peak just after the monsoon and before winter (Kaul 1993, Shrestha and Pant 1994, Dinesh et al. 2001). Studies conducted on P. papatasi Scopoli populations, the proven vector of L. major in the old world, indicate two population peaks per year (pre- and post-monsoon) (Shrestha and Pant 1994). No publications regarding the population dynamics of Sergentomyia babu Annandale were found by the authors. S. babu is not a known vector of zoonotic Leishmania species, however the sand fly species is known to spread L. tarentolae in lizard species (Mukherjee et al. 1997). The focus of this study was to determine the spatial distribution of sand flies within villages that lie within the VL endemic area of Bihar. This study is part of an overall project focused on developing low-cost sand fly control products for the rural farmer and adding to the current integrated vector control program for VL in India. There is a need for breaking the life cycle of VL transmission by reducing vector (P. argentipes) populations to a level where transmission of VL is reduced. The data presented in this paper provide information on the seasonal abundance and distribution of the most common phlebotomine sand flies in a VL endemic area of Bihar: P. argentipes, P. papatasi,

2 Vol. 36, Supplement 1 Journal of Vector Ecology S107 and Sergentoymia species. The knowledge of phlebotomine ecology gained will increase ability to develop effective integrated vector control programs in VL endemic areas of the Indian subcontinent. Data on the annual cycles of sand flies, in particular P. argentipes, the vector of VL, are essential in launching more effective control efforts as timing for control methods will be critical to ensure maximum efficacy of any products used. MATERIALS AND METHODS Study area The study was conducted in the Saran District, Bihar State, India, in northeastern India bordering Nepal. Bihar is cool in the winter months of December and January, with temperatures ranging from 4 to 10 C. Summers are hot, with average highs around C during April to July. During the monsoon months of June, July, August, and September, rainfall averages about 120 cm per year. Three long-term study villages approximately 5 km apart and within the VL endemic area of Bihar were selected for monitoring. These included Rasulpur, Mohammadpur, and Mahesia, all within the Saran District northwest of Patna, the capital city of Bihar. The villages share the same bioclimatic and agricultural characteristics. The villages were initially selected through the examination of aerial photographs based upon a variety of factors including size (between 175 and 275 houses), proximity to other villages, and abundance of adjacent natural vegetation and cropland. Site visits were made to approximately 50 potential villages and surveyed for potential monitoring. The final criteria for selection were based on human population size, the number of recent or active cases of VL (more than two), diversity and population density of livestock, absence of recent DDT spraying, and a minimum of one active poultry house. Basic environmental conditions, including temperature and relative humidity, were recorded daily in each village with digital min-max thermometer/hygrometers (Fisher Scientific, Thermo-Hygro Model ). One thermometer/hygrometer was set up in a representative home, a second in a combined dwelling, and a third in a cattle shed. Rainfall was recorded to the nearest mm using a conventional rain gauge calibrated in mm. Sand fly collection CDC light traps (Bioquip Products and John W. Hock Company) were used to collect sand flies in sample villages. Twenty-one CDC light traps were set in each village for a total of 63 trap-nights each week. Traps were placed at random in five different sand fly habitat types: human households, combined dwellings (structures shared by humans and livestock), cattle sheds, and adjacent vegetation areas. As poultry houses are not abundant, only one additional trap in each village was set in a poultry house. All traps were hung with the light source approximately one m above the ground. Traps in vegetation and poultry houses were provided with protective lids to protect electronic components from rain and debris, such as bird feces from above. Traps were identified by a unique identification code and GPS location. Sand flies were collected weekly from 28 October 2009 through 21 October Traps were activated Wednesdays at 18:00 and run until 06:00 the following morning. At that time the baskets containing insects were collected and transported to the laboratory in Patna. Collected trap baskets containing insects were stored at -20 C to kill collected insects and preserve them until sorting and identification began. Each trap was individually numbered and notes recorded on collection date, the total number of sand flies collected, phlebotomine species composition, sex ratios, number of blood-fed females, and the number of gravid females. Blood-fed P. argentipes females were removed to ethanol-filled mini-centrifuge containers for future use in bloodmeal analyses. Male and unfed female sand flies were stored in sealed Petri dishes at -20 C. Sand flies were dissected and examined under a microscope. Slides were made of head and genitalia for species confirmation of the Phlebotomus spp. Sergentomyia spp. are not vectors of VL but were counted and segregated by sex. All species were identified based on training and keys provided by the U.S. Department of the Army, Walter Reed Army Institute of Research, and Hebrew University sand fly experts. Data were collected and entered into Excel spreadsheets. Sand flies (Phlebotomus spp. only) were sorted and the mean number of sand flies captured per trap per night (trap-night) was plotted on graphs. Village census surveys were conducted to obtain basic information regarding human and livestock populations and living conditions. Each household was noted, including the number of adults and children, the general construction material of the home itself, type and number of livestock, and the history of VL over the past ten years. RESULTS There was no rainfall in the study villages from study initiation, 28 October 2009, until May 2010, when 12 mm was recorded (Table 1). A second rain occurred on 7 May, also in the amount of 12 mm. The coolest temperature recorded was in Mohammadpur on 13 January 2010, at 9 C. Temperatures during the month of January were consistently 8 C in cattle sheds, suspected breeding sites for sand flies. Temperatures in homes ranged between 9-16 C, while homes varied from 9-25 C. Combined dwelling temperatures ranged from 9-21 C in January. Temperatures increased rapidly in March initiating an increase in sand fly emergence. By April the maximum temperatures reached 35 C, and initiating a steady increase in numbers. The warmest temperatures recorded were in April, reaching 41 C in homes and 43 C in cattle sheds. Temperatures only varied within a few degrees among houses (24-29 C), cattle sheds (21-21 C), and combined dwellings (22-42 C). Many homes had no doors or window coverings. During the same period there was no rainfall.

3 S108 Journal of Vector Ecology March 2011 Table 1. Monthly average rainfall, temperature, and humidity records for study area. Month Temperature Rainfall Humidity C (mm) Min Max Min Max Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Humidity varied greatly among the structures. Within homes, as an example, in January humidity ranged from 49-88%. In cattle sheds the spread was greater, ranging from 25-97%. Combined dwellings had more consistent humidity, which recorded between 25-91%. Peridomestic vegetation was rich in organic matter and shade and was conducive to higher humidity. Sand fly collection A total of 3,276 trap-nights yielded the capture of 52,653 sand flies over the 12-month study period (Table 2). For the villages of Mahesia, Rasulpur, and Mohammadpur, the total flies captured were 25,651, 14,334, and 12,668, respectively. Sand fly species differed in abundance (P<0.0003). These included P. argentipes (72.1%), Sergentomyia spp. (27.1%), and P. papatasi (0.8%). Figure 1 shows the monthly capture records for total sand flies. Overall, this amounted to 15.7 sand flies per trap-night over the sampling period. November proved to be the end of a population peak. As ambient air temperature slowly dropped to a low of 17.3 C, and sand fly numbers declined steadily. Capture of P. argentipes was most frequent in comparison to other sand fly species in the three villages throughout the sampling period. The distribution of captures as per trap site within the three villages is shown in Tables 2 and 3 and Figure 2. After poultry houses, the highest mean sand fly capture collection site was combined dwellings, followed by vegetation, cattle sheds, and housing (Table 3). There were slight significant differences in the number of P. argentipes between building types (P>0.086). When considering all trapping habitats, less poultry houses, the difference in trap success was not significant (P>0.84). The poultry house had the fewest total numbers because each village had only one chicken house with a single CDC light trap set vs five traps used in each other trap location type. In examining the mean number of sand flies per trap per trap-night, collection was highest in the poultry house (mean = 36.3), compared to 14.1, 12.9, 8.9, and 5.5 sand flies for combined dwellings, vegetation, cattle sheds, and housing, respectively. Total sand fly captures over the year reflected peaks in November, March, and from June through August (Figure 1). Among the villages, Figure 2 shows a similar trend, with Mahesia being the exception and having many more flies in August and September. Sand fly numbers decreased in late November to mid- February as the climate became cooler. No sand flies were trapped for a five-week period during the cooler months of January and February in Mohammadpur (6 January-3 Feb), four weeks in Rasulpur (6-27 January), and three weeks with no flies in Mahesia (6-20 January). A detailed breakdown of P. argentipes captures from the different trap sites is presented in Figures 3-7. Within homes, capture rates were similar for the three villages (Figure 3). Capture numbers showed peaks in November, March, and June-July. In cattle sheds (Figure 4), the number of sand flies captured per trap-night was lower than expected, since cattle are a known source for blood meals. Mahesia was the exception with a sharp increase in sand flies in September and October. Combined dwellings, which are shared by livestock and humans at night, had peaks in activity in November, March, and June. Mahesia, unlike the other two villages, had a sharp increase in sand fly in numbers in September and October. Traps set in various vegetation types had results showing peaks in captures in November, March, and June (Figure 6). Mahesia continued to increase in sand fly numbers in June while the remaining two villages had a drop in numbers. In poultry houses, sand fly captures were similar in the three villages from October to July (Figure 7). A sharp increase in P. argentipes began in July through September, and numbers declined in October. The village of Mahesia was consistently high in sand fly numbers. For all but two weeks during the 52-week sampling period, Mahesia had more sand flies captured than other villages. Village surveys Information gathered from each homeowner or landlord regarding the livestock within each village is presented in Table 4. Figure 8 presents the distribution of domestic livestock within the three villages. These are sources of blood meals for sand flies, in addition to humans. Rasulpur had the highest number of goats and buffalo compared to the other two villages. Housing type varied among villages, with brick being most common in Rasulpur (Figure 9). Rasulpur also had the most number of thatched homes. This type of construction, along with mud brick, is conducive to harboring sand flies in cracks and crevices. Rasulpur had the highest number of cases of VL of the villages surveyed (Figure 10). As of 20 October 2010, there were nine confirmed cases. Mohammadpur had more homes of concrete, is more affluent based on housing type, and has very few cases of VL. How these factors involving livestock species, housing type, and vegetation type contribute to the number of cases of VL remains to be

4 Vol. 36, Supplement 1 Journal of Vector Ecology S109 Table 2. Total sand fly captures from three villages in Bihar, India. The table codes are H= House, CD= Cattle Shed, CD= Combined Dwelling, V= Vegetation/Outdoor, E-1= Poultry House, SF=Sand fly, Pa= P. argentipes, Pp= P. papatasi, S= Sergentomyia spp. UF= Unfed, BF= Blood fed, G= Gravid, W=trap worked, F=Trap failure. Mohammadpur (T)SF (T)P.A (M) (F) UF (F) BF (F)G (T)P.P (M) (F) UF (F)BF (F)G (T)S (M) (F) UF (F) BF (F) G E CD CS H V Totals Rasulpur (T)SF (T)P.A (M) (F) UF (F) BF (F) G (T)P.P (M) (F) UF (F)BF (F) G (T)S (M) (F) UF (F) BF (F) G E CD CS H V Totals Mahesia (T)SF (T)P.A (M) (F) UF (F) BF (F) G (T)P.P (M) (F) UF (F)BF (F) G (T)S (M) (F) UF (F) BF (F) G E CD CS H V Totals

5 S110 Journal of Vector Ecology March 2011 Figure 1. The average number of sand flies trapped in all villages over a one-year period. Figure 2. The average number of sand flies collected over a one-year period for the three villages.

6 Vol. 36, Supplement 1 Journal of Vector Ecology S111 Figure 3. The average number of P. argentipes, the only known vector of VL in India, collected in homes in the study villages over a one-year period. These data include all construction types from thatched to brick. Figure 4. The mean number of P. argentipes collected per trap-night from cattle sheds, considered major breeding sites, within the three villages.

7 S112 Journal of Vector Ecology March 2011 Figure 5. Combined dwelling, a structure shared by livestock and people, P. argentipes sand fly collection data over a one-year period. Figure 6. Sand fly collection data from various types of vegetation within and within 30 meters of the study area village sites.

8 Vol. 36, Supplement 1 Journal of Vector Ecology S113 Figure 7. Data from trapping sand flies inside poultry houses in the villages. Most villages have a minimum of one poultry house, which are sources of sand flies. Figure 8. The number of domestic animals within the study villages which serve as a source of blood meals for sand flies.

9 S114 Journal of Vector Ecology March 2011 Figure 9. The various types of housing construction in the three villages, which influences the distribution of sand flies within a village. Figure 10. The number of reported cases of VL within the study villages in recent years.

10 Vol. 36, Supplement 1 Journal of Vector Ecology S115 Table 3. Distribution of P. argentipes captures according to habitat type. Habitat Type No. Sand Flies Trap-nights Mean # Sand Flies/ trap/night Poultry House 5, Combined Dwelling 10, Cattle Shed 6, Housing 4, Vegetation 10, Totals 37,957 3, determined. DISCUSSION Placement of the CDC light traps began in mid- October, 2009 and indicated a downward trend in sand fly numbers as part of a larger October peak. Temperatures continued to cool, diminishing sand fly abundance. No sand flies were trapped from the villages in much of January and February because of climatic conditions. The results presented here demonstrate a non-uniform distribution of sand flies within the villages studied in Bihar. Vector abundance is dependent on multiple factors such as building construction type, proximity and amount of vegetation, number and type of livestock available for blood meals, and climatic conditions. Warming trends in late February triggered sand fly emergence and numbers rose steadily to a peak in March. As the humidity remained low towards the end of March, sand fly numbers dropped steadily. With the onset of rain in April after an almost five-month dry period, sand fly emergence began again. In all three villages, the downside of a large peak was noted in late autumn 2009 (Figure 2). An increase in sand fly numbers was observed in March. After the warmer temperatures in early March, the increased emergence in sand flies was particularly evident in cattle sheds, known to be vector habitats. Combined dwellings were similar in numbers to the cattle sheds, since both had livestock during the night as blood meal sources. The vegetation in Rasulpur yielded numerous sand flies in November and again the following March. We suspect that since cattle are harnessed under trees and bamboo groves during the day, sufficient organic matter is available as potential breeding sites for the sand flies. The village survey results yielded very useful information to enable the initial examination of building type and sand fly distribution. The poorer villages have more rudimentary housing construction, mainly thatched and mud brick. It is no surprise that more cases of VL were noted from Rasulpur than the other two villages. Rasulpur also had more sand fly captures from cattle sheds than did Mohammadpur and Mahesia. Although the data have not been collected as of yet, it is probable that the combination of building construction types and the number of cattle sheds contribute to the larger population of sand flies. When examining the data from the combined dwellings, sand fly numbers were higher in Rasulpur and Mahesia. Bringing cattle into the house during the night serves as an attractant for sand flies, and this becomes unfortunate for humans sleeping in the same area. Since the nights in Bihar are typically warm from April through October, it is commonplace to have beds placed outside the home in an attempt to escape the heat. Farmers will sometimes keep cattle tied to posts in the yard overnight, often within several meters of the sleeping area. Rasulpur had the highest number of buffalo and goats compared to the other two villages. Thatched and mud brick housing are more common than in the other villages and provide more microhabitats in the form of cracks and crevices for sand flies to retreat to during daylight hours. As shown in Figure 8, Rasulpur has a much higher number of goats compared to the other villages. Studies in Nepal (Khanal et al. 2010, Bhattarai et al. 2010), allude to the role goats may play in the transmission of Leishmania donovani. This may be reflected in the greater number of cases of Kalaazar in Rasulpur over the other two villages. The unanticipated abundance of captured sand flies Table 4. Mean livestock per household by species in villages 1-3. Rasulpur Mohammadpur Mahesia Livestock Per Household 3.98 ± ± ± 0.14 Cattle ± ± ± Buffalo ± ± ± Goats ± ± ± Oxen ± ± ± Poultry (non-cooped) ± ± ± Households Owning Zero (0) Livestock 30 (12.6%) 76 (30.6%) 79 (36.7%)

11 S116 Journal of Vector Ecology March 2011 in vegetation will require further examination for creative control measures. Since many livestock owners tie their cows or buffalo under shady trees or bamboo groves, a layer of organic matter develops and is conducive to larval sand flies. In a separate study (unpublished data), sand flies were trapped from the canopy of coconut palms, 18.5 m above ground level. These data suggest that conventional indoor spraying may have limited success since the role of palm trees in the ecology of sand flies in Bihar needs to be researched more fully. Control strategies will have to take into consideration that more sand flies may exist in vegetation groves, such as palm trees, banana plantations, and bamboo thickets, than previously suspected. Sand flies trapped from vegetation were presumed to be seeking sugar meals, rather than inhabiting dense cover which provides resting sites and organic matter. The analysis of blood meals from flies collected from palm tree canopies identified the sources as approximately 90% human (unpublished data). This would suggest that in areas of Bihar, P. argentipes may be more exophagic. Sand flies would tend to feed on the nearest source of blood, including those people sleeping outside the homes. Based on observations made during this study, we suspect sand fly movements are localized. Typically, villages are separated by km, with most of the land between them under cultivation or fallow, depending on the time of year. Sand flies have abundant food sources within and surrounding villages or single structures and are not required to fly long distances for blood meals or sugar. Plant sugar sources provide the energy requirements and are available both in the villages and peridomestically. Although there are few reports of long distance movements of sand flies, we suspect that such occurrences within Bihar are rare. Recent studies by Khanal et al. (2010) and Bhattarai et al. (2010), suggested that domestic animals in Nepal, goats in particular, may play a role in the distribution of L. donovani. In an ecosystem such as found in Bihar, sand fly control may have to be reevaluated to consider such approaches as topical application or feed-through applications of adult and larval control products (Poché 2010). More sand flies living outside would necessitate an alternate means, rather than indoor residual spraying alone. The findings presented in this study have contributed to a better understanding of sand fly distribution within and around villages in northeastern India. These data will be of importance and may contribute to a broader integrated sand fly control program. Acknowledgments This project was funded by the Bill and Melinda Gates Foundation, Grant No Special thanks go to Alon Warburg, Dia Elnaiem, and Edgar Rowton for onsite training assistance at the initiation of the project. 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