The role of Palmyra palm trees (Borassus flabellifer) and sand fly distribution in northeastern India

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1 148 Journal of Vector Ecology June 2012 The role of Palmyra palm trees (Borassus flabellifer) and sand fly distribution in northeastern India Richard M. Poché 1*, Rajesh Garlapati 2, Dia-Eldin A. Elnaiem 3, Diana Perry 1, and David Poché 1 1 Genesis Laboratories, Inc., Wellington, CO 80549, U.S.A., richard@genesislabs.com 2 Boring Canal Road, Patna, India 3 University of Maryland Eastern Shore, Princess Anne, MD 21853, U.S.A. Received 2 August 2011; Accepted 2 February 2012 ABSTRACT: Visceral leishmaniasis (VL), known as Kala-azar in India, is a parasite transmitted by the bite of the sand fly vector Phlebotomus argentipes. Published information on the species indicates it is a poor flyer, mainly hopping and gliding. This study describes the vector as more arboreal than previously documented. Data collected indicate the ability of P. argentipes and Sergentomyia spp to attain vertical heights in Palmyra palm trees Borassus flabellifer up to 18.4 m above ground level. To determine if sand flies were either climbing the tree trunk to rest in the canopy or flying, sticky traps were set around the tree trunk and checked for captures overnight. CDC traps set in the palm tree canopy resulted in the capture of 5,067 sand flies, 3,990 of which were P. argentipes. Traps were set during daylight hours to determine if sand flies remained and rested in the canopy. A total of 128 sand flies were trapped over 29 trap days in the palm trees. With the CDC traps, 130 P. argentipes and no Sergentomyia spp were captured. The converse was true for the sticky traps set around tree trunks 3 m below the CDC traps. Of the 105 sand flies collected, only one was P. argentipes and 104 were Sergentomyia spp. As reported elsewhere, this indicates Sergentomyia spp tend to climb and hop, wheareas P. argentipes are capable of longer and more sustained flight. Data presented herein suggest that P. argentipes is more exophylic and exophagic than previously reported. These findings have implications for sand fly control. Journal of Vector Ecology 37 (1): Keyword Index: Visceral leishmaniasis, palm tree, Kala-azar, Bihar, India, sand fly. INTRODUCTION Visceral leishmaniasis (VL) is endemic to northeastern India, particularly in the state of Bihar. The disease is transmitted by Phlebotomus argentipes and 90% of the cases are from the Indian subcontinent (Desjeaux 2001, Bern et al. 2005, Singh et al. 2006). Conservative estimates by the World Health Organization place the number of VL cases near 100,000. The sand fly vector P. argentipes is abundant throughout much of northeastern India. Studies involving the vertical distribution of sand flies have been conducted in the Americas (Basimike et al and Chaniotis et al. 1974). In India, one study examined the longitudinal distribution of the vector at different heights in cattle sheds (Hati et al. 1991). A recent study focused on sand fly distribution and density in areas within and outside building structures and in peridomestic vegetation around villages (Poché et al. 2011). No published data from India are available on sand fly movements vertically in vegetation. Identification of blood meals in hematophagous arthropods plays a vital role in epidemiological studies and also helps to define control strategies (Boreham 1975, Tempelis 1975). Blood meal identification is also used to determine host preferences in natural habitats. The anthroponotic index (percentage feeding on humans) is a crux component of vector capacity and the knowledge of other hosts in turn helps to identify the reservoirs of vectorborne diseases (Boakye et al. 1999). A wide range of serological methods was used to identify blood meals of insects (Washino and Templis 1983). Introduction of polymerase chain reaction (PCR)- based technology using mitochondrial DNA helped to eliminate various hindrances in serological methods. PCRbased technology is a more direct approach in identifying host species. A reverse line blot assay was developed to identify the blood meal of ticks (Humair et al. 2007). In the current study, we used blood meal identification based on amplification of the mitochondrial cytochrome b (cyt b) followed by reverse line blot analysis in phlebotomine sand flies described in the literature (Abbasi et al. 2008). After observing several palm squirrels (Funambulus pennati) scamper down palm trees in a small, VL-endemic village, we decided to investigate atypical resting areas of sand flies, in particular P. argentipes. Roof rats (Rattus rattus) are known to nest in the canopy of coconut trees in many countries and are responsible for extensive loss by damaging the husks (Hoque and Fiedler 1988, Dolbeer et al. 1988). Since VL is anthroponotic in India, we wanted to determine if nesting rodents might be a source of blood meals for the sand flies. This study represents the first attempt to look into collecting sand flies at higher elevations in India, and to determine the source of blood meals from the vector at those higher elevations. Data will contribute to an integrated vector control program which may impact the epidemiology of VL in India.

2 Vol. 37, no. 1 Journal of Vector Ecology 149 MATERIALS AND METHODS Study area The study was conducted in the village of Sutihaar, located in the Saran District, Bihar, India. Sutihaar has had numerous cases of VL in recent years and has an abundance of goats and wild rodents. The socio-economic status of village inhabitants is quite low and reflected by high unemployment with the majority of homes constructed from thatch or homemade brick. The village is populated with over 500 palmera palm trees and occupies an area of approximately 0.4 km 2. Sand fly trapping Trapping was conducted from 1 September to 3 December 2010 (19 weeks). Initially, two CDC light traps (BioQuip Products, Inc.) were set to discern the presence of sand flies in the palmyra palm tree canopy. As sand fly captures were confirmed, the number of CDC traps was increased to eight. On a weekly basis, traps were positioned in the canopy of palm trees within the village. Palm trees were selected on the basis of obtaining permission to climb trees from the owners. Obtaining permission proved to be a difficult task. An experienced palm tree climber was hired to attach CDC traps just under the canopy approximately 0.5 to 1 m away from the trunk of palm trees. Traps were attached to the branches using thin rope. The position of the trap ranged from 10 to 18.4 m above the ground. The 6-volt battery operating the trap was securely placed at the base of the branches. The sex of the palm tree was based on morphological characteristics. It was not until the field work was completed that we examined the differences in sand fly abundance based on tree sex. Three trees were male and five were female. Initially, traps were set at sunset and removed near sunrise on the following morning. Several weeks later, traps were set during the daytime hours to monitor fly activity near the palm tree canopy, which would indicate flies were resting in the fronds. Day time trapping was conducted after night time collections ceased. In early November, a 2-kg pressurized CO 2 cylinder with regulator was used in an attempt to attract more sand flies to CDC traps during daytime hours. A small 6 mm diameter plastic tube regulated the gas up into the CDC trap, with the hose outlet taped near the intake fan. Initially, the cylinder was placed on the ground at the base of the tree. Later, for security, the cylinder was mounted on a wooden platform attached to the tree trunk approximately 4 m above the ground. Traps were removed and the specimens transported to a laboratory in Patna and stored at -20 C. Sand flies were separated as to genus and the Phlebotomus identified as to species. The Sergentomyia were not identified further since these are not known vectors of VL. Specimens were retained and stored in the -20 C freezer upon completion. Hopping vs flying To ascertain if sand flies were climbing or hopping up the tree trunk rather than flying to the tree canopy, a 1-m band of sealing wrap coated with sesame oil was applied to tree trunks about 2 m below the CDC light traps. Six separate trees were selected and the trapping was done weekly between 16 March and 18 April (6 weeks). The oil was evenly spread on the sealing wrap in sufficient amounts to trap insects that crawled, hopped, or flew onto it. The sticky bands were set up in the early evening at the same time the CDC traps were put into operation, carefully removed the following morning along with the CDC traps, and transported to the lab where the sand flies were sorted and identified. Canopy-level sticky paper traps From 16 March to 13 April 2011 (five trap nights), four previously unused palm trees were selected based on availability and permission by the owner to climb, for sticky paper trap sand fly collection. Copier paper was submerged in castor oil for 3 min and hung to drip clean. A flexible metal rod was forced through the paper and bent at both ends such that the sticky trap could be hung in the tree canopy from sunset to sunrise. Each tree had four sticky traps hung overnight and spaced evenly under the canopy high above the ground. DNA extraction and amplification Sand flies collected from the traps were identified and the blood-fed sand flies were stored in a -20 C freezer for future processing. Whole DNA from the blood-fed sand flies was extracted using an Epicentre Biotechnologies (Epicentre, Madison, WI) DNA extraction kit. The extracted DNA was stored at -80 o C until further processed. A 344 base pairs conserved region of the cytochrome-b gene was amplified using bio-tinilated universal primers designed by Abbasi et al. (2008). The required region was amplified in a total reaction of 50 µl consisting of 25 µl of Hot start Taq Master mix that consisted of 1.5 mm MgCl 2, 200 µm each dntp and 75 mm KCl, 10 mm Tris HCl ph 8.8 (Qiagen, Valencia CA), and 25 pmoles of each primer and genomic DNA. The thermo-cyclic conditions consisted of 95 C for 15 min, 35 cycles at 94 C for 30 s, 55 C for 30 s, and 72 C for 1 min, followed by an elongation step at 72 C for 10 min. The amplified PCR products were used as probes in RLB hybridization reactions and followed by chromogenic detection. Species-specific human, cow, buffalo, goat, dog, brown rat, and avian probes developed by Abbasi et al. (2008) were used in this study. These selected oligonucleotides were covalently linked to a nylon membrane through the formation of amide bonds between the carboxyl groups on the nylon and amino groups linked to the oligonucleotides. The procedure for immobilization, hybridization, and detection were followed according to their method. RESULTS Sand fly trapping The morning after the initial two traps were set, one CDC trap had 19 sand flies and the other had none. All were

3 150 Journal of Vector Ecology June 2012 Table 1. The results of sand fly captures from the Palmyra palm tree canopy using CDC light traps during the fall of The total trap nights/days for night and daytime trapping were 132 and 29, respectively. Set T (SF) T(Pa) M UF BF T (S) M UF BF Night 4, ,941 2,286 1, Day Total 5,062 3,990 2,312 1, , Set=when traps were set; T(SF)=Total sand flies; T(Pa)= Total P. argentipes; M=Male; F=Female; UF=Unfed Females; BF= Blood-fed; T(S)=Total Sergentomyia spp. 1 Five P. papatasi were collected and not included. Sergentomyia spp. However, as the collections continued, number and diversity of species increased. During the fall of 2010, a total of 5,602 sand flies was collected over 131 trap nights and trap days: 3,990 P. argentipes (2,312 M, 1,677 F), 1,064 Sergentomyia spp. (358 M, 709 F), and five P. papatasi (3 M, 2 F) (Table 1). An average of 37.3 and 4.4 sand flies were collected per trap per day for nights and days, respectively. Thirteen P. argentipes females were blood-fed and ten Sergentomyia spp. Of the P. argentipes collected, 57.94% were males and 43.03% females. Data from the fall collection of P. argentipes exhibited a difference in numbers trapped between male and female palm trees (t=0.041). Table 2 presents data showing a total of 1,138 P. argentipes trapped with CDC traps from male trees and 2,774 collected from female palms. An average of 23.7 and 34.2 sand flies for males and females, respectively, were collected per trap per night. Traps enhanced with CO 2 in November captured 17 Sergentomyia spp and only one P. argentipes during the day. Nighttime captures were 12 Sergentomyia spp and 17 P. argentipes. Hopping vs flying Information obtained from the sticky traps below the canopy and associated CDC light traps is presented in Table 3. A total of 130 sand flies was collected from CDC traps and 105 from sticky traps. All of the sand flies collected from the CDC traps were P. argentipes. Conversely, only one P. argentipes was trapped from the sticky traps and 104 Sergentomyia spp. These results suggest that the Sergentomyia hopped up the tree trunk towards the canopy, while P. argentipes flew directly to traps in the canopy. Sticky paper traps Sticky paper traps set in the palm tree canopy yielded captures of sand flies, but much fewer than the CDC light traps. A total of 290 sand flies was collected from the CDC traps. These included 52 male and 37 female P. argentipes, three female P. papatasi, and 106 male and 92 female Sergentomyia spp. The sticky traps in the canopy yielded the capture of two male and four female P. argentipes and five male and ten female Sergentomyia. These data demonstrate that both P. argentipes and Sergentomyia spp. occupy palm tree canopies. CDC traps set in tree canopies captured sand flies during the day. Over eight trapping days, 115 sand flies were collected from the tree canopies, of which 18 were P. argentipes and 97 were Sergentomyia spp. None contained blood meals. These data, in addition to the sticky trap data, suggest that sand flies in Bihar are more arboreal than previously published. Sand fly blood meal analysis Blood-fed sand flies collected in palm trees were processed to identify the blood meal using cytochrome b PCR and reverse line blotting. The source of the blood was successfully identified in all the sand flies tested and presented in Table 5. Eighty-five percent of the blood meals were from humans. Garlapati et al. (2011) published more detailed sand fly blood meal data with more than 50% of the blood meals of human origin. DISCUSSION Seventy percent of the houses in the study village were thatched and the rest mud brick with thatched roofs. Grass Table 2. Total number of P. argentipes trapped from the canopy of male and female Palmyra palm trees using CDC light traps during Sept Oct Nov Dec Totals TN SF/T/TN Male Trees , Female Trees 1, , , Total 1, , , TN= Trap Night; SF/T/TN = Sand flies per trap per night.

4 Vol. 37, no. 1 Journal of Vector Ecology 151 Table 3. Comparison of sand flies captured from the canopy of eight palmyra palm trees using CDC traps and sticky traps set on the tree trunks 3 m beneath the live traps in December, Set T(SF) T(Pa) M UF BF T(S) M UF BF CDC STICKY TOTAL Set=type trap set; T(SF)=Total sand flies; T(Pa)= Total P. argentipes; M=Male; F=Female; UF=Unfed Females; BF= Bloodfed; T(S)=Total Sergentomyia spp. Table 4. Total number of female P. argentipes trapped from the canopy of male and female Palmyra palm trees using CDC light traps during Trapped from Sept Oct Nov Dec Totals TN SF/T/TN Male Trees Female Trees , Total ,638 TN= Trap Night; SF/T/TN = Sand flies per trap per night. Table 5. Results of blood meal analysis of sand flies collected from Palymra palm tree canopies in the village of Sutihaar during None of the sand flies collected during daylight hours had taken blood meals. Collection Date Trap Number Species Host Using RLB 22 Sept S-5(palm) P. argentipes Human 22 Sept S-2(palm) P. argentipes Human 20 Oct B-4 P. argentipes Human 15 Sept S-1 Sergentomyia Human 15 Sept S-1 P.argentipes Human,Buffalo 28 Oct N-2 P.argentipes Human,Buffalo 3 Nov N-5 P.argentipes Human,Goat 6 Oct N-8 P.argentipes Human 6 Oct N-8 P.argentipes Human 2 Sept 1(palm tree) P.argentipes Human 3 Sept 4B(palm) Sergentomyia Human 15 Sept S-7 (palm) P.argentipes Human 15 Sept S-5(palm) Sergentomyia Human 15 Sept S-5(palm) Sergentomyia Human 29 Sept S-3(Palm) P.argentipes Buffalo 29 Sept S-7 (palm) P.argentipes Buffalo

5 152 Journal of Vector Ecology June 2012 homes of this type may result in a reduction in potential resting areas and suitable microhabitats for sand flies. The lack of possessions in this impoverished village resulted in a lack of nesting places within the homes for sand flies. A typical village in the region may have 10-20% thatched homes. The exact role palm trees play in the ecology of sand flies remains to be studied in detail. In this survey, 65% of trapped sand flies were P. argentipes, the vector of VL in India. The palm tree canopy contains enough organic matter to provide potential breeding sites and provides daytime cover for sand flies. The pattern of sand fly movement is unclear at this stage. There are several published reports of sand flies in South America utilizing tree canopies. To our knowledge this is the first report of sand flies occupying an arboreal habitat in south Asia. Most references state that Asian species tend to hop rather than fly and rarely travel more than a few meters vertically. A significant finding with many implications is that P. argentipes are more arboreal in flight behavior than previously documented. This study is the first to document arboreal flight behavior of P. argentipes in densely populated palmyra palm trees distributed throughout a small village. Sutihaar has a human population of about 1,600. Scattered across the approximate 0.4 km 2 village are some 500 palm trees. The sand fly genus Phlebotomus is known as a hopper, or low flying glider (Lawyer and Perkins 2004). The fact that the vector for VL in Bihar occupies vegetation as high as 18.4 m above the ground opens a new dimension of sand fly ecology in the region. The fact that sand flies were collected consistently from palm tree canopies up to 18.4 m suggests that the species may be more exophilic than previously thought. Poche et al. (2011) reported, in a study conducted in three Bihar villages, a higher number of sand fly captures in peri-domestic vegetation (banana groves, bamboo, and fruit orchards) than in man-made structures. Blood meal analysis also confirmed that 85% fed on humans. This indicated the flies were resting at lower elevations in homes or vegetation or flew down at night from the canopy, took a blood meal from villagers sleeping outside (exophagic), then flew/hopped/ climbed up the tree to resting areas within the tree bark and canopy. Blood meal analysis from females captured in the canopy were identified as human, demonstrating the exophagic behavior of P. argentipes. Sand flies do not have to fly into homes or cattle sheds as frequently to obtain a source of blood if humans are sleeping outdoors. Also, cattle tend to be tethered outside during the warmer months. A survey report revealed that 95% of residents from Sutihaar and another larger village nearby move their beds outside to escape the heat (Perry et al., unpublished data). Fewer than 0.5% of people in the region use bed nets because of the extreme heat for eight months of the year. These sleeping conditions result in increased human exposure to biting sand flies and mosquitoes. Garlapati et al. (unpublished data) found that the majority of blood-fed sand flies collected from cattle sheds fed on humans rather than livestock, showing the potential preference for human blood over domestic animals. Extensive research is required to further identify the distribution of sand flies in other vegetation, such as bamboo thickets, banana plantations and groves, and other plant species. The study conducted in Kenya by Basimike et al. (1989) on Sergentomyia species resulted in captures of sand flies as high as 11 m. Sticky traps were attached to poles erected in woodland areas. The authors suggested that this genus of sand fly inhabiting forested areas is characterized by hopping rather than direct flight. Our data confirm similar findings in Bihar for Sergentomyia spp when comparing CDC light traps and sticky traps. Our data suggest that phlebotomine sand flies, in particular P. argentipes, are better fliers than previously documented. The same may be true for sand flies in Bihar. There is probably flight between trees and down to the ground for blood meals, then back up into the dense tree canopy to rest. Although difficult to prove, sand flies probably travel more at diagonals in flight from tree to tree using each as a stepping stone en route to the upper tree canopy. This is a subject for further research. The mechanism by which a blood-fed P. argentipes reaches the near crown of an 18.4-m tree remains to be studied. Although the wing structure is not conducive to long-term flight, how these flies make it to such a height may indicate we do not give the species enough credit for its flight capabilities. In the Kenya study cited above, few Sergentomyia spp males were trapped over 2 m above the ground. With Lutzomyia spp in Honduras, Williams (1970) showed that the majority of sand flies remained near to the ground and that females moved near the tree tops. Data from this current study showed that 57% of the captures of P. argentipes were males and 43% female. This might suggest that breeding may also take place in the higher canopy of palm trees. No other studies with sand flies collected from tree canopies have reported blood-fed females with human blood meals (Basimike et al. 1989). It is particularly interesting that P. argentipes will feed on humans near ground level and navigate considerable heights to find resting places. The fact that female trees host more sand flies than male palms raises the question of preferred arboreal habitat. Additional research proposes to examine palms as possible breeding sites for sand flies. Female trees, because of increased organic matter, may prove to be more conducive to such behavior. Sand flies may feed on these trees as an energy source since the female palm trees may have a higher nutritive content. In Bihar, previous studies have focused on indoor structures such as homes and cattle sheds (Hati et al. 1991). The study focused on sand fly abundance in cattle sheds to a level of 2.74 m. This was more in line with the concept of indoor residual spraying and the maximum height required to spray walls within cattle sheds. The standard sand fly collection technique in Bihar is with the use of aspirators. The research into feed-through products for livestock to control both adult and larval sand flies is showing promise with such compounds as ivermectin, fipronil,

6 Vol. 37, no. 1 Journal of Vector Ecology 153 and diflubenzuron (Ingenloff et al., unpublished data). A comprehensive sand fly management program is being researched and will consider cultural practices, the use of insect growth regulators and insecticidal sprays on suspected breeding sites, the administration of drugs to livestock as feed-through larval control, and indoor residual spraying. Effective sand fly control may be difficult to attain, given the results of sand fly blood meal analysis and their slight preference to human blood, coupled with P. argentipes exhibiting a greater peridomestic habitat preference (Garlapati et al., unpublished data). This may have important implications for population management of the vector. A more comprehensive integrated sand fly management plan may be required and may necessitate treatment of suspected breeding sites, with insect growth regulators, not only livestock or IRS in homes and other buildings. Acknowledgments This project was funded by a grant from the Bill and Melinda Gates Foundation, Grant No We extend special thanks to Mukesh Roy and Sanjay Singh for assisting with the field work. REFERENCES CITED Abbasi, I., R. Cunio, and A. Warburg Identification of blood meals imbibed by phlebotamine sand flies using cytochrome b PCR and reverse line blotting. Vect. Borne Zoonot. Dis. DOI: /vbz Basimike M, M.J. Mutuku, and C.M. Mutero Vertical distribution of Phlebotomine sandflies in two habitats in Marigat Leishmaniases endemic focus, Baringo District, Kenya. Insect Sci. Appl. 10: Bern, C., A. Hightower, R. Chowdhury, M. Ali, J. Amann, Y. Wagatsuma, R. Haque, K..Kurkljan, L.E. Vaz, M. Begum, T. Akter, C.B. Centre-Sossah, I.B. Ahluwalia, E. Dotson, W.E. Secor, R.F. Breiman, and J.H. Maguire Risk factors for kala-azar in Bangladesh. Emerg. Infect. Dis. 11: Boakye, D.A., J. Tang, P. Truc, A. Merriweather, and T.R. Unnasch TR Identification of blood meals in haematophagous Diptera cytochrome B heteroduplex analysis. Med. Vet. Entomol. 1: Boreham, P.F Some applications of bloodmeal identifications in relation to the epidemiology of vector-borne tropical diseases. Am. J. Trop. Med. Hyg. 78: Chaniotis, B.N., M.A. Correa, R.B. Tesh RB, and K.M. Johnson Horizontal and vertical movements of Phlebotomine sandflies in Panamanian rain forest. J. Med. Entomol. 11: Desjeux, P The increase in risk factors for leishmaniasis worldwide. Trans. R. Soc. Trop. Med. Hyg. 95: Dolbeer, R.R., L.R. Fiedler, and H. Rasheed Management of fruit bat and rat populations in the Maldive Islands, Indian Ocean. Proc 13 th Vert. Pest. Conf. 13: Hati, A.K., S. Sur, N. De, H.N. Dwivedi, J. Bhattacharyya, H. Mukherjee, and G. Chandra Longitudinal study on distribution of Phlebotomus argentipes sandflies at different heights in cattleshed. Indian J. Med. Res. 93: Hoque, M.M. and L.A. Fiedler Rat control in Phillipines. NCPC Technical Bulletin 3: 20 pp. Humair, P.F., V. Douet, F.M. Cadenas, L.M. Schouls, I. Van De Pol, and L. Gern Molecular identification of blood meal source in Ixodes ricinus ticks using 12S rdna as a genetic marker. J. Med. Entomol. 44: Lawyer, P.G. and P.V. Perkins Leishmaniasis and trypanosomiasis. In: B.F. Eldridge and J.D. Edman (eds). Medical Entomology. pp Kluwer, The Netherlands. Poché, D, R. Garlapati, K. Ingenloff, J. Remmers, and R. Poché Bionomics of phlebotomine sand flies from three villages in Bihar, India. J. Vector Ecol. 36 (Supplement 1): S106-S117. Singh, S.P., D.C.S. Reddy, R.N. Mishra, and S. Sundar Knowledge, attitude, and practices related to kala-azar in rural areas of Bihar State, India. Am. J. Trop. Med. Hyg. 75: Tempelis, C.H Host-feeding patterns of mosquitoes, with a review of advances in analysis of blood meals by serology. J. Med. Entomol. 11: Washino, R.K. and C.H. Tempelis Mosquito host blood meal identification : methodology and data analysis. Annu. Rev. Entomol. 28: Williams, P Phlebotomine sandflies and leishmaniasis in British Honduras (Belize). Trans. R. Soc. Trop. Med. Hyg. 64: