The phlebotomine fauna (Diptera: Psychodidae) of Guaraí, state of Tocantins, with an emphasis on the putative vectors of American cutaneous leishmaniasis in rural settlement and periurban areas.

Phlebotomine sandflies were captured in rural settlement and periurban areas of the municipality of Guaraí in the state of Tocantins (TO), an endemic area of American cutaneous leishmaniasis (ACL). Forty-three phlebotomine species were identified, nine of which have already been recognised as ACL vectors. Eleven species were recorded for the first time in TO. Nyssomyia whitmani was the most abundant species, followed by Evandromyia bourrouli, Nyssomyia antunesi and Psychodopygus complexus. The Shannon-Wiener diversity index and the evenness index were higher in the rural settlement area than in the periurban area. The evaluation of different ecotopes within the rural area showed the highest frequencies of Ev. bourrouli and Ny. antunesi in chicken coops, whereas Ny. whitmani predominated in this ecotope in the periurban area. In the rural settlement area, Ev. bourrouli was the most frequently captured species in automatic light traps and Ps. complexus was the most prevalent in Shannon trap captures. The rural settlement environment exhibited greater phlebotomine biodiversity than the periurban area. Ps. complexus and Psychodopygus ayrozai naturally infected with Leishmania (Viannia) braziliensis were identified. The data identified Ny. whitmani as a potential ACL vector in the periurban area, whereas Ps. complexus was more prevalent in the rural environment associated with settlements.

American cutaneous leishmaniasis (ACL) has undergone a clear geographical expansion in Brazil in recent decades, which is likely associated with environmental and climatic changes. In the context of this novel distribution, human cases have been recorded in environmentally impacted and deforested rural areas, including periurban regions of some Brazilian towns (MS/SVS 2007).

In most Brazilian endemic areas, ACL is associated with Leishmania (Viannia) braziliensis infection , which is transmitted by several sandfly species, including Psychodopygus wellcomei and Psychodopygus complexus , which are involved in the sylvatic cycle in the Amazon Basin, and Nyssomyia whitmani, Nyssomyia intermedia, Nyssomyia neivai and Migonemyia (Migonemyia) migonei , which are associated with the outskirts of cities and areas that have suffered from environmental impact in the Northeast, Southeast, South and Central Regions (Rangel & Lainson 2009).

In the state of Tocantins (TO), ACL presents an occupational epidemiological profile, affecting mostly males and young adults. In this context, the local transmission patterns are associated with deforestation for the construction of highways, railroads, hydroelectric dams and agricultural expansion, which has favoured the establishment of settlements and villages (Graser 2008, SESAU/TO 2010). A total of 6,497 cases of ACL were recorded in the period from 2001-2012 (Information System for Notifiable Diseases) (dtr2004.saude.gov.br/sinanweb/index.php).

The purpose of this paper is to contribute to the current knowledge of phlebotomine fauna in TO and to identify putative ACL vectors in a rural settlement area and in the periurban environment of Guaraí.

MATERIALS AND METHODS

Sandfly capture sites - Guaraí is located in the northwestern TO (Supplementary data) at coordinates S08°50'03'' W48°30'37'' and at an altitude of 259 m. The local population is estimated to be 23,445 inhabitants distributed over 2,268,155 km2 with a demographic density of 10.23 inhabitants/km2. The inhabitants' main source of income is agricultural activity (Brazilian Institute of Geography and Statistics) (ibge.gov.br/cidadesat/link.php?uf=to). The city lies along the BR-153 highway, which connects the cities of Belém and Brasília, and is the major link between the Central and Northeast Regions of Brazil. The traffic along this highway is heavy, as it constitutes the main conduit of economic activity in the region (Government of the state of Tocantins) (atm-to.org.br/cidade.php?l=e6149e89399dba56fa890afff1b0f138) (to.gov.br/tocantins/guarai/891).

Guaraí is within the Cerrado biome, which has a continuous canopy and tree cover ranging from 50-90%, with the most cover in the rainy season and least during the dry season (Brazilian Agricultural Research Corporation) (agencia.cnptia.embrapa.br/Agencia16/AG01/arvore/AG01_58_911200585234.html). The sandfly capture sites are referred to as monitoring stations (MSs). In the rural settlement area, the MSs bordered the forests of the Agricultural Project Pedra Branca, MS 1 (S08°40'08'' W48°24'76'') and MS 2 (S08°39'99'' W48°24'73''), approximately 40 km from the town centre (Supplementary data). In the periurban area, two residences were selected, MS 3 (S08°48'55.9” W48°30'25”) and MS 4 (S08°49'09” W48°30'21.6”). These MSs have rural characteristics, such as the presence of animal shelters and a small area with banana plants (Supplementary data).

Sandfly capture and taxonomy - Sandflies were captured from January 2005-June 2008 using CDC light traps (HP model) (Pugedo et al. 2005) and Shannon traps (Sudia & Chamberlain 1962). The light traps were used in peridomestic areas near animal shelters or in the forest every month for three consecutive nights from 06:00 pm-06:00 am. Light traps were placed in each ecotope 1 m above the ground. The captured specimens were fixed in 70% alcohol and identified in accordance with Galati (1995, 2003) ; the abbreviation of the species' names conforms to Marcondes (2007) . Shannon traps were used in MS 1 in a forested part of the rural settlement area over a period of 12 h (06:00 pm-06:00 am) monthly from March-June 2008. The anthropophily of the species was also evaluated in these captures. A single capture with a Shannon trap was made in November 2008 on four consecutive nights to search for natural Leishmania spp infection in phlebotomine females using multiplex polymerase chain reaction (PCR). The insects were kept alive in nylon cages (Barraud 1929) and were taken to the laboratory (Entomology Laboratory of the Center for Zoonosis Control of Guaraí). For sandfly identification, males and the last abdominal segments of the females were cleared and mounted on slides. Males were identified by analysing the morphology of the genitalia (gonostyle, gonocoxite, structures of the parameres, pump and ejaculatory ducts) as well as the colour of the thighs and thoracic pleurae; the spermathecae, individual ducts and common duct were also observed in females. After identification, the insects were grouped into pools of 10 females and 10 males by species and locality. Male insects were used in diagnostic assays as negative controls. The pools of sandflies were kept at -20°C for DNA extraction.

Statistical analysis - The index of species abundance (ISA) and the standardised index of species abundance (SISA) (Roberts & Hsi 1979) were calculated for the phlebotomine species collected in the rural and periurban MSs. Excel 2010 and Diversidade de Espécies v. 2.0 software (DivEs) were used to analyse the data. The data were entered into Tables organised by MS, species and period of capture. Each column was classified separately according to the number of specimens of each species. The values in each column were ranked, with the highest value classified as 1, the second as 2 and so on. ISA was calculated according to this formula:

ISA = a + Rj/k

where k is the number of columns in the table (number of months of collection), a is the number of columns in which the species was absent multiplied by c, c is the highest value of the ranking obtained in all columns + 1 and Rj is the sum of the values for each species.

The minimum and maximum limits of the index were determined according to the highest distribution classification, where the limit is different in each series of data. To avoid this variation and to standardise the index we used a scale of values between 0-1 to calculate SISA:

SISA = c-ISA/c-1

Species abundance was considered high when the SISA value was close to the maximum value of 1. The results provide information on the relative abundance of species and on the temporal distribution of the collected individuals.

To analyse and compare the overall number of phlebotomine insects captured in the rural and urban areas, the DivEs software was used to analyse the data using the Shannon-Wiener diversity index (H) and the evenness index (J) (Rodrigues 2005).

PCR multiplex studies - To detect Leishmania spp infection, molecular analysis was performed on a total of 250 females and 40 males, which were grouped into pools of 10 specimens each. DNA was extracted as previously described (Pita-Pereira et al. 2005). Multiplex PCR was designed to simultaneously amplify the cacophony gene IVS6 region in Neotropical sandflies, which was used as an internal control for the polymerase enzyme activity and DNA extraction and the conserved kinetoplast DNA minicircle from Leishmania spp. The amplified products were further analysed by dot blot hybridisation with a biotinylated Leishmania ( Viannia )-specific probe (Pita-Pereira et al. 2005). Rigorous procedures were used to prevent contamination: negative control groups (male sandflies) were included in the DNA extraction step, instrument and working areas were decontaminated with diluted chloride solution and ultraviolet light and artificially infected females were added as positive controls.

RESULTS

The Guaraí exhibited diverse phlebotomine fauna with 43 identified species represented in 3,530 total specimens, 1,872 of which were male and of which were 1,658 female (male/female ratio = 1.12:1.0). Eleven phlebotomine species that had never been found in TO were recorded: Pintomyia (Pintomyia) damascenoi , Pressatia choti , Psathyromyia (Forattiniella) runoides , Psathyromyia (Xiphomyia) dreisbachi , Ps. complexus , Psychodopygus davisi , Psychodopygus llanosmartinsi , Psychodopygus hirsutus hirsutus , Psychodopygus ayrozai , Psychodopygus paraensis and Trichophoromyia ubiquitalis .

Some species identified in both areas are considered to be putative vectors of leishmaniasis: Nyssomyia antunesi, Ny. whitmani, Ny. intermedia, Bichromomyia flaviscutellata , Ps. complexus, Ps. ayrozai, Ps. paraensis, Ps. hirsutus hirsutus, T. ubiquitalis and Mig. (Mig.) migonei , which are vectors of ACL, and Lutzomyia (Lutzomyia) longipalpis and Mg. (Mig.) migonei , which are vectors of American visceral leishmaniasis (AVL).

The SISA of the Phlebotominae showed that Ny. whitmani was the species with the highest index (0.9524) followed by Evandromyia bourrouli (0.9444), Ny. antunesi (0.9206), Ps. complexus (0.9206) and Lu. longipalpis (0.8571) ( Table I). With regard to species abundance, Ev. bourrouli was the most represented species (1,000) in the rural settlement area, followed by Ny. antunesi (0.9682) and Ps. complexus (0.9206). In the periurban area, Ny. whitmani was the most abundant (1.000), followed by Lu. longipalpis (0.9512) and Ps. complexus (0.878) (Table II).

TABLE I

Index of species abundance (ISA) and standardised index of species abundance (SISA) obtained from the captures with automatic light traps in municipality of Guaraí, state of Tocantins, Brazil, January 2005-June 2008

Species	ISA	SISA	Position
Brumptomyia brumpti	25.875	0.2103	25th
Micropygomyia (Sauromyia) peresi	28.625	0.1230	29th
Micropygomyia (Sauromyia) longipennis	29.250	0.1032	34th
Micropygomyia (Sauromyia) quinquefer	31.875	0.0198	40th
Micropygomyia (Sauromyia) rorotaensis	29.500	0.0952	35th
Micropygomyia (Sauromyia) villelai	20.375	0.3849	17th
Sciopemyia sordellii	12.500	0.6349	10th
Lutzomyia (Lutzomyia) longipalpis a	5.500	0.8571	5th
Lutzomyia. (Tricholateralis) sherlocki	30.250	0.0714	39th
Migonemyia (Migonemyia) migonei a	15.000	0.5556	12th
Pintomyia (Pintomyia) christenseni	32.250	0.0079	41st
Pintomyia (Pintomyia) damascenoi	30.250	0.0714	38th
Pressatia choti	28.250	0.1349	27th
Trichopygomyia dasydopogeton	14.750	0.5635	11th
Evandromyia (Aldamyia) carmelinoi	15.625	0.5357	13th
Evandromyia (Aldamyia) lenti	17.875	0.4643	16th
Evandromyia (Aldamyia) evandroi	17.000	0.4921	15th
Evandromyia (Aldamyia) termitophila	22.000	0.3333	20th
Evandromyia (Aldamyia) walker	23.625	0.2817	21st
Evandromyia (Evandromyia) bourrouli	2.750	0.9444	2nd
Evandromyia (Evandromyia) pinottii	20.875	0.3690	18th
Evandromyia (Evandromyia) begonae	10.000	0.7143	8th
Evandromyia (Evandromyia) saulensis	22.000	0.3333	19th
Psathyromyia (Forattiniella) runoides	25.000	0.2381	22nd
Psathyromyia (Forattiniella) aragaoi	30.250	0.0714	37th
Psathyromyia (Forattiniella) lutziana	29.250	0.1032	33rd
Psathyromyia (Xiphomyia) hermanlenti	25.375	0.2262	24th
Psathyromyia (Xiphomyia) dreisbachi	32.250	0.0079	43rd
Psathyromyia (Psathyromyia) shannoni	28.625	0.1230	30th
Psathyromyia (Psathyromyia) dendrophyla	32.250	0.0079	42nd
Viannamyia furcate	28.750	0.1190	31st
Bichromomyia flaviscutellata a	7.875	0.7817	7th
Psychodopygus complexus a	3.500	0.9206	4th
Psychodopygus davisi	11.750	0.6587	9th
Psychodopygus claustrei	26.375	0.1944	26th
Psychodopygus llanosmartinsi	6.750	0.8175	6th
Psychodopygus hirsutus hirsutus a	28.250	0.1349	28th
Psychodopygus ayrozai a	15.750	0.5317	14th
Psychodopygus paraensis a	29.625	0.0913	36th
Nyssomyia antunesi a	3.500	0.9206	3rd
Nyssomyia whitmani a	2.500	0.9524	1st
Nyssomyia intermedia a	28.875	0.1151	32nd
Trichophoromyia ubiquitalis a	25.000	0.2381	23rd

a : vector species.

TABLE II

Index of species abundance (ISA) and standardised index of species abundance (SISA) obtained from the captures with automatic light traps for environment in municipality of Guaraí, state of Tocantins, Brazil, January 2005-June 2008

	Rural settlement area			Periurban area
Species	ISA	SISA	Position	ISA	SISA	Position
Brumptomyia brumpti	25.75	0.2142	25th	20.50	0.0487	21th
Micropygomyia (Sauromyia) peresi	-	-	-	20.50	0.0487	23th
Micropygomyia (Sauromyia) longipennis	32.00	0.0158	39th	21.00	0.2439	26th
Micropygomyia (Sauromyia) quinquefer	31.25	0.0396	34th	-	-	-
Micropygomyia (Sauromyia) rorotaensis	-	-	-	21.00	0.2439	28th
Micropygomyia (Sauromyia) villelai	24.00	0.2698	23th	16.75	0.2317	17th
Sciopemyia sordellii	14.00	0.5873	13th	11.00	0.5121	8th
Lutzomyia (Lutzomyia) longipalpis a	9.00	0.746	9th	2.00	0.9512	2th
Lutzomyia (Tricholateralis) sherlocki	28.00	0.1428	30th	-	-	-
Migonemyia (Migonemyia) migonei a	10.00	0.7142	10th	20.00	0.0731	20th
Pintomyia (Pintomyia) christenseni	32.00	0.0158	35th	-	-	-
Pintomyia (Pintomyia) damascenoi	28.00	0.1428	29th	-	-	-
Pressatia choti	24.00	0.2698	20th	-	-	-
Trichopygomyia dasydopogeton	9.50	0.7301	9th	20.00	0.0731	19th
Evandromyia (Aldamyia) carmelinoi	23.75	0.2777	19th	7.50	0.6829	7th
Evandromyia (Aldamyia) lenti	24.00	0.2698	22th	11.75	0.4756	11th
Evandromyia (Aldamyia) evandroi	21.00	0.365	18th	13.00	0.4146	13th
Evandromyia (Aldamyia) termitophila	-	-	-	11.50	0.4878	10th
Evandromyia (Aldamyia) walker	14.75	0.5634	14th	-	-	-
Evandromyia (Evandromyia) bourrouli	1.00	1.000	1th	4.50	0.8292	4th
Evandromyia (Evandromyia) pinottii	15.25	0.5476	15th	21.00	0.2439	27th
Evandromyia (Evandromyia) begonae	7.50	0.7936	7th	12.50	0.439	12th
Evandromyia (Evandromyia) saulensis	17.50	0.4761	16th	21.00	0.2439	29th
Psathyromyia (Forattiniella) runoides	28.50	0.1269	31th	16.00	0.2682	16th
Psathyromyia (Forattiniella) aragaoi	28.00	0.1428	28th	-	-	-
Psathyromyia (Forattiniella) lutziana	-	-	-	20.50	0.0487	22th
Psathyromyia (Xiphomyia) hermanlenti	32.00	0.0158	38th	13.25	0.4024	14th
Psathyromyia (Xiphomyia) dreisbachi	32.00	0.0158	37th	-	-	-
Psathyromyia (Psathyromyia) shannoni	30.75	0.0555	32th	21.00	0.2439	30th
Psathyromyia (Psathyromyia) dendrophyla	32.00	0.0158	36th	-	-	-
Viannamyia furcate	25.00	0.238	24th	-	-	-
Bichromomyia flaviscutellata a	4.50	0.8888	5th	11.25	0.5	9th
Psychodopygus complexus a	3.50	0.9206	3th	3.50	0.878	3th
Psychodopygus davisi	9.50	0.7301	10th	14.00	0.3658	15th
Psychodopygus claustrei	26.25	0.1984	26th	21.00	0.2439	24th
Psychodopygus llanosmartinsi	7.00	0.8095	6th	6.50	0.7317	6th
Psychodopygus hirsutus hirsutus a	24.00	0.2698	21th	-	-	-
Psychodopygus ayrozai a	11.50	0.6666	12th	20.00	0.0731	18th
Psychodopygus paraensis a	26.75	0.1825	27th	-	-	-
Nyssomyia antunesi a	2.00	0.9682	2th	5.00	0.8048	5th
Nyssomyia whitmani a	4.00	0.9047	4th	1.00	1.000	1th
Nyssomyia intermedia a	31.25	0.0396	33th	21.00	0.2439	25th
Trichophoromyia ubiquitalis a	17.50	0.4761	17th	-	-	-

a : vector species.

The H and the J analyses revealed that the indexes recorded in the rural settlement environment (H = 0.9641 and J = 0.6059) were higher than those of the periurban environment (H = 0.7758 and J = 0.5252) (Table III).

TABLE III

Total number, percentage and Shannon-Wiener diversity (H) and evenness (J) indexes of phlebotomines captured in light traps at rural settlement and periurban areas in the municipality of Guaraí, state of Tocantins, Brazil, January 2005-June 2008

	Rural settlement area			Periurban area
Species	M (n)	F (n)	Total n (%)	M (n)	F (n)	Total n (%)
Brumptomyia brumpti	2	2	4 (0.16)	1	0	1 (0.10)
Micropygomyia (Sauromyia) peresi	-	-	-	0	1	1 (0.10)
Micropygomyia (Sauromyia) longipennis	1	0	1 (0.04)	1	0	1 (0.10)
Micropygomyia (Sauromyia) quinquefer	0	1	1 (0.04)	-	-	-
Micropygomyia (Sauromyia) rorotaensis	-	-	-	1	0	1 (0.10)
Micropygomyia (Sauromyia) villelai	2	2	4 (0.16)	4	0	4 (0.39)
Sciopemyia sordellii	4	11	15 (0.60)	6	7	13 (1.28)
Lutzomyia (Lutzomyia) longipalpis a	53	21	74 (2.94)	15	42	57 (5.62)
Lutzomyia (Tricholateralis) sherlocki	0	1	1 (0.04)	-	-	-
Migonemyia (Migonemyia) migonei a	8	10	18 (0.72)	1	1	2 (0.20)
Pintomyia (Pintomyia) christenseni	0	1	1 (0.04)	-	-	-
Pintomyia (Pintomyia) damascenoi	0	2	2 (0.08)	-	-	-
Pressatia choti	0	3	3 (0.12)	-	-	-
Trichopygomyia dasydopogeton	20	32	52 (2.07)	0	2	2 (0.20)
Evandromyia (Aldamyia) carmelinoi	1	5	6 (0.24)	8	14	22 (2.17)
Evandromyia (Aldamyia) lenti	2	2	4 (0.16)	7	6	13 (1.28)
Evandromyia (Aldamyia) evandroi	1	4	5 (0.20)	4	5	9 (0.89)
Evandromyia (Aldamyia) termitophila	-	-	-	2	10	12 (1.18)
Evandromyia (Aldamyia) walkeri	4	10	14 (0.56)	-	-	-
Evandromyia (Evandromyia) bourrouli	538	212	750 (29.82)	83	23	106 (10.44)
Evandromyia (Evandromyia) pinottii	8	0	8 (0.32)	1	0	1 (0.10)
Evandromyia (Evandromyia) begonae	0	94	94 (3.74)	0	8	8 (0.79)
Evandromyia (Evandromyia) saulensis	2	5	7 (0.28)	0	1	1 (0.10)
Psathyromyia (Forattiniella) runoides	1	1	2 (0.08)	3	6	9 (0.89)
Psathyromyia (Forattiniella) aragaoi	1	0	1 (0.04)	-	-	-
Psathyromyia (Forattiniella) lutziana	-	-	-	1	0	1 (0.10)
Psathyromyia (Xiphomyia) hermanlenti	0	1	1 (0.04)	7	8	15 (1.48)
Psathyromyia (Xiphomyia) dreisbachi	0	1	1 (0.04)	1	0	1 (0.10)
Psathyromyia (Psathyromyia) shannoni	0	1	1 (0.04)	-	-	-
Psathyromyia (Psathyromyia) dendrophyla	0	1	1 (0.04)	-	-	-
Viannamyia furcata	0	4	4 (0.16)	-	-	-
Bichromomyia flaviscutellata a	109	60	169 (6.72)	7	4	11 (1.08)
Psychodopygus complexus a	47	215	262 (10.42)	17	78	95 (9.36)
Psychodopygus davisi	13	50	63 (2.50)	4	5	9 (0.89)
Psychodopygus claustrei	0	9	9 (0.36)	0	1	1 (0.10)
Psychodopygus llanosmartinsi	11	73	84 (3.34)	6	23	29 (2.86)
Psychodopygus hirsutus hirsutus a	0	4	4 (0.16)	-	-	-
Psychodopygus ayrozai a	5	16	21 (0.83)	0	2	2 (0.20)
Psychodopygus paraensis a	0	3	3 (0.12)	-	-	-
Nyssomyia antunesi a	124	422	546 (21.71)	11	28	39 (3.84)
Nyssomyia whitmani a	244	33	277 (11.01)	478	70	548 (53.99)
Nyssomyia intermedia a	1	0	1 (0.04)	1	0	1 (0.10)
Trichophoromyia ubiquitalis a	0	1	1 (0.04)	-	-	-
Total	1,202	1,313	2,515 (100)	670	345	1,015 (100)
H		0.9641			0.7758
J		0.6059			0.5252

a : vector species; F: female; M: male.

In the rural settlement area, 2,515 specimens representing 39 species were captured, 13 of which were found exclusively in this environment: Micropygomyia (Sauromyia) quinquefer , Lutzomyia (Tricholateralis) sherlocki , Pintomyia (Pintomyia) christenseni , Pi. (Pin.) damascenoi , Pr. choti , Evandromyia (Aldamyia) walkeri , Psathyromyia (Forattiniella) aragaoi , Pa. (Xiphomyia) dreisbachi , Psathyromyia (Psathyromyia) dendrophyla , Viannamyia furcata , Ps. hirsutus hirsutus , Ps. paraensis and T. ubiquitalis . In this environment, Ev. bourrouli had the highest frequency among the collected species (29.82%), followed by Ny. antunesi (21.71%), Ny. whitmani (11.01%) and Ps. complexus (10.42%) (Table III). In the periurban area, 1,015 specimens were collected, belonging to 30 species, four of which were exclusive to this environment, including Micropygomyia (Sauromyia) peresi , Micropygomyia (Sauromyia) rorotaensis, Evandromyia (Aldamyia) termitophila and Psathyromyia (Forattiniella) lutziana . Ny. whitmani was the most predominant species, representing more than half of the collected individuals (53.99%), followed by Ev. bourrouli (10.44%) and Ps. complexus (9.36%) (Table III).

When evaluating the overall number of specimens captured per ecotope, it was observed that the MS 2 forested area had the most individuals (n = 848), followed by other environments in which animals were present, such as chicken coops: MS 1 (n = 707), MS 2 (n = 695) and MS 4 (n = 581) (Supplementary data).

The H and the J indexes of the evaluated ecotopes showed the highest values in the banana grove ecotope at MS 4 (H = 1.1238 and J = 0.7063), followed by the forest ecotope at MS 1 (H = 1.0850 and J = 0.6819), the forest ecotope at MS 2 (H = 0.9755 and J = 0.6131) and the chicken coop ecotope at MS 1 (H = 0.9563 and J = 0.6011) (Supplementary data).

The analysis of species frequency per ecotope showed that Ev. bourrouli was present in all ecotopes and predominated in the rural settlement area, as well as in the banana grove ecotope at MS 4 in the periurban area. Ny. antunesi was the predominant species in the rural settlement area chicken coop at MS 2 (42.01%). Ny. whitmani was the most prevalent in the other ecotopes near domestic animal shelters in the periurban area, such as the chicken coop at MS 1 (44.62%), the pig sty at MS 2 (49.52%) and the chicken coop at MS 3 (56.63%), followed by Lu. longipalpis in all ecotopes (Supplementary data).

The Shannon trap captures at the MS1 rural settlement area yielded 1,096 specimens, including 190 males (18%) and 906 (82%) females, resulting in a female/male ratio of 4.55:1.0. Among the 14 species identified, those from the Psychodopygus genus were the most prevalent, including Ps. complexus , Ps. llanosmartinsi and Ps. ayrozai . All of these species were observed biting humans during the captures. Among these species, Ps. comple-xus (42.61%) was predominant (Table IV).

TABLE IV

Number of phlebotomine according to sex, captured monthly in Shannon traps at rural settlement area - monitoring station 1, municipality Guaraí, state of Tocantins, Brazil, March-June 2008

	March		April		May		June
Species	M (n)	F (n)	M (n)	F (n)	M (n)	F (n)	M (n)	F (n)	Total n (%)
Sciopemyia sordellii	-	-	-	-	-	1	-	-	1 (0.09)
Lutzomyia (Lutzomyia) sherlocki	-	1	-	-	-	-	-	-	1 (0.09)
Evandromyia (Aldamyia) walkeri	-	-	-	-	-	-	-	1	1 (0.09)
Evandromyia (Evandromyia) bourrouli	-	-	-	-	-	-	-	1	1 (0.09)
Psathyromyia (Psathyromyia) dendrophyla	-	-	-	-	6	2	-	-	8 (0.72)
Bichromomyia flaviscutellata a	-	-	-	-	-	-	-	3	3 (0.27)
Psychodopygus complexus a	42	280	-	41	-	1	7	96	467 (42.60)
Psychodopygus davisi	21	17	1	3	-	2	5	18	67 (6.11)
Psychodopygus claustrei	-	7	-	3	-	-	-	1	11 (1)
Psychodopygus llanosmartinsi	78	165	-	60	-	-	-	7	310 (28.28)
Psychodopygus ayrozai a	19	130	-	17	-	-	1	6	173 (15.78)
Psychodopygus paraensis a	-	-	-	2	-	-	-	-	2 (0.18)
Nyssomyia antunesi a	1	8	-	1	8	30	-	1	49 (4.47)
Nyssomyia whitmani a	-	-	-	-	1	1	-	-	2 (0.18)
Total	161	608	1	127	15	37	13	134	1,096 (100)

a : vector species; F: female; M: male.

A total of 290 specimens were collected and used for PCR and dot blot hybridisation. Sandflies of the same species were analysed in pools of 10; there were 23 Ps. complexus pools (3 male and 20 female pools) and six Ps. ayrozai pools (1 male and 5 female pools) (Table V). The results showed that four of 25 (16%) female pools were positive for Leishmania ( Viannia ) sp. infection. In the Ps. complexus pools, only three of 20 were found to be positive (15%). In the Ps. ayrozai pools (n = 5), one pool (20%) was positive for infection (Supplementary data).

TABLE V

Phlebotomine molecular diagnostic evaluation for infection with Leishmania (Viannia) braziliensis and positive polymerase chain reaction hybridisation results with female pools of Psychodopygus complexus and Psychodopygus ayrozai by traps in the municipality of Guaraí, state of Tocantins, Brazil

Traps	Ps. complexus (p/n)	Ps. ayrozai (p/n)	Females (p/n)
CDC light - HP model	1/52	0/0	2/5
Shannon	2/151	1/51	2/20
Total	3/203	1/51	4/25

the numbers in superscript represent the groups of females positive related to each species of phlebotomine. n: total number of females evaluated; p: number of positive females.

The municipality of Guaraí, state of Tocantins, Brazil.

Monitoring stations (MS) in rural settlement area, Agricultural Project Pedra Branca, municipality of Guaraí, state of Tocantins, Brazil. A, B: MS 1; C, D: MS 2.

Monitoring stations (MS) in periurban area, municipality of Guaraí, state of Tocantins, Brazil. A, B: MS 3; C, D: MS 4.

Products from polymerase chain reaction (PCR) multiplex assays submitted to hybridisation analysis for the diagnosis of naturally Leishmania (Viannia) braziliensis infection in Psychodopygus complexus and Psychodopygus ayrozai collected in the municipality of Guaraí, state of Tocantins, Brazil. A: ethidium bromide-stained 2% agarose gel revealing the 220 bp product form the cacophony gene amplification and the 120 bp fragment corresponding to the conserved region of kinetoplast minicircles from Leishmania spp [M: molecular weight marker (100 bp DNA ladder); Lane 1: amplification reaction without added DNA (PCR negative control); 2-4: negative controls for the DNA extraction step (male insect pools): 5-8: female sandfly pools (5-7: Ps. complexus positive pools; 8 : Ps. ayrozai positive pool); 9: PCR positive control (DNA extracted from a mixture of male insect pool containing L. (V.) braziliensis promastigotes)]; B: dot hybridisation using a biotinylated probe specific for parasites from the Viannia subgenus.

DISCUSSION

Initial studies of phlebotomine fauna in TO focused on the description of species (Barreto 1946, Martins et al. 1962, 1964, 1975); further entomological studies identified 32 sandfly species in the state (Lustosa et al. 1968, Andrade Filho et al. 2001), including the new species Micropygomyia (Silvamyia) echinatopharynx and Martinsmyia reginae (Andrade Filho et al. 2004, Carvalho et al. 2010). These findings indicate a great diversity of sandfly species in TO. Recent investigations of ACL and AVL vectors in rhe municipality of Porto Nacional recorded 48 sandfly species, 22 of which were the first records of these species in the state (Vilela et al. 2011).

The present study identified 43 phlebotomine species in Guaraí, 11 of which were newly recorded species, underscoring the rich biodiversity of the Brazilian Cerrado . Among the identified sandfly species, Ev. bourrouli was predominant in the rural settlement area and presented the second highest percentage in the periurban area. This species was found in various environments and ecotopes, such as preserved forest and animal shelters (chicken coops and pig sties), which underscores its eclectic behaviour. In previous studies carried out in TO, this species was the most frequent at Porto Nacional along with Evandromyia sallesi (Andrade Filho et al. 2001). However, it is important to emphasise that this species has not been reported to transmit pathogens to humans.

Of the species identified in this study, 11 were recorded in TO for the first time, including Ps. complexus , Ps. davisi , Ps. hirsutus hirsutus , Ps. ayrozai , Ps. paraensis and T. ubiquitalis , which are putative ACL vectors in Brazil; other sandfly vectors of leishmaniasis that had already been recorded in TO were also found in Guaraí, including Ny. antunesi, Nyssomyia flaviscutellata, Ny. whitmani, Ny. intermedia, Mig. (Mig.) migonei and Lu. longipalpis .

Previous studies developed in TO identified Ny. whitmani as the main vector of L. (V.) braziliensis , especially in areas affected by hydroelectric construction and agricultural activities, where this sandfly has been found inside and outside residences, near animal shelters and even within forest boundaries (Carvalho 2008, Vilela et al. 2008, 2011). Ny. whitmani is associated with the transmission of L. (V.) braziliensis in most Brazilian endemic areas, occupying different types of plant cover and modified environments (da Costa et al. 2007, Rangel 2010).

Ny. whitmani was the most abundant species among the species collected in the present study. Furthermore, it was the most abundant ACL vector in periurban areas and one of the most represented species in the rural settlement areas. This species was found in all ecotopes, with a particularly high density in animal shelters (chicken coops and pig stys) from residences in the periurban environment. Studies performed in Porto Nacional discussed the capacity of Ny. whitmani to adapt to environmental changes by expanding its transmission cycle (Vilela et al. 2011 ).

Of the ACL vectors observed in Guaraí, it is important to highlight the identification of Ps. complexus , an important L. (V.) braziliensis vector in the low-altitude forests of the state of Pará (PA) in the Amazon Region of northern Brazil (Souza et al. 1996, Lainson & Shaw 2005, Garcez et al. 2009, Rangel & Lainson 2009). This sandfly species has been recorded outside of the Amazon Region, but there has been no evidence to suggest its involvement in L. (V.) braziliensis transmission in the states of Pernambuco (Andrade et al. 2005) and Mato Grosso (Azevedo et al. 2002, Ribeiro et al. 2007 ).

The present study provides evidence for the presence of Ps. complexus in all evaluated ecotopes, including animal shelters in the periurban area. Although Ny. whitmani is thought of as the most important ACL vector in TO (Rangel & Lainson 2009, Vilela et al. 2011), the discovery of naturally L. (V.) braziliensis- infected Ps. complexus , the spatial distribution of this sandfly in forest and peridomiciliary ecotopes, its predominance in Shannon traps and its relatively high abundance in settlements and periurban areas, together with epidemiological evidence reported in the literature, suggest that Ps. complexus may also play a role in the transmission cycle of ACL in the rural settlement areas of Guaraí.

Ps. ayrozai , another sandfly species that is naturally infected by L. (V.) braziliensis in Guaraí, has been suggested as a putative vector of Leishmania (Viannia) naiffi in PA (Lainson et al. 1994, Lainson & Shaw 2005). Ps. ayrozai is an anthropophilic species (de Aguiar et al. 1985, Gomes & Galati 1989) and experimental infection studies have revealed its susceptibility to Leishmania (Leishmania) forattinii (Barretto et al. 1986, Lainson & Shaw 2005). Despite reports showing the vector competence of Ps. ayrozai , this species was not found frequently in this study and no further evidence was found of its participation in local transmission cycles.

Ny. antunesi, which was found in the rural settlement area, has been proven to be a Leishmania (Leishmania) lindenbergi vector in the northern PA and has been identified as anthropophilic in previous studies conducted in TO by Andrade Filho et al. (2001); however, cases of human infection by L. (V.) lindenbergi have not been reported in this study area thus far.

The ecoepidemiology of ACL in Brazil is strongly related to the transmission cycle of L. (V.) braziliensis and the vector Ny. whitmani , particularly in environmentally impacted areas, rural environments and the periphery of cities from all geographical regions. This new epidemiological profile has resulted from drastic environmental changes and the capacity of this sandfly to adapt to new ecological niches.

The Cerrado biome has experienced significant environmental impacts caused by deforestation. In this context, the establishment of highly populated, poor rural settlement groups and periurban areas without adequate infrastructure typically results in close contact between people and pathogen vectors.

This scenario has been frequently observed in TO and in this context, Ny. whitmani and Ps. complexus , as putative L. (V.) braziliensis vectors, likely maintain two transmission cycles in Guaraí: one related to the periurban area and the other in settlements in rural environments.

Total number, percentage and Shannon-Wiener diversity (H) and evenness (J) indexes of phlebotomines captured in light traps in different ecotopes in the municipality of Guaraí, state of Tocantins, Brazil, January 2005-June 2007

	Rural settlement area				Periurban area
	Monitoring station 1		Monitoring station 2		Monitoring station 3		Monitoring station 4
Species	Chicken coop n (%)	Forest n (%)	Chicken coop n (%)	Forest n (%)	Chicken coop n (%)	Pig sty n (%)	Chicken coop n (%)	Banana grove n (%)
Brumptomyia brumpti	-	1 (0.28)	-	3 (0.35)	1 (0.77)	1 (0.32)	-	-
Micropygomyia (Sauromyia) peresi	-	-	-	-	-	1 (0.32)	-	-
Micropygomyia (Sauromyia) longipennis	-	1 (0.28)	-	-	-	-	-	1 (1.28)
Micropygomyia (Sauromyia) quinquefer	-	-	-	1 (0.12)	-	-	-	-
Micropygomyia (Sauromyia) rorotaensis	-	-	-	-	-	-	1 (0.17)	2 (2.56)
Micropygomyia (Sauromyia) villelai	-	2 (0.56)	1 (0.14)	1 (0.12)	-	1 (0.32)	1 (0.17)	2 (2.56)
Sciopemyia sordellii	4 (0.57)	3 (0.84)	6 (0.86)	2 (0.24)	3 (2.31)	4 (1.29)	-	5 (6.41)
Lutzomyia (Lutzomyia) longipalpis a	17 (2.40)	9 (2.52)	29 (4.17)	2.12)	20 (15.38)	39 (12.54)	80 (13.77)	5 (6.41)
Lutzomyia (Tricholateralis) sherlocki	1 (0.14)	-	-	-	-	-	-	-
Migonemyia (Migonemyia) migonei a	60 (8.49)	5 (1.40)	2 (0.29)	7 (0.83)	-	1 (0.32)	1 (0.17)	-
Pintomyia (Pintomyia) christenseni	-	1 (0.28)	-	-	-	-	-	-
Pintomyia (Pintomyia) damascenoi	-	2 (0.56)	-	-	-	-	-	-
Pressatia choti	1 (0.14)	1 (0.28)	-	1 (0.12)	-	-	-	-
Trichopygomyia dasydopogeton	12 (1.70)	21 (5.88)	5 (0.72)	14 (1.65)	-	1 (0.32)	1 (0.17)	-
Evandromyia (Aldamyia) carmelinoi	1 (0.14)	-	3 (0.43)	2 (0.24)	5 (3.85)	4 (1.29)	6 (1.03)	5 (6.41)
Evandromyia (Aldamyia) lenti	1 (0.14)	1 (0.28)	-	2 (0.24)	2 (1.54)	6 (1.93)	4 (0.69)	2 (2.56)
Evandromyia (Aldamyia) evandroi	3 (0.42)	-	-	2 (0.24)	1 (0.77)	4 (1.29)	3 (0.52)	1 (1.28)
Evandromyia (Aldamyia) termitophila	-	-	-	-	5 (3.85)	6 (1.93)	1 (0.17)	-
Evandromyia (Aldamyia) walkeri	2 (0.28)	1 (0.28)	1 (0.14)	8 (0.94)	-	-	-	-
Evandromyia (Evandromyia) bourrouli	219 (30.98)	68 (19.05)	248 (35.68)	213 (25.12)	9 (6.92)	3 (0.96)	79 (13.60)	14 (17.95)
Evandromyia (Evandromyia) pinottii	-	3 (0.84)	7 (1.01)	2 (0.24)	-	-	-	1 (1.28)
Evandromyia (Evandromyia) begonae	41 (5.80)	15 (4.20)	10 (1.44)	27 (3.18)	-	2 (0.64)	3 (0.52)	4 (5.13)
Evandromyia (Evandromyia) saulensis	2 (0.28)	-	-	5 (0.59)	-	-	1 (0.17)	-
Psathyromyia (Forattiniella) runoides	-	-	1 (0.14)	1 (0.12)	2 (1.54)	7 (2.25)	-	-
Psathyromyia (Forattiniella) aragaoi	1 (0.14)	1 (0.28)	-	-	-	-	-	-
Psathyromyia (Forattiniella) lutziana	-	-	-	-	-	1 (0.32)	-	-
Psathyromyia (Xiphomyia) hermanlenti	1 (0.14)	-	-	-	2 (1.54)	14 (4.50)	-	-
Psathyromyia (Xiphomyia) dreisbachi	-	1 (0.28)	-	-	-	-	-	-
Psathyromyia (Psathyromyia) shannoni	-	1 (0.28)	-	1 (0.12)	-	-	1 (0.17)	-
Psathyromyia (Psathyromyia) dendrophyla	-	1 (0.28)	-	-	-	-	-	-
Viannamyia furcata	-	-	3 (0.43)	2 (0.24)	-	-	-	-
Bichromomyia flaviscutellata a	42 (5.94)	49 (13.73)	12 (1.73)	92 (10.85)	1 (0.77)	2 (0.64)	4 (0.69)	4 (5.13)
Psychodopygus complexus a	80 (11.32)	61 (17.09)	42 (6.04)	81 (9.55)	12 (9.23)	38 (12.22)	36 (6.20)	9 (11.54)
Psychodopygus davisi	10 (1.41)	19 (5.32)	8 (1.15)	26 (3.07)	-	1 (0.32)	5 (0.86)	3 (3.85)
Psychodopygus claustrei	-	5 (1.40)	-	3 (0.35)	-	-	-	1 (1.28)
Psychodopygus llanosmartinsi	21 (2.97)	14 (3.92)	12 (1.73)	37 (4.36)	2 (1.54)	8 (2.57)	14 (2.41)	5 (6.41)
Psychodopygus hirsutus hirsutus a	1 (0.14)	1 (0.28)	1 (0.14)	1 (0.12)	-	-	-	-
Psychodopygus ayrozai a	2 (0.28)	10 (2.80)	5 (0.72)	7 (0.83)	-	1 (0.32)	-	1 (1.28)
Psychodopygus paraensis a	-	2 (0.56)	1 (0.14)	-	-	-	-	-
Nyssomyia antunesi a	115 (16.27)	49 (13.73)	292 (42.01)	91 (10.73)	7 (5.38)	13 (4.18)	10 (1.72)	4 (5.13)
Nyssomyia whitmani a	65 (9.19)	9 (2.52)	6 (0.86)	194 (22.88)	58 (44.62)	154 (49.52)	329 (56.63)	11 (14.10)
Nyssomyia intermedia a	-	-	-	1 (0.12)	-	-	1 (0.17)	-
Trichophoromyia ubiquitalis a	5 (0.71)	-	-	3 (0.35)	-	-	-	-
Total	707 (100)	357 (100)	695 (100)	848 (100)	130 (100)	311 (100)	581 (100)	78 (100)
H	0.9563	1.0850	0.6936	0.9755	0.7933	0.8149	0.6963	1.1238
J	0.6011	0.6819	0.4359	0.6131	0.4986	0.5517	0.4376	0.7063

a : vector species.