Journal of Medical Entomology, 53(6), 2016, 1488–1491

doi: 10.1093/jme/tjw107
Advance Access Publication Date: 24 June 2016
Short Communication Short communication

Sand Flies (Diptera: Psychodidae) From the Brazilian

Atlantic Forest Domain Collected With Malaise Traps
Paloma Helena Fernandes Shimabukuro,1,2 Jéssica Adalia Costa Moreira,3 and
Tiago Silva da Costa4
1
Centro de Refer^encia Nacional e Internacional para Flebotomıneos, Centro de Pesquisas René Rachou, Fiocruz/MG, Avenida

Downloaded from https://academic.oup.com/jme/article/53/6/1488/2658151 by guest on 26 January 2022

Augusto de Lima 1715, Barro Preto, 31190-002, Belo Horizonte, Minas Gerais, Brazil (palomahfs@gmail.com), 2Corresponding
author, e-mail: palomahfs@gmail.com, 3Departamento de Ci^ encias Biol
ogicas. Universidade Federal de Minas Gerais (jessicada-
liamoreira@gmail.com), and 4Laborat orio de Arthropoda. Universidade Federal do Amap a. (tiago_sc@hotmail.com)

Received 15 April 2016; Accepted 30 May 2016

Abstract
Here, we present the results of a 2-yr sampling using malaise traps along the Atlantic Forest domain from the
northeast to the south of Brazil. In total, 217 sand flies were collected, of which the most abundant species was
Bichromomyia flaviscutellata (Mangabeira, 1942) (60.4%), followed by Psychodopygus ayrozai (Barretto &
Coutinho, 1940) (11%) and Micropygomyia schreiberi (Martins, Falca ~ o & Silva, 1975) (4.1%), and the remaining
less abundant species comprised 10.1% of the total of sand flies collected. We report the occurrence for the first
time of: 1) B. flaviscutellata, Pintomyia fischeri (Pinto, 1926), Ps. ayrozai, and Psychodopygus carreirai (Barretto,
1946) in the state of Alagoas; 2) Psychodopygus claustrei (Abonnenc, Lèger & Fauran,1979), Psychodopygus
amazonensis (Root, 1934), and Sciopemyia sordellii (Shannon & del ponte, 1927) in the state of Bahia; 3)
Nyssomyia anduzei (Rozeboom, 1942) in the state of Pernambuco; and 4) B. flaviscutellata, M. schreiberi, Ps.
ayrozai, and Psychodopygus davisi (Root, 1934) in the state of Sergipe. Our results present novel records of
sand flies collected with malaise traps in the Atlantic Forest domain demonstrating that different collecting
methods such as malaise traps can provide new interesting data about these insects that are natural vectors of
many pathogens.

Key words: biodiversity survey, inventory, leishmaniasis, sand fly

Phlebotomine sand flies (Diptera: Psychodidae) are insects with
great importance to public health, as they play a role in the transmis-
sion of pathogens between human and nonhuman animals (Rangel
and Lainson 2003). There are approximately 990 described species
of sand flies, of which 273 are recorded in Brazil (Bates et al. 2015,
Shimabukuro et al. 2016).
Phlebotomine sand flies are usually collected with light traps, of
which the lightweight CDC model has been the most widely used
(Young and Duncan 1994). Other collecting methods include: 1)
Shannon traps, in which both a light source and the presence of
humans, act as baits to collect anthropophilic species (Pérez et al.
1988); and 2) Disney traps, which have been used to collect species
attracted to rodents, especially Bichromomyia flaviscutellata
(Dorval et al. 2007). Only a few studies have previously used mal-
aise traps for sand fly collection (Alexander et al. 2001, Andrade-
Filho et al. 2008). These traps are low-cost and easy to use, that
allow: 1) operation for long continuous periods without the pres-
ence of a researcher in situ; and 2) insect detection during daytime
activity (Andrade-Filho et al. 2008). The Brazilian Atlantic forest
originally covered 1,300,000 km2 distributed over 17 states in
Brazil. Nowadays, this biome is reduced to only 22% of its original
range (MMA 2016), but this complex biome shows a species diver-
sity higher than most of the Amazon forests. Species richness, the ex-
tremely high levels of endemism, and the small fraction of the
original forest left make this biome one of the most vulnerable and it
is considered one of the top biodiversity hotspots in the world
(Myers et al. 2000).
It is in Atlantic forest areas that the most populated areas in
Brazil have developed since the period of European colonization.
Landscapes and biomes undergo significant changes as a conse-
quence of urbanization and human incursion into new environ-
ments, which are regarded among the biggest promoters of
changes in disease dynamics, as well as their increasing
prevalence in humans, domestic animals, and wildlife (Brearley et al.
2012).
Here, we present the distribution of sand flies collected using
malaise traps in the Atlantic Forest domain during the BIOTA-
FAPESP project.

C The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America.
V
All rights reserved. For Permissions, please email: journals.permissions@oup.com 1488
Journal of Medical Entomology, 2016, Vol. 53, No. 6 1489

Table 1. Localities, geographical coordinates, and dates for collection of sand flies in Brazilian Atlantic Forest domain

State Municipality Collection site Geographical coordinates Collection dates Collectors

 0 00  0 00
AL Quebrangulo Reserva Biol ogica Pedra Talhada *09 13 57.75 S, 36 24 53.2 1W 08-11.IX.2002 Penteado-Dias et al.
AL Quebrangulo Reserva Biol ogica Pedra Talhada *09 130 57.7500 S, 36 240 53.200 1W 11-14.IX.2002 Penteado-Dias et al.
BA Ilhéus Mata Esperança *14 470 12.3800 S, 39 040 03.9100 W 15-18.V.2002 no data
BA Ilhéus Mata Esperança *14 470 12.3800 S, 39 040 03.9100 W 15-18.V.2002 no data
BA Mata de S~ ao Jo~ao Reserva do Sapiranga 12 330 3700 S, 38 020 46.800 W 19-22.VII.2001 M.T. Tavares et al.
BA Mata de S~ ao Jo~ao Reserva do Sapiranga 12 330 36.400 S, 38 020 57.200 W 19-22.VII.2001 M.T. Tavares et al.
BA Mata de S~ ao Jo~ao Reserva do Sapiranga 12 330 37.200 S, 38 020 55.300 W 22-25.VII.2001 M.T. Tavares et al.
BA Mata de S~ ao Jo~ao Reserva do Sapiranga 12 330 37.200 S, 38 020 55.300 W 19-22.VII.2001 M.T. Tavares et al.
BA Mata de S~ ao Jo~ao Reserva do Sapiranga 12 330 37.400 S, 38 020 46.800 W 22-25.VII.2001 M.T. Tavares et al.
BA Mata de S~ ao Jo~ao Reserva do Sapiranga 12 330 38.500 S, 38 020 57.200 W 21-25.VII.2001 M.T. Tavares et al.
BA Mata de S~ ao Jo~ao Reserva do Sapiranga 12 330 41.100 S, 38 020 51.000 W 22-25.VII.2001 M.T. Tavares et al.
BA Mata de S~ ao Jo~ao Reserva do Sapiranga 12 330 41.100 S, 38 020 51.000 W 19-22.VII.2001 M.T. Tavares et al.
BA Mata de S~ ao Jo~ao Reserva do Sapiranga 12 330 41.700 S, 38 020 42.900 W 22-25.VII.2001 M.T. Tavares et al.

Downloaded from https://academic.oup.com/jme/article/53/6/1488/2658151 by guest on 26 January 2022

BA Mata de S~ ao Jo~ao Reserva do Sapiranga 12 330 42.900 S, 38 020 42.900 W 22-25.VII.2001 M.T. Tavares et al.
BA Mata de S~ ao Jo~ao Reserva do Sapiranga 12 330 42.900 S, 38 020 42.900 W 19-22.VII.2001 M.T. Tavares et al.
BA Porto Seguro Estaç~ao Ecol
ogica Pau-Brasil 16 230 17.600 S, 39 100 55.600 W 20.V.2002 C.O. Azevedo et al.
BA Porto Seguro Estaç~ao Ecol
ogica Pau-Brasil 16 230 17.600 S, 38 100 55.600 W 20.V.2002 C.O. Azevedo et al.
BA Porto Seguro Estaç~ao Ecol
ogica Pau-Brasil 16 230 17.600 S, 39 100 55.600 W 20.V.2002 C.O. Azevedo et al.
BA Porto Seguro Estaç~ao Ecol
ogica Pau-Brasil 16 230 17.600 S, 39 100 55.600 W 17.V.2002 C.O. Azevedo et al.
ES Linhares Reserva Biol ogica Sooretama 18 580 02.500 S, 40 080 11.100 W 24-27.III.2002 C.O. Azevedo et al.
ES Linhares Reserva Biol ogica Sooretama 18 580 02.800 S, 40 070 53.600 W 21-24.III.2002 C.O. Azevedo et al.
ES Santa Teresa Estaç~ao Biol
ogica Santa Lucia 19 580 19.500 S, 40 320 10.300 W 09-12.IV.2001 C.O. Azevedo et al.
ES Santa Teresa Estaç~ao Biol
ogica Santa Lucia 19 580 18.500 S, 40 320 07.600 W 06-09.IV.2001 C.O. Azevedo et al.
PE Recife Parque dos Dois Irm~aos *08 000 41.5000 S, 34 560 41.0500 W 17-20.VII.2002 S.T.P. Amarante et al.
PE Recife Parque dos Dois Irm~aos *08 000 41.5000 S, 34 560 41.0500 W 20-23.VII.2002 S.T.P. Amarante et al.
PR Morretes Parque Estadual do Pau-Oco 25 340 27.900 S, 48 530 46.700 W 07-10.IV.2002 M.T. Tavares et al.
PR Morretes Parque Estaç~ao do Pau-Oco *25 340 27.900 S, 48 530 46.700 W 10-13.IV.2002 M.T. Tavares et al.
RJ Nova Iguaçu Reserva Biol ogica do Tingua 22 340 2700 S, 43 260 11.400 W 08-11.III.2002 S.T.P. Amarante et al.
RJ Nova Iguaçu Reserva Biol ogica do Tingua 22 340 3700 S, 43 260 0500 W 05-08.III.2002 S.T.P. Amarante et al.
SE Santa Luzia do Itanhy Crasto 11 220 32.800 S, 37 250 0.0000 W 01-04.VIII.2001 M.T. Tavares et al.
SE Santa Luzia do Itanhy Crasto 11 220 31.400 S, 37 240 53.900 W 29.VII.2001-VIII.2001† M.T. Tavares et al.
SE Santa Luzia do Itanhy Crasto 11 220 37.900 S, 37 250 01.800 W 28.VII-01-VIII.2001† M.T. Tavares et al.
SP Peruıbe Estaç~ao Ecol
ogica Juréia-Itatins 24 310 0600 S, 47 120 0600 W 05.V.2002 N.W. Perioto et al.
SP Peruıbe Estaç~ao Ecol
ogica Juréia-Itatins 24 310 0600 S, 47 120 0600 W 03.V.2002 N.W. Perioto et al.
SP Peruıbe Estaç~ao Ecol
ogica Juréia-Itatins 24 310 0600 S, 47 120 0600 W 06.V.2002 N.W. Perioto et al.
SP Ribeir~ ao Grande Parque Estadual Intervales 24 150 S, 48 1000 W 13-16.XII.2000 M.T. Tavares et al.
SP Ubatuba Parque Estadual Serra do Mar 23 210 4300 S, 44 490 2200 W 21.I.2002 N.W. Perioto et al.
SP Ubatuba Parque Estadual Serra do Mar 23 210 4300 S, 44 490 2200 W 24.I.2002 N.W. Perioto et al.

*Approximate geographical coordinates. †Traps were installed continuously for a year

Material and Methods Results and Discussion

Study Area
The insects were collected between December 2000 and November
2002 as part of the BIOTA/FAPESP “Riqueza de Hymenoptera e
Isoptera ao longo de um gradiente latitudinal da Mata Atl^ antica”
project, which is a multi-institutional effort to sample the insects
from orders Hymenoptera and Isoptera from the Atlantic Forest do-
main. The insects were originally sampled in 17 nearly equidistant
localities, extending from northern (07 080 25.000 S) to southern (26
190 25.600 S) Brazil. The specimens studied here were collected from
13 of these sampled localities (Table 1 and Fig. 1) using malaise
traps. All Diptera were separated and deposited at the Diptera
Collection of the Museu de Zoologia of the Universidade de S~ ao
Paulo (MZUSP) and made available to others for further studies.
Details about sampling with malaise traps can be found in the article
by Perioto and Lara (2003).
The insects were kept in 70% alcohol until processing. In the la-
boratory, the insects were prepared according to the clarification
method described by Forattini (1973), and their identification was per-
formed using the keys by Young and Duncan (1994) and Galati
(2003a). The specimens were mounted on microscope slides with
Canada balsam. The classification of the species follows Galati (2003b),
and the abbreviation of the generic names follows Marcondes (2007).
The map was produced using SimpleMppr (Shorthouse 2010).
In total, 217 sand flies were collected from eight states ranging from
the north to the south of Brazil, of which 187 were identified to spe-
cies level (Table 2). It was not possible to identify to species level 30
(13.8%) specimens collected, due to loss of morphological struc-
tures used for taxonomic identification. Accordingly, these latter in-
dividuals were only identified to genus level. Overall, there were
103 females and 114 males collected.
Overall, the most abundant species was B. flaviscutellata
(Mangabeira, 1942) (n ¼ 131, 60.4%), followed by Psychodopygus
ayrozai (Barretto & Coutinho, 1940) (n ¼ 24, 11.0%) and
Micropygomyia schreiberi (Martins, Falc~ ao & Silva, 1975) (n ¼ 9,
4.1%). The remaining less abundant species comprised 10.1%
(n ¼ 22) of the total amount of sand flies collected. Bichromomyia
flaviscutellata is an incriminated vector of Leishmania amazonensis
Lainson & Shaw, 1972, while Ps. ayrozai and Psychodopygus ama-
zonensis (Root, 1934) are suspected vectors in known transmission
areas of Leishmania braziliensis Vianna, 1911 (Rangel and Lainson
2009). The genera with the largest number of species collected were
Psychodopygus (5) and Pintomyia (3).
Here, we report the occurrence for the first time of 1) B. flaviscu-
tellata, Pintomyia fischeri (Pinto, 1926), Ps. ayrozai, and
Psychodopygus carreirai (Barretto, 1946) for the state of Alagoas; 2)
Psychodopygus claustrei (Abonnenc, Lèger & Fauran,1979), Ps.
1490 Journal of Medical Entomology, 2016, Vol. 53, No. 6

Downloaded from https://academic.oup.com/jme/article/53/6/1488/2658151 by guest on 26 January 2022

;
Fig. 1. Map of Brazil indicating collection sites per state. State abbreviations: AL – Alagoas; BA – Bahia; ES – Espırito Santo; PE – Pernambuco; PR – Parana
RJ – Rio de Janeiro; SE – Sergipe; SP – Sa~o Paulo.

Table 2. Phlebotominae sand flies collected with Malaise traps between December 2000 and November 2002 in Brazilian Atlantic Forest
domain

States Species AL BA ES PE PR RJ SE SP Total

Sex M F M F M F M F M F M F M F M F
Genus

Bichromomyia flaviscutellata 1 – 32 33 – 1 – 1 – – – – 2 5 46 10 131

Evandromyia cortelezzii – – – – – – – – – – – – – – 1 – 1
Evandromyia sp. – – – 4 – – – – – – – – – 2 – – 6
Micropygomyia schreiberi – – – 7 – – – – – – – – – 2 – – 9
Nyssomyia anduzei – – – – – – 2 – – – – – – – – – 2
Nyssomyia whitmani – – 1 – – – – – – – – – – – – – 1
Nyssomyia sp. – – 1 1 – 2 – 2 – – – 1 – 1 1 – 9
Pintomyia fischeri 1 – – – – – – – – – – – – – – – 1
Pintomyia misionensis – – – – – – – – – – – – – – 1 – 1
Pintomyia pessoai – – – – – 1 – – – 2 – – – – – – 3
Pressatia sp. – – – – – – – – – – – – – 1 – – 1
Psychodopygus sp. – – – 1 – 2 – – – – – 2 – 7 – – 12
Psychodopygus amazonensis – – 1 – – – – – – – – – – – – – 1
Psychodopygus ayrozai 2 – 2 2 2 – – – – – 2 1 5 6 2 – 24
Psychodopygus carreirai 1 – 1 1 – – – – – – – – – – – – 3
Psychodopygus claustrei – – 1 – – – – – – – – – – – – – 1
Psychodopygus davisi – – – – – – – – – – 2 – – 1 – – 3
Sciopemyia servulolimai – – 1 – – – – – – – – – – – – – 1
Sciopemyia sordellii – – 1 2 – – – – – – – – – – – – 3
Sciopemyia sp. – – – – – – – – – – – – – – – 1 1
Trichopygomyia trichopyga – – 1 – – – – – – – – – – – – – 1
Trichopygomyia viannamartinsi – – 1 – – – – – – – – – – – – – 1
Trichopygomyia sp. – – 1 – – – – – – – – – – – – – 1
Total 5 95 8 5 2 8 32 62 217

First record for the state.

State abbreviations: AL, Alagoas; BA, Bahia; ES, Espırito Santo; PE, Pernambuco; PR, Paran
a; RJ, Rio de Janeiro; SE, Sergipe; SP, S~
ao Paulo.

amazonensis, and Sciopemyia sordellii (Shannon & del Ponte, 1927) Sergipe. The higher number of new records for the states of Alagoas
for the state of Bahia; 3) Nyssomyia anduzei (Rozeboom, 1942) for (4) and Sergipe (4) is due to the scarcity of previous studies carried
the state of Pernambuco; 4) B. flaviscutellata, M. schreiberi, Ps. ayr- out in these localities, and the new records for the states of
ozai, and Psychodopygus davisi (Root, 1934) for the state of Pernambuco (1) and Bahia (2) might be related to the use of
Journal of Medical Entomology, 2016, Vol. 53, No. 6
malaise traps rather than light traps, the most commonly used for
sand flies.
Malaise traps used in this study were placed at low height,
touching the ground, which might have accounted for the collection
of high numbers of B. flaviscutellata and Psychodopygus spp. Both
B. flaviscutellata and Psychodopygus spp. have been described feed-
ing at ground level and use leaf litter and low shrubs as resting sites
during the day (Shaw et al. 1972, Alexander 1999). As both B. fla-
viscutellata and at least some species of Psychodopygus are involved
in the transmission of Leishmania parasites between man and other
animals (Rangel and Lainson 2009), the use of malaise traps might
be an additional tool for the investigation of sand fly fauna and
leishmaniases eco-epidemiological studies.
In the present study, we showed novel records of sand flies col-
lected with malaise traps in the Atlantic Forest domain.
Phlebotomine sand flies are distributed throughout different biomes
in Brazil, and their investigation using different collecting methods,
such as flight interception traps, can provide new and interesting
data about these insects and the pathogens they transmit. In add-
ition, our data provide information for areas where there have been
few previous sand fly studies.
Acknowledgments
We wish to thank the BIOTA-FAPESP Project, and Professor Carlos J. E.
Lamas, curator of Museu de Zoologia da Universidade de S~ ao Paulo (MZUSP),
for making the specimens available for our study. Financial support was given
by Fundaç~ao de Amparo  a Pesquisa de Minas Gerais (FAPEMIG - APQ-
001811-12) and Fundaç~ ao Oswaldo Cruz (FIOCRUZ). We also thank Luke
Baton for revising and commenting on draft versions of this manuscript.
References Cited
Alexander, B. 1999. Tatuquiras of the terra firme: ecological and public health
significance of the subgenus Lutzomyia (Psychodopygus) Mangabeira,
1941. Mem. Entomol. Int. 14: 519–542.
Alexander, B., J. M. Freitas, and L. W. Quate. 2001. Some psychodidae
(Diptera) from Atlantic Forest in South-Eastern Brazil, with descriptions of
Trichomyia dolichopogon sp. nov. and Trichomyia riodocensis sp. nov.
Braz. J. Biol. 6: 467–474.
Andrade-Filho, J. D., R. C. Oliveira, and A. R. Fonseca. 2008. Flebotomıneos
(Diptera: Psychodidae) Coletados com Armadilha Malaise no Centro-Oeste
de Minas Gerais. Neotrop. Entomol. 37: 104–106.
Bates, P. A., J. Depaquit, E. A. Galati, S. Kamhawi, M. Maroli, M. A.
McDowell, A. Picado, P. D. Ready, O. D. Salom on, J. J. Shaw, et al. 2015.
Recent advances in phlebotomine sand fly research related to leishmaniasis
control. Parasit Vectors 27:131. (doi: 10.1186/s13071-015-0712-x)
1491
Brearley, G., J. Rhodes, A. Bradley, G. Baxter, L. Seabrook, D. Lunney, Y.
Liu, and C. McAlpine. 2012. Wildlife disease prevalence in human-modified
landscapes. Biol. Rev. 88: 427–442.
Dorval, M. E., T. P. Alves, A. G. Oliveira, R. P. Brazil, E. A. Galati,
and R. V. Cunha. 2007. Modification of Disney trap for capture of
sand flies (Diptera: Psychodidae: Phlebotominae). Mem Inst Oswaldo
Cruz. 102: 877–878.
Forattini, O. P. 1973. Entomologia Médica. Psychodidae. Phlebotominae.
Leishmanioses. Bartonelose, v. 4. Edgar Blücher Editora Ltda/Ed. da
Universidade de S~ao Paulo, S~
ao Paulo, SP.
Galati, E.A.B. 2003a. Morfologia, terminologia de adultos e identificaç~ ao
dos taxons da América. pp. 53–175. In: E. F. Rangel and R. Lainson,
(eds.), Flebotomıneos do Brasil, Fundaç~ ao Oswaldo Cruz, Rio de
Janeiro, RJ.
Galati, E.A.B. 2003b. Morfologia e taxonomia: Classificaç~ ao de
Downloaded from https://academic.oup.com/jme/article/53/6/1488/2658151 by guest on 26 January 2022
Phlebotominae. pp. 23–51. In: E. F. Rangel, and R. Lainson, (eds.),
Flebotomıneos do Brasil, Fundaç~ao Oswaldo Cruz, Rio de Janeiro, RJ.
Marcondes, C. B. 2007. A proposal of generic and subgeneric abbreviations
for phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) of the
world. Entomol News 118: 351–356.
#Ministério do Meio Ambiente. MMA. 2016. Mata Atl^ antica. (http://www.
mma.gov.br/biomas/mata-atlantica)
Myers, N., R. A. Mittermeier, C. G. Mittermeier, G.A.B. Fonseca, and J. Kent.
2000. Biodiversity hotspots for conservation priorities. Nature 403:
853–858.
Pérez, J. E., A. Villaseca, A. Llanos-Cuenta, M. Campos, and H. Guerra.
1988. Técnicas para colectar “titiras” (Lutzomyia spp., Diptera:
Psychodidae), en ambientes altoandinos peruanos. Rev. Peru. Ent. 30:
77–80.
Perioto, N. W., and R.I.R. Lara. 2003. Himen opteros parasit
oides (Insecta:
Hymenoptera) da Mata Atl^ antica. I Parque Estadual da Serra do Mar,
Ubatuba, SP, Brasil. Arq. Inst. Biol. 70: 441–445.
Rangel, E. F., and R. Lainson. 2003. Flebotomıneos do Brasil. Rio de Janeiro,
Fundaç~ao Oswaldo Cruz, Rio de Janeiro, RJ.
Rangel, E. F., and R. Lainson. 2009. Proven and putative vectors of American
cutaneous leishmaniasis in Brazil: Aspects of their biology and vectorial
competence. Mem. Inst. Oswaldo Cruz 104: 937–954.
Shaw, J. J., R. Lainson, and R. D. Ward. 1972. Leishmaniasis in Brazil. VII.
Further observations on the feeding habitats of Lutzomyia flaviscutellata
(Mangabeira) with particular reference to its biting habits at different
heights. Trans. R. Soc. Trop. Med. Hyg. 66: 718–723.
Shimabukuro, P.H.F., A. J. Andrade, and E.A.B. Galati. 2016. Phlebotominae
in Catalogo Taxonômico da Fauna do Brasil. PNUD. (http://fauna.jbrj.gov.
br/fauna/faunadobrasil/3297) (accessed 12 May 2016).
Shorthouse, D. P. 2010. SimpleMappr, an online tool to produce publication-
quality point maps (http://www.simplemappr.net) (accessed 2 May 2016).
Young, D. G., and M. A. Duncan. 1994. Guide to the identification and geo-
graphic distribution of Lutzomyia sand flies in Mexico, the West Indies,
Central and South America (Diptera: Psychodidae). American
Entomological Institute, Gainesville, FL.