Immature stages of two species of Evandromyia (Aldamyia) and the systematic importance of larval mouthparts within Psychodidae (Diptera, Phlebotominae, …

Accepted by J. Moulton: 21 Feb. 2008; published: 2 Apr. 2008 1

ZOOTAXAISSN 1175-5326 (print edition)

ISSN 1175-5334 (online edition)Copyright © 2008 · Magnolia Press

Zootaxa 1740: 1–14 (2008) www.mapress.com/zootaxa/

Immature stages of two species of Evandromyia (Aldamyia) and the systematic importance of larval mouthparts within Psychodidae (Diptera, Phlebotominae, Psychodinae)

FELIPE ARLEY COSTA PESSOA1, MARLISSON AUGUSTO COSTA FEITOSA2, ELOY GUILLERMO

CASTELLÓN-BERMÚDEZ2, CLAUDIA MARÍA RÍOS-VELÁSQUEZ1 & RICHARD DOUGLAS WARD3

1Biodiversidade em Saúde, Instituto Leônidas & Maria Deane - FIOCRUZ, Rua Terezina, 476, 69.057-070 Manaus – AM, Brazil. E-mail: [email protected], [email protected]ção de Pesquisas em Ciências da Saúde, Instituto Nacional de Pesquisas da Amazônia, Manaus, AM, Brazil. E-mail: [email protected] of Life Sciences, Centre of Applied Entomology and Parasitology Keele University, Staffordshire, UK. E-mail: [email protected]

Abstract

Phlebotomine sand flies (Diptera: Psychodidae) are vectors of Leishmania, Bartonella and several arboviruses. Sand flytaxonomy has been mainly based on adult morphological characters and few larval characters have been used. In thiswork the egg and all larval instars of Evandromyia carmelinoi (= Lutzomyia carmelinoi migonei group of authors) aredescribed, as well as the fourth instar of E. lenti, two morphologically similar species. Scanning electron microscopy(SEM) and light microscopy were used to describe the species. The sand flies E. carmelinoi and E. lenti can be differen-tiated most readily by the antennae and the shoulder accessory b setae on the thoracic segments. Some information on themouthpart morphology of Phlebotominae and Psychodinae that could be useful for future phylogenetic and systematicstudies is also provided.

Key words: larva, chaetotaxy, larval ontogeny

Introdution

Phlebotomine sand flies (Diptera: Psychodidae) have medical and veterinary importance as the vectors ofLeishmania, Bartonella and some arboviruses. As with many insect groups, the morphological charactersused to distinguish sand fly species are those of the adults, the immature stages of most species beingunknown and/or undescribed. The larval stages of less than 70 species of the 400 New World sand flies havebeen described, not always adequately (Young & Duncan 1994). In the past few decades, descriptions of lar-vae of many Neotropical species have been possible due to advances in rearing techniques. The larval descrip-tion presented was based on chaetotaxy and some additional information on the antennal form (e.g. Barretto1941; Hanson 1968; Ward 1976a; 1976b).

The use of scanning electron microscopy (SEM) improved the descriptions of New World larvae, allow-ing details of larval chaetotaxy (Leite & Williams 1996; 1997; Bahia et al. 2007); ontogeny (Secundino &Pimenta 2000); spiracles (Fausto et al. 1999; Pessoa et al. 2000); antennal, mouthpart sensillae and caudalsetae (Pessoa et al. 2001). However descriptions of immature forms of sand flies are still limited to the refer-ences cited above. Larval characters have been used for phylogenetic approaches to the study of medicallyimportant insects, especially Culicidae (e.g. Judd 1996; Harbach & Kitching 1998) and Simuliidae (e.g. Cos-

TERM OF USEThis pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website site is prohibited.

PESSOA ET AL.2 · Zootaxa 1740 © 2008 Magnolia Press

carón 1987; Adler et al. 2004). Phylogenetic analysis of phlebotomine sand flies is still rarely attempted andthere is just a single proposal (Galati 2003), which suggests the need to describe other semaphoronts besidesadults to corroborate or to strengthen new proposals in the systematic of sand flies.

The sand fly Evandromyia carmelinoi (Ryan, Frahia, Lainson & Shaw) [= Lutzomyia carmelinoi migoneigroup, unplaced subgenus, according to Young and Duncan (1994)] is very similar to E. lenti (Mangabeira).Those species have slight differences in the length and the shape of the genital filament tips, used to distin-guish the males (Young & Duncan 1994). Ryan et al. (1986) distinguished them by the ratio of the widths ofthe common and individual sperm ducts. Spiegel et al. (2000; 2002) used SEM to observe interesting varia-tion in the sensillae numbers and arrangements in males of both E. carmelinoi and E. lenti. Variations in thesex pheromone disseminating structures of the two species have been considered to have little or no taxo-nomic value. Females of the two species are very similar in structure.

Eggshell morphology may be used as a marker to differentiate related species of Diptera (Hinton 1981).The insect eggshell consists of an internal layer (endochorion) in contact with the embryo and an externallayer (exochorion). The exochorion itself presents distinct layers, generally forming elaborated sculptures onthe egg surface (Hinton 1969). SEM has been used to analyze the egg surface of different phlebotomine spe-cies in the New World (Ward & Ready 1975; Zimmerman et al. 1977; Endris et al. 1987; Feliciangeli et al.1993; Queiróz 1995; Perez & Ogusuku 1997; Fausto et al. 2001; Almeida et al. 2004).

The objectives of this paper are to (a) describe the egg and all larval instars of E. carmelinoi, (b)

redescribe the 4th instar larva of E. lenti and (c) provide more information on morphological characters usefulfor future phylogenetic and systematic studies of immature stages.

Material and methods

Larvae of E. carmelinoi and E. lenti were obtained from laboratory colonies, established and maintained fol-lowing the methods of Killick-Kendrick and Killick-Kendrick (1991) and reared from adults originally col-

lected in Santarém, Pará State, Brazil (02o 24’52’’S, 54o42’36’’W, for E. carmelinoi) and Baturité, Ceará State,

Brazil (04º 15’ 48" S, 38º 55’ 59" W, for E. lenti). Four slides of each larval instar (1st to 4th for E. carmelinoi,

4th for E. lenti) were slide-mounted in Berlese’s fluid. Mouthparts of three larvae were dissected and thenplaced on 10% KOH for two days, then slide-mounted in Berlese (Ward 1976a). Measurements of the bodybristles and morphological structures (mouthparts, setae, spiracles) were made under light microscopy. Dorsaland ventral views and the mouthparts were drawn and photographed. Four mature larvae were slide-mountedand examined by light microscopy for both species. Morphology and chaetotaxy of the head were observedfollowing Arrivillaga et al. (1999) who described the morphology and setae of the mouthparts and drew atten-tion to their taxonomic importance. The chaetotaxy of the body followed the system used by Ward (1976a),with some modifications (some setae were added). Systematic classification follows that proposed by Galati(2003). Fourth instar larvae of E. lenti from Baturité reared in a laboratory colony were studied and photo-

graphed under scanning electron microscopy (SEM): the larvae were killed in hot water (70oC), fixed in 3%glutaraldehyde and then washed thoroughly in phosphate-buffered saline, the solution being changed every 30min during 6 h. They were then pos-fixed in osmium tetroxide, dehydrated in a series of ethyl alcohol concen-trations, submitted to critical-point drying in carbon dioxide and spattered with 25 MA colloidal gold. The

specimens were examined with a Hitachi S 4500 SEM®. In order to search for some previously unrecognized structures of sand fly larvae and to better investigate

new probable apomorphies for mouthpart structures for the family, we slide-mounted mature larvae of thecommon moth fly Clogmia albipunctata (Williston) (Psychodidae: Psychodinae).


Zootaxa 1740 © 2008 Magnolia Press · 3EVANDROMYIA SAND FLY IMMATURES (PSYCHODIDAE)

Results

Egg of E. carmelinoi: The egg is elongated, with one side slightly flattened, measuring 342 (330–345) μm inlength and 101 (98–103) μm in width (n=4). The exochorion is formed by a thin surface that supports sculp-tures with polygonal reticulation, which is comprised of ridges, some discontinuous, forming alternatingtransverse rows of generally rectangular parallel cells or square to polygonal cells (Fig. 1).

FIGURE 1. Exochorion of egg of Evandromyia carmelinoi, bar = 50µm.

General appearance of the fourth instar larva: The larva is caterpillar-like, with a well sclerotized hypog-nathous non-retractile head with very short antennae; thorax with the anterior spiracle borne laterally on ashort tubercle in the prothorax, meso and metathoracic segments; and a nine segmented abdomen, covered bypale brown setae and the body tegument yellowish, with a pair of posterior spiracles borne laterally on a shorttubercle. Caudal setae dark. The larva is 3.14 mm from the head to end of ninth abdominal segment) and withmaximum width of 0.40 mm at the metathorax. Head and body tegument covered with small spines and scat-tered tubercles. Head dark. Body pale, with darker eighth and ninth abdominal segments and tiny spines on allsegments. Two types of paired setae, i.e., barbed brush-like and simple naked, distributed over the larval body(Fig. 2). Setal sizes and types summarized in Table 1.

Head capsule-like, broader than high. Dorsal cephalic tagma (Fig. 2a) with the following setae: frontocli-peal anterior setae (1) small and barbed, frontoclipeal posterior setae (2) barbed, the genal anterior setae (3)simple and spiniform. Genal medial (4) and posterior (5) setae barbed and brush-like. Ventral postgenal (6)and subgenal (7) setae simple. All setae inserted on small tubercles.



TABLE 1. Corresponding number and size of the setae of each segment (size in μm, N=4) of fourth to first instar larvaeof Evandromyia carmelinoi and the fourth instar larva of E. lenti.

Part of the larva body E. carmelinoi E. lenti

Number of setae Type of setae Size of the setae of the correspondentlarval instar

Head bristle 4th 3rd 2nd 1st 4th

Frontoclipeal anterior 1 little barbed 92,3 75,1 43,0 35,0 80,0

Frontoclipeal posterior 2 little barbed 76,9 66,3 45,3 34,8 50,0

Genal anterior 3 simple 92,3 72,3 55,6 39,0 62,5

Genal medial 4 barbed 100 77,6 42,1 35,0 75,0

Genal posterior 5 barbed 84,6 62,5 43,2 25,0 76,2

Postgenal 6 simple 92,3 72,5 48,3 35,0 72,5

Subgenal 7 simple 48,5 37,3 31,1 18,0 50,0

Prothorax

Dorsal internal 1 barbed 77,0 45,9 40,2 27,2 72,5

Dorsal intermediate 2 barbed 61,5 37,5 32,1 22,0 75,0

Dorsal external 3 barbed 92,3 55,3 42,6 NO 92,5

“Shoulder”accessory a simple 26,1 24,3 22,2 20,0 30,0

“Shoulder”accessory b Trifid or bifid 8 5,7 5,3 NO NO

Anterior ventrolateral 4 Barbed 92,3 55,6 37,9 35,0 92,5

Ventral external 5 Barbed 92,3 62,8 38,1 30,0 80,0

Ventral internal 6 Barbed 107,7 74,9 41,5 29,0 84,3

Dorsal submedian 7 Barbed 80,2 52,1 35,8 20,0 84,5

Mid – dorsal 8 Barbed 80,7 48,3 41,1 20,9 87,5

Dorsolateral 9 Barbed 76,9 42,6 38,2 20,4 70,0

Basal 10 Barbed 46,1 29,3 12,6 NO 37,5

Post-ventrolateral 11 Barbed 77,9 59,4 30,6 NO 62,5

Post-ventral 12 Spine 10,7 07,5 5,2 05,0 20,0

Mid ventral 13 Barbed 61,5 50,3 24,6 19,8 52,5

Ventral intermediate 14 Barbed 18,5 13,0 11,4 NO 32,5

Ventral submedian 15 Barbed 36,9 32,1 25,3 NO 45,0

Meso and metathorax

“Shoulder”accessory a Simple 10,7 08,2 07,0 07,0 10,7

“Shoulder”accessory b trifidid or bifid 8 07,5 07,0 NO NO





Basal 10 little barbed 18,7 10,0 08,8 05,5 12,5

Post-ventrolateral 11 Barbed 75,4 60,3 52,1 14,3 62,5


Mid ventral 13 Barbed 69,3 47,5 20,3 14,1 55,0

Ventral intermediate 14 Barbed 18,5 15,3 11,8 NO 22,5

to be continued.



“A”— Setae not observed on dorsal side of pseudopodium; NO- not observed, probably absent.

Antennae (Fig. 3a) with short basal segment, small and cylindrical segment fused at second distal ovalsegment, following the antenna pattern described by Pessoa et al. (2001) for Migonomyia migonei (França).This segment presents an antennal organ equipped with a longitudinal furrow on the posterior surface andthree short structures at its base, as well as a long sensilla that begins at the base of the first segment and isinserted in the furrow. Small digitiform projection on central apex of distal segment.

Each segmented mandible bearing two simple long setae in the mid-dorsum (S1 and S2); and a simplesetae (S6) in the superior margin of the mandible. Three setae on the ventral side, one in the proximal upperside (S5), and two in the middle central part of the mandible (S3 and S4). Three strong apical teeth on lower

TABLE 1. (continued.)

Part of the larva body E. carmelinoi E. lenti

Number of setae Type of setae Size of the setae of the correspondentlarval instar

Ventral submedian 15 Barbed 36,9 32,1 29,3 NO 37,5

Abdominal segments 1–7

Dorsal intermediate 2 Barbed 23,2 12,5 5,0 NO 25,3





Post-ventrolateral 11 Spine 17,3 15,8 11,2 05,3 08,0


Ventral submedian 15 Spine 50,0 45,1 29,3 12,3 100,0

“A” - Spine 15,4 7,5 NO NO NO

Abdominal segment 8




Dorsolateral 9 Barbed 100 72,6 45,7 25,1 112,5

Post-ventrolateral 11 little barbed 53,8 29,3 20,1 NO 55,0



“should” accessory A Spine 7,7 07,0 NO NO 12,5

Abdominal segment 9

Anterior ventrolateral 7 Simple 184,6 143,2 88,3 52,1 187,5

Dorsal submedian 8 Simple 92,3 59,9 31,9 41,0 62,4



Post-ventrolateral 12 Simple 69,2 42,3 15,6 NO 45,0

Post-ventral 12 Simple 46,1 33,9 15,2 NO 45,3


Internal caudal IC Simple 928 650 396 250 800

External caudal EC Simple 1142 863 500 342 1001



part of the mandible as well as a slightly protuberant mandibular lobe, with a brush inserted in its margin. Twolatero-mandibular processes (lmp) inserted on middle part of mandible can only be observed of mandible iswell clarified (Fig. 3b). Each maxilla with three simple setae, a S1 in the apical dorsal part and two (S2 andS3) in the proximal part. Maxillary process in the middle of this structure. Margin of the dorsal part with asmall, sparse comb of spines, very similar to those found in the maxilla of E. lenti (Fig. 3c). Apex, with papil-liform and trichodea sensillae (spinous hairs). Row of small setae on dorsum.

FIGURE 2. a–c. Schematic drawing and pictures of the chaetotaxy of larva of Evandromyia carmelinoi. 2a. Maturelarva: DS—dorsal side; VS—ventral side, cl—clypeus, m—mentum, fs—frontal suture, 1–7: setae; b —thorax, as—anterior spiracle, PT—prothorax, MS and MT—meso and metathorax, metathorax not drawn because is similar of themesothorax, 1–15: setae; c—abdomen, AS 1–7—abdominal segments first to seven, 2–15—setae; d—AS 8—abdominalsegment eight, – ec—external caudal seta, ic—internal caudal seta, ps— posterior spiracle, 2–15—setae; 2b and 2c—details of setae of the thoracic and abdominal setae.



FIGURE 3. a–c. Larva of Evandromyia carmelinoi. a—Antenna; b—mandible, c—dorsal side of the maxilla, at: apicalteeth, lmp: latero-mandibular process, mb: mandibular brush, mdl: mandibular lobe, s1 – s6: setae; maxilla—ma: maxil-lary process; ds—digitiform sensillae, ps—papiliform sensillae, s1-s3: setae; ts—trichodeal sensillae; d—Antenna of thelarva of E. lenti.

The hyaline labrum and the more sclerotized clypeus follow the descriptions of Arrivillaga et al. (1999)and Ward (1976a). Ventral surface of labrum covered with parallel, transverse rows of digitiform setal combs;dorsal side with two pairs of very small simple setae. Clypeus with two pairs of simple setae, the distal pairsmaller than the apical.

Prothorax appears divided in two segments, meso- and metathorax homologous with the posterior setae ofthe prothorax. Chaetotaxy follows the classification system of Ward (1976a) (Fig. 2a–b). Ventral part of eachthoracic segment bearing a seta, next to seta 4, well-developed bifid or trifid spine and similar-sized shoulderaccessory (a) seta, denominated here as b (Fig. 2b), and not included in the numerical chaetotaxy of Ward(1976a) although predicted by Forattini (1973). Anterior spiracles conical with eight papillae.

Chaetotaxy of prothorax: Tergite with two rows of setae, the first bearing three pairs (dorsal internal, dor-sal intermediate and dorsal external) and the second row of two setae (dorsal submedian and mid-dorsal).Pleura with two setae (anterior ventrolateral and dorsolateral) that appear to change position in the larva.However this is merely an artifact occurring when the larva is flattened during slide-mounting. These setaesimilar, barbed or brush-like with only very small differences in size (Fig. 2a–b, Table 1). Hyaline spiniformseta between the first and second rows of setae, usually near ventrolateral setae. Sternite also with two rows ofsetae. The first with two similar pairs of setae (ventral external and the ventral internal), somewhat less barbedthan those on the dorsum. Second row with seven pairs of setae, including seta b, distinguishable from the oth-ers based on its size and shape (Figs. 2a–c, Table 1). Basal, post-ventrolateral, post-ventral, mid ventral, ven-tral intermediate, ventral submedian, and b setae present.



FIGURE 4. Head, part of thorax and last segments of the abdomen of first instar larva of Evandromyia carmelinoi. a—Head and prothorax, m—mandible, l— labrum, s—spur or egg buster, 1–9 —setae; b—abdominal eight and nine seg-ments, cs—caudal setae, e—spiracle, – tb—tubercle of insertion of the caudal setae, 7–15—setae

Abdominal setae with almost the same distribution proposed by Ward (1976a), seta 10 of each segmentbeing absent. Segments 1–7 are homologous, with similar size and shape. Anterior pseudopodium with simplepair of setae, similar in size to setae 11 and 12 and not considered by other authors as taxonomically impor-



tant. This seta is denominated here as “c” (Fig. 2a, c). Eighth and ninth segments darker. Posterior spiraclesare conical with 14 papillae. Shape and measurements of the setae presented in Fig. 2a and Table 1. Chaetot-axy of abdominal segments 1–7 as follows: tergite with ungrouped pairs of setae, the anterior dorsal interme-diates being much smaller than the others. Anterior ventrolateral setae located at the border with the pleura,together with a row of paired dorsal submedian, mid-dorsal and dorsolateral setae. All these setae barbed andvarying in size (Table 1). Sternites with large pseudopodia bearing a few simple setae (post-ventrolateral andpost-ventral), both being large, and ventral submedian setae. Anterior pseudopodia bearing pair of setae (c),very similar to post-ventrolateral and post-ventral ones. Abdominal segments eight and nine lacking ofpseudopodia. Segment nine ending in two tubercles each one bears a caudal filament (Fig. 2a). Mammiliformsensilla at base of each caudal lobe, also found in E. lenti (Fig. 6). Posterior spiracle with 14–15 papillae.

FIGURE 5. Scanning electron microscopy of the thorax of Evandromyia lenti . a—prothorax and mesothorax, ventralsurface, 4–15 setae, b and c- increased areas with small setae 12, and 14.

Other larval instars: Length of larval instars 1, 2 and 3 from head to the end of the ninth abdominal seg-ment 1.2, 1.78 and 2.5 mm, respectively. Maximum width at metathorax of these instars 0.18, 0.23 and 0.36,respectively. First instar easily identified by presence of a unique pair of caudal setae (Fig. 4b), absence ofsome bristles on the prothorax alone (seta 3) and on pro-, meso- and metathorax (b, 14 and 15) as well as thepresence of an egg tooth on the head (Fig. 4). Shoulder accessory seta very well developed in first instar (Fig.4a), almost as long as in other larval instars, size increasing only very slightly (Table 1). Prothoracic seta 5simple in this instar and barbed in the others. Chaetotaxy for the other instars same as in the fourth, with cleardifferences in size (Table 1).

E. lenti – General appearance of the fourth instar larva and main differences of E. carmelinoi: Larvaof E. lenti very similar to that of E. carmelinoi. Total body length 3.55 (3.44 – 3.58 mm, n = 4) from head toend of ninth abdominal segment and maximum width 0.42 (0.40–0.49, n = 4) mm at metathorax. Main differ-ences between species the size of setae of head and body (not a good character), and absence of the shoulder



accessory b setae on thoracic segments. Size and type of setae summarized in Table 1. Pattern of the setaeshown in Figs. 5–7 and similar to the schematic figure of E. carmelinoi (Fig. 2). Antenna already described byPessoa et al. (2001) and differing from E. carmelinoi in the smaller transverse furrow of the distal segmentand its less accentuated sensillae. Digitiform appendix of this species, normally apical in E. carmelinoilocated somewhat above the centre of the antenna, within another sensillum and showing two transversal fur-rows separated by a layer of tegument (Fig. 3d).

FIGURE 6. Scanning electron microscopy of last abdominal segments of Evandromyia lenti, dorsal side. 2–15—setae,increased areas: 2 —seta 2, asterisk—mamiliform sensillae of the tubercle of the caudal seta. Bar—300µm.

Ornaments of the mouthparts of the larva of C. albipunctata: Mandible (Fig. 8) with a single latero-mandibular process (lmp), large, well developed and bifid in the distal part. Apical prominence present, cov-ered by a setal brush on the dorsal mandible, homologous to the adoral brushes of simuliid larvae (Crosskey1990) or mandibular brushes of culicid larvae (Harbach & Knight 1980). One pair of conspicuous setae abovethe most apical teeth, known in simuliid larvae as frontal setae (Py-Daniel 1990) and in culicid larvae as setae1 and 2 Mn (Harbach & Knight 1980). Setal brush on the ventral surface called as mandibular brush in sandflies (Arrivillaga et al. 1999), ventral setae in the Bruchomyiine Nemapalpus nearticus Young (Mahmood &Alexander 1992), prosthecal brushes in simuliids (Crosskey 1990) and mandibular sweepers in culicids (Har-bach & Knight 1980). Three very strong and dark apical teeth, the first stronger and more prominent than theothers. Projection above first apical tooth, homologous to external tooth of simuliid larvae (Py-Daniel 1990)and dorsal tooth of culicids (Harbach & Knith 1980), smaller and more slender than other apical teeth. Veryprominent mandibular lobe distal to third tooth, with one forwardly apical projection, itself resembling atooth. Labrum of C. albipunctata with well-developed brush of long setae in distal part.



Discussion

The exochorionic pattern of E. carmelinoi is similar to the polygonal ones of E. lenti (Queiróz 1995) and E.evandroi (Costa Lima & Antunes) (Almeida et al. 2004). Although Almeida et al. (2004) noted that at leastanother 25 species of neotropical sand flies in different genera showed this polygonal pattern of exochorionicsculpturing, they noted that the exochorion of E. evandroi appeared to be a distinct variation and as Galati(1995) included all three species in the subgenus Aldamyia Galati, this pattern may have a phylogenetic sig-nificance.

Despite the existence of several papers on larval morphology of phlebotomine sand flies, described usinglight or scanning electron microscopy, some morphological characters require further attention. Due to theoblique position of the mandible and maxilla, characteristic of the family Psychodidae (Stehr 1991) mouth-parts need to be dissected and clarified, to allow some of the setae that are obscured in whole mounted speci-mens. By following this procedure it is possible to find interesting characters such as the lmp. We observedthe lmp for the first time in Psychodidae, noting that it was smaller and less well-developed in Phlebotominaethan in the Psychodinae. This structure is common in Simuliidae and Chironomidae, both inserted in theinfraorder Culicomorpha, and has taxonomic importance. The projection found in the mandible of C. albi-punctata is similar to that described in Culicidae (Harbach & Knight 1980). There is a similar projection insimuliid larvae, named mandibular serrations by Crosskey (1990). The maxilla of E. carmelinoi has a row ofsmall setae, similar in the shape, described for L. longipalpis by Arrivillaga et al. (1999), but in L. longipalpisis distributed only on the dorsal surface, but for E. carmelinoi the row arises in the middle part of the dorsalsurface to the distal part.

We used the chaetotaxy system of Arrivillaga et al. (1999) for the head setae, due to the lack of uniformityshown among other such systems and the apparent confusion of several authors who consider some mouthpartsetae as representative and others as invalid for taxonomic purposes. There is however consensus that thelabral setae have little or no taxonomic importance (Ward 1976a; Arrivillaga et al. 1999). The chaetotaxo-nomic terminology of Ward (1976a) was very useful in describing thoracic and abdominal setae during thepresent study, although Leite and Williams (1996) suggested it was inadequate for L. longipalpis. All but twoof the setae (b and c, Fig. 2) for E. carmelinoi and E. lenti were represented and numbered by Ward’s system.

According to Sæther (2000) larvae of the infraorder Culicomorpha (which includes black flies, mosqui-toes, biting and non-biting midges and other related families), all possess a pair of labral brushes, each in theform of a convex, cushion-like area of cuticle covered with parallel, transverse rows of long setae”, and a“mandible with multitoothed apical comb”. Although the transverse rows of setae are not long in sand flies,they are visible under light microscopy. Mahmood and Alexander (1992) described the labrum of Nemapalpusnearcticus Young (Psychodidae: Bruchomyiinae) as being similar to that in sand flies. The labral brushes of C.albipunctata have long setae but are not clearly divided. These characters are considered in the most recentphylogenetic analyses of larval mouthpart morphological characters (Wood & Borkent 1989; Oosterbroeck &Courtney 1995), as being homologous within Nematocera, particularly the members of the Culicomorpha.Further phylogentic studies based on examination of these structures could change the present dendrogramsfor Nematocera.

The mamiliform sensillum found in the base of the caudal lobe was also described by Leite and Williams(1996; 1997) in L. longipalpis (Lutz & Neiva) and although not described, is easily visible in the picture ofdescription of the last segment of Phlebotomus tobbi Adler, Theodor & Lourie by Killick-Kendrick et al.(1989). It is probably a common structure to all sand fly genera. Although the high similarity of morphologi-cal characters of adults and larvae of E. carmelinoi and E. lenti, there are clear apormorphies which can beused to distinguish these semaphoronts.



FIGURE 7. a: lateral part of the head of larva of Evandromyia lenti, l—labrum, md—mandible, mx—maxilla, 3–7—setae, bar—60µm; b—labrum, bar 20µm; c—apex of the maxilla, showing trichoidal or spine like probable sensillae,bar—4,5µm ; d—part of the labrum, showing the transverse row of finger-like combs of setae, bar—1,64µm.

FIGURE 8. Mouth part of the larva of Clogmia albipunctatus: a- mandible, ab—adoral brush, fs—frontal setae to theapical teeth, ml—mandibular lobe, pb—prosthecal brush; b—detail of the mandible. Et—external tooth, at—apical teeth,plm latero-mandibular process, c—part of the head, l—labrum, m—mandible; d— labral brush.



Acknowledgments

The late Raul de Queiróz, adviser of Felipe Pessoa, provided the photo of the eggs of E. carmelinoi. Threeanonymous reviewers are thanked for offering valuable comments that improved this article.

References

Adler, P.H., Currie, D.C. & Wood, D.M. (2004) The black flies (Simuliidae) of North America. ROM / Comstock - Cor-nell University Press, New York, USA. 941p.

Almeida, D.N., Oliveira, R.S., Brazil, B.G. & Soares M.J. (2004) Patterns of exochorion ornaments on eggs of sevensouth American species of Lutzomyia sand flies (Diptera: Psychodidae). Journal of Medical Entomology, 41, 819–825.

Arrivillaga, J., Navarro, J.C. & Feliciangeli, M.D. (1999) Morfología y quetotaxia del tagma cefálico larval de LutzomyiaFrança 1924 (Diptera: Psychodidae): proposición de un sistema de nomenclatura. Boletín de Entomolgia Vene-zolana, 14, 1–13.

Bahia, A.C., Secundino, N.F., Miranda, J.C., Prates, D.B., Souza, A.P., Fernandes, F.F., Barral, A. & Pimenta, P.F. (2007)Ultrastructural comparison of external morphology of immature stages of Lutzomyia (Nyssomyia) intermedia andLutzomyia (Nyssomyia) whitmani (Diptera: Psychodidae), vectors of cutaneous leishmaniasis, by scanning electronmicroscopy. Journal of Medical Entomology, 44(6), 903–14.

Barretto, M.P. (1941) Morfologia dos ovos, larvas e pupas de alguns flebótomos de São Paulo. Anais da Faculdade deMedicina da Universidade de São Paulo, 17, 357–427.

Coscarón, S. (1987) El género Simulium Latreille en la región Neotropical: análisis de los grupos supraespecíficos,especies que los integran y distribución geográfica (Simuliidae, Diptera). Belém, Museu Paraense Emílio Goeldi,112 p.

Crosskey, R.W. (1990) The natural history of blackflies. John Wiley & Sons Ltd, Chichester, 711pp. Endris, R.G., Young, D.G. & Perkins, P.V. (1987) Ultrastructural comparison of egg surface morphology of five Lut-

zomyia species (Diptera: Psychodidae). Journal of Medical Entomology, 24, 412–415.Fausto, A.M., Taddei, A.R., Mazzini, M. & Maroli, M. (1999) Morphology and ultrastructure of spiracles in phlebotom-

ine sandfly larvae. Medical and Veterinary Entomology, 13, 101–9.Fausto, A.M., Feliciangeli, M.D., Maroli, M. & Mazzini, M. (2001). Ootaxonomic investigation of five Lutzomyia spe-

cies (Diptera: Psychodidae) from Venezuela. Memórias do Instituto Oswaldo Cruz, 96, 197–204.Feliciangeli, M.D., Castejon, O.C. & Limongi, J. (1993) Egg surface ultrastructure of eight New World phlebotomine

sandfly species (Diptera: Psychodidae). Journal of Medical Entomology, 30, 651–656.Forattini O.P. (1973) Entomologia Médica, vol. 4, São Paulo, Editora da Universidade de São Paulo.Galati, E.A.B. (1995) Phylogenetic systematic of Phlebotominae (Díptera, Psychodidae) with emphasis on American

groups. Boletin de la Direccion de Malariología y Saneamento Ambiental, 35, 133–142.Galati, E.A.B. (2003) Classificação de Phlebotominae. In EF Rangel, R Lainson (eds), Flebotomíneos do Brasil, Fiocruz,

Rio de Janeiro, p. 23–51.Hanson, W.J. (1968) The immature stages of the subfamily Phlebotominae in Panama (Diptera: Psychodidae). PhD The-

sis, Ann Arbor, University of Kansas, Michigan, 105pp. Harbach, R.E. & Knight, K.L., (1980) Taxonomists’s glossary of mosquito anatomy. Plexus Publishing, Marlton, New

Jersey, 415pp.Harbach, R.E. & Kitching, I.J. (1998) Phylogeny and classification of the Culicidae (Diptera). Systematict Entomology,

23, 327–370.Hinton, H.E. (1969) Respiratory systems of insect eggs shell. Annual Review of Entomology, 14, 343–368.Hinton, H.E. (1981) Biology of insect eggs, vol.1. Pergamon, Oxford, UK. Judd, D.D. (1996) Review of the systematics and phylogenetic relationships of the Sabethini (Diptera: Culicidae). Sys-

tematic Entomology, 21, 129–150.Killick-Kendrick, R., Killick-Kendrick, M., Leger, N., Pesson, B., Madulo-Leblond, G. & Page, A.M. (1989) Absence of

outer caudal setae on all larval instars of Phlebotomus tobbi from Ionian Greek islands. Medical and VeterinaryEntomology, 3, 131–135.

Killick-Kendrick,. M. & Killick-Kendrick, R. (1991) The initial establishment of sandfly colonies. Parassitologia, 33,315–320.

Leite, A.C. & Williams, P. (1996) Description of the fourth instar larva of Lutzomyia longipalpis under scanning electronmicroscopy. Memórias do Instituto Oswaldo Cruz, 9, 571–578.

Leite, A.C.R. & Williams, P. (1997) The first instar larva of Lutzomyia longipalpis (Diptera: Psychodidae). Memórias do



Instituto Oswaldo Cruz, 92, 197–203.Mahmood, F. & Alexander, J.B. (1992) Immature stages of Nemapalpus nearticus (Diptera: Psychodidae). Florida Ento-

mologist, 75, 171–178. Oosterbroek, P. & Courtney, G. (1995) Phylogeny of the nematocerous families of Diptera (Insecta). Zoological Journal

of Linnean Society, 115, 267–311. Pérez, E.J. & Ogusuku, E. (1997) Chorion patterns on eggs of Lutzomyia sand flies from the Peruvian Andes. Medical

and Veterinary Entomology, 11, 127–133.Pessoa, F.A.C., Queiroz, R.G. & Ward, R.D. (2000) Posterior spiracles of fourth instar larvae of phlebotomine sand flies

(Diptera: Psychodidae) under scanning electron microscopy. Memórias do Instituto Oswaldo Cruz 95, 689–691.Pessoa, F.A.C., Queiroz, R.G. & Ward, R.D. (2001) External morphology of fourth instar larvae of neotropical species of

phlebotomine sand flies (Diptera: Psychodidae) under scanning electron microscopy. Memórias do InstitutoOswaldo Cruz, 96, 1103–1108.

Py-Daniel, V. (1990) Revisão da tribo Prosimuliini seg. Crosskey (Diptera, Culicomorpha, Simuliidae) nas regiões zoo-geográficas neártica e neotropical (larvas e pupas). Phd thesis, Instituto Nacional de Pesquisas da Amazônia, 320pp.

Queiróz, R.G. (1995) Phlebotomine sand flies of a leishmaniasis focus in Baturité, Brazil. PhD thesis, London Univer-sity, 224 pp.

Ryan, L., Frahia, H., Lainson, R. & Shaw, J.J. (1986) New phlebotomine sandflies of the walkeri group (Diptera: Psy-chodidae) from Pará state, Brazil. Memórias do Instituto Oswaldo Cruz, 81, 323–331.

Sæther, O.A. (2000) Phylogeny of Culicomorpha (Diptera). Systematic Entomology, 25, 223–234. Secundino, N.F.C. & Pimenta, P.F.P. (2000) Scanning electron microscopy study of the egg and imature stages of the

sandflies Lutzomyia longipalpis. Acta Microscópica, 8, 33–38. Spiegel, C.N., Brazil, R.P. & Soares, M.J. (2000) Sensilla on the terminalia of Lutzomyia spp. (Diptera: Psychodidae)

sand flies. Journal of Medical Entomology, 37(6), 860–863.Spiegel, C.N., Brazil, R.P. & Soares, M.J. (2002) Ultrastructure of male sex pheromone glands in abdominal tergites of

five Lutzomyia sandfly species (Diptera: Psychodidae). Arthropods Structure Development, 30, 219–227.Stehr, F.W. (1991) Immature insects, vol. 2. Dubuque, Iowa: Kendall/Hunt Publishing Company, 930 pp.Ward, R.D. (1976a) A revised numerical chaetotaxy for neotropical Phlebotomine sandfly larvae (Diptera: Psychodidae).

Systematic Entomology, 1, 89–94. Ward, R.D. (1976b) The immature stages of some Phlebotomine sandflies from Brazil (Diptera: Psychodidae). System-

atic Entomology, 1, 227–240.Ward, R.D. & Ready, P.A. (1975) Chorionic sculpturing in some sand fly eggs (Diptera: Psychodidae). Journal of Ento-

mology, 50, 127–134.Wood, D.M. & Borkent, A. (1989) Phylogeny and classification of the Nematocera. In: McAlpine JF, Wood DM, eds.

Manual of Neartic Diptera 3. Research Branch, Agriculture Canada, Monograph 32, 1333–1370.Young, D.G., Duncan, M.A. (1994) Guide to identification and geographic distribution of Lutzomyia sandflies in México,

the West Indies, Central and south America (Diptera: Psychodidae). Memoirs of American Entomological Institute,1–881.

Zimmerman, J.H., Newson, H.D., Hooper, G.R. & Christensen, H.A. (1977) A comparison of the egg surface structure ofsix anthropophilic phlebotomine sand flies (Lutzomyia) with the scanning electron microscope (Diptera: Psychod-idae). Journal of Medical Entomology, 13, 574–579.


Documents

Immature stages of two species of Evandromyia (Aldamyia) and the systematic importance of larval mouthparts within Psychodidae (Diptera, Phlebotominae, …