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Journal of Medical Entomology logoLink to Journal of Medical Entomology
. 2020 Aug 6;57(6):1722–1734. doi: 10.1093/jme/tjaa124

Ultrastructure of the Antennae and Sensilla of Nyssomyia intermedia (Diptera: Psychodidae), Vector of American Cutaneous Leishmaniasis

Fernando de Freitas Fernandes 1,2, Ana Beatriz F Barletta 3, Alessandra S Orfanó 1, Luciana C Pinto 1, Rafael Nacif-Pimenta 1, Jose Carlos Miranda 4, Nágila F C Secundino 1, Ana Cristina Bahia 5,#, Paulo F P Pimenta 1,#,✉
Editor: Carlos Marcondes
PMCID: PMC7899269  PMID: 32761144

Abstract

The antennal sensilla and the antenna of females Nyssomyia intermedia, one of the main vectors of American cutaneous leishmaniasis, were studied by scanning electron microscopy. The main goal was to characterize the quantity, typology, and topography of the sensilla with particular attention to the olfactory types. The insects were captured in the city of Corte de Pedra, State of Bahia, Brazil, by CDC-type light traps and raised in a laboratory as a new colony. Fourteen well-differentiated sensilla were identified, among six cuticular types: trichoidea, campaniformia, squamiformia, basiconica, chaetica, and coeloconica. Of these, six sensilla were classified as olfactory sensilla due to their specific morphological features. Smaller noninnervated pilosities of microtrichiae type were also evidenced by covering all antennal segments. The antennal segments differ in shapes and sizes, and the amount and distribution of types and subtypes of sensilla. This study may foment future taxonomic and phylogenetic analysis for a better evolutionary understanding of the sand flies. Besides, it may assist the targeting of future electrophysiological studies by Single Sensillum Recording, and aim to develop alternative measures of monitoring and control of this vector.

Keywords: Phlebotominae, sand fly, scanning electron microscopy, sense organs, sensillary typology

The leishmaniasis is one major infectious parasitic disease, spreading in four continents being endemic in 97 countries and territories (WHO 2018). It affects mainly poor populations in Africa, Asia, and Latin America. The different clinical forms of this disease are included within the most neglected group of tropical diseases, accounting for significant economic losses related to diagnosis, treatment, and time of work in various parts of the World. In Brazil, Nyssomyia intermedia (Lutz and Neiva) has been incriminated as one of the main vectors of Leishmania (Viannia) braziliensis (Vianna) (Trypanosomatida: Trypanosomatidae) (Vieira 2015), an etiological agent of American cutaneous leishmaniasis and mucocutaneous leishmaniasis. The Ny. intermedia s.s. is widely distributed in Brazil, having been reported in the states of the Northeast Region of the country, in the states of the Southeast Region, Espírito Santo, Rio de Janeiro, in eastern Minas Gerais, and on the northern coast of São Paulo, in the states of the Midwest Region, Mato Grosso do Sul and Goiás, close to the border with Bahia and Minas Gerais, and in the South Region, in the state of Paraná, and in the North Region, in the state of Tocantins (Marcondes 1998, 2007; Rangel and Lainson 2003; Andrade Filho et al. 2007, Santos et al. 2009, Vilela et al. 2013). The Ny. intermedia is completely adapted to the anthropic environment, mainly in the intra- and peri-domestic environments. The vector has a high degree of anthropophily, but with an eclecticism regarding the hosts (Vieira 2015). The Ny. intermedia is also present in primary and secondary forests and transition areas between forests and anthropogenic environments (Souza et al. 2002).

It stands out as an essential target to be attacked by the complex epidemiological chain of leishmaniasis, the combat against its vectors. Chemical insecticides are still the main products used to reduce vector populations and control outbreaks of vector-borne diseases worldwide. However, the number of reports of the development of DDT-resistance and pyrethroid-tolerance by sand flies in endemic areas of leishmaniasis has been steadily increasing in several countries (Dhiman and Yadav 2016, Fawaz et al. 2016, Gomes et al. 2017). This fact, coupled with the toxicity of these products, the accumulation of their residues in the food chain, and the severe ecological damage caused by chemical insecticides raises the researchers’ efforts to develop more effective and eco-friendly sand flies’ control methods. With this aim, the use of traps containing as attractive semiochemical baits for insect vector control and monitoring stands out as a promising alternative, based on favorable results obtained with other harmful insects (Bray et al. 2010, Witzgall et al. 2010, Lorenzo et al. 2014). Through single sensillum recording (SSR), an efficient method for isolating potential insect attractants, the action potentials of odor receptor neurons (ORNs) present in each type of olfactory sensilla can be recorded in situ. This method allows knowing the sensitivity and response to specific odor stimuli, as well as enabling to encode the stimulatory quality of odor, the response–concentration relationship, as well as temporal changes in odor concentration and spatial distribution (De Bruyne et al. 1999, Li et al. 2018). The SSR coupled to gas chromatography (SSR-GC) goes further and is employed to identify key ligands of odorant molecules to neuronal receptors, directly from natural collections of odors (Ghaninia et al. 2008). The SSR-GC is the most effective method for localizing semiochemicals components in physiologically and environmentally relevant quantities within a complex array of chemicals in natural extracts (Bray et al. 2010, Logan et al. 2010).

However, conducting such accurate in vivo electrophysiological assays is greatly hindered by the extremely small size of certain olfactory sensilla. The variety of shapes they can present, often ‘hidden’ in cavities and between large number and density of sensilla, of various types and sizes, in the antennae of certain dipterans, such as mosquitoes and sand flies, e.g., in which there is no evidence of regionalized sensory distributions. Therefore, before these electrophysiological assays, it is necessary to thoroughly observe by scanning electron microscopy (SEM) the entire surface extension of the antennae. So, it is possible to locate the sites where sensilla with olfactory morphofunctional characteristics to enable the precise introduction of the electrodes and the potential targeting of the odor pulse tested.

Most behavioral studies and of chemical ecology, related to sand flies, were conducted with Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae), the most important invertebrate host of L. chagasi (=Leishmania infantum chagasi) (Nicolle) (Trypanosomatida: Trypanosomatidae), the causative agent of visceral leishmaniasis in Brazil and other countries of South and Central America (Dougherty et al. 1999, Bray et al. 2010). Many other species of sand flies of epidemiological importance have been ignored in this field of science. It is believed that this is partly due to the difficulty and cost of maintaining colonies to provide specimens for such studies. Much remains to be elucidated about the sensory organs used in the communication, behavior, and complex ecological relationships of sand flies in general.

This work aimed to analyze the anatomy and ultrastructure of the antennae of female Ny. intermedia, to locate, classify, and quantify the different morpho-functional types and subtypes of sensilla. It also was focused on the topographic location of olfactory sensilla. The olfactory sensilla detect communication chemicals, such as pheromones, kairomones and apneumones, involved in copula, feeding, and oviposition. This study may support the performance of the aforementioned electrophysiological studies, and, also, they may foment future taxonomic and phylogenetic analysis to understand the evolution of Diptera: Phlebotominae better.

Material and Methods

Sand Fly

Nyssomyia intermedia insects were captured live with CDC light traps (Sudia, and Chamberlain 1962) at Corte de Pedra (13°32′S, 39°25′W) in the State of Bahia, Brazil, and transferred to a colony cage where they were blood-fed on hamsters and provided a 50% sugar solution ad libitum (Bahia et al. 2007). The predominant sand fly species was Ny. intermedia. Taxonomic identification was performed using the Galati taxonomic key (2018), with special attention to the characteristics of the spermathecae e dos spermathecal ducts, previously described in Marcondes (1996). The Ny. intermedia sand flies were held in the colony cages at 26C and 60% RH to allow for mating and digestion of the bloodmeal. Three- or four-day postfeeding, the blood-fed females were transferred to oviposition containers (Modi and Tesh 1983), where they laid their eggs. Upon hatching, the larvae were fed the appropriate diet (Secundino and Pimenta 1999) to complete their life cycle.

Specimen Preparation and SEM Examination

Twenty specimens of Ny. intermedia females were selected and cleaned as described in Fernandes et al. (2002). The heads were dissected, together with part of the thorax, washed in PBS, and immediately fixed overnight in 2.5% glutaraldehyde in 0.1M cacodylate buffer, pH 7.2, at room temperature. After this, the samples were postfixed in 1% osmium tetroxide and 0.8% potassium ferrocyanide, prepared in a cacodylate buffer, pH 7.2 (Fernandes et al. 2008, 2020). Subsequently, these were washed in buffer and dehydrated in an increasing series of concentrations of ethanol, alternating the concentration of alcohol at 6-h intervals, using 100% ethanol three times (Fernandes and Pimenta 2018). The samples were then critical-point dried in CO2 and mounted in various positions on SEM stubs using conductive silver paint or conductive double-sided adhesive carbon tabs. After sputtering with platinum (Fernandes et al. 2004), the preparations were examined at an accelerating voltage of 5–20 kV in JSM-5600 scanning electron microscope (JEOL, Tokyo, Japan).

Structure Analysis

Sensillary classification was performed according to the descriptions of morphological types and by comparison with illustrations in specialized literature in insect sense organs. The sensillary types were designated according to the nomenclature originally created by the first researchers in insect sensory organs (Keil and Steinbrecht 1984; McIver 1982; Zacharuk 1985; Steinbrecht 1998, 1999; Hallberg, and Hansson 1999; Shanbhag et al. 1999), derived from the Latin and Greek languages. For the sensory subtypes, we have updated the nomenclature to English, currently more used for this purpose. The typology and the distribution of antennal sensilla, as well as the general aspects of the antennal segments of females Ny. intermedia, have been discussed with previous studies carried out with other sand fly species (Ilango 2000, Zayed et al. 2002, Fernandes et al. 2008).

The Number of Sensilla

The average number of sensilla was considered counting three-fourth of each segment of the antennae. The other one-fourth of the antennae was adhered to the carbon tape, as required for SEM observation.

Measurement of Structures

Each observed antennal segment and the evidenced sensilla chaetica were measured with the SEM analytical software program (JEOL JSM 5600).

Statistic Methods

To compare the length of the segments and the chaetic sensilla, and the number of sensilla found on the antennae of sand flies, the analysis of variance (ANOVA) test with multiple comparisons of Tukey was used. When the parametric model (ANOVA) was not adequate, we used the Kruskal–Wallis test with multiple comparisons of Dunn. All the analyzes were realized with a reliable level of 95% (α = 0.05). The statistical analysis and graphical presentation of the results were done using the GraphPad Prism software (San Diego, CA).

Results

General External Anatomical Characteristics of Sand Fly Antennae

The Ny. intermedia females have a pair of filiform antennae composed of 16 subsegments: a short scape (basal segment) with a shape that resembles a triangle, with the upper surface larger than the lower; a globular pedicel (second antennal segment); and a flagellum (Fig. 1) consisting of 14 filiform flagellomeres (Figs. 1–4).

Fig. 1.

Fig. 1.

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Electron micrographs of the antennae of females Nyssomyia intermedia. (A) Latero-internal view of the antennae of the showing the first two segments: scape (S) and pedicel (P), with short sharp-tip trichoid (st), arranged in a set of two on the scape and a set of three on the pedicel, long blunt-tipped trichoid (bt) sensilla, next to some ommatidia (om) of one of the sandfly´s compound eyes; and squamiform sensillum (sq; scale bar = 28.2 µm). (B) Latero-external view of the scape and pedicel showing short sharp-tip trichoid (st), arranged in a set of two on the Scape (S) and in a set of three on the pedicel (P; scale bar = 10.4 µm). (C and D) Short sharp-tip trichoid (st) observed in higher magnification on the scape ([B] circle area) and pedicel (B box area), respectively, and microtrichia (m) covering all the surface of these antennal segments (scale bars = 3.9 µm, both). (E) Dorsoexternal view of antenna showing the scape (S) with a set of three short sharp-tip trichoid (st) (scale bar = 6.8 µm). (F) Two campaniform sensilla, surrounded by microtrichia (m), laterally observed on the Pedicel (P; scale bar = 2.6 µm). (G) Higher magnification of one campaniform sensillum (scale bar = 1.3 µm).

Fig. 2.

Fig. 2.

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Scanning electron micrographs of the flagellomeres of females Nyssomyia intermedia. (A) Flagellomere I showing long blunt-tipped trichoid (bt), and squamiform sensilla (sq; scale bar = 20 µm). (B) Higher magnification showing two squamiform sensilla (s), and microtrichia (m; scale bar = 10 µm). (C) Box area showing squamiform (sq), basiconic (b), and short blunt-tip trichoid (sb) sensilla, in higher magnification, besides microtrichia (m; scale bar = 10 µm). (D) The same sensilla in higher magnification (scale bar = 5 µm). (E) Flagellomere VI showing long blunt-tipped trichoid (bt) and chaetic (ch) sensilla, and microtrichia (m; scale bar = 20 µm).

Fig. 3.

Fig. 3.

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Electron micrographs of the flagellomeres of females Nyssomyia intermedia. (A) Flagellomere XI showing long blunt-tipped trichoid (bt), short prominent-base trichoid (sp), and chaetic sensilla, single-base subtype (ch; scale bar = 20 µm). (B) Box area showing long blunt-tipped trichoid (bt), and short prominent-base trichoid (sp; scale bar = 6.35 µm). (C) Higher magnification of the antennal flagellomere V showing a base of a chaetic sensillum, single-base subtype (ch), with the cuticle perforated with wall pores (po), and microtrichia (m; scale bar = 1.8 µm). (D) Flagellomere I showing chaetic sensillum, forked-base subtype (chfb), and of grooved coeloconic sensillum, palmately lobed subtype (gcpl; scale bar = 14.6 µm). (E) Higher magnification of the antennal segment showing grooved coeloconic (gc) sensillum, palmately lobed subtype, with structures fused at its base, but distally lobed, arranged around its inner cone, which has longitudinal wall grooves (lg); and microtrichia (m; scale bar = 4 µm).

Fig. 4.

Fig. 4.

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Scanning electron micrographs of the flagellum of females Nyssomyia intermedia. (A) Flagellomere XIII showing implantation sites of common grooved coeloconic sensilla (gc), a short prominent-base trichoid (sp) sensillum, chaetic sensilla, single-base subtype (ch), long blunt-tipped trichoid (bt), and medium pointed-tipped trichoid (pt) sensilla (scale bar = 10 µm). (B) Three common grooved coeloconic sensilla (gc), surrounded by microtrichia (m) on flagellomere XII, beyond the base of implantation of two medium pointed-tipped trichoid (pt) sensilla, the apex of two long blunt-tipped trichoid (bt) sensilla (scale bar = 10 µm). (C) Flagellomere XII in higher magnification sowing a common grooved coeloconic sensilla (gc), showing longitudinal wall grooves (lg), surrounded by microtrichia (m; scale bar = 5 µm). (D) Last antennal flagellomere (XIV) showing long blunt-tipped trichoid (bt), medium pointed-tipped trichoid (pt), grooved coeloconic (gc) sensilla, short fixed-base sensilla (sf), and the apical trichoid sensillum (ap; scale bar = 10 µm). (E) The apex of the last flagellomere in higher magnification showing medium pointed-tipped trichoid (pt), long blunt-tipped trichoid (bt), short fixed-base sensilla (sf), and the apical trichoid sensillum (ap; scale bar = 5 µm).

In order to present the results concisely, similar electromicrographs, of flagellomeres and sensilla with the same morphological characteristics, obtained in different antennal subsegments, were not included in this work.

The flagellomeres I to IX have more evident filiform aspects, with flagellomere I being the most filiform of all. (Fig. 2A). It was observed that the flagellomeres II to XI are morphologically similar (Figs. 2 and 3). The flagellomeres XII, XIII, and XIV are more globular (less filiform) and slightly wrinkled, being this characteristic generally more evident on the flagellomere XII (Fig. 4).

Sensillar Typology

Fourteen well-differentiated sensilla were identified, among six types of cuticular sensilla: trichoidea, campaniformia, squamiformia, basiconica, chaetica, and coeloconica. The detailed description of each type and subtypes of sensilla is presented:

  1. Sensilla trichoidea—Hair or bristle shaped sensory structures featuring single wall (SW-sensilla or single-walled sensillum), with or without pores, and flexible, partially flexible or fixed insertion. Subtypes observed:

    • a. long blunt-tipped trichoid sensillum—cylindrical-shaped bristle of varying thickness and size, long in the vast majority of antennal subsegments (Figs. 1A, 2A and E, and 4A, D, and E), but may also be of medium size (Fig. 3A and B). Presents pilosities covering its entire not porous surface (NP-sensillum or aporous sensillum) and a blunt tip and flexible insertion base (with a cuticular collar around your implantation base);

    • b. short sharp-tip trichoid sensillum—small hair with flexible implantation base, and thin tip (Fig. 1A–E);

    • c. short blunt-tip trichoid sensillum—small hair, even lower than the subtype 2, with flexible implantation base (with cuticular collar), with a slightly blunt tip, and wall pores (WP-sensillum or multiporous sensillum) (Fig. 2C and D);

    • d. short prominent-base trichoid sensillum—small hair, apparently with a flexible insertion base and porous wall, but implanted just above the cuticle level (in a slight protuberance), and with a thinner tip (Fig. 3A and B);

    • e. medium pointed-tipped trichoid sensillum—medium size hair, without a full cuticular collar (with partially flexible fitting), and with sharped tip. (Fig. 4A, B, D, and E).

    • f. short trichoid sensillum, fixed-base subtype—small hair of sharp-tipped and with a nonflexible insertion base (Fig. 4D and E);

    • g. short trichoid sensillum, apical subtype—small hair very similar to anterior subtype, in tip size and shape, but differing somewhat in the form of their implantation base occurring only one at the apex of each antenna (Fig. 4D and E).

  2. Grooved sensilla coeloconica—Small sensilla presenting an internal grooved cone (double-walled sensilla or DW-sensilla), implanted in a shallow pit, and surrounded by a protective covering structure. The inner cone of this sensilla type is characterized by the presence of longitudinal grooves, which distally are more evident, formed by the junction of fingers-like projections of one hand.

    • a. grooved coeloconic sensillum, common subtype—with isolated microtrichia encircling its grooved internal cone (Fig. 4A–D);

    • b. grooved coeloconic sensillum, palmately lobed subtype—whose structures around its grooved cone are fused at its base, but with distal divisions or lobes, resembling palmately lobed leaves (Fig. 3D and E).

  3. Sensilla chaetica—Long and more robust hair with a flexible base (Figs. 4A and E and 3A–D), and presenting wall pores (WP-sensillum) (Fig. 3C). Some are symmetrical with their peers showing a lyre shape (Fig. 3A).

Subtypes observed

  • a. single-base chaetic sensilla—presents a single deployment base. Its more common subtype, showing a unique implantation base (Figs. 2F, 3A–C and 4A).

  • b. forked-base chaetic sensilla—in a single Ny. intermedia specimen, a bifurcated base chaetic sensilla, was observed (Fig. 3D).

  • 4. Sensilla squamiformia—Hair with a trichoid-like flexible implantation base, but which widens and then narrows distally, with the dorsal surface covered by pilosities, showing a shape similar to dorsally hairy scale or a lanceolate brush. The pilosities were observed covering the whole dorsal surface of this sensillum similar to the pilosities seen on the surface of long blunt-tipped trichoid sensillum (Figs. 1A and 2A–C).

  • 5. Sensilla campaniformia—This sensillum resembles the external view of a circumvallate papilla, consisting of a flexible cuticular dome (cap) embedded in cuticle within a slightly high circular collar (Fig. 1F and G)

  • 6. Sensilla basiconica—Small sensillum with wall pores (WP-sensillum) characterized by presenting a base implanted directly into the cuticle, without flexible fitting, and similar to a little finger (Fig. 2C and D).

Sensilla Distribution in Each Antennal Segment

Flagellomeres presented a greater variety of sensilla types and subtypes when compared to the scape and pedicel. Also, smaller noninnervated pilosities of microtrichiae type were seen along with all antennal segments (Figs. 1–4). The sensilla distribution is detailed below and in Table 1:

Table 1.

Antennal sensilla of Nyssomyia intermedia females: typology and distribution

Antennal segments Sensillary classification
Types Subtypes
Scape Sensilla trichoidea Short sharp-tipped trichoid
Long blunt-tipped trichoid
Pedicel Sensilla trichoidea  
 Sensilla squamiformia  
 Sensilla campaniformia 		Short sharp-tipped trichoid
Long blunt-tipped trichoid
Flagellum Flagellomere I Sensilla trichoidea  
 Sensilla chaetica  
 Sensilla coeloconica  
 Sensilla squamiformia  
 Sensilla basiconica 		Short sharp-tipped trichoid
Long blunt-tipped trichoid
Short blunt-tipped trichoid
Single-base chaetic
Forked-bae chaetica
palmately lobed grooved coeloconic
Flagellomere II Sensilla trichoidea  
 Sensilla chaetica  
 Sensilla coeloconica 		Long blunt-tipped trichoid
Single-base chaetic
Palmately lobed grooved coeloconic
Flagellomere III to VIII Sensilla trichoidea  
 Sensilla chaetica 		Long blunt-tipped trichoid
Single-base chaetic
Flagellomere IX Sensilla trichoidea  
 Sensilla chaetica 		Long blunt-tipped trichoid
Short prominent-base trichoid
Single-base chaetic
Flagellomere X Sensilla trichoidea  
 Sensilla chaetica 		Long blunt-tipped trichoid
Single-base chaetic
Flagellomere XI Sensilla trichoidea  
 Sensilla chaetica 		Long blunt-tipped trichoid
Short prominent-base trichoid
Single-base chaetic
Flagellomere XII Sensilla trichoidea  
 Sensilla coeloconica 		Long blunt-tipped trichoid
Medium pointed-tipped trichoid
Common grooved coeloconic
Flagellomere XIII Sensilla trichoidea  
 Sensilla coeloconica 		Long blunt-tipped trichoid
Medium pointed-tipped trichoid
Short prominent-base trichoid
Common grooved coeloconic
Flagellomere XIV Sensilla trichoidea  
 Sensilla coeloconica 		Long blunt-tipped trichoid
Medium pointed-tipped trichoid
Short fixed-base trichoid
Short apical trichoid
Common grooved coeloconic
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aObserved in only one specimen.

  • 1. Scape—In this first antennal segment, long blunt-tipped trichoid sensilla were observed distributed in the middle of the segment and short sharp-tipped trichoid sensillum in its basal region.

  • 2. Pedicel—In this second antennal segment, a group of three short sharp-tipped trichoid sensilla also were observed, proximally. Arranged in line in their central region, where were seen long blunt-tipped trichoid, and squamiformia sensilla, while two campaniformia sensilla were found in their distal region.

  • 3. Flagellum:

    • a. Flagellomere I—In this antennal segment, the most significant number and diversity of sensilla were observed: chaetica, coeloconica, squamiformia, basiconica, and trichoidea sensilla (long blunt-tipped, short sharp-tip, and short blunt-tip subtypes). In their basal region were evidenced short sharp-tip trichoid and squamiformia sensilla. This latter was also observed in large numbers in the middle region of this segment. Next to a grouping of three basiconica sensilla was found one short trichoid sensillum, blunt-tip subtype, and at another angle were observed in four sensilla units, but without the presence of basiconica sensilla. In their apical region, several long blunt-tipped trichoid and one or two subtypes of chaetic sensilla were found on the proximal third of the flagellomere: simple-base and forked-base subtypes. This latter subtype was observed in only one specimen among all studied. One palmately lobed grooved coeloconic sensillum was observed at the apex of this segment.

    • b. Flagellomere II—Approximately 20 long blunt-tipped trichoid and two single-base chaetic sensilla were observed along the segment, and one palmately lobed grooved coeloconic sensillum in their apex.

    • c. Flagellomere III—In this segment, we observed the same sensillary cover saw in flagellomere II, except for the palmately lobed grooved coeloconic sensillum that was not found.

    • d. Flagellomeres IV to VIII—approximately twenty long blunt-tipped trichoid and two single-base chaetic sensilla were found along each of these subsegments.

    • e. Flagellomere IX—The sensilla found in this antennal segment were similar to those observed in flagellomeres IV to VIII, however, presenting a short trichoid sensillum, prominent-base subtype in its apical region.

    • f. Flagellomere X—The sensilla found in this antennal segment were similar to those observed in flagellomeres IV to VIII.

    • g. Flagellomere XI—The sensilla found in this antennal segment were identical to those noted in flagellomeres IV to VIII, however, presenting a short trichoid sensillum, prominent-base subtype in its distal region.

    • h. Flagellomere XII—In this antennal subsegment, long blunt-tipped trichoid, medium pointed-tipped trichoid, common grooved coeloconic sensilla were found. One or two smaller simple-base chaetic sensilla were also observed.

    • i. Flagellomere XIII—presented the same sensory typology seen in flagellomere XII, except for the presence of short prominent-base trichoid sensillum near at its apex.

    • j. Flagellomere XIV—The following sensilla were noted: long blunt-tipped trichoid sensilla, mainly in the proximal region, medium pointed-tipped trichoid sensilla distributed throughout most of the segment, except the proximal region, and common grooved coeloconic sensilla distributed in the middle region of the segment.

This segment presents a cylindrical extension in its distal region that exhibits near its base two short fixed-base trichoid sensilla, and at its apex, similar sensilla, designated short apical trichoid sensillum.

Dimension of the Antennal Segments

The Ny. intermedia antennae had an average length of 1.354 ± 0.037 mm. The first flagellomere is the most extensive among all others. The significantly longer length was also seen in the IX–XI flagellomeres of the antennae (Fig. 5A).

Fig. 5.

Fig. 5.

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Number and size of the antennal segments of Nyssomyia intermedia females and of some of their antennal sensilla (P < 0.05). (A) Dimensions of the antennal segments. (B) Dimensions of the single-base chaetic sensilla found on different subsegments of the antenna. (C) The total number of sensilla observed on the antennal segments (D) Number of long blunt-tipped trichoid sensilla found on antennal segments. (E) The number of medium pointed-tipped trichoid sensilla found on the last three antennal subsegments. (F) Number of common grooved coeloconic sensilla located on the last three antennal subsegments.

Chaetica Sensilla Size

Variations were evidenced in the dimension of the chaetica sensilla in all antennal flagellomeres in that this sensillum was present. However, significant variations in the size of this sensillum were not observed in the present study. The length of these sensilla on female Ny. intermedia antennae ranged from 58 to 68.6 µm. It was seen that the dimensions of the chaetica sensilla in flagellomeres from I to XI were very similar and that it decreased significantly from flagellomere XII, with this sensory subtype absent in the last flagellomere (Fig. 5B).

Number of Sensilla

The flagellomeres have a higher number of sensilla when compared with the scape and the pedicel. The flagellomere I present approximately four times more sensilla than the other flagellomeres (Fig. 5C). There was a progressive decrease in the number of long blunt-tipped trichoid sensilla observed from the X to XIV flagellomeres (Fig. 5D). There was no significant difference between the number of medium pointed-tipped trichoid and common grooved coeloconic sensilla found in subsegments XII and XIII, but the number of these sensilla was higher in the last flagellomere (Fig. 5E and F).

Discussion

The shape and number of the antennal subsegments found in the present study, in female Ny. intermedia, coincide with that previously described by Fernandes et al. (2008), in females and males Lu. longipalpis, and by Ilango (2000) in Phlebotomus argentipes (Annandale & Brunetti) (Diptera: Psychodidae).

Sensilla chaetica are commonly known as ‘antennal ascoid’ in the Phlebotomine sand flies and are used to differentiate within and between species (Ilango 2000; Galati 2003, 2018). These sensilla can present themselves, in different species of sand flies, simple or forked, with long or short posterior prolongation. The chaetic sensilla of a single-base subtype are the more common subtype present in antennae of sand flies (Ilango 2000, Zayed et al. 2002, Fernandes et al. 2008) that presents a unique implantation base. However, several species of sand flies belonging to different taxa, such as the genera Brumptomyia (França & Parrot) (Diptera: Psychodidae), Psathyromyia (Barretto) (Diptera: Psychodidae), and some species of Lutzomyia s.s., e.g., show forked ascoids (Galati 2013, 2018).

In a single Ny. intermedia specimen, a bifurcated base chaetic sensilla was observed (Fig. 3D) and classified as a forked-base subtype. Would an individual malformation motivate the rare occurrence of this subtype? Or could it be a remnant character of evolutionary differentiation still present only in a smaller percentage of this population of sand flies? Future studies with different populations of this species and other sand flies will be useful to ratify any of these hypotheses.

Although significant variations in the size of this sensilla were not observed in the present study (Fig. 5B), this result on the length of the sensilla chaetica can be used as a taxonomic character that, when compared with others obtained later, related to different populations and species of sand flies, may favor future taxonomic and phylogenetic studies for better evolutionary elucidation of Diptera Phebotominae.

It was previously described trichoidea (two subtypes), coeloconica, chaetica, basiconica, and auricillica on Phlebotomus argentipes antennae (Ilango 2000). Zayed et al. (2002) observed trichoidea (three subtypes), chaetica, basiconica, campaniformia, squamiformia, ‘Bohm’ and ‘falcate’ sensilla on Phlebotomus bergeroti adult antennae, and the first six of these sensillary types also on Phlebotomus papatasi. Later on, Fernandes et al. (2008) described 11 subtypes of sensilla in Lu. longipalpis adults, including 5 subtypes of trichoidea sensilla; 2 coeloconica sensilla (grooved and ‘praying hands’ subtypes); and campaniformia, chaetica, basiconica, and squamiformia sensilla.

In the present study, all these sensilla were evidenced in the antennae of Ny. intermedia females except the Bohm, falcate, auricillica, and praying hands coeloconic sensilla. In contrast, the forked-base chaetic sensillum, the palmately lobed grooved coeloconic sensillum, and the short fixed-base trichoid sensillum were firstly described in the present work, in the antennae of females Ny. intermedia, as well as a greater diversity of trichoidea sensilla (seven subtypes) were observed in Phlebotominae. It needs to emphasize that Phlebotomus argentipes, Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) and Phlebotomus bergeroti (Parrot) (Diptera: Psychodidae) are species of Old World sand flies, while Ny. intermedia is present in the New World. Therefore, they are species continentally separated for millions of years. Lutzomyia longipalpis, although it is also a New World species, differs from Ny. intermedia in some aspects as distribution, frequented environments, hematophagous behavior, degree of anthropophilic, species used as reservoirs and or hosts, and even in the vectored Leishmania species and, consequently, in the clinical types of transmitted Leishmaniasis. It is possible that such sensilla differences observed between the sand fly may have been motivated by the evolutionary differentiation that commonly occurs between individuals of the same group that are originated from a common ancestor, but which change behaviors and then, consequently, also the morphology and typology, since they are subjected to different environments (ecosystems), climate, shelters, altitude, ecological relationships, food sources (plant and animal hosts), predators, etc.

However, according to our knowledge, sensillum quantification in sand flies was performed only in this study and, previously, by Fernandes et al. (2008) in Lu. longipalpis. Further studies on the distribution and density of sensilla types and subtypes in sand fly vectors should be done to establish better taxonomic comparative parameters between different species and populations with different ecological behaviors and relationships.

The grooved coeloconic sensilla observed in the present study, previously commonly known as ‘papillae’, has been used as a character for species identification by light microscopy (Galati 2003). Unlike the evidenced in Lu. longipalpis, on the antennae of Ny. intermedia, the praying hands coeloconic sensillum was not observed. Possibly the new type of grooved coeloconic sensilla observed in the antennae of Ny. intermedia in this study, the palmately lobed grooved coeloconic sensillum, is equivalent to the praying hands coeloconic sensillum, found in Lu. longipalpis antennae (Fernandes et al. 2008), probably originated by the evolution and taxonomic differentiation of these species and their group. This hypothesis is based on the moderate ultrastructural similarity seen between the two sensory subtypes as well as their similar distribution (both present in the flagellomeres I and II of their own sand fly species) and the nonoccurrence of the first subtype in Ny. intermedia, and the nonoccurrence of the second subtype in Lu. longipalpis. All these observed differences in the sensilla, their typology, quantification, as well as their distribution can also be used as characters in Phlebotomine taxonomic and phylogenetic studies.

Although advanced electrophysiological studies are needed to determine chemicals molecules that interact with each chemoreceptor sensilla, some sensilla types have very particular and evident ultrastructural characters that allow a general classification (functional morphology). These characters were thoroughly studied and classified (Keil and Steinbrecht 1984; McIver 1982; Zacharuk 1985, Hallberg and Hansson 1999; Steinbrecht 1998, 1999; Shanbhag et al. 1999).

The morpho-functional classification determines as gustatory the sensilla with a single apical pore (uniporous sensilla), as olfactory the sensilla with a wall that presents multiporous or longitudinal grooves and, as mechanoreceptor the sensilla with a wall without pores (NP-sensilla or aporous sensilla). Therefore, the general function of the most common types of sensilla is deduced from its microstructure (Zhang et al. 2016). However, other more complex functions can only be identified with behavioral and electrophysiological studies. For example, to confirm that a sensillum acts as an extraocular photoreceptor, or as a sensor for infrared or ultraviolet radiation. Nevertheless, even in these more complex functional diagnosis cases, the previous ultrastructural analysis by SEM of the surface of the main body parts of the insect with sense organs, such as antennae, maxillary palps, labella, and tarsi, is fundamental to foment the location of probable sensilla with these essential sensory functions and, consequently, in the precise targeting of the electrodes, odor pulses, or emitters of electromagnetic radiation, during the accomplishment of theses assays among others advanced electrophysiological studies as SSR (De Bruyne et al. 1999, Bray et al. 2010, Logan et al. 2010, Li et al. 2018).

The sensillary subtypes of grooved coeloconic sensilla observed in this work have evident ultrastructural characters such as a grooved inner cone that was previously described as a type of double-walled sensillum (DW-sensillum) presenting slit-like pores with radial-channel (spoke channels). Usually, this short type of sensillum has longitudinal grooves (most evident from the middle to the distal region of the sensilla) on its outer cuticular wall. Through transmission electron microscopy (Hunger and Steinbrecht 1998, Hallberg and Hansson 1999, Steinbrecht 1999), it is observed that each longitudinal groove consists of the junction of partially fused cuticular fingers. These finger-like structures leave open slits that penetrate the cuticle, allowing access to molecules of sex pheromones, host-associated odors, and other types of volatile involved in communication and chemical ecology. From these slits, hollow cuticular spoke channels connect the grooves of the external wall to the internal cylindrical wall of the sensilla, allowing the lymph of the innervated lumen to reach the bottom of the grooves. Thus, it can be said that spoke channels act as wall pores in these double-walled sensilla. In addition to olfactory function, it was observed that this sensillum type also acts on the thermal perception in some insect species—bimodal sensilla (Hunger and Steinbrecht 1998, Steinbrecht 1999). The finding here of these olfactory sensilla and in Lu. longipalpis (Fernandes et al. 2008) that has protective covering in its structure that sometimes is covering the grooved internal cone or sometimes open, exposing this cone suggests movement dynamic in living sand flies. It is possible that this sensilla protective structure occurs, controlling the exposure of the sensilla to odor stimuli avoiding excessive saturation of the odorant-binding proteins or the neuron receptors of the sensilla, by odorant molecules (Steinbrecht 1998). The opening/closing mechanism for pore protection on sensillum types has previously been described in other insects based on SEM images (Van der Wolk 1978). Also, studies have associated the selective directional movement of sensilla, or structures attached to the sensilla, to the quantitative control of the reception of odor stimuli (Wang et al. 2018) and the depolarization and generation of action potentials for signal transmission (Smith 2009).

At least six different sensilla found in this study are olfactory sensilla considering the morpho-functional classification. Of these, four distinct ones were seen with multiporous single walls (of wall-pore type)—basiconic, short blunt-tipped trichoid (Fig. 4A), and the chaetic sensilla, single-base, and forked-base subtypes) (Fig. 5A, C, and D)—and two that showed double-walled with longitudinal grooves—the palmately lobed grooved coeloconic and the common grooved coeloconic sensilla (Figs. 3D and E and 4B–D). Besides these well-classified sensilla, other ones seen in this study need further investigation to have possible chemosensory functions. Although they are similar to the chemoreceptors sensilla described in other species, was not noted the presence of wall-pores. It is possible that additional analysis with a field emission scanning electron microscope that provides higher resolution images can clarify the existence or not of wall pores.

The short sharp-tipped trichoid, and short prominent-base trichoid sensilla, are similar to the chemoreceptors trichoid sensilla previously observed in other Diptera, such as Dermatobia hominis (Linnaeus Jr.) (Oestridae: Cuterebrinae), Haematobia irritans (Linnaeus) (Diptera: Muscidae), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae), Phormia regina (Meigen, 1826) (Diptera: Calliphoridae), and Lu. longipalpis sand fly (Van der Wolk 1978; Fernandes et al. 2002, 2004, 2005, 2008, 2020). Also, it is possible that sharp-tipped trichoid sensilla and campaniform sensilla may be mechanoreceptors associated with proprioceptions. The former one was observed very close to the junctions between the head and scape or between the scape and pedicel, while the second one was seen near the articulation of the pedicel with the flagellomere I, similar to the previous observation in Lu. longipalpis (Fernandes et al. 2008). Sensilla similar to these were also found in articulations as well as in the scape and pedicel of mosquitoes and other insects (Zacharuk 1985). They play an essential role in detecting antenna movements to relocate their antennal segments during flight, for better exposure of the olfactory sensilla of their antennae, while tracking plumes of attractive odors in the air (McIver 1982). It is possible a similar role of these sensilla in the Ny. intermedia antennae.

The trichoidea sensilla are known as the most common sensory organ of the filo Arthropoda. They can be found even in immature stages of insects, as previously observed in larvae of Ny. whitmani (Antunes & Coutinho) (Diptera: Psychodidae) and Ny. intermedia, e.g. (Bahia et al. 2007). Interestingly, a similar medium pointed-tipped trichoid sensillum found here in Ny. intermedia was previously detected on the adult palpal segment of Lu. longipalpis sand fly and by the silver staining method seen wall pores, suggesting an olfactory function (Spiegel et al. 2005). Also, this sensillary type in Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) has been recognized with olfactory function based on the SSR method since exhibit responses to the pheromone component, the cis-vaccenyl acetate (De Bruyne et al. 1999).

Nevertheless, it has been well known that arthropod sensilla may be associated with more than one sensory function (e.g., bimodal or multimodal sensillum), when associated with different types of sensory neurons or when associated with a neuron genuinely bimodal. The apical trichoid sensilla, due to its strategic position at the tip of the antennae, are probably a tactile (mechanoreceptors) and or contact-chemoreceptor sensilla, with unimodal or bimodal function. This type of sensilla, strategically found at the ends of many insect antenna, is often essential in short-range communication for the recognition of semiochemicals signals, such as sex pheromones, host odors, and harmful substances (Hunger and Steinbrecht 1998, Mitchell et al. 1999, Steinbrecht 1999, Fernandes et al. 2005, 2008).

The squamiformia and the long blunt-tipped trichoid sensilla of Ny. intermedia are similar to the mechanoreceptor sensilla identified in several species of the Insecta class (Mitchel et al. 1999, Merivee et al. 2004, Fernandes et al. 2008). Specifically, about the scale-shaped sensillum observed in this work, the squamiformia type, studies using SEM, and computational fluid dynamics methods (Wang et al. 2018) demonstrated that in the filiform antennae of the male Heliozelidae moth specimens, the scales play a filter-like role. The scales increase the antennae diameter and, consequently, use the aerodynamic forces opposing the airflow. This fact, besides avoiding contamination with environmental microparticles, it creates an area with slow airflow around the antennae; thus, concentrating sex pheromone molecules (nanoparticles) near the olfactory sensilla. The detection of the olfactory signal by the action of the antennal scales increases by about 25–48%. These authors observed that high concentration areas of female sex pheromone molecules that are promoted by the antennal scales are coincident with the olfactory multiporous trichoid sensilla. This function is designated as a ‘helper role in olfaction’. Further studies using SEM coupled to computational fluid dynamics method would be needed to confirm the hypothesis that squamiformia sensilla in sand fly vectors may play a similar role.

Conclusion

The large number and diversity of sensilla observed in the antennae of Ny. intermedia sand fly vector, including typically olfactory types, enforce the importance of these structures in the olfaction, communication, and field-ecological relationships, including with vertebrate hosts. The information about the typology and topography of the Ny. intermedia antennal sensilla may support future electrophysiological studies to isolate and synthesize attractive volatile semiochemicals. This information can be used to develop trap baits for effective vector control measures against leishmaniasis. Moreover, this knowledge can assist future taxonomic and phylogenetic studies to a better understanding of the origin, evolution, and species differentiation of the Diptera: Phlebotominae.

Acknowledgments

The following Brazilian agencies partially funded this study: Foundation of the Institute Oswaldo Cruz (FIOCRUZ); Brazilian Council for Scientific and Technological Development (CNPq/MCTI/CAPES/SCTIE/MS); Coordination of Improvement of Higher Level Personnel (CAPES/MEC); Strategic Programme for Supporting Health Research (PAPES V); National Institutes of Science and Technology—INCT Elimina and INCT Molecular Entomology; Minas Gerais State Research Support Foundation (FAPEMIG) and Amazonas State Research Support (FAPEAM). We thank Professor Dr. José Dilermando Andrade Filho for reviewing the nomenclature and geographical distribution of Nyssomyia intermedia. FFF is postdoctoral researcher at the Laboratory of Medical Entomology (LEM) of FIOCRUZ, MG. NFCS and PFPP are senior research fellows supported by CNPq.

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