JOURNAL OF MORPHOLOGY 228:195-201 (1996)

Sensilla Species

Associated With of Triatominae

the Rostrum of Eight

SILVIA CATALA Catedra de Biologia Celular, Facultad de Ciencias Exactas Fisicas y Naturales, Universidad Nacional de Cdrdoba, Cdrdoba 5000, Argentina

ABSTRACT Scanning electron microscopy (SEMI permits description of 10 types of rostral sensilla of eight species of Triatominae. The chaetica sensilla are the most abundant over all the rostral surface. On the second and third rostral segments, these sensilla are arranged in six columns along the main axis. A distinctive sensory field lies on the third segment very close to the emergence of the stylet. The total number of chaetica sensilla increases significantly on the last segment. The number and distribution of sensilla do not differ between nymphs and adults. The pattern of rostral sensilla of five species-Triatoma sordida, T. platensis, T. protracta, T. infestans, and T. guasayana-were analyzed, and the results were compared by multivariate discriminant analysis. Interspecific variability was found. The method is pro- posed as a taxonomic tool. o 1996 Wiley-Liss, Inc.

Triatomine bugs are widely distributed in Latin America where they are vectors of Try- panosoma cruzi, the etiologic agent of Cha- gas disease or American trypanosomiasis. All triatomine suck blood from vertebrates, mainly birds and mammals. The subfamily comprises 118 species within 14 genera (Schofield, '94). Species of Triatoma, Rhod- nius, and Panstrongylus are the most impor- tant vectors of the parasite, although a large number of species can be infected and thus transmit the disease.

As in other Hemiptera, the rostrum of the triatomine consists of a labium modified to protect a stylet, essential tools for sucking organic humors from animals. A triatomine extends its rostrum straight forward when it is close to a host. This behavior is elicited by stimuli emitted by the host, mainly by those detected by the antennal receptors (Lazzari, '90). The triatomine rostrum has sensilla that can sense the movements of the host and probably odors and temperature (Ber- nard, '74). However, they are used to per- ceive the host during the last phase of their feeding behaviour, just before and during the haematophagic act, when the bug is very close to its prey. A brief description of Tri- atoma infestans rostral sensilla and a more detailed study of some receptors placed on its maxillary stylet were presented by Bernard ('74) and Bernard et al. ('70). No more is

known about these sensory receptors that are strongly involved with hematophagism.

The number and distribution of sensilla form a pattern that might be characteristic of each species and thus very useful in tax- onomy (Ismail and Hammoud, '68; Meineke, '75; Walther, '78) as well as in studies of the specific relationships between insects and their hosts (Chapman, '82; McIver, '82). A recent work about antennal sensilla in the genus Rhodnius (Catala and Schofield, '94) presented data supporting the concept of spe- cies-specific patterns of antennal sensilla. Lent and Wygodzinsky ('79) found that the large mechanoreceptors on the triatomine rostrum differed among species and encour- aged more detailed studies on the distribu- tion of hairs. The aim of the present work is to provide new information on the sensilla types and patterns on the rostra of eight species of triatomine. The rostra of fifth in- star nymphs were also analyzed in order to reveal similarities with those of adults.

MATERIALS AND METHODS

Triatomine bugs were provided by the in- sectary of the Servicio Nacional de Chagas (SNCH, Cbrdoba, Argentina). One-fifth in- star nymph and three females of each of the following species were used: Triatoma sor- dida, T. platensis, T. protracta, T. infestans, and T. guasayana. One-fifth instar and one female of T. delpontei, also from SNCH, one

G 1996 WILEY-LISS, INC.

196 s. CATALA

female of T. matzunoi, and one female of Rhodnius neglectus, from the Natural His- tory Museum (London, UK) were also ana- lyzed.

Araldite was used to attach the three- segmented rostrum of each insect in a verti- cal position to an aluminium SEM stub. The specimen was sputter-coated with three lay- ers of gold-palladium and observed with a Hitachi S-2500 scanning electron micro- scope. Each segment was micrographed in different positions to identify and count the different types of sensilla. The last three spe- cies cited were not included in counts of sensilla.

We used multivariate linear discriminant analysis (Morrison, '76) to check the possibil- ity of using density of sensilla as means to distinguish species. The analysis considered three specimens for each of the five species analyzed. The following variables were evalu- ated from the third segment of each speci- men: the length of the third segment and the densities of sensilla on the dorsal, ventral and lateral faces.

RESULTS Sensilla types

The three rostral segments have hairs of variable distribution and length (Fig. lA,B). The external characteristics shown in scan- ning electron micrographs permitted the iden- tification of 10 types of sensilla. These sen- silla were classified in four groups following the guidelines established by Altner and Prill- inger ('80).

Nonporous sensilla with flexible sockets Chaetica type 1 (Figs. 2B, 3A-D) are the

most frequently found sensilla. Only the first segment lacks these hairs. Bernard ('74) found that they are phasic mechanoreceptors (MP sensilla). The shaft has soft longitudinal grooves and the tip is pointed and faintly curved to the rostrum surface. Lengths vary among species (100-700 ym). The socket is a double rim of cuticle.

Chaetica type 2 (Fig. 2E) are found only on the first segment within a triangular depres- sion that articulates with the labrum. In Triatoma infestans, the shaft is 50 ym long and sited in a well-developed socket, 7.6 pm in diameter. This sensillum was not found in other areas of the rostrum.

Chaetica type 3 (Fig. 2F) are very similar to Bristles I1 found on triatomine antennae (McIver and Siemicki, '84; Catala and

Fig. 1. Scanning electron micrographs of rostrum of two Triatoma species. A. Triatoma platensis. Long chae- tica type 1 sensilla are seen covering the second and third (terminal) segments. ~ 2 5 . B. Triatoma guasuyana. View of first (f), second (s), third (t) segments showing sensilla. x30.

Schofield, '94). Each sensillum has a curved shaft 60-120 pm in length with vertical folds. Near the tip, on the outer face, the hair displays small tubercles. The socket has a double rim, which is higher in the nymphal stage than in adults.

Chaetica type 4 (Fig. 2B) are small, cone- shaped hairs 5 ym in length with a double rim of cuticle at the base. They occur only on the third rostral segment.

Chaetica type 5 (Fig. 2A) is a single, cone- shape sensillum - 25 ym long, placed on the dorsal face of the third rostral segment very close to the articulation with the second seg- ment where the tip is pointed. The cuticle around the base has radial folds. The sensilla probably function as proprioceptors, similar to the tapered hairs of the antennae of Rhod- nius (Catala and Schofield, '94).

Nonporous sensilla in inflexible sockets Pitpegs type 1 (Fig. 2D) are small sensilla 1

ym in length that are inserted in round de-

ROSTRAL SENSILLA OF TRIATOMINAE 197

Fig. 2. Types of sensilla on the rostrum of Triatomi- nae. A. Triatorna rnatzunoi. Chaetica type 5 on the third segment. x 300. B. T. infestans, Distal end of the third segment showing the socket and base of one chaetica type 1 (ql) , several campaniformia (c), one chaetica type 4 (q4) and the orifice of two dermal glands (d) surrounded by glandular secretions, x 1,500. C . T. protracta. Trichoidea sensillum on the third segment. ~2,000. D. Rhodnius

neglectus, Pit peg type 1 on the third segment. ~4 ,000 . E. T. infestans. Sensillum chaetica type 5 on the third segment. x 1,700. F. 2’. guasayana, Chaetica type 3 on the first segment of a fifth stage nymph. x 1,000. G. T. infestans, Pit peg type 2 on the third segment of a fifth stage nymph. ~6,000. H. R. neglectus, Two porous pegs showing pores (p) on the third segment. ~6,000.

pressions no more than 2 pm in diameter in Rhodnius neglectus. They are found fre- quently on the tip of the third rostra1 seg- ment. The morphological features of these receptors suggest that they probably func- tion as thermo-hygroreceptors.

Pit pegs type 2 (Fig. 2G) are also restricted to the tiD of the rostrum. These arise from

Sensilla campaniformia (Fig. 2Bc) are oval plates 6-7 pm in diameter. Each plate is surrounded by a fold with a small transverse groove. These sensilla have central pores that may be ecdysial channels that are not related to their sensory function (Zacharuk, ’85). Sensilla campaniformia are restricted to the distal end of the third segment.

Y

high soikets placed in the middle of round pits. Their dimensions are variable in differ- ent species. In Triatoma infestans, the diam- eter of the pit is - 6 pm, the socket is 1.5 pm high, and the peg is 4.5 pm long. Bernard (’74) referred to this sensillum as “S”, but its function has not been studied.

Uniporous sensilla in flexible sockets Sensilla trichoidea (Fig. 2C) on the ros-

trum of Triatoma protracta are long (20-25 pm) hairs. The curved shaft rests within an irregular socket 2-5 km in diameter, with the distal walls higher than the others. It was

198 s. CATALA

Fig. 3. Views of third rostral segment of Triatominae. A. Triatona guasayana, lateral view x200. B. 2'. protructa, lateral view, ~ 2 0 0 . C. Rhodnius neglectus lateral view. ~130. D. T. delpontei, fifth instar nymph, view of tip of rostrum showing the six columns of chaetica type 1. x 170. E. 2'. guasayana, view of tip of rostrum with uniporous pit pegs (u) and pit pegs type 2 (p), and trichoidea (t). X 1,800.

not possible to verify the presence of the terminal pore, but the characteristics and location of the hairs suggest that they could be uniporous sensilk which are found very frequently on the rostrum of insects.

~n iporous sensilla in inflexible sockets

outer face, suggesting a gustatory function. Sometimes the tip is curved like a hook. Bernard ('74) proposed that the hook-like receptors placed as a crown around the distal end of the third segment be considered as MT (tonic-mechanoreceptors) sensilla.

porousPegs (Fig. 2H) are cone-shape recep- Distribution and number of sensilla on rostral segments tors placed exclusively on the distal end of

the third rostral segment. They have quite variable sizes (2-20 urn) in different sDecies. Table 1 shows the distribution of different The tip of each sensillurn is pointed,-and a pore is clearly visible in the middle of the

sensilla over the three rostral segments of the eight species analyzed.

ROSTRAL SENSILLA OF TRIATOMINAE 199

The first segment shows a small number of sensilla on its ventral face. Over the dorsal face, chaetica type 2 sensilla are restricted mainly to the zone of articulation with the

TABLE 1. Sensilla types on each rostral segment in several Triatominae species

Segment Sensillum ~

First Second Third Chaetica types 1,3

Chaetica types 2 and 3 Chaetica types 1 and 3

and 4 Chaetica type 5 Pit peg types 1 and 2

Campaniformia Trichoidea

Pegs

Probable function

Mechanoreception Mechanoreception Mechanoreception

Proprioception Thermo-hygrorecep-

Proprioception Contact chemorecep-

Contact chemorecep-

tion

tion

tion

. -.

Fig. 4. Triatoma guasayana. Drawing showing distri- bution of sensilla on the tip of the rostrum. u, uniporous pegs; p, pit peg type 2; t , trichoidea.

120

100

P 8 0 E 5 a * v) 2 w v, 40

20

0

labrum, over its edges. Sensilla chaetica types 1 andlor 3 cover the lateral faces of the segment.

The second segment, commonly the long- est, shows chaetica type 1 sensilla, alone or mixed with chaetica type 3. No other sensilla were observed on the first two segments.

The main characteristic of the third seg- ment is the presence of the longest chaetica type 1 and a diversity of sensilla, especially on the distal end of the segment. A distinctive sensory field lies very close to the emergence of the stylet (Fig. 3C). This group consists of pit pegs, campaniformia sensilla, trichoidea sensilla, and porous pegs (Fig. 4). The position, number, and types of sensilla in this area are very similar in all of the species studied. Al- though nothing is known about this senso- rial field, the morphology of the receptors seen by SEM strongly suggests that taste (porous pegs) and thermo-perception (pit pegs) might be the main functions of the field.

Over the entire rostral surface, the chae- tica types 1 and 3 sensilla are the most abun- dant. The second and third rostral segments show these sensilla arranged in six columns along the main axis: two ventral, two lateral, and two dorsal. Each column bears 1-3 longi- tudinal groups of hairs. The ventral face shows the longest chaetica sensilla (Fig. 3A). An analysis of the total number of chaetica sensilla (type 1 and/or 3) showed a signifi- cant increase on the last segment (ANOVA: p < 0.05) (Fig. 5).

A simple comparison between the density of chaetica type 1 on the nymphal and adult

FIRST SECOND THIRD ROSTRAL SEGMENT

Fig. 5 . Sensilla number on the three rostral segments. Values are means for Triatoma sordida, T. guasayana, T. infestans, T . protracta and T. platensis. Standard deviations are shown as vertical bars.

200 S. CATALA

TABLE 2. Density of sensilla chaetica type 1 on the third rostral segment of many species of Triatominae:

Comparison between adult and fifth instar nymph (expressed as number of sensilla by millimeter

of segment)'

Suecies Adult Nymph Triatoma sordida 188 200 Triatoma delpontei 134 136 Triatoma platensis 172 200 Triatoma protracta 320 318 Triatoma infestans 230 232

'Figures are counts on single specimens of nymphs and averages of counts on three specimens for each species of adults.

third segments demonstrated the lack of in- traspecific variation (Table 2). Only differ- ences based on their smaller size could be attributed to this nymphal stage.

Pattern of rostral sensilla as a taxonomic tool

The pattern of rostral sensilla (Table 3) was analyzed to evaluate the possibility of its

use as a taxonomic tool. The five species analyzed were clearly separated by multivar- iate linear discriminant analysis (Morrison, '76) (Fig. 6). Three discriminant functions were derived, of which two were significant and together accounted for 97% of the total variability. The length of the third segment and the density of hairs on the lateral face were the most important variables. These two functions allowed clear discrimination among the five species.

DISCUSSION

The type, distribution, and number of sen- silla establish a pattern. Using the antennal pattern of sensilla, Catala and Schofield ('94) identified six Rhodnius species. The first in- spection of the triatomine rostrum by SEM suggested that the number, distribution, and length of sensilla chaetica 1 and 3 are quite variable among species, as was previously suggested by Lent and Wygodzinsky ('79) and Cobben ('78) (Figs. 1, 3A,B,C). This as-

TABLE 3. Third rostral segment parameters: Length in millimeters and number of total sensilla on different faces in five species o f Triatoma'

Parameter T. protracta T. sordzda T. infestans T. guasayana T. platensis

Length 0.59 0.81 1.16 1.16 1.08 N" lateral 62 40 84 46 46 No dorsal 82 60 90 48 70 No ventral 70 78 112 88 70

'Figures are averages of counts on three specimens for each species.

I

T.protracta

I

I =T.p'aqnsis a m

T.sordlda

I I a

T.guaeayana

-16 -1 1 -6 -1 4 9 14 DISCRIMINANT FUNCTION 1

Graph showing discriminant analysis of the five species of Triatoma. Fig. 6.

ROSTRAL SENSILLA OF TRIATOMINAE 201

sumption was proved by discriminant analy- sis: each species has a different pattern of sensilla on the rostrum.

These results are congruent with findings in others groups of insects. There are varia- tions in feeding strategies in phytophagous hemipterans. They are strongly correlated with taxonomic status and therefore evolved in distinct patterns. The sensory systems that mediate feeding also evolved in patterns. The sensilla used to detect external plant cues to orientation and host acceptance (an- tennal and labial sensilla), are highly vari- able among species (Backus, '88). Similar conclusions were obtained for Acridoidea (Chapman, '88; Chapman and Thomas, '78).

Interspecific, but not intraspecific, variabil- ity can be explained by the role played by the rostrum. The function of the rostrum is basi- cally similar in nymphs and adults of the same species the habitats and hosts of which are identical. However, it can be assumed that different species show different respon- siveness to host movements (Catala, '93b), which might be related to the types and num- bers of sensilla present. When a triatomine bug sucks blood, the rostrum, mainly the last segment with long chaetica type 1, rests upon the host skin anchored by the stylet. Bernard ('74) demonstrated that the long MP hairs (chaetica type 1) are phasic mechanorecep- tors. Their high density, mainly in the last rostral segment, probably contributes to the perception of host movements during feed- ing. Interspecific variability and intraspecific similarity in the numbers of rostral sensilla indicate that numerical analysis of rostral sensilla has potential usefulness as a taxo- nomic tool. In comparison with the anten- nae, however, the rostrum possesses larger mechanoreceptors that are easier to analyze by light microscopy.

ACKNOWLEDGMENTS

This work was carried out at the Medical and Veterinary Entomology Division, Natu- ral History Museum (NHM), London, with the support of a grant given to Dr. David Gorla by the British Council and CONICOR. I acknowledge the assistance of Drs. S. McIver and D. Gorla who read the manuscript, Di- ana Abal for Figure 4 drawing, and the staff of the Electron Microscopy Unit and Medical and Veterinary Entomology Division, for pro- viding facilities in support of this work. This

project received partial financial support from SECYT Universidad Nacional de Cordoba (Argentina).

LITERATURE CITED Altner, H., and L. Prillinger (1980) Ultrastructure of

invertebrate chemo-, thermo-, and hygroreceptors and its functional significance. Int. Rev. Cytol. 67t69-139.

Backus, E.A. (1988) Sensory systems and behaviours which mediate Hemipteran plant-feeding: a taxonomic overview. J. Insect Physiol. 34/3):151-165.

Bernard, J. (1974) Etude electrophysiologique des re- cepteurs impliques dans le orientation vers l'hote et dans l'acte hemophage chez un hemipthre Triatoma infestans, thesis, Universite de Rennes.

Bernard, J., J.M. Pinet, and J . Boistel(1970) Electrophysi- ologie des recepteurs des stylets maxillaires de Tri- atorna infestanst Action de la temperature et la teneur en eau de l'air. J. Insect Physiol. 16t2157-2180.

Catala, S. (1993a) El habitat y las poblaciones de sensilla antenales en Triatominae. Medicina supl. 1,53:75.

Catala, S. (1993b) Transmision vectorial de Trypano- soma cruzi: Define el hospedador su propio riesgo de infeccion? Medicina supl. 1,53:27-28.

Catala, S., and C. Schofield (1994) Antenna1 sensilla of Rhodnius. J . Morphol. 219t193-204.

Chapman, R.F. (1982) Chemoreception: The significance of receptor numbers. Adv. Insect Physiol. 16t247-336.

Chapman, R.F., and J.G. Thomas (1978) The numbers and distribution of sensilla on the mouth parts of Acridoidea. Acrida 7t115-148.

Cobben, R.H. (1978) Evolutionary trends in Heteroptera. Part 11. Mouthpart-structures and feeding strategies. Meded. Landbouwhogesschool Wageningen 78:1407.

Ismail, I.A.H., and E.I. Hammoud (1968) The use of coeloconic sensilla on the female antennae in differenti- ating the members of the Anopheles gambiae Giles complex. Bull WHO 385314-821.

Lazzari, C. (1990) Fisiologia del comportamiento de Tri- atorna infestans (Klug, 1834) (Heteroptera: Reduvi- idae), Orientacion termica, thesis, Univ. Buenos Aires, 161 pp.

Lent, H., and P. Wygodzinsky (1979) Revision of the Triatominae (Hemiptera, Reduviidae) and their signifi- cance as vectors of Chagas' disease. Bull. Am. Mus. Nat. Hist. 163:l-520.

McIver, S.B. (1982) Sensilla of mosquitoes (Diptera: Cu- licidae). J. Med. Entomol. 19t489-535.

McIver, S., and R. Siemicki (1984) Fine structure of antenna1 mechanosensilla of adult Rhodnius prolixus Stal (Hemiptera: Reduviidae). J . Morphol. 18Ot19-28.

Meinecke, C. (1975) Riechsensillen und Systematik der Lamellicornia (Insecta, Coleoptera). Zoomorphologie 82t1-42.

Morrison, D.F. (1976) Multivariate Statistical Methods. Tokyo: McGraw-Hill Kogakusha.

Schofield, C.J. (1994) Triatominae: Biologia y Control. Bognor Regis, UK: Eurocommunica Publications, 1-80 PP.

Walther, J.R. (1978) Vergleichende morphologische Be- trachtung der antennalen Sensillenfelder einiger aus- gewahlter Aculeata (Insecta; Hymenoptera). Z. Zool. Syst. Evolution-forsch. 17:30-56.

Zacharuk, R.Y. (1985) Antennae and sensilla. In: L.I. Gilbert and G.A. Kerkut (eds): Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 6. Oxford: Pergamon Press, pp 1-70.