Fine structure of antennal sensilla coeloconica of culicine mosquitoes

T I S S U E & CELL 1973 5 (1) 105 112 Published by Longman Group Ltd. Printed #t Great Britain

SUSAN B. MclVER

FINE STRUCTURE OF ANTENNAL SENSILLA COELOCONICA OF CULICINE MOSQUITOES ABSTRACT. The sensilla coeloconica (pegs in pits) previously mis-identified as campamform organs, at the tip of the antennae of female Aedes aegypti L. and Culex pipiens (L.) are described. Each sensillum is innervated by three bipolar neurons: the dendrites of two are unbranched whereas the distal portion of the third is folded into tightly packed lamellae. One unbranched dendrite extends to the tip of the peg and the other ends near the base of the peg. The lamellae-bearing dendrite terminates 4 5/z beneath the base of the peg. Chemo- and thermoreception are the proposed functions for the sensillum.

Introduction M o s q u i t o e s have various types of a n t e n n a l a n d palpal sensilla which are responsible for the reception of e n v i r o n m e n t a l stimuli, including those involved in host finding and selection. N u m e r o u s studies with the light microscope have determined the types, n u m b e r s , a n d distribution of these sensilla. Studies on the sensilla o f adult mosquitoes with the electron microscope, however, have been sparse (McIver, 1972a,b; M c l v e r a n d H u t c h i n s o n , 1972; Slifer a n d Sekhon, 1962). A t the tip of the a n t e n n a are structures which were referred to as 'papillae' by Slifer a n d S e k h o n (1962) a n d identified as sensilla c a m p a n i f o r m i a by investigators using the light microscope (lsmail, 1962, 1964; Steward a n d A t w o o d , 1963). T h e w o r k presented herein d e m o n s t r a t e s these structures o n female Aedes aegypti L. a n d Culex pipiens (L.) to be sensilla coeloconica, i.e. pegs in pits. A fine structure description o f these sensilla is given which aids in p r o v i d i n g the m o r p h o l o g i c a l basis for u n d e r s t a n d i n g sensillar function a n d eventually m o s q u i t o behavior.

Department of Parasitology, School of Hygiene, University of Toronto, Toronto 181, Ontario, Canada. Received 30 October 1972.

Materials and Methods A n t e n n a e f r o m specimens reared in l a b o r a tory colonies were r e m o v e d while the m o s q u i t o was i m m e r s e d in K a r n o v s k y ' s fixative ( K a r n o v s k y , 1965) at p H 7 a n d 4°C a n d left in fixative for 6-18 hr. T h e tissue was washed o v e r n i g h t in 0.05 M Sorensen's 10 9o sucrose buffer, p H 7, a n d post fixed in I °,"o OsO4 in veronal acetate buffer, at p H 5 a n d at r o o m t e m p e r a t u r e for 2 hr. D e h y d r a t i o n t h r o u g h e t h a n o l was followed by e m b e d d i n g in S p u r r ' s low viscosity epoxy m e d i u m (Spurr, 1969). Sections were cut using a d i a m o n d knife o n a P o r t e r Blum ultramicrotome, stained in uranyl acetate a n d lead citrate, a n d e x a m i n e d in a Zeiss E M 9 electron microscope. F o r s c a n n i n g electron microscopy specimens were fixed in 5 % formalin a n d deh y d r a t e d t h r o u g h a graded series of e t h a n o l s to xylene (Slifer, 1972), attached to the stubs with silver conductive paint, coated with gold d u r i n g spin rotation, a n d e x a m i n e d in a C a m b r i d g e Stereoscan Microscope. P e r m e a b l e areas in the cuticle were d e m o n s t r a t e d by staining whole specimens with crystal violet (Slifer, 1960). Results T h e a n t e n n a e of a n o p h e l i n e a n d culicine mosquitoes consist o f 13 flagellar segments 105

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set into a cup-shaped pedicel and attached to the head by the scape. The flagellar segments are similar, although some differences in

Fig. 1. Diagram of tip of antenna of female Aedes aegypti with cut-away view of one sensillum coeloconicum showing arrangement of the neurons. A, neuron which extends into peg (p); B, neuron which terminates near base of peg; C, neuron with lamellated outer segment of dendrite; cs, cuticular sheath : dc, dark inner component ofcuticular sheath which extends below base of sheath; cr, ciliary region; m, mitochondria; n, nucleus of neuron; SC, sensilla chaetica ; I, approximate level of Fig. 3 ; 2, approximate level of Fig. 4; 3, approximate level ot" Fig. 5: 4, approximate level of Fig. 8.

segment size and m n n b e r of sensilla occur. The m o s t distal segment, number 13, is distinctive, however, in that it tapers to a tip which is free of trichoid and basiconic sensilla (Fig. 1). At the tip of segment 13 are located sensilla coeloconica: two in A. aegypti and three in C. pipiens. The following description o f these sensilla pertains to b o t h species of mosquitoes, unless otherwise stated. Externally each sensillum appears as a peg located within a pit (Figs. 1, 2), the walls o f which are scalloped (Fig, 3). The sensillum is permeable to crystal violet stain and as perforations in the lateral wails o f the pegs were not observed in electron micrographs (Fig. 3), communication with the exterior must be restricted to one, or possibly a few, pores at the tip o f the peg. Three bipolar neurons innervate each sensillum. The cell bodies, axons, and proximal portion (inner segment) of the three dendrites are similar to one another, but distinctive features in location or m o r phology occur distally to the ciliary region (outer segment). Two n e u r o n s (A a n d B, Fig. 1) have undivided dendrites. The dendrite o f neuron A extends into the lumen of the peg (Fig. 3), whereas that o f n e u r o n B terminates near the base o f the peg. Fig. 4 shows the two u n b r a n c h e d dendrites in the region below the peg where the cuticular sheath is present. N e u r o n C t e r m i n a t e s 4 5/x below the base o f the peg near the proximal end o f the cuticular s h e a t h (Fig. 1). The outer segment o f the d e n d r i t e is divided into n u m e r o u s closely packed lamellae between which conspicuous septate d e s m o s o m e s occur

Fig. 2. Scanning electron micrograph of tip of antenna of A. aeg)7)ti with two sensilla coeloconica (arrows). >, 12,000. Fig. 3. A cross section of peg in pit (level I, Fig. 1). Only one neuron is present in peg. Note neurotubules (nt) within neuron and scalloped walls of pit. A. aegypti. × 66,500. Fig. 4. A cross section below base of peg (level 2, Fig. 11 in which two dendrites with neurotubules (nt), a cuticular sheath (cs) with a dark inner component (dc), and a crystalline structure (q), presumably a honeycomb desmosome, are present. A. aegypti x 106,000.

pr ~ ~. ~,~!~i ~!I~~,

ANTENNAL SENSILLA COELOCONICA

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(Fig. 5). N e u r o t u b u l e s , which average 200/~ in d i a m e t e r in A. aegypti, are p r o m i n e n t in the outer segment of all three dendrites (Figs. 3, 4, 5). A few neurofilaments were also observed. T h e distal p o r t i o n of dendrites A a n d B is encased in a cuticular sheath (Fig. 4) which in A . aeL:ypti averages 0.12 ** in width and 3"6 ~, in length. O n the inner surface of the cuticular s h e a t h is a d a r k (more electrondense) c o m p o n e n t (Figs. 1, 4) which extends proxirnally from the base o f the sheath and s u r r o u n d s the distal part o f dendrite C, as well as the adjacent portions of dendrites A a n d B (Fig. 1). In A. a e g y p t i the dark layer averages 5 0 0 / ~ in width in the region proximal to the base of the cuticular sheath. Each dendrite possesses a basal body with the usual 9 + 0 a r r a n g e m e n t of double tubules. Fig. 6 shows fibrils radiating laterally from the doublets to the peripheral p l a s m a m e m b r a n e . E x t e n d i n g proximally f r o m the basal body are b a n d e d ciliary rootlets which in A . a e g y p t i have a period length o f 7 1 5 ~ (Fig. 7). Periodicity is similar in the three n e u r o n s and in each, remains c o n s t a n t along the rootlet. A l t h a u g h differences in d i a m e t e r occur, the inner segment of the three dendrites have a similar structure. A t this level the dendrites are in close association with one a n o t h e r a n d are most likely connected by d e s m o s o m e s (Fig. 8). In some regions along the cell m e m b r a n e , including those adjacent to a n o t h e r dendrite, there is a concentration of granules or filaments cut in cross section

(Fig. 8). T h e inner scgmcnt of the dendrites c o n t a i n s n u m e r o u s m i t o c h o n d r i a , indicating t h a t it is a site of high metabolic activity. T h e three cell bodies are located a b o u t 20 tt from the distal end of flagellar segment 13 a n d near a ring of sensilla chaetica located at the base o f the a n t e n n a l tip (Fig. 1). T h e nuclei o f the n e u r o n s are s o m e w h a t irregular in outline, contain large peripheral clumps of c h r o m a t i n with smaller clumps m o r e centrally situated, a n d average 3 ×4"25 t~ in d i a m e t e r in A. aegypti. Each cell b o d y gives rise to one axon, thus f o r m i n g the beginning of the a n t e n n a l nerve. Associated with each sensillum are two sheath cells, the cell bodies of w h i c h lie slightly distal to those of neurons. T h e inner s h e a t h cell or trichogen cell, is w r a p p e d a r o u n d the dendrites f r o m their origin to the p r o x i m a l end of the cuticular sheath. A n o u t e r cell, the t o r m o g e n cell, s u r r o u n d s the inner one a n d extends distally a r o u n d the cuticular sheath and occupies the space between the sheath a n d the segment wall. Distal to the cell bodies b o t h sheath cells are p r o d u c e d into n u m e r o u s tightly p a c k e d lamellae which contain m a n y m i t o c h o n d r i a . These m i t o c h o n d r i a are consistently larger t h a n the ones f o u n d in the i n n e r segment of the dendrites (Fig. 8).

Discussion T h e w o r k presented herein clearly shows t h a t the terminal receptors o n the a n t e n n a e of

Fig. 5. A cross section at level 3 (Fig. 1) showing the two unbranched dendrites and the third dendrite which is divided into lamellae containing neurotubules (nt) and connected by septate desmosomes (sd). A. aegypti. / 85,500. Fig. 6. A section cut slightly above basal body with the typical doublet arrangement of tbe 9 peripheral tubules. Note fibrils (arrow) extending from tubules to plasma membrane. A. aegypti. × 71,250. Fig. 7. A section of ciliary region of dendrite showing basal body (bb) and banded rootlets (r). Mitochondria (m) and neurotubules (nt) are visible in surrounding cytoplasm. A. aegypti. "<36,400. Fig. 8. A cross section through inner segment (level 4, Fig. l) of the dendrites. Mitochondria (m) desmosomes (dJ, neurotubules (nt), and granules or filaments cut in cross section (arrows) along the plasma membrane are visible. Note differepce in size between the mitochondria in the dendrites (m) and those in the surrounding trichogen cell (ram). A. aegypti. >'.36,400.

110 female A. ae,gypti and C. pipiens are sensilla coeloconica and provides information pertinent to identifying their function. Ismail (1962), using the light microscope, referred to the structures at the tip of the antenna of Anopheles maculipemlis atroparvus (v. Thiel) as sensilla campaniformia, as did Steward and Atwood (1963) working with A. aegypti. In subsequent light microscope studies on both anophelines and culicines, usage of the term was continued (Ismail, 1964; McIver, 1969, 1970, 1971 ; Mclver and Hudson, 1972). Relatively little is known about the function of sensilla coeloconica. Schneider and Steinbrecht (19681) state that neurons associated with sensilla coeloconica on various insects have been demonstrated electrophysiologically to respond to carbon dioxide, odors, and temperature and/or humidity. Although definitive proof of function awaits electrophysiological studies, some reasonable speculation can be made about the function of the sensilla coeloconica on the antennae of A. aegypti and C. pipiens. Neuron A with the unbranched dendrite that extends into the peg is most likely a chemoreceptor. The structure of the peg with its terminal pore(s) is similar to that known for contact chemoreceptors: a function difficult to envisage for a receptor sunken in a pit unless the substrate sampled is liquid. Deinocerties cancer Theobald males, which repeatedly touch the surface of the water with their antennae in quest of female pupae, is an example of a situation in which the terminal receptors could function as contact chemoreceptors. The tips of the antennae of male D. cancer are enlarged (Provost and Haegar, 1967), but whether or not they bear sensilla coeloconica remains to be determined. In my observations of female A. aegypti feeding on blood and a sugar solution the tips of the antennae do not come in contact with the substrate indicating that the terminal sensilla coeloconica do not function as contact chemoreceptors during feeding. Dethier (1972) has shown that structures known to be contact chemoreceptors may respond to compounds in the gaseous state as well as the aqueous state. Perhaps neuron A possesses such a dual sensitivity. Quite interestingly the sensilla coeloconica have two neurons (B & C) the dendrites of which do not contact the exterior and do not have the characteristics of mechanoreceptors.

MclVER The dendrite of neuron B terminates near the base of the peg and may possibly receive molecules which enter the peg and pass between the dendritic membrane of neuron A and the peg wall. Neuron C terminates in tightly packed lamellae, 4-5 t~ from the base of the peg. Assuming this neuron to be functional, it must be sensitive to a factor which does not require direct contact with the exterior. Heat seems the most likely stimulus to effect a neuron surrounded by several microns of tissue. Evidence from behavioral and morphological studies exists which supports the proposed heat reception function of neuron C. Roth (1951) reported from behavioral studies that the antennae of female A. aegypti function as directional distance thermoreceptors. Ismail (1962) found that removal of the terminal segment of the antennae of female Anopheles maculipennLs" atroparvus altered the mosquitoes' response to heat. On the basis of these results lsmail (1962) suggested that the terminal sensilla coeloconica, which he thought to be campaniform organs and consequently mechanoreceptors, were also sensitive to heat. On the antennae of cave dwelling beetle larvae Corbibre-Tichan6 (1971) has described a hair-like sensillum which is innervated by three neurons: two enter the hair and one terminates at the base in a lamellar arrangement strikingly similar to that of neuron C. (Compare Fig. 5 with Figs. 12a, b and 13a, b in Corbi6re-Tichan6 and Bermond, 1972.) Cave dwelling species have a more extensive development of the lamellae-bearing neuron than surface species, which suggests a specific function in this environment (Corbi6re-Tichan6 and Bermond, 1972). As the fine structure of the lamellae is closely related to that of photosensory cells, a similar function in the perception of infrared radiation is postulated for the neuron (Corbi~re-Tichan6, 1971; Corbi~reTichan~ and Bermond, 1972). Based on similarity in morphology, neuron C in A. aegypti and C. pipiens may also have such a function. A crystalline structure of questionable identity is visible in Fig. 4. The structure is composed of units with an electron-dense center, 100 A in diameter which is surrounded by an electron-lucid layer, 30 A in width.

ANTENNAL

SENSILLA

COELOCONICA

E x c e p t f o r t h e reversal o f e l e c t r o n densities, p e r h a p s a t t r i b u t a b l e to p e c u l i a r i t i e s o f t h e staining technique, the structures resemble septate desmosomes cut tangentially (Locke, 1965) a n d s h o w t h e h o n e y c o m b p a t t e r n for w h i c h D a n i l o v a et al. (1960) h a s r e c o m m e n d e d t h e t e r m ' c o m b d e s m o s o m e ' to distinguish these types of desmosomes p e c u l i a r to insects, f r o m t h o s e f o u n d in other invertebrates. The possibility of the

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s t r u c t u r e b e i n g o f viral o r i g i n is d i m i n i s h e d b y t h e s m a l l size o f its u n i t s ( W i l d y , 1971).

Acknowledgements T h e a u t h o r t h a n k s M i s s S. A . H u t c h i n s o n f o r able technical assistance. This investigation was supported by Medical Research Council Grant number M A 2909 a n d D e f e n s e R e s e a r c h B o a r d Grant number 6801-50.

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MCIVER, S. B. 1972a. Fine structure of pegs on the palps of female culicine mosquitoes. Can. J. Zool., 50, 571-576. MCIVER, S. B. 1972b. Fine structure of the sensilla chaetica on the antennae of Aedes aegypti (Diptera : Culicidae). Ann. ent. Soc. Am. In press. MCIVER, S. and HUDSON, A. 1972. Sensilla on the antennae and palps of setected Wyeomyia mosquitoes. J. Med. Entomol., 9, 337-345. MclVER, S. B. and HUTCHINSON, S. A. 1972. Coeloconic sensilla on the antennae of the yellow fever mosquito, Aedes aegypti (L.). Experientia, 28, 323. PRovosT, M. W. and HAE~ER, J. S. 1967. Mating and pupal attendance in Deinocerites cancer and comparisons with Opifexjilscus (Diptera : Culicidae). Ann. ent. Soc. Am., 60, 565 574. ROTIJ, L. M. 1951. Loci of sensory end-organs used by mosquitoes (Aed,,s aegypti (L.) and Anopheles quadrimaeulatus Say) in receiving host stimuli. Ann. ent. Soe. Am., 44, 59-74. SCHNEIDER, D. and STETNBRECHT, R. A. 1968. Check list of insect olfactory sensilla. In Invertebrate receptors. Syrup. zooL Soc. Lond., 23, 279-297. SL1FER, E. H. 1960. A rapid and sensitive method for identifying permeable areas in the body wall of insects. Entomol. News, 71, 179 182. SL~FER, E. H. 1972. Pores in the thin-walled chemoreceptors of the grasshopper. Acrida, 1, 1-5. SUFER, E. H. and SEKHON, S. S. 1962. The fine structure of the sense organs on the antennal flagellum of the yellow-fever mosquito Aedes aegypti (Linnaeus). & Morph., 111, 49 67. SPUJ~R, A. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct, Res., 26, 31-43. STEWARD, C. C. and ATWOOD, C. E. 1963. The sensory organs of mosquito antennae. Can. J. Zool., 41, 577-594. W~LDY, P. 1971. Classification and nomenclature of viruses. Monographs in virology, 5 (ed. J. L. Melnick), 81 pp. S. Karger, New York.