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Fine structure of surface and sunken grooved pegs on the antenna of female Anopheles stephensi (Diptera: Culicidae) K. S. Boo AND S. B. MCIVER Department of Microbiology and Parasitology, FitzGerald Building, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1Al Received June 16,1975 Boo, K. S., and S. B. MCIVER.1976. Fine structure of surface and sunken grooved pegs on the antenna of female Anopheles stephensi (Diptera: Culicidae). Can. J. Zool. 54: 235-244. The antenna of female Anopheles stephensi Liston bears three types of sensilla with grooved pegs: those sunken in pits and subtypes A and B of those located on the flagellar surface. The sunken peg sensilla are innervated by four or five neurons with branching dendrites. The dendrites are exposed to the exterior by means of longitudinal clefts at the bases of the grooves in the peg wall. Surrounding the dendrites and extending into the clefts is an extracellular material of medium electron density. Three sheath cells are associated with each sunken peg sensillum. Subtype-A surface peg sensilla are generally similar to the sunken peg sensilla, except that they are located on the antennal surface and are innervated by two neurons with unbranched dendrites. Subtype-B surface peg sensilla have three or four neurons, the dendrites of which do not branch and are exposed less to the exterior than those in the other peg sensilla because the clefts in the peg wall are smaller and less frequent. Only trace amounts of electron-dense material occur in the clefts of the subtype-B surface peg sensilla. The sunken peg and both subtypes of the surface peg sensillaare probably olfactory receptors. Boo, K. S., et S. B. MCIVER.1976. Fine structure of surface and sunken grooved pegs on the antenna of female Anopheles stephensi (Diptera: Culicidae). Can. J. Zool. 54: 235-244. Chez Anopheles stephensi Liston, il y a trois types de sensilles a batonnets cannelts: certains sont enfonces dans une cavite et d'autres, de deux sous-types, A et B, sont localises a la surface du flagelle. Les sensilles enfonces sont innerves par quatre ou cinq neurones portant des dendrites ramifites. Les dendrites sont en contact avec I'exterieur par le truchement de fissures longitudinales situies a la base des cannelures de la paroi du bkonnet. Tout autour des dendrites et s'ttendant jusque dans les fissures, on observe une substance extracellulaire d'opacitt moyenne au microscope electronique. Chaque sensille enfonce s'associe trois cellules enveloppes. Les sensilles a biitonnets de surface, du sous-type A, sont, de faqon generale, semblables aux sensilles enfoncts, sauf pour ce qui est de leur position a la surface de I'antenne et de leur innervation par deux neurones a dendrites simples. Les sensilles de surface du sous-type B ont trois ou quatre neurones dont les dendrites ne sont pas ramifiees et sont moins expostes a I'exterieur que celles des autres types, car les fissures de la paroi du bltonnet sont plus petites et plus rares. On ne trouve que des traces de substance extracellulaire opaque au microscope electronique dans les fissures des sensilles de surface du sous-type B. Les sensilles enfoncts et les deux types de sensilles de surface servent probablement de recepteurs olfactifs. [Traduit par le journal]
Introduction In mosquitoes many external stimuli which elicit behavioral patterns are detected by various types of antennal sensilla. These sensilla occur as three general morphological types : bristles (sensilla chaetica), hairs (sensilla trichodea), and pegs. Several types of peg sensilla may be distinguished on the basis of location and structure of the peg wall. Mosquitoes in both subfamilies Anophelinae and Culicinae have (a) smoothwalled pegs in inconspicuous pits (sensilla coeloconica) at the tip of the antenna and at infrequent
intervals along the flagellum (McIver and Hutchinson 1972; McIver 1973; Boo and McIver 1975), (b) smooth-walled pegs situated in a chamber at the end of a long tube (sensilla ampullacea) on the basal flagellar segments (Boo and McIver 1975), and (c) grooved walled pegs located on the flagellar surface. The latter type has been called sensillum basiconicum (e.g., Ismail 1964; McIver 1970), type A3 seta (Steward and Atwood 1963), and thorn-shaped hairs (Slifer and Sekhon 1962). The fine structure of the grooved surface pegs on female Aedes aegypti
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has been described by McIver (1974). Occurring only on the antennae of anophelines are grooved walled pegs situated in conspicuous pits (sensilla coeloconica) (Ismail 1962, 1964; Steward and Atwood 1963). This work was conducted to describe the fine structure of the grooved pegs which occur on the flagellar surface and in conspicuous pits on the antennae of female Anopheles stephensi Liston (Diptera: Culicidae). The results aid in providing the morphological basis for understanding sensillar function and eventually mosquito behavior.
Materials and Methods Antennae of specimens obtained from a laboratory colony were fixed in either Karnovsky's fixative (Karnovsky 1965) or in 3% glutaraldehyde, postfixed in 1% OsO,, block-stained with 0.5% uranyl acetate, dehydrated through a graded series of ethanols, and embedded in Spurr's low-viscosity epoxy medium (Spurr 1969). For details see Boo and Mcher (1975). Flagellar segments 1-6, and 12 and 13 were serially sectioned using a diamond knife. Specimens for scanning electron microscopy were fixed in 5% formalin for several days, dehydrated through a graded series of ethanols, and placed in a series of 100% ethanol and isoamvl acetate mixtures with increasing concentrations of the latter to 100%. After critical-point drying from CO,, specimens were coated with goldpalladium during spin rotation
Results The antenna of female Anopheles stephensi consists of 13 flagellar segments set into a cupshaped pedicel and attached to the head by a ring-shaped scape. Ismail (1964) reported that the sunken pegs (Fig. 2) occur on segments 1-7
and the surface pegs (Figs. 2 and 3) on all flagellar segments, except segment 1 (the most basal one). In one antenna of the six we examined using transmission electron microscopy, one surface,peg occurred on segment 1. In thin sections we observed the surface pegs to be present on all aspects of the segments on which they occur, whereas the sunken pegs are restricted to the lateral side of the distal half of the segments on which they are located. Surface pegs are more prevalent, with an average of 107 per antenna, than sunken ones, with 31 per antenna (Ismail 1964). The external surface of the peg of both the surface and sunken peg sensilla appeared similar, with deep grooves in the wall beginning slightly above the base and terminating just proximal to the tip. Internally, however, the sensilla were distinctly different. In addition, differences were found in the internal features of the surface peg, which led us to conclude that they occur as two subtypes, henceforth designated as subtypes A and B. Table 1 gives a summary of the characteristics of each type of peg sensillum.
Sunken Pegs Each peg arises from the center of the floor of a pit which narrows at the orifice (Figs. 1, 2, 4). Externally only the tip of the peg is visible (Fig. 2). The pegs average 4-5 p (microns) in length and 1.6 p in diameter in the middle region. From 13 to 16 grooves occur in the peg wall. Each sensillum is innervated by four or five neurons, the dendrites of which divide within the peg lumen to form 7-1 3 branches and terminate
TABLE 1. Characteristics of sunken and surface pegs on the antenna of female Anopheles stephensi
Sensillar type Surface pegs Characteristic Location on flagellar segments Peg length, p No. grooves No. neurons Dendritic branching Electron-dense material in grooves Degree of exposure of dendrites to exterior
Sunken pegs
Subtype A
Subtype B
Lateral side of distal half 4-5 13-16 4-5 Yes
All aspects
All aspects
Yes
Yes
Traces
Large
Large
Small
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FIG.1. Diagram of sunken peg sensillum with two of the four or five neurons shown. bb, basal bodies with ciliary rootlets; c, cuticle; cs, cuticular sheath; ds, desmosomes; is, inner segment of dendrite; m, mitochondria; n, nucleus; nl, neurilemma cell; nr, neuron; os, outer segment of dendrite; pt, pit; re, receptacle cavity; rlc, receptor lymph cavity; sp, sunken peg; to, tormogen cell; and tr, trichogen cell.
slightly proximal to the peg tip. Each dendrite is divided into an inner and an outer segment (Figs. 1 and 6) by a region with a 9 x 12 0 configuration of microtubules. Distal to this region additional microtubules appear (Fig. 7). The outer segment is encased within a cuticular sheath, 7-8 p in length. The only inclusions observable within the outer segment are microtubules, whereas the inner segment contains prominent mitochondria, vesicles, microtubules, two basal bodies displaying the 9 x 3 + 0 configuration of microtubules (Fig. 8), and periodically banded rootlets (Figs. 1, 6, and 9).
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The cell body of each neuron contains an oblong nucleus with scattered clumps of dark chromatin and a prominent nucleolus with associated chromatin (Fig. 9), and the usual organelles, viz., mitochondria, Golgi bodies, endoplasmic reticulum, microtubules, and ribosomes. Extending proximally from the cell body is an axon which joins one of the two flagellar nerve bundles. Along the length of the peg the relationship between the peg wall and the cuticular sheath changes. Basally, where the grooves in the wall are absent or just beginning, the cuticular sheath
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and peg wall are separated by an extension of the receptor lymph cavity, an extracellular space containing microtubules in a finely granular matrix (Fig. 10). Distally the wall invaginates and the sheath evaginates to form separate columns, each enclosing a sinus which is continuous with the receptor lymph cavity (Figs. 10 and 11). Longitudinal clefts in the peg wall occur at the cuticle-free bases of the grooves. An extracellular material of medium electron density, presumably the residue of a liquid in the living state, is present in the clefts (Figs. 10 and 11). Basally the ridges (outer aspect of the columns) are round (Fig. 10). Distally the ridges become progressively more pointed until just proximal to the peg tip, where they appear as V-shaped projections (Figs. 11 and 12). At the peg tip the ridges disappear, leaving the peg wall smooth (Fig. 13).
Near the peg tip where the dendrites terminate, the columns merge to re-form a continuous cuticular sheath and peg wall, both without perforations. This separation of the wall and the sheath results in the sinuses becoming confluent in one extracellular space, which is still continuous with the receptor lymph cavity below. Just proximal to the tip the cuticular sheath fuses to one side of the peg wall (Fig. 12). Beyond this point the peg contains only a small extracellular space (Fig. 13). Three sheath cells are associated with each sensillum. The neurilemma cell, which is the most proximal, distally encloses the extracellular receptacle cavity (Fig. 1) and surrounds the neurons from just distal to the ciliary regions, to at least the distal part of the axons. A trichogen cell encases the distal portion of the neurilemma cell and the dendrites from proximal to
FIGS.2-5. Female Anopheles stephensi. Fig. 2. Scanning electron micrograph showing sunken pegs (sp), surface pegs (p), hairs (h), and base of a scale (s) on the antenna. x 3375. Fig. 3. Scanning electron micrograph of a surface peg (subtype undeterminable) on antenna. Note grooves, which begin above the peg base and terminate proximal to the tip. x 14 000. Fig. 4. Nearly longitudinal section through sunken peg sensillum showing sunken peg (sp), pit (pt), cuticular sheath (cs), dendrites (d), tormogen cell (lo), trichogen cell (tr), and neurilemma cell (nl). x 5400. Fig. 5. Nearly longitudinal section through surface peg (subtype undeterminable) showing peg (p), cuticular sheath (cs), dendrites (d), tormogen cell (to), trichogen cell (tr), and neurilemma cell (nl). x 7125. FIGS.6-9. Female Anopheles stephensi. Fig. 6. Section through junction region between inner (is) and outer (0s) segments of a dendrite in a sunken peg sensillum. cr, ciliary rootlets; dbb, distal basal body; m, mitochondria; mt, microtubules; pbb, proximal basal body; and x , area with 9 x 2 0 arrangement of microtubules. x 61 750. Fig. 7. Cross section just distal to region x in Fig. 6 of dendrite in a sunken peg sensillum. At this level a central and a peripheral microtubule are present in addition to the 9 x 2 + 0 configuration. x 114 000. Fig. 8. Cross section through a proximal basal body with the 9 x 3 + 0 configuration of microtubules. Sunken peg sensillum. x 114 000. Fig. 9. Section through inner segment of dendrite and cell body of a sunken peg sensillum. Visible are the basal bodies (bb), ciliary rootlets (cr), mitochondria (m), nucleus (n), nucleolus (nu), nucleolus-associated chromatin (nc), and a small portion of the flagellar nerve (fn). x 15 000. FIGS.10-17. Female Anopheles stephensi. Fig. 10. Section through peg of a sunken peg sensillum near the base where the grooves in the wall are beginning. Note electron-dense material in clefts (arrow), microtubules in sinuses (s) within columns, and confluent extracellular space (es) (extension of receptor lymph cavity), and dendritic branches (d) in lumen. x 38 000. Fig. 11. Cross section through middle region of peg of a sunken peg sensilla. At this level the columns with sinuses (s) and clefts (arrow) with electron-dense material are completely formed. d, dendritic branches. x 38 000. Fig. 12. Section near the tip of the peg of a sunken peg sensillum. At this level the grooves in the wall are disappearing, the sinuses have merged to form a confluent extracellular space (es), the dendrites have terminated, and the cuticular sheath (cs) is merging with the peg wall. x 38 000. Fig. 13. Section just below peg tip of a sunken peg sensillum. The wall of the peg lacks grooves and the lumen contains only a tiny extracellular space (es). x 38 000.Fig. 14. Cross section through middle region of peg of a subtype-A surface peg. Note electron-dense material which surrounds the two unbranched dendrites (d) and extends into the clefts (arrow). Small sinuses (s) are visible in some of the columns. x 38 000. Fig. 15. Section near tip of peg of a subtype-A surface peg sensillum. The cuticular sheath and peg wall are beginning to separate and the sinuses to merge into a confluent extracellular space (es). x 38 000. Fig. 16. Cross section through middle region of peg of a subtype-A surface peg sensillum. Only a few clefts (arrow) are formed completely and several of the sinuses (s) are confluent. x 38 000.Fig. 17. Section near tip of peg of a subtypeB peg sensillum. At this level the dendrites are beginning to disappear and the cuticular sheath and peg wall are beginning to separate, causing the sinuses to merge into a common extracellular space (es). x 38 000.
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the ciliary regions to the base of the peg. Surrounding the distal part of the trichogen cell is the tormogen cell. In all sheath cells, especially the trichogen and tormogen cells, the surfaces which contact extracellular space bear numerous slender lamellae containing mitochondria. Septate desmosomes connect the sheath cells to one another and the neurilemma cell to the dendrite. The dendrites are also connected to one another by septate desmosomes (Fig. 1). Surface Pegs Both subtypes A and B of the surface pegs average 5-6 p in length, are located on small prominences, have unbranched dendrites encased in sturdy cuticular sheaths, and like the sunken pegs, have three sheath cells. Figures 3 and 5 illustrate the general features of the surface pegs. In the light microscope and scanning electron microscope the two subtypes cannot be differentiated. Sections examined in the transmission electron microscope, however, reveal distinguishing characteristics in the structure of the peg wall and in the number of neurons present. Subtype A Each subtype-A peg is innervated by two, rarely one or three, morphologically similar neurons, the dendrites of which do not branch, and which terminate slightly proximal to the peg tip. The peg wall bears 12 or 13 grooves (Fig. 14), which begin above the base of the peg and terminate just proximal to the tip. As with the sunken pegs, the cuticular sheath of the subtype-A pegs evaginates and the peg wall invaginates to form distinct columns containing sinuses. The sinuses in the subtype-A pegs (Fig. 14) are smaller than those in the sunken pegs (Figs. 10 and 11). Near the tip, the peg wall and the cuticular sheath separate and the sinuses reform a continuous extracellular space (Fig. 15). In cross section the columns of the subtype-A pegs are mallet-shaped (Figs. 14 and 15), whereas those in the sunken pegs are shaped like button mushrooms (Fig. 10) or spades (Fig. 1I), depending upon the level in the peg. A material of medium electron density surrounds the dendrites and extends into the clefts (Fig. 14). Subtype B Subtype-B pegs are innervated by three or four morphologically similar neurons with unbranched dendrites (Fig. 16) which terminate proximal to the peg tip. The peg wall bears 9-1 1 grooves (Figs. 16 and 17). The relationship
between the peg wall and the cuticular sheath changes along the length of the peg, but in a manner different from that in subtype-A and the sunken pegs. At the base of the peg the cuticular sheath and the wall are separate. Distally the wall invaginates towards the sheath. The sheath does not evaginate and remains a distinct entity along the length of the peg (Figs. 16 and 17). As the invagination of the peg wall is not always complete, only occasionally are distinct columns formed (Figs. 16 and 17). Consequently, the receptor lymph cavity is frequently not separated into sinuses and is common between two or among more of the partial columns. Where the invagination of the wall goes completely to the sheath, there are perforations in the latter which form clefts. The clefts are fairly infrequent, with the result that the dendrites in subtype-B pegs are exposed much less to the external environment than those in the other peg sensilla. In some of the clefts are traces of a material of medium electron density (Fig. 16).
Discussion On the basis of morphological similarities with sensilla of known function in other insects (Schneider and Steinbrecht 1968; Slifer 1970; Kaissling 1971), the sunken pegs and both subtypes of the surface pegs on female Anopheles stephensi are probably olfactory receptors. Ismail (1962) concluded from behavioral studies with A. maculipennis atroparvis that the sunken pegs, which he called sensilla coeloconica, respond to odors. Electrophysiological studies have demonstrated that neurons of the grooved pegs of female A. aegypti are sensitive to water vapor, ammonia, acetone, acetic acid, anisole (Kellogg 1970), fatty acids, essential oils (Lacher 1967), lactic acid (Davis, personal communication), and commercial repellents (Lacher 1971 ; Davis and Rebert 1972). Whether or not the grooved surface pegs on A. stephensi respond to the same stimuli awaits experimental verification. Superficial grooved pegs (sensilla styloconica) occur on the antennae of Simulium spp. (Mercer and McIver 1973), and both superficial (sensilla basiconica) and sunken (sensilla coeloconica) ones on the antennae of Culicoides furens (ChuWang et el. 1975). These grooved pegs have many fine-structural features in common with those on A. stephensi, especially the subtype-B peg sensilla. This suggests that for blackflies, sand flies, and mosquitoes similar olfactory
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BOO AND McIVER: GROOVED PEGS ON ANTENNA OF A. STEPHENS1
stimuli are involved in eliciting behavioral patterns, perhaps host finding and discrimination. Sunken pegs with grooved walls are found in mosquitoes in the subfamily Anophelinae, but not in those in the Culicinae (Steward and Atwood 1963; Ismail 1964). This suggests either that different types of olfactory stimuli are important in the behavior of the individuals within the two subfamilies or that morphologically different sensilla are used for the reception of the same stimuli. The fine structure of grooved surface pegs on the antennal flagellum of Culex pipiens molestus and Aedes aegypti has been described respectively by Elizarov and Chaika (1972) and McIver (1974). Although externally quite similar, the pegs on the culicine species have a different internal morphology from that of the surface pegs on Anopheles stephensi. In Aedes aegypti each peg has a terminal pore, lacks perforations at the bases of the grooves, and is innervated by usually three, but occasionally four or five, neurons (McIver 1974). The structure of the pegs on C . pipiens molestus is similar to that of A. aegypti, except that they are innervated by two neurons. In neither A . aegypti nor C . pipiens were subtypes of the grooved pegs found. These differences among the grooved pegs of the three species of mosquitoes studied indicate that even within the same family (Culicidae) of insects, superficially similar sensilla may indeed be quite different internally. This finding emphasizes the need for transmission electron microscopy to prevent misinterpretation of sensillar structure, which could occur if only scanning electron microscopy is used. In the sensilla studied herein a spectrum of morphological adaptation for functional sensitivity is displayed. Branching of the dendrites, a feature indicated by Lewis (1970) to increase sensitivity, occurs in the sunken pegs, but not in either subtype of the surface pegs. In the sunken pegs and subtype-A surface pegs the dendrites are exposed to the exterior considerably more than in subtype B. The sensilla with the least adaptation for sensitivity are the subtype-B pegs and those with the greatest, the sunken pegs. In the latter this degree of adaptation may be related to their being located in pits and (or) being restricted to the basal seven flagellar segments. Cross sections of the sunken pegs and subtype A of the surface pegs reveal the presence of
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extracellular electron-dense material surrounding the dendrites and extending into the clefts between the columns of the peg wall. Traces of a similar material were found in the subtype-B pegs. Somewhat similar observations have been made on antennal sensilla of locusts (Steinbrecht 1969), stable flies (Lewis 1971), cockroaches (Lambin 1973), and beetles (Corbitre-TichanC 1974). In Figs. 10, 11, and 14 a distinct dark line can be seen on the outer surface of the extracellular electron-dense material. As this material is extracellular, the line cannot be a cell membrane. Rather, the line is probably an artefact formed upon initial contact of the extracellular electrondense material with the fixative. In A. stephensi the peg lumen is continuous with the extracellular receptacle cavity delineated by the neurilemma cell, which shows evidence of secretory activities, i.e., numerous long slender microvilli and many mitochondria, and apparently produces the fluid found within the cavity and the peg lumen. At the level in the peg where clefts occur this fluid is exposed to the exterior. Consequently, the interaction between the stimulating molecules and the dendrites must occur through the fluid, which probably plays an important role in sensory transduction.
Acknowledgments Financial support by Medical Research Council (Canada) grant No. MA 2909 and Defence Research Board (Canada) grant No. 6801-50 is gratefully acknowledged. Boo, K . S . , and S . B. MCIVER.1975. Fine structure of sunken thick-walled pegs (sensilla ampullacea and coeloconica) on the antennae of mosquitoes. Can. J. Zool. 53: 262-266. I.W., R. C. AXTELL, and D. L. KLINE. 1975. CHU-WANG, Antennal and palpal sensilla of the sand fly Culicoides furens (Poey) (Diptera: Ceratopogonidae). Int. J . Insect Morphol. Embryol. 4: 131-149. G . 1974. Fine structure of an antenCORBIERE-TICHANE, nal sensory organ ('vesicule olfactive') of Speophyes lucidulus Delar. (Cave Coleoptera of the Bathysciinae subfamily). Tissue Cell, 6: 535-550. DAVIS,E. E . , and C. S. REBERT.1972. Elements of olfactory receptor coding in the yellow fever mosquito. J . Econ. Entomol. 65: 1058-1061. 1972. UltrastrucELIZAROV, Yu. A , , and S . Yu. CHAIKA. ture of olfactory sensillae on antennae and palps of the mosquitoes Culexpipiens moleslus (Diptera, Culicidae). (In Russian, English summary.) Zool. Zh. 51: 1665-1674. ISMAIL, I. A. H . 1962. Sense organs in the antennae of Anopheles maculipennis atroparvus (v. Thiel) and their
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possible function in relation to the attraction of female mosquito to man. Acta Trop. 19: 1-58. 1964. Comparative study of sense organs in the antennae of culicine and anopheline female mosquitoes. Acta. Trop. 21: 155-168. KAISSLING, K. E. 1971. Insect olfaction. In Handbook of sensory physiology IV. Chemical senses 1 Olfaction. Edited by L . M. Beidler. Springer-Verlag, Berlin, Heidelberg, New York. pp. 351-431. KARNOVSKY, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27: 137-138A. KELLOGG,F. E. 1970. Water vapor and carbon dioxide receptors in Aedes aegypti (L.). J . Insect Physiol. 16: 99-108. LACHER, V. 1967. Elektrophysiologische Untersuchungen an Einzelnen Geruchsrezeptoren auf den Antennen weiblicher Moskitos (Aedes aegypti L.). J . Insect Physiol. 13: 1461-1470. 1971. Arbeitsbereiche von Geruchsrezeptoren auf der Moskitoantennae (Aedes aegypti). J. Insect Physiol. 17: 507-517. LAMBIN, M. 1973. Les sensillesde I'antenne chez quelques blattes et en particulier chez Blaberus craniifer (Burm.). Z. Zellforsch. Mikrosk. Anat. 143: 183-206. LEWIS,C. T. 1970. Structure and function in some external receptors. In Insect ultrastructure. R. Entomol. Soc. London Symp. 5: 59-76. 1971. Superficial sense organs of the antennae of the fly, Stomoxys calcitrans. J . Insect Physiol. 17: 449461. MCIVER,S. B. 1970. Comparative study of the antennal sense organs of female culicine mosquitoes. Can. Entomol. 102: 1258-1268.
-1973. Fine structure of antennal sensilla coeloconica of culicine mosquitoes. Tissue Cell, 5: 105112. -1974. Fine structure of antennal grooved pegs of the mosquito, Aedes aegypti. Cell Tissue Res. 153: 327-337. MCIVER,S. B., and S. A. HUTCHINSON. 1972. Coeloconic sensilla on the antennae of the yellow fever mosquito, Aedes aegypti (L.). Experientia, 28: 323. MERCER,K. L., and S. B. MCIVER.1973. Studies on the antennal sensilla of selected blackflies (Diptera: Simuliidae). Can. J. Zool. 51: 729-734. SCHNEIDER, D., and R. A. STEINBRECHT. 1968. Checklist of insect olfactory sensilla. Symp. Zool. Soc. London, 23: 279-297. SLIFER, E. H. 1970. The structure of arthropod chemoreceptors. Annu. Rev. Entomol. 15: 121-142. SLIFER,E . H., and S. S. SEKHON.1962. The fine structure of the sense organs on the antennal flagellum of the yellow-fever mosquito Aedes aegypti (Linnaeus). J . Morphol. 111: 49-67. SPURR,A. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26: 3143. STEINBRECHT, R. A. 1969. Comparative morphology of olfactory receptors. 111. Int. Symp. Olfaction Taste. Edited by C. Pfaffmann. Rockefeller University Press, New York. pp. 3-21. STEWARD, C. C., and C . E . ATWOOD.1963. The sensory organs of mosquito antennae. Can. J. Zool. 41: 577-594.
Fine structure of surface and sunken grooved pegs on the antenna of female Anopheles stephensi (Diptera: Culicidae) K. S. Boo AND S. B. MCIVER Department of Microbiology and Parasitology, FitzGerald Building, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1Al Received June 16,1975 Boo, K. S., and S. B. MCIVER.1976. Fine structure of surface and sunken grooved pegs on the antenna of female Anopheles stephensi (Diptera: Culicidae). Can. J. Zool. 54: 235-244. The antenna of female Anopheles stephensi Liston bears three types of sensilla with grooved pegs: those sunken in pits and subtypes A and B of those located on the flagellar surface. The sunken peg sensilla are innervated by four or five neurons with branching dendrites. The dendrites are exposed to the exterior by means of longitudinal clefts at the bases of the grooves in the peg wall. Surrounding the dendrites and extending into the clefts is an extracellular material of medium electron density. Three sheath cells are associated with each sunken peg sensillum. Subtype-A surface peg sensilla are generally similar to the sunken peg sensilla, except that they are located on the antennal surface and are innervated by two neurons with unbranched dendrites. Subtype-B surface peg sensilla have three or four neurons, the dendrites of which do not branch and are exposed less to the exterior than those in the other peg sensilla because the clefts in the peg wall are smaller and less frequent. Only trace amounts of electron-dense material occur in the clefts of the subtype-B surface peg sensilla. The sunken peg and both subtypes of the surface peg sensillaare probably olfactory receptors. Boo, K. S., et S. B. MCIVER.1976. Fine structure of surface and sunken grooved pegs on the antenna of female Anopheles stephensi (Diptera: Culicidae). Can. J. Zool. 54: 235-244. Chez Anopheles stephensi Liston, il y a trois types de sensilles a batonnets cannelts: certains sont enfonces dans une cavite et d'autres, de deux sous-types, A et B, sont localises a la surface du flagelle. Les sensilles enfonces sont innerves par quatre ou cinq neurones portant des dendrites ramifites. Les dendrites sont en contact avec I'exterieur par le truchement de fissures longitudinales situies a la base des cannelures de la paroi du bkonnet. Tout autour des dendrites et s'ttendant jusque dans les fissures, on observe une substance extracellulaire d'opacitt moyenne au microscope electronique. Chaque sensille enfonce s'associe trois cellules enveloppes. Les sensilles a biitonnets de surface, du sous-type A, sont, de faqon generale, semblables aux sensilles enfoncts, sauf pour ce qui est de leur position a la surface de I'antenne et de leur innervation par deux neurones a dendrites simples. Les sensilles de surface du sous-type B ont trois ou quatre neurones dont les dendrites ne sont pas ramifiees et sont moins expostes a I'exterieur que celles des autres types, car les fissures de la paroi du bltonnet sont plus petites et plus rares. On ne trouve que des traces de substance extracellulaire opaque au microscope electronique dans les fissures des sensilles de surface du sous-type B. Les sensilles enfoncts et les deux types de sensilles de surface servent probablement de recepteurs olfactifs. [Traduit par le journal]
Introduction In mosquitoes many external stimuli which elicit behavioral patterns are detected by various types of antennal sensilla. These sensilla occur as three general morphological types : bristles (sensilla chaetica), hairs (sensilla trichodea), and pegs. Several types of peg sensilla may be distinguished on the basis of location and structure of the peg wall. Mosquitoes in both subfamilies Anophelinae and Culicinae have (a) smoothwalled pegs in inconspicuous pits (sensilla coeloconica) at the tip of the antenna and at infrequent
intervals along the flagellum (McIver and Hutchinson 1972; McIver 1973; Boo and McIver 1975), (b) smooth-walled pegs situated in a chamber at the end of a long tube (sensilla ampullacea) on the basal flagellar segments (Boo and McIver 1975), and (c) grooved walled pegs located on the flagellar surface. The latter type has been called sensillum basiconicum (e.g., Ismail 1964; McIver 1970), type A3 seta (Steward and Atwood 1963), and thorn-shaped hairs (Slifer and Sekhon 1962). The fine structure of the grooved surface pegs on female Aedes aegypti
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has been described by McIver (1974). Occurring only on the antennae of anophelines are grooved walled pegs situated in conspicuous pits (sensilla coeloconica) (Ismail 1962, 1964; Steward and Atwood 1963). This work was conducted to describe the fine structure of the grooved pegs which occur on the flagellar surface and in conspicuous pits on the antennae of female Anopheles stephensi Liston (Diptera: Culicidae). The results aid in providing the morphological basis for understanding sensillar function and eventually mosquito behavior.
Materials and Methods Antennae of specimens obtained from a laboratory colony were fixed in either Karnovsky's fixative (Karnovsky 1965) or in 3% glutaraldehyde, postfixed in 1% OsO,, block-stained with 0.5% uranyl acetate, dehydrated through a graded series of ethanols, and embedded in Spurr's low-viscosity epoxy medium (Spurr 1969). For details see Boo and Mcher (1975). Flagellar segments 1-6, and 12 and 13 were serially sectioned using a diamond knife. Specimens for scanning electron microscopy were fixed in 5% formalin for several days, dehydrated through a graded series of ethanols, and placed in a series of 100% ethanol and isoamvl acetate mixtures with increasing concentrations of the latter to 100%. After critical-point drying from CO,, specimens were coated with goldpalladium during spin rotation
Results The antenna of female Anopheles stephensi consists of 13 flagellar segments set into a cupshaped pedicel and attached to the head by a ring-shaped scape. Ismail (1964) reported that the sunken pegs (Fig. 2) occur on segments 1-7
and the surface pegs (Figs. 2 and 3) on all flagellar segments, except segment 1 (the most basal one). In one antenna of the six we examined using transmission electron microscopy, one surface,peg occurred on segment 1. In thin sections we observed the surface pegs to be present on all aspects of the segments on which they occur, whereas the sunken pegs are restricted to the lateral side of the distal half of the segments on which they are located. Surface pegs are more prevalent, with an average of 107 per antenna, than sunken ones, with 31 per antenna (Ismail 1964). The external surface of the peg of both the surface and sunken peg sensilla appeared similar, with deep grooves in the wall beginning slightly above the base and terminating just proximal to the tip. Internally, however, the sensilla were distinctly different. In addition, differences were found in the internal features of the surface peg, which led us to conclude that they occur as two subtypes, henceforth designated as subtypes A and B. Table 1 gives a summary of the characteristics of each type of peg sensillum.
Sunken Pegs Each peg arises from the center of the floor of a pit which narrows at the orifice (Figs. 1, 2, 4). Externally only the tip of the peg is visible (Fig. 2). The pegs average 4-5 p (microns) in length and 1.6 p in diameter in the middle region. From 13 to 16 grooves occur in the peg wall. Each sensillum is innervated by four or five neurons, the dendrites of which divide within the peg lumen to form 7-1 3 branches and terminate
TABLE 1. Characteristics of sunken and surface pegs on the antenna of female Anopheles stephensi
Sensillar type Surface pegs Characteristic Location on flagellar segments Peg length, p No. grooves No. neurons Dendritic branching Electron-dense material in grooves Degree of exposure of dendrites to exterior
Sunken pegs
Subtype A
Subtype B
Lateral side of distal half 4-5 13-16 4-5 Yes
All aspects
All aspects
Yes
Yes
Traces
Large
Large
Small
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FIG.1. Diagram of sunken peg sensillum with two of the four or five neurons shown. bb, basal bodies with ciliary rootlets; c, cuticle; cs, cuticular sheath; ds, desmosomes; is, inner segment of dendrite; m, mitochondria; n, nucleus; nl, neurilemma cell; nr, neuron; os, outer segment of dendrite; pt, pit; re, receptacle cavity; rlc, receptor lymph cavity; sp, sunken peg; to, tormogen cell; and tr, trichogen cell.
slightly proximal to the peg tip. Each dendrite is divided into an inner and an outer segment (Figs. 1 and 6) by a region with a 9 x 12 0 configuration of microtubules. Distal to this region additional microtubules appear (Fig. 7). The outer segment is encased within a cuticular sheath, 7-8 p in length. The only inclusions observable within the outer segment are microtubules, whereas the inner segment contains prominent mitochondria, vesicles, microtubules, two basal bodies displaying the 9 x 3 + 0 configuration of microtubules (Fig. 8), and periodically banded rootlets (Figs. 1, 6, and 9).
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The cell body of each neuron contains an oblong nucleus with scattered clumps of dark chromatin and a prominent nucleolus with associated chromatin (Fig. 9), and the usual organelles, viz., mitochondria, Golgi bodies, endoplasmic reticulum, microtubules, and ribosomes. Extending proximally from the cell body is an axon which joins one of the two flagellar nerve bundles. Along the length of the peg the relationship between the peg wall and the cuticular sheath changes. Basally, where the grooves in the wall are absent or just beginning, the cuticular sheath
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and peg wall are separated by an extension of the receptor lymph cavity, an extracellular space containing microtubules in a finely granular matrix (Fig. 10). Distally the wall invaginates and the sheath evaginates to form separate columns, each enclosing a sinus which is continuous with the receptor lymph cavity (Figs. 10 and 11). Longitudinal clefts in the peg wall occur at the cuticle-free bases of the grooves. An extracellular material of medium electron density, presumably the residue of a liquid in the living state, is present in the clefts (Figs. 10 and 11). Basally the ridges (outer aspect of the columns) are round (Fig. 10). Distally the ridges become progressively more pointed until just proximal to the peg tip, where they appear as V-shaped projections (Figs. 11 and 12). At the peg tip the ridges disappear, leaving the peg wall smooth (Fig. 13).
Near the peg tip where the dendrites terminate, the columns merge to re-form a continuous cuticular sheath and peg wall, both without perforations. This separation of the wall and the sheath results in the sinuses becoming confluent in one extracellular space, which is still continuous with the receptor lymph cavity below. Just proximal to the tip the cuticular sheath fuses to one side of the peg wall (Fig. 12). Beyond this point the peg contains only a small extracellular space (Fig. 13). Three sheath cells are associated with each sensillum. The neurilemma cell, which is the most proximal, distally encloses the extracellular receptacle cavity (Fig. 1) and surrounds the neurons from just distal to the ciliary regions, to at least the distal part of the axons. A trichogen cell encases the distal portion of the neurilemma cell and the dendrites from proximal to
FIGS.2-5. Female Anopheles stephensi. Fig. 2. Scanning electron micrograph showing sunken pegs (sp), surface pegs (p), hairs (h), and base of a scale (s) on the antenna. x 3375. Fig. 3. Scanning electron micrograph of a surface peg (subtype undeterminable) on antenna. Note grooves, which begin above the peg base and terminate proximal to the tip. x 14 000. Fig. 4. Nearly longitudinal section through sunken peg sensillum showing sunken peg (sp), pit (pt), cuticular sheath (cs), dendrites (d), tormogen cell (lo), trichogen cell (tr), and neurilemma cell (nl). x 5400. Fig. 5. Nearly longitudinal section through surface peg (subtype undeterminable) showing peg (p), cuticular sheath (cs), dendrites (d), tormogen cell (to), trichogen cell (tr), and neurilemma cell (nl). x 7125. FIGS.6-9. Female Anopheles stephensi. Fig. 6. Section through junction region between inner (is) and outer (0s) segments of a dendrite in a sunken peg sensillum. cr, ciliary rootlets; dbb, distal basal body; m, mitochondria; mt, microtubules; pbb, proximal basal body; and x , area with 9 x 2 0 arrangement of microtubules. x 61 750. Fig. 7. Cross section just distal to region x in Fig. 6 of dendrite in a sunken peg sensillum. At this level a central and a peripheral microtubule are present in addition to the 9 x 2 + 0 configuration. x 114 000. Fig. 8. Cross section through a proximal basal body with the 9 x 3 + 0 configuration of microtubules. Sunken peg sensillum. x 114 000. Fig. 9. Section through inner segment of dendrite and cell body of a sunken peg sensillum. Visible are the basal bodies (bb), ciliary rootlets (cr), mitochondria (m), nucleus (n), nucleolus (nu), nucleolus-associated chromatin (nc), and a small portion of the flagellar nerve (fn). x 15 000. FIGS.10-17. Female Anopheles stephensi. Fig. 10. Section through peg of a sunken peg sensillum near the base where the grooves in the wall are beginning. Note electron-dense material in clefts (arrow), microtubules in sinuses (s) within columns, and confluent extracellular space (es) (extension of receptor lymph cavity), and dendritic branches (d) in lumen. x 38 000. Fig. 11. Cross section through middle region of peg of a sunken peg sensilla. At this level the columns with sinuses (s) and clefts (arrow) with electron-dense material are completely formed. d, dendritic branches. x 38 000. Fig. 12. Section near the tip of the peg of a sunken peg sensillum. At this level the grooves in the wall are disappearing, the sinuses have merged to form a confluent extracellular space (es), the dendrites have terminated, and the cuticular sheath (cs) is merging with the peg wall. x 38 000. Fig. 13. Section just below peg tip of a sunken peg sensillum. The wall of the peg lacks grooves and the lumen contains only a tiny extracellular space (es). x 38 000.Fig. 14. Cross section through middle region of peg of a subtype-A surface peg. Note electron-dense material which surrounds the two unbranched dendrites (d) and extends into the clefts (arrow). Small sinuses (s) are visible in some of the columns. x 38 000. Fig. 15. Section near tip of peg of a subtype-A surface peg sensillum. The cuticular sheath and peg wall are beginning to separate and the sinuses to merge into a confluent extracellular space (es). x 38 000. Fig. 16. Cross section through middle region of peg of a subtype-A surface peg sensillum. Only a few clefts (arrow) are formed completely and several of the sinuses (s) are confluent. x 38 000.Fig. 17. Section near tip of peg of a subtypeB peg sensillum. At this level the dendrites are beginning to disappear and the cuticular sheath and peg wall are beginning to separate, causing the sinuses to merge into a common extracellular space (es). x 38 000.
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the ciliary regions to the base of the peg. Surrounding the distal part of the trichogen cell is the tormogen cell. In all sheath cells, especially the trichogen and tormogen cells, the surfaces which contact extracellular space bear numerous slender lamellae containing mitochondria. Septate desmosomes connect the sheath cells to one another and the neurilemma cell to the dendrite. The dendrites are also connected to one another by septate desmosomes (Fig. 1). Surface Pegs Both subtypes A and B of the surface pegs average 5-6 p in length, are located on small prominences, have unbranched dendrites encased in sturdy cuticular sheaths, and like the sunken pegs, have three sheath cells. Figures 3 and 5 illustrate the general features of the surface pegs. In the light microscope and scanning electron microscope the two subtypes cannot be differentiated. Sections examined in the transmission electron microscope, however, reveal distinguishing characteristics in the structure of the peg wall and in the number of neurons present. Subtype A Each subtype-A peg is innervated by two, rarely one or three, morphologically similar neurons, the dendrites of which do not branch, and which terminate slightly proximal to the peg tip. The peg wall bears 12 or 13 grooves (Fig. 14), which begin above the base of the peg and terminate just proximal to the tip. As with the sunken pegs, the cuticular sheath of the subtype-A pegs evaginates and the peg wall invaginates to form distinct columns containing sinuses. The sinuses in the subtype-A pegs (Fig. 14) are smaller than those in the sunken pegs (Figs. 10 and 11). Near the tip, the peg wall and the cuticular sheath separate and the sinuses reform a continuous extracellular space (Fig. 15). In cross section the columns of the subtype-A pegs are mallet-shaped (Figs. 14 and 15), whereas those in the sunken pegs are shaped like button mushrooms (Fig. 10) or spades (Fig. 1I), depending upon the level in the peg. A material of medium electron density surrounds the dendrites and extends into the clefts (Fig. 14). Subtype B Subtype-B pegs are innervated by three or four morphologically similar neurons with unbranched dendrites (Fig. 16) which terminate proximal to the peg tip. The peg wall bears 9-1 1 grooves (Figs. 16 and 17). The relationship
between the peg wall and the cuticular sheath changes along the length of the peg, but in a manner different from that in subtype-A and the sunken pegs. At the base of the peg the cuticular sheath and the wall are separate. Distally the wall invaginates towards the sheath. The sheath does not evaginate and remains a distinct entity along the length of the peg (Figs. 16 and 17). As the invagination of the peg wall is not always complete, only occasionally are distinct columns formed (Figs. 16 and 17). Consequently, the receptor lymph cavity is frequently not separated into sinuses and is common between two or among more of the partial columns. Where the invagination of the wall goes completely to the sheath, there are perforations in the latter which form clefts. The clefts are fairly infrequent, with the result that the dendrites in subtype-B pegs are exposed much less to the external environment than those in the other peg sensilla. In some of the clefts are traces of a material of medium electron density (Fig. 16).
Discussion On the basis of morphological similarities with sensilla of known function in other insects (Schneider and Steinbrecht 1968; Slifer 1970; Kaissling 1971), the sunken pegs and both subtypes of the surface pegs on female Anopheles stephensi are probably olfactory receptors. Ismail (1962) concluded from behavioral studies with A. maculipennis atroparvis that the sunken pegs, which he called sensilla coeloconica, respond to odors. Electrophysiological studies have demonstrated that neurons of the grooved pegs of female A. aegypti are sensitive to water vapor, ammonia, acetone, acetic acid, anisole (Kellogg 1970), fatty acids, essential oils (Lacher 1967), lactic acid (Davis, personal communication), and commercial repellents (Lacher 1971 ; Davis and Rebert 1972). Whether or not the grooved surface pegs on A. stephensi respond to the same stimuli awaits experimental verification. Superficial grooved pegs (sensilla styloconica) occur on the antennae of Simulium spp. (Mercer and McIver 1973), and both superficial (sensilla basiconica) and sunken (sensilla coeloconica) ones on the antennae of Culicoides furens (ChuWang et el. 1975). These grooved pegs have many fine-structural features in common with those on A. stephensi, especially the subtype-B peg sensilla. This suggests that for blackflies, sand flies, and mosquitoes similar olfactory
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BOO AND McIVER: GROOVED PEGS ON ANTENNA OF A. STEPHENS1
stimuli are involved in eliciting behavioral patterns, perhaps host finding and discrimination. Sunken pegs with grooved walls are found in mosquitoes in the subfamily Anophelinae, but not in those in the Culicinae (Steward and Atwood 1963; Ismail 1964). This suggests either that different types of olfactory stimuli are important in the behavior of the individuals within the two subfamilies or that morphologically different sensilla are used for the reception of the same stimuli. The fine structure of grooved surface pegs on the antennal flagellum of Culex pipiens molestus and Aedes aegypti has been described respectively by Elizarov and Chaika (1972) and McIver (1974). Although externally quite similar, the pegs on the culicine species have a different internal morphology from that of the surface pegs on Anopheles stephensi. In Aedes aegypti each peg has a terminal pore, lacks perforations at the bases of the grooves, and is innervated by usually three, but occasionally four or five, neurons (McIver 1974). The structure of the pegs on C . pipiens molestus is similar to that of A. aegypti, except that they are innervated by two neurons. In neither A . aegypti nor C . pipiens were subtypes of the grooved pegs found. These differences among the grooved pegs of the three species of mosquitoes studied indicate that even within the same family (Culicidae) of insects, superficially similar sensilla may indeed be quite different internally. This finding emphasizes the need for transmission electron microscopy to prevent misinterpretation of sensillar structure, which could occur if only scanning electron microscopy is used. In the sensilla studied herein a spectrum of morphological adaptation for functional sensitivity is displayed. Branching of the dendrites, a feature indicated by Lewis (1970) to increase sensitivity, occurs in the sunken pegs, but not in either subtype of the surface pegs. In the sunken pegs and subtype-A surface pegs the dendrites are exposed to the exterior considerably more than in subtype B. The sensilla with the least adaptation for sensitivity are the subtype-B pegs and those with the greatest, the sunken pegs. In the latter this degree of adaptation may be related to their being located in pits and (or) being restricted to the basal seven flagellar segments. Cross sections of the sunken pegs and subtype A of the surface pegs reveal the presence of
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extracellular electron-dense material surrounding the dendrites and extending into the clefts between the columns of the peg wall. Traces of a similar material were found in the subtype-B pegs. Somewhat similar observations have been made on antennal sensilla of locusts (Steinbrecht 1969), stable flies (Lewis 1971), cockroaches (Lambin 1973), and beetles (Corbitre-TichanC 1974). In Figs. 10, 11, and 14 a distinct dark line can be seen on the outer surface of the extracellular electron-dense material. As this material is extracellular, the line cannot be a cell membrane. Rather, the line is probably an artefact formed upon initial contact of the extracellular electrondense material with the fixative. In A. stephensi the peg lumen is continuous with the extracellular receptacle cavity delineated by the neurilemma cell, which shows evidence of secretory activities, i.e., numerous long slender microvilli and many mitochondria, and apparently produces the fluid found within the cavity and the peg lumen. At the level in the peg where clefts occur this fluid is exposed to the exterior. Consequently, the interaction between the stimulating molecules and the dendrites must occur through the fluid, which probably plays an important role in sensory transduction.
Acknowledgments Financial support by Medical Research Council (Canada) grant No. MA 2909 and Defence Research Board (Canada) grant No. 6801-50 is gratefully acknowledged. Boo, K . S . , and S . B. MCIVER.1975. Fine structure of sunken thick-walled pegs (sensilla ampullacea and coeloconica) on the antennae of mosquitoes. Can. J. Zool. 53: 262-266. I.W., R. C. AXTELL, and D. L. KLINE. 1975. CHU-WANG, Antennal and palpal sensilla of the sand fly Culicoides furens (Poey) (Diptera: Ceratopogonidae). Int. J . Insect Morphol. Embryol. 4: 131-149. G . 1974. Fine structure of an antenCORBIERE-TICHANE, nal sensory organ ('vesicule olfactive') of Speophyes lucidulus Delar. (Cave Coleoptera of the Bathysciinae subfamily). Tissue Cell, 6: 535-550. DAVIS,E. E . , and C. S. REBERT.1972. Elements of olfactory receptor coding in the yellow fever mosquito. J . Econ. Entomol. 65: 1058-1061. 1972. UltrastrucELIZAROV, Yu. A , , and S . Yu. CHAIKA. ture of olfactory sensillae on antennae and palps of the mosquitoes Culexpipiens moleslus (Diptera, Culicidae). (In Russian, English summary.) Zool. Zh. 51: 1665-1674. ISMAIL, I. A. H . 1962. Sense organs in the antennae of Anopheles maculipennis atroparvus (v. Thiel) and their
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