Antennal olfactory sensilla of the face fly, Musca autumnalis Degreer (Diptera : Muscidae)

Int. J. Insect MorphoL & Embryol. 5(1): 1-16. 1976. Pergamon Press. Printed in Great Britain.

A N T E N N A L OLFACTORY SENSILLA OF T H E FACE FLY, M U S C A A U T U M N A L I S D E G R E E R (DIPTERA • MUSCIDAE)* D. E. BAY~ and C. W. P1TTS+ Department of Entomology, Kansas State University, Manhattan, Kansas 66506, U.S.A. (Accepted 7 October 1975)

Abstract--We used scanning and transmission electron microscopy to study the olfactory sensilla of the basal flagellar subsegment, or funiculus, of the face fly, Musca autumnalis. Olfactory sensilla on the surface consist of thick-walled sensilla trichodea and 2 types of thin-walled sensilla basiconica; all have a typical pore-tubule system. In addition, the funicular surface has grooved, or fluted, sensilla. Olfactory pits on the ventral surface have numerous thin-walled sensilla basiconica morphologically similar to those on the surface. Female face flies have 2 ventral pits while males have only one. The dorsolateral surface of the funiculus of both sexes also has a single pit, which contains several grooved sensilla and a previously undescribed sensillum with a filamentous tip whose method of communicating with the exterior we could not determine. Index descriptors (in addition to those in title):

Basiconicum, trichodeum, chemoreceptor, scanning and transmission electron microscopy.

INTRODUCTION BEHAVIORALTESTShave recently shown antennae to play a p r i m a r y role in olfactory response o f the face fly, M . autumnalis to bovine feces (Bay and Pitts, in press). However, the nature o f the funicular sensiUa has not been determined although detailed studies have been made o f receptors on the labellum (Moherek, 1964) and ovipositor ( H o o p e r et al., 1972). The study reported here was designed to determine the types and distribution o f olfactory sensilla on the basal flagellar subsegment by using scanning (SEM) and transmission electron m i c r o s c o p y (TEM). Greenberg and A s h (1972), who have studied the sense organs on the antennal pedicel o f the face fly and n u m e r o u s other muscoid species by SEM, concluded by behavorial tests that receptors on the antennal pedicel have no olfactory function. The presence o f a pore-tubule system is n o w considered p r o o f o f an olfactory function (Slifer, 1970); however specific odors to which a given receptor is sensitive m a y be determined only by behavioral or electrophysiological means (Schneider and Steinbrecht, 1968). Basic knowledge o f the olfactory sensory system is necessary for future research on face fly attractants and repellents. * Contribution No. 1155, Department of Entomology, Kansas Agricultural Experiment Station, Manhattan, Kansas 66506. Research supported in part by NIH Training Grant No. AI-00328 from National Institute of Allergy and Infectious Diseases. t Present address: Assistant Professor, Department of Entomology, Texas A&M University, College Station, Texas. ++Research entomologist. I.M.E. 5/1--A

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D.E. BAY and C. W. P1TTS

MATERIALS AND METHODS Excised antennae for SEM study were fixed in 5 ~ formalin for 24 h r, then slowly dehydrated through a series of ethanols. Specimens were transferred to 85 ~ amyl acetate and dried in a Denton Vacuum DCP-1 critical point dryer. Fixed, dried antennae were coated in vacuo with gold-palladium in a Kinney KSE-2A-M evaporator and examined in an Etec Autoscan SEM. Antennae for TEM examinations were excised in a 4°C solution of 3 ~o glutaraldehyde buffered to pH 7.4 with Millonig's phosphate buffer containing 0"02 M sucrose (Millonig, 1962). Excised sepcimens were subsequently transferred to fresh fixative at 4°C for 4 hr, rinsed several times in phosphate buffer over 2 hr, and postfixed 1 hr in a 4°C phosphate-buffered 1 ~ solution of osmium tetroxide containing 0.02 M sucrose. Tissues were dehydrated through a series of graded, cold ethanols, transferred to propylene oxide, and embedded in Epon 812 (Luft, 1961). After polymerization, antennal tissue was sectioned with a DuPont diamond knife mounted in a Westfall-Healey section mounter on a LKB-Huxley ultramicrotome. Sections were mounted on parlodion-carbon grids, stained in a saturated solution of uranyl acetate and lead citrate (Reynolds, 1963), and examined in a Philips 201 TEM. OBSERVATIONS AND DISCUSSION Face fly antenna consists of a scape, pedicel, and enlarged basal flagellar subsegment (termed the funiculus) with the plumose arista attached. Cross sections o f the funiculus approximate a triangle with the longest side lying against the antennal fossa when the antennae are held at rest. I n that position, the other 2 sides are exposed frontally with the arista extending along the lateral edge. F o r orientation, surfaces o f the flagellar subsegment m a y be divided into ventral, dorso-lateral (with attached arista), and dorsomedian regions. Male and female funiculi are similar in size, type, and distribution of sensilla. The funiculus o f b o t h is approximately 500/zm long, 175 tzm dia, and densely covered with noninnervated cuticular spines interspersed with 4 morphological types of sensilla (Fig. 3): (1) thick-walled sensilla trichodea; (2) thin-walled sensilla basiconica, type I; (3) thinwalled sensilla basiconica, type I I ; and (4) grooved, or fluited, sensilla. Several olfactory pits are also present and contain 3 additional kinds of sensory pegs (Figs. 14, 17): (1) thinwalled sensilla basiconica; (2) grooved sensilla; and (3) a previously undescribed sensillum with a filamentous tip. Tactile hairs do not occur on the funiculus n o r are chemosensory organs present on the arista. The distribution and description o f the various sense organs follow. Surface sensiIla Thick-wailed sensilIa trichodea. The most conspicuous sensilla on the funicular surface are approximately 25/~m long and range in diameter f r o m near 3 t~m at the base to approximately 1 t~m distally (Fig. 4). These sensilla m a y be identified by a characteristic distal curvature with the long axis tending to parallel that of the antennal surface. They are evenly distributed over the entire dorsolateral and dorsomedian funicular surface (Fig. 1). Thick-walled sensilla trichodea also are evenly distributed over the distal 1/3 of the ventral surface but rarely on the proximal 2/3 o f the ventral surface (Fig. 2). The cuticular walls o f the sensilla are thick ranging f r o m 1-0 tzm basally to 0.3 t~m at the distal end (Fig. 5), Pores, discussed in detail later, are uniformly distributed over the surface o f the peg. The external openings o f the pores range in diameter f r o m 150 ~ to 200 ,~, and are separated by approximately 0.25 /~m so their density is about 20 pores per square micron. Internally, each pore widens into a c h a m b e r approximately 500 A across. The cuticular wall o f the sensillum is interrupted below the c h a m b e r by a channel filled with an electron dense material continuous with that surrounding the dendrites within the peg lumen. Pore tubules extend f r o m the pore c h a m b e r approximately 1000/~ into the channel.

Antennal Olfactory Sensilla of the Face Fly

FIG. 1. Dorsolateral funicular surface of female face fly, left antenna showing single pit opening. × 250 FIG. 2. Ventral funicular surface of female face fly, left antenna showing openings into 2 ventral pits. x 2 0 0

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D . E . BAY and C. W. PITTS

FI~. 3, Ventral funicular surface showing 4 morphological types of olfactory sensilla: sensillum trichodeum (ST); sensillum basiconicum, type I (SB1); sensillum basiconicum, type II (SB2); stellate sensillum (SS). x 3200 FIo. 4. Thick-walled sensillum trichodeum, x 9000

Antennal Olfactory Sensilla of the Face Fly

5

There appear to be 2 to 4 neurons associated with each thick-walled sensillum trichodeum. Dendritic branching occurs only at the extreme distal end of the peg. Fine structures of the sensory neurons are discussed later. Thin-walled sensilla basiconica, type I and type II. The 2 types of thin-walled sensilla basiconica are sufficiently similar so they are described together. The type I peg is approximately 15/~m long and 1.5/~m dia throughout most of its length (Fig. 6). It occurs on all 3 funicular surfaces but is most abundant on the ventral surface in the region where thickwailed sensiUa trichodea are scarce. The type II sensillum is only about 10/zm long and about 1/xm dia (Fig. 7). Type II sensilla are the least abundant of the surface sense organs and occur predominantly throughout the same general area on the ventral funicular surface as the type I peg. Both peg types are slightly curved distally but not so strongly as are thick-walled trichodea. The cuticular walls of both sensillum types are similar with average thicknesses of approximately 0.2/~m. The surfaces of both pegs are perforated by many minute pores with external openings from 350 to 400 A in diameter. Pores of the type I peg are about 0-15/zm apart resulting in a desnity of about 35 pores per square micron. Pores of the type II sensillum are only 0.1/zm apart; their density is nearly 60 pores per square micron. Each pore widens just below the cuticular surface to form a small chamber, the "pore kettle" of Ernst (1969), that is nearly 0.1 /~m across (Figs. 8-11). The floor of the chamber is concave and appears to be continuous with the epicuticular wall of the sensillum. Leading from the chamber are 15 to 20 individual pore tubules t h a t e x t e n d approximately 1000 A into the peg lumen. Diameters of the pore tubules are approximately 150 A, within the accepted 100 to 200 A range of other insect species studied (Steinbrecht, 1969). A single sensory neuron is commonly associated with the type I and type II thin-walled sensilla basoconica although 2 occasionally have been observed. Immediately above the cell body of the neuron, the dendrite abruptly narrows to about 0.3 ~m dia and assumes the typical ciliary ultrastructure with 9 pairs of symmetrically arranged peripheral fibrils. At the base of the ciliary region are the expected proximal and distal basal bodies with rootlets extending into the cell body of the neuron. The ciliary region of the face fly sensory neuron is shorter (only about 0.5/~m long) than that of most insect species studied. Above the very short ciliary collar, the dendrite again increases in diameter to approximately 0.5/xm. Numerous microtubules are the only organelles in this distal dendritic segment. The distal dendritic segments o f type I and type II thin-walled sensilla basiconica and the thick-walled sensilla trichodea previously described extend to t h e funicular surface unprotected by a cuticular sheath. Both the ciliary and distal dendritic segments are enclosed by a vacuole containing an electron dense material which may be the coagulum of a liquid (Slifer and Sekhon, 1964). The paired fibrils in the ciliary region of the dendrite apparently separate and become indistinguishable from the numerous microtubules throughout the distal dendritic segment. Although the number of microtubules increases beyond the 18 expected from separation of the 9 pairs of ciliary fibrils, there is some evidence for their continuity (Slifer and Sekhon, 1969). We observed paired and single tubules in the same cross section. The dendrite in the peg lumen also is surrounded by an electron dense material (Figs. 8-11). Since the peg lumen is continuous with the previously described vacuole below, the material probably also is the coagulated residue of a fluid, the "sensillenliquor" of Ernst (1969). As the dendrite enters the peg lumen, the microtubules assume a position on the periphery of the dendrite (Fig. 8). From that point, branching commences at various levels

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FtG. 5. C. S. of thick-walled sensillum trichodeum, Pore (P) penetrating cuticular wall and 2, unbranched dendrites (D) containing numerous microtubules. × 34,200 FIG. 6. Thin-walled sensillum basiconicum, type I. x 13,500

Antennal Olfactory Sensilla of the Face Fly

FIG. 7. Thin-walled sensillum basiconicum, type II. × 20,300 FIG. 8, C. S. of thin-walled sensillum basiconicum, type I, showing arrangement of microtubules (MT) at periphery of dendrite (D) before branching, x 33,100

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throughout the dendritic process (Figs. 9, 10). Each dendritic branch receives one microtubule as a central core. Branching continues until there are no remaining microtubules within the former dendritic process, which then decreases in size and becomes vacuolated (Fig. 11). Grooved Sensilla. The longitudinally grooved or fluted pegs, referred to be Dethier et aL (1963) as stellate or coronal pegs from their appearance in cross section, appear to be rather evenly distributed over the 3 funicular surfaces. They are less abundant than the thick-walled sensiUa trichodea or thin-walled sensilla basiconica, type I; but more numerous than the thin-walled sensilla basiconica, type II. Each peg has a short, smooth-walled base, about 1/~m long and 1 /zm dia, from which the fluted portion arises (Fig. 12). The entire sensillum averages approximately 5 tLm in length. The number of grooves around the peg circumference is constant at 16 basally, but all do not extend the entire length of the peg. Although fluted sensilla are usually described as being double cuticular-walled structures (Steinbrecht, 1969), face fly funicular fluted sensilla appear to have single cuticular walls approximately 0.1 /zm thick (Fig. 13). The fluted cuticular wall is interrupted between grooves by narrow canals 300/~ dia that appear in electron micrographs to be filled with an electron dense substance apparently continuous with that lining the peg lumen. Serial cross sections do not indicate a longitudinal continuity of canals throughout the entire length of the grooved portion of the sensillum. Steinbrecht (1969) offered proof of the olfactory nature of the fluted sensilla and tentatively speculated that such canals serve the same function as the pore-tubule system of other olfactory receptors. No dendritic filaments, as speculated by Slifer and Sekhon (1964), were detected entering the canals. The grooved sensilla of the face fly funiculus are innervated by 2 bipolar sensory neurons A ciliary region, 0.3 tzm dia and 0.5 tzm long, is located a short distance above the neuron cell body. Distal and proximal basal bodies, with rootlets extending toward the cell body, lie at the base of the cilium. The dendrite, containing numerous microtubules, increases in diameter to approximately 1/~m distal to the ciliary region. In the area of the ciliary collar, the dendrites of the 2 sensory neurons enter a cuticular sheath that surrounds the distal dendritic segments as they proceed toward the antennal surface. At the inner cuticular surface, the sheath expands in diameter to the approximate size of the peg base and continues into the peg lumen approximately 1/~m to a point on the peg wall apparently coinciding with the origin of the grooved portion of the peg. It is probable that the cuticular sheath is invaginated from the sensillum wall at that point. Pit sensilla

Compared with other muscoid flies studied, the face fly antenna has few olfactory pits. Antenna of the female contains 3 such pits; that of the male, only 2. This observation apparently conflicts with the general hypothesis of Liebermann (1926) that dung-feeding muscids which rely largely on odor to locate food contain numerous olfactory pits. Moreover, Liebermann reported males to be more abundantly supplied with pits than females, apparently because males use the olfactory sense to search for females. According to Dethier et al. (1963), from 9 to 11 pits are present on the basal flagellar subsegment of Phormia regina males and from 11 to 16 on that of females. The number is much greater for Sarcophaga argyrostoma as approximately 52 pits were counted on the antenna of a male and 261 on a female (Slifer and Sekhon, 1964). Two of the olfactory pits on the female face fly basal flagellar subsegment and one of those on the male are on the ventral surface (Fig. 2). That contrasts with the flesh fly whose

Antennal Olfactory Sensilla of the Face Fly

FIG. 9. C. S. of thin-walled sensillum basiconicum, type I, showing beginning of dendritic branching (D]3). x 34,000 FIG. 10. C. S. of thin-walled sensillum basiconicum, type I, showing further dendritic branching (DB). × 35,600

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FIG. 11. C. S. of thin-walled sensillum basiconicum, type I, showing numerous dendritic branches (DB). × 40,700 FIG. 12. Grooved sensillum. ×30,000

Antennal Olfactory Sensilla of the Face Fly

Fro. 13. C. S. of grooved sensillum containing 2 unbranched dendrites (D). × 72,000 Fta. 14. Large pit on ventral funicular surface of female face fly antenna. × 2,000

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pits were located more frontally (Slifer and Sekhon, 1964). All pits on the face fly atenna are on the proximal half of the basal flagellar subsegment. The ventral pits of both males and females contain numerous thin-walled sensilla basiconica similar to the thin-walled sensilla basiconica, type I and type II, on the funicular surface. Apparently, the antennae must be erected horizontally to permit maximal air flow to the pits as well as over the entire ventral surface. Such behavior has been observed by female face flies in earlier olfactometer tests with bovine feces. The other pit on the funiculus of both male and female flies is adjacent to the arista on the dorsolateral surface (Fig. 1). It contains only grooved sensilla and several pegs with filamentous tips not previously described. Ventralpits. The 2 pits on the ventral funicular surface of the female are usually in close proximity, although occasionally separated considerably. The size of the pit openings, especially the anterior ventral pit, also varies. It may be asymmetric, measuring from 20/zm wide to 50/xm long and enlarging internally to 30 #m wide and deep, or it may be nearly circular measuring approximately 20 tzm dia. Some cuticular ridges internally are frequent, especially in the more asymmetric pits where they create a multichambered effect. The circular pits contain up to 40 thin-walled sensilla basiconica morphologically similar to the thin-walled sensilla basiconica on the funicular surface, while the asymmetric shaped pits may contain up to 200 such sensilla (Fig. 14). In contrast to the anterior ventral pit, the posterior ventral pit nearly always has a circular opening approximately 15/zm dia (Fig. 1). Internally, the pit again enlar~ges to a width and depth of 25 to 30/xm, respectively, with neither cuticular infolding nor internally connected pits. The smaller posterior pit contains up to 20 thin-walled sensilla. The single pit on the ventral funicular surface of the male may correspond to the smaller posterior pit of the female. The thin-walled sensilla in the ventral pits of male and female flies appear morphologically identical. Individual sensilla are approximately 10/zm long and 1.5 tzm dia. The epicuticular wall is extremely thin, averaging only about 0.1 ~m thick. The sensillum surface is perforated by many minute pores (Figs. 15, 16), whose dimensions parallel those of the thinwalled sensilla basiconica on the surface. Pore density is approximately 50 per square micron. One bipolar sensory neuron is associated with the thin-walled pit sensilla whose fine structure is the same as previously described. Dorsolateral pit. The single pit adjacent to the arista on the dorsolateral funicular surface has a nearly circular opening with a diameter of 10 to 15 ~m (Fig. 2). Internally, the pit enlarges to about 20 ~m wide and approximately 25 tzm deep (Fig. 17). Four grooved sensilla, morphologically indistinguishable in size and structure from those on the funicular surface, are located in the pit along with 2 sensilla with filamentous tips not previously described (Figs. 17, 18). These 2 sensilla measure 1 t~m dia basally and are approximately 5/zm in length up to the region of the filamentous tip which is an additional 1"5 tzm long. Two unbranched dendrites are present in the peg lumen and a cuticular sheath appears to be invaginated from the peg base (Fig. 18). We could not determine the relationship of the dendritic processes to the external environment. An olfactory nature for such morphological construction is difficult to envision, but the location of the sensillum in the pit suggests no feasible alternative function. In summary, morphological evidence presented suggests that the basal flagellar subsegments of male and female face fly antennae bear only olfactory sense organs whose morphology is summarized in Table 1. Olfactory sensilla on the funicular surface consist of thick-walled sensilla trichodea and 2 types of thin-walled sensilla basiconica, all of

Antennal Olfactory Sensilla of the Face Fly

FIG. 15. Thin-walled pit sensiUa basiconica showing numerous pores. × 8,800 FIG. 16. Base of thin-walled pit sensillum showing dendrite (D) and dendritic branches (DB) within peg lumen. × 36,300

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FIG. 17. Pit on dorsolateral funicular surface of female face fly. Note sensillum with slender, filamentous tip (arrow). x 3,300 FI~. 18. L. S. of filamentous tip sensillum. Two unbranched dendrites (D) are within a cuticular sheath (CS). No opening evident, x 9,300

Antennal Olfactory Sensilla of the Face Fly

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TABLE ] . TYPES AND FINE STRUCTURE OF THE SENSILLA LOCATED ON THE FUNICULAR SURFACE OF THE FACE FLY ANTENNA

Sensillum

Sensillum

Pore

diameter

diameter

Pore density

(u)

(u)

(A)

(per u 2)

25

1-3

150-200

20

2-4

15

1.5

350-400

35

1-2

i0

1

350-400

60

1-2

!.5

350-400

50

1

length

Sensillum

No. of sense cells (per sensillum)

Surface

S. triehodeum Surface

S. basiconicum, Type I Surface

S. basieonicum, Type II Surface Grooved

sensillum

5

Pit S. basiconicum

i0

Pit Grooved

sensillum

5

1

6.5

1

Pit sensillum with filamentous tip

w h i c h have typical pore-tubule systems. In addition, fluted sensilla occur o n the surface o f the funiculus. Olfactory pits on the ventral surface have n u m e r o u s thin-walled sensilla basiconica m o r p h o l o g i c a l l y similar to those on the surface. A pit on the dorsolateral surface bears several fluted sensilla and 2 previously undescribed receptors with filamentous tips w h o s e m e t h o d o f exterior c o m m u n i c a t i o n could n o t be determined. As excising the basal ftagellar s u b s e g m e n t s destroys response to bovine feces (Bay and Pitts, in press), one or m o r e o f the receptors m u s t detect the olfactory stimuli elicitating attraction. The final identity o f the olfactory function of each sensillum m a y be resolved only electrophysiologically. REFERENCES BAY, D. E. and C. W. PtTTS. Olfactory responses of the face fly to bovine feces. Ann. EntomoL Soc. Amer. (In press.) DETmER, V. G., J. R. LARSENand J. R. ADAMS. 1963. The fine structure of the olfactory receptors of the blowfly, pp. 105-114. In Y. Zotterman (ed.), Olfaction and Taste. Pergamon Press, Oxford. ERNST, K. D. 1969. Die Feinstruktur yon Riechsensillen auf der Antenne des Aask~iffers, Necrophoms. Z. Zetl forsch. Mikrosk. Anat. 94: 72-102. GREENBERO, B. and N. AsH. 1972. Setiferous plaques on antennal pedicels of muscoid Diptera: appearance in various species and tests of function. Ann. Entomol. Soc. Amer. 65: 1340-46. HOOPER, R. L., C. W. PITTS, and J. A. WESTFALL.1972. Sense organs on the ovipositor of the face fly, Musca autumnalis. Ann. EntomoL Soc. Amer. 65: 577-86.

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LIEBnR~qN, A. 1926. Correlation zwischen den antennalen Deruchsorganen und der Biologic der Musciden. Z. Morphol. OkoL Tiere 5: 1-97. LUFT, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochern. CytoL 9: 409-14. MmLONIO, G. 1962. Further observations on a phosphate buffer for osmium solutions in fixation. Proc. 5th Int. Congr. Electron Microsc. 2: 8. MOHER~X¢, E. A. 1964. A study of the labellar contact chemoreceptors of the face fly, Musca autumnalis DeGeer. Unpublished Ph.D. Dissertation. Rutgers University. 113 pp. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17: 208-12. SCHNEIDER, D. and R. A. STEINBRECrIT. 1968. Checklist of insect olfactory sensilla. Syrup. ZooL Soc. Lond. 23: 279-97. SLIr~R, E. H. 1970. The structure of arthropod chemoreceptors. Annu. Rev. EntomoL 15: 121-42. SLIrER, E. H. and S. S. SEKHON. 1964. Fine structure of the sense organs on the antennal flagellum of a flesh fly, Sarcophaga argyrostoma R.-D. (Diptera, Sarcophagidae). or. MorphoL 114:185-207 SLIrER, E. H. and S. S. SE~ON. 1969. Some evidence for the continuity of ciliary fibrils and microtubules in the insect sensory dendrite, or. Cell Sci. 4: 525-40. STEINBRECHT, R. A. 1969. Comparative morphology of olfactory receptors, pp. 3-21. In C. Pfaffman (ed.), Olfaction and Taste, III. Rockefeller University Press, New York.