- Email: [email protected]
Permeability of tarsal sensilla in the tick Amblyomma Americanum L. (Acarina, Ixodidae)
TISSUE & CELL 1972 4 (1) 130-135 Published by Longman Group Ltd. Printed ilt Great Britahz
RAINER F. FOELIX*
PERMEABILITY OF TARSAL SENSILLA IN THE TICK A M B L Y O M M A A M E R I C A N U M L. (ACARINA, IXODIDAE) ABSTRACT. Ticks were submerged in silver-protein solution, prior to fixation for electron microscopy, in order to trace the pathway of molecules in supposed tarsal chemoreceptors. Sensilla with radially arranged cuticular canals (100 200 A in diameter) leading to the centrally located dendrites show silver granules insidc the canals and in the central lumen, thus directly making contact with the dendrites. Sensilla with large, plugged pores (1200 A_) exhibit an accumulation of silver granules in the pore openings but no granules (about 50 A. in diameter) were observed penetrating into the lumen. Apparently silver granules could diffuse in, but not through the material which suspends the pore plugs. It is suggested that this material corresponds to the 'pore tubules" in inscct olfactory sensilla and that it may play an essential role in transmitting a chcmical stimulus from the environment to the dendrites.
Introduction
RECENT fine structural studies of tarsal sensilla in the tick Amblyomma americammt L. (Foelix and Axtell, 1971, 1972) revealed several types of sensilla which most likely function as chemoreceptors, since pores could be demonstrated in their cuticular walls. The most unusual type sensillum has numerous large pores which are centrally plugged by a cuticle lens. It was of particular interest to find the pathway through which molecules could penetrate into the sensillum in order to stimulate the dendrites. A method using silver granules as a tracer substance was recently introduced by Ernst (1969) and successfully applied to olfactory sensilla of a beetle. The diffusion pathway of the silver granules can be verified in ultrathin sections upon inspection with the electron microscope Materials and Methods
The original method of Ernst (1969 and personal communication) using a 1-2% silver* Department of Entomology, North Carolina State University, Raleigh, N.C. 276607. Manuscript received 29 July 1971. I
protein solution (Protargol '~) at atmospheric pressure was modified by exposing the animals to a 'vacuum" while the silver-protein solution (1-5% ; Etablissement Roques, Paris) was slowly flowing into the container with live ticks (Amblyomma americanum, nymphs). The objective of this procedure was to avoid the formation of small air bubbles and thus to facilitate direct contact of the solution with the receptors. The aqueous silverprotein solution had been ultra-sonicated prior to use in order to produce silver granules of about 50 N in diameter. Ticks were kept submerged for 10 min (Exp. 2 : 6 0 rain) under very low pressure ('vacuum'), then returned to atmospheric pressure and remained submerged for another 15-30 min (Exp. 2 : 3 0 - 4 5 min). All specimens survived this treatment. After briefly rinsing in distilled water, they were immediately transferred into cold 5% glutaraldehyde, where the tarsi of the first legs were cut off and stored for 15 hours. Several changes of cacodylate buffer and postfixation in 1% OsO4 followed, before dehydration in ethanol series and embedding in hard Epon 812 over propylene oxide. Unstained sections were examined with a
129
30
FOEL1X
Fig. l. Small pore sensillnm from anterior pit of Haller's organ; cross-section, double-stained. Note spoke-wheel structure of the cuticle, the two dendrites in the central lumen and fine canals (arrow) passing through the spokes to the pore opening (P). lnterspaces (i) between cuticuJar spokes are tilled by an electron-dense fluid and by processes of an enveloping cell..:42,000. Fig, 2, Same type sensillum as in Fig, I, but simultaneously fixed in glutaraldehyde and OsOv Radially arranged canals appear more distinct: apparently pore canals can also be cross-connected (arrow). > 45,000. Fig. 3, Unstained cross-section of the same type sensillum as in Figs. 1 and 2. This sensillum had been exposed to a silver-protein solution prior to fixation. Note the silver granules utz route from the pore (P) to the central lumen (EL ,' 70,000. Fig. 4. Slit pore sensillum of the "4-group' on the tick's tarsus. A hexagonal pattern of slit pores connects one centrally located dendrite to the environment. Cross-section, double-stained. :. 47,000. Fig. 5. Unstained cross-section of same type sensillum as in Fig. 4 after exposure to silver-protein solution. Silver granules have accumulated in the central l~men and in the middle part of the canals. Due to the level of sectioning the inner part of the canals is not visible in this particular preparation. >, 46,000. Fig. 6. Large, plug-pore sensillum from the anterior pit of Haller's organ; crosssection, double stained. Numerous dendritic branches occupy the lmnen while the thick cuticle wall is perforated by plug-pores. ×23,000. Inset: Plug-pore at higher magnification. A cuticular lens is centrally suspended in the wide pore opening. The material connecting this plug to the pore wall is of lower electron density than the cuticle and shows short projections toward the inside. :49,000. Fig. 7. Large, plug-pore sensillum after exposure to silver-protein solution: crosssection, unstained. Note accumulation of silver granules in pore necks. -34,000. Inset: Plug-pore at higher magnification with silver granules accumulated in the pore neck region. >:85,000. Figs. 8-12. Thin-walled capsule sensilla with large plug-pores after exposure to silver-protein solution, All sections unstained. Fig. 8. Longitudinal section, All silver granules are retained in the pore necks and none of them contacts the dendritic branches (db) inside the lumen. Arrows indicate extensions of the material that suspends the pore plugs. , 83,000. Fig. 9. Tangential section. Pore plug (p) with surrounding suspending material, seen et1/'ace. Note that silver granule distribution is restricted to the area of the suspending material. "<97,000. Fig. 10. In some cases the material suspending the pore plug (p) extends far into the lumen and those extensions are in close apposition with dendritic branches (db). Silver granules do not penetrate into those extensions. > 108,000. Fig. 1 I. Heavy accumulation of silver granules in the pore neck region and in the material that suspends the plug (p). Apparently the pore is also filled with some extracellular substance (compare Fig. 6, inset). :,, 105,000. Fig. 12. Extended exposure time to silver-protein solution leads to break-down of certain pores (arrows) and silver granules can freely diffuse into the lumen. Note that silver granules remain accumulated in intact pores. >:20,000.
®
,h
/:)h
®
! !ii .,i
,
j
~
..~•
i'~' ......
~
~
~ ,,~.~:~ ~
~
~,~:~i.~,~~ ~ ;~ ~i~,~ ~ ,~ .: ~ ~ ~ :~ ~ ~,.~:7.~~ i~ ~ .... ~
.....
....
~,~::
•
~
: ~ ':,~!~,,,~~~::'~:,~,-'~" ~,
~:~
~~
~' ~
~:,:~,~, ~
~
~i/~
~::~ . . . . . . . .
~:~ .... :~,~,~19~i ~,~' ..... •
~
:!~
~
~,,,.~i~:,.~i:~~ ~ ....... :~ ~;:ii~:.~ ~, ~
~
db
~,: ~ ~ :~ : ~
~ ~ ~ ~ ~:i~,::,,iiii ~' :;.~-~:,.~i~,~::,.~: ~: ~
~
~i~i~iii~'i~:'i ~.'I~QIIQ/~ ~ ~ ~iii~~
•
~i:i~-:,~ • i~:~ ~ , ~ ' ~
~"~i
PERMEABILITY OF TICK SENSILLA Siemens Elmiskop 1A at 80 kV: silver deposits can be localized as solid black granules, which stand out clearly against the pale background of the tissue.
Results
Among the variety of tarsal sensilla only the two types of poroas sensilla were considered: (a) sensilla with small pores of 100-200 A width, which may form convoluted cuticular canals or narrow slit pores, and (b) sensilla with large, plugged pores (about 1200 /~ in diameter). (a) Small pore sensilla Sensilla with small tubular pores occur in the anterior pit of Haller's organ (A3, A5 ; Foelix and Axtell, 1972). Externally they resemble the "grooved pegs' of certain insects (Slifer, 1970), that is, the surface has several ridges and furrows. The cuticle of the bristle shaft forms a spoke-wheel structure; the center ('hub') is occupied by 2-3 dendrites, while canals pass through the 'spokes" to the furrows on the surface, thus establishing a connection between dendrites and the environment (Figs. 1 and 2). Those sensilla, which had been exposed to silver-protein solutions prior to fixation, show silver granules inside the canals, en route to the center (Fig. 3). There, srnall silver granules apparently can aggregate and form large, irregular masses. Another form of small pore sensilla is found proximal to Haller's organ on the dorsal side of the tick's tarsus ('4-group'; Foelix and Axtell, 1971). In this case the pores run as narrow slit openings (100-200 ~ ) along the bristle shaft; they are pentagonally or hexagonally arranged and have a peculiar 'vase'-shape (Fig. 4). One or two unbranched dendrites occupy the central lumen of this receptor. Silver granules can enter through the narrow slit pores and reach the central lumen (Fig. 5), where they can make contact with the dendrites. Although all ticks survived the manipulation of being submerged in a silver-protein solution for 30-105 rain, one must bear in mind that this exposure is quite unphysiological and certainly detrimental to at least some receptors. Indeed, many electron micrographs show severe effects on the
133 cellular parts, i.e. dendrites or processes of enveloping cells may become distorted or completely destroyed, Such sensilla exhibit a heavy accumulation of silver granules in all interspaces (i, Figs. 1 and 2) standing between the ~spokes', but this is thought to be an artifact. Sensilla which have been exposed for longer than 30-45 rain (Exp. 2) to the silver-protein solution under low pressure generally show more of such artifacts. (b) Large phtg-pore sensilla All large pores (1200 /~ in diameter) are provided with a central cuticle plug which is suspended by fine strands at the pore wall (Fig. 6). Plug-pore sensilla can be either thick-walled (0.5 t~; AI and A2 sensiIla of the anterior pit of Haller's organ) or extremely thin-walled (0"1-0.2 t~; capsule sensil la of Haller's organ) ; both types possess numerous branches of several dendrites (3-9) inside the lumen. In sensilla, which have been submerged in silver-protein solution for 25-40 rain (Exp. 1) the silver granules accumulate in the neck region of the pore, just above and around the cuticle plug (Figs. 7 and 9). No granules were ever found to diffuse into the lumen, though they apparently penetrate the material which suspends the pore plugs (Figs. 10 and ll). Exposure for 96105 min (Exp. 2) did not yield further penetration of the silver granules, except in those instances, where the pore plugs were destroyed; in these cases, of course, silver granules could freely diffuse into the sensillar lumen (Fig. 12). A striking difference between olfactory sensilla of insects (Ernst, 1969; Slifer, 1970; Steinbrecht, 1970) and the thin-walled capsule sensilla of ticks was the apparent lack of 'pore tubules' in tick sensilla (Foelix and Axtell, 1972). In insects, silver granules penetrate into the pore tubules and can be traced en route (Ernst, 1969; Chu and Axtell, 1971). At least in some micrographs (Figs. 8 and 10) it now appears as if similar structures are also present in the plug-pore sensillum of the tick. In rare cases extensions (about 140 in diameter) of the material that suspends the pore plug even seem to contact dendritic branches (Fig. 10). However, these structures do not occur consistently, nor are they clearly tubular. Perhaps most important, these extensions were never penetrated by even the smallest silver granules.
FOELIX
134
Discussion In small pore sensilla silver granules can penetrate into the central lumen via long cuticular canals. It can hence be assumed that molecules from the outside could directly interact with dendritic membranes. In contrast, silver granules were not found to enter the lumen of the plug-pore sensilla, but rather were retained in the pore necks: thus a direct contact with the dendrites is nowhere indicated. The silver-protein procedure is, of course, an artificial test situation, using relatively large particles. From previous experiments (Foelix and Axtell, 1972) it is known that the large, plug-pore sensilla are permeable to dyes and it might well be possible that odour molecules can diffuse into the lumen of the sensillum. Ernst (1969) considered the pore tubules as a stimulus transmitting system but did not decide whether odour molecules affect the dendrites directly or whether they diffuse first into the sensillar fluid ('Sensillenliquor'). There is now evidence that the pore tubules are often in direct connection with dendrites (Myers, 1968; Steinbrecht, 1970) and Steinbrecht (1970) has pointed out that the odour molecules do not necessarily have to diffuse into the sensillar fluid. This suggestion is in good agreement with Ernst's experiments (1969) using silver-protein and hemolymph, respectively, none of which entered the lumen but were retained in the pore tubule system. In very recent experiments Borg and Norris (1971b) used a radioactive labelled feeding stimulant (Ha-catechol) as a tracer on olfactory sensilla of a beetle. They claim that the stimulant actually penetrates into the lumen via the pore tubules. However, since the application of Ha-catechol was extremely artificial (beetles were bathed in 80°,; ethanol with Ha-catechol added) it is difficult to draw such explicit conclusions. Also, the limited resolution of autoradiographic techniques (Rogers, 1969) does not allow one to precisely identify the site of stimulation. The pore tubules, formerly believed to be
fine dendritic extensions (Slifer, 1967), have been shown to be an extracellular part of the cuticle wall (Ernst, 1969). More recently, Borg and Norris (1971a) described the pore tubules as invaginations of a continuous membrane which lines the inner side of the sensillum wall. We do not know the chemical nature of this "membrane" but it apparently differs from the normal cuticle. Ernst (1969) describes a swelling effect on the pore tubule material after exposure to distilled water; tick sensilla which had been submerged in an aqueous silver-protein solution for a longer period of time (Exp. 2) often show ruptured pores, due to damage to the material which suspends the pore plugs. It seems reasonable to draw an analogy between the material composing the pore tubules in insects and the substance that suspends the pore plugs in tick sensilla. At least in the thin-walled tick sensilla this material forms a separate layer on the inside of the cuticle wall and appears to be continuous; furthermore, extensions of this material protrude into the lumen, which are similar to the pore tubules in insects with respect to their diameter and relationship to dendrites. In any event, stimulants have to penetrate into this material and the experiments using small silver granules indicate this pathway. Whether stimulation in vivo occurs directly at those dendrites which are closely opposed to the material suspending the pore plug, or whether the molecules have to penetrate further into the lumen ('Sensillenliquor'), cannot be decided at present.
Acknowledgements This work was supported in part by a contract with the Office of Naval Research (R. C. Axtell, principal investigator) and PHS Training Grant ES 00069. Paper no. 3535 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh. The author thanks Drs R. C. Axtell, L. B. Coons and M. A. Roshdy for reviewing the manuscript.
PERMEABILITY
OF TICK
SENSILLA
135 Ret~renees
B~)RC,, T. K. and NORRIS, D. M. 1971a. Ultrastructure of sensory receptors on the antennae of Scolytus multistriatus (Marsh.). Z. ZellJorsch. mikrosk. Anat., 113, 13-28. BoaG, T. K. and NORRJS, D. M. 1971b. Penetration of H~-catecho}, a feeding stimulant, into chemoreceptor sensilla of Scolytus multistriatus (Coleoptera: Scolytidae). Ann. Entomol Soc. Amer., 64, 544-547. Cn~3, I-Wu and AXTHa., R. C. 1971. Fine structure of the dorsal organ of the house fly larva, Musca domestica L. Z. Zel![brsch. mikrosk, Anat., 117, 17 34. ERNST, K. D. 1969. Die Feinstruktur yon Riechsensillen auf der Antenne des Aaskfif~rs Necrophorus (Coleopteral. Z. Zellforsch. mikrosk. Anat., 94, 72-102. FOEL1X,R. F. and AXrELL, R. C. 1971. Fine strtmture of tarsal sensilla in the tick Amblyomma atttericanum (L.). Z. ZellJorsch. mikrosk. Anat., 114, 22 37. FowLlX, R. F. and AXTELL, R. C. 1972. Ultrastructure of Haller's organ in the tick Ambh, omma americattttm (L.I.Z. Zel(/brsch. mikrosk. Anat., 124, 275 292. MYERS, J. 1968. The structure of the antennae of the Florida queen butterfly, Danaus gi[ippe berenice (Cramer). J. Morph., 125, 315-328. ROGERS, A. W. 1969. Techniques o/'autoradiography, p. 62. Elsevier, Amsterdam, London, New York. SLIWR, E. H. 1967. Thin-walled olfactory sense organs on insect antennae. In htsects and Physiology (J. W. L. Beament and J. E. Treherne, editors). Oliver and Boyd, Edinburgh and London. SLIWR, E. H. 1970. The structure of arthropod chemoreceptors. Ann. Re)'. EtltomoL, 15, 121-142. STWINBRECHT, R. A. 1970. Stimulus transferring tubules in insect olfactory receptors. 7idme Congr. Int. Micr. Eh, ctr., Grenoble, 947 948.
RAINER F. FOELIX*
PERMEABILITY OF TARSAL SENSILLA IN THE TICK A M B L Y O M M A A M E R I C A N U M L. (ACARINA, IXODIDAE) ABSTRACT. Ticks were submerged in silver-protein solution, prior to fixation for electron microscopy, in order to trace the pathway of molecules in supposed tarsal chemoreceptors. Sensilla with radially arranged cuticular canals (100 200 A in diameter) leading to the centrally located dendrites show silver granules insidc the canals and in the central lumen, thus directly making contact with the dendrites. Sensilla with large, plugged pores (1200 A_) exhibit an accumulation of silver granules in the pore openings but no granules (about 50 A. in diameter) were observed penetrating into the lumen. Apparently silver granules could diffuse in, but not through the material which suspends the pore plugs. It is suggested that this material corresponds to the 'pore tubules" in inscct olfactory sensilla and that it may play an essential role in transmitting a chcmical stimulus from the environment to the dendrites.
Introduction
RECENT fine structural studies of tarsal sensilla in the tick Amblyomma americammt L. (Foelix and Axtell, 1971, 1972) revealed several types of sensilla which most likely function as chemoreceptors, since pores could be demonstrated in their cuticular walls. The most unusual type sensillum has numerous large pores which are centrally plugged by a cuticle lens. It was of particular interest to find the pathway through which molecules could penetrate into the sensillum in order to stimulate the dendrites. A method using silver granules as a tracer substance was recently introduced by Ernst (1969) and successfully applied to olfactory sensilla of a beetle. The diffusion pathway of the silver granules can be verified in ultrathin sections upon inspection with the electron microscope Materials and Methods
The original method of Ernst (1969 and personal communication) using a 1-2% silver* Department of Entomology, North Carolina State University, Raleigh, N.C. 276607. Manuscript received 29 July 1971. I
protein solution (Protargol '~) at atmospheric pressure was modified by exposing the animals to a 'vacuum" while the silver-protein solution (1-5% ; Etablissement Roques, Paris) was slowly flowing into the container with live ticks (Amblyomma americanum, nymphs). The objective of this procedure was to avoid the formation of small air bubbles and thus to facilitate direct contact of the solution with the receptors. The aqueous silverprotein solution had been ultra-sonicated prior to use in order to produce silver granules of about 50 N in diameter. Ticks were kept submerged for 10 min (Exp. 2 : 6 0 rain) under very low pressure ('vacuum'), then returned to atmospheric pressure and remained submerged for another 15-30 min (Exp. 2 : 3 0 - 4 5 min). All specimens survived this treatment. After briefly rinsing in distilled water, they were immediately transferred into cold 5% glutaraldehyde, where the tarsi of the first legs were cut off and stored for 15 hours. Several changes of cacodylate buffer and postfixation in 1% OsO4 followed, before dehydration in ethanol series and embedding in hard Epon 812 over propylene oxide. Unstained sections were examined with a
129
30
FOEL1X
Fig. l. Small pore sensillnm from anterior pit of Haller's organ; cross-section, double-stained. Note spoke-wheel structure of the cuticle, the two dendrites in the central lumen and fine canals (arrow) passing through the spokes to the pore opening (P). lnterspaces (i) between cuticuJar spokes are tilled by an electron-dense fluid and by processes of an enveloping cell..:42,000. Fig, 2, Same type sensillum as in Fig, I, but simultaneously fixed in glutaraldehyde and OsOv Radially arranged canals appear more distinct: apparently pore canals can also be cross-connected (arrow). > 45,000. Fig. 3, Unstained cross-section of the same type sensillum as in Figs. 1 and 2. This sensillum had been exposed to a silver-protein solution prior to fixation. Note the silver granules utz route from the pore (P) to the central lumen (EL ,' 70,000. Fig. 4. Slit pore sensillum of the "4-group' on the tick's tarsus. A hexagonal pattern of slit pores connects one centrally located dendrite to the environment. Cross-section, double-stained. :. 47,000. Fig. 5. Unstained cross-section of same type sensillum as in Fig. 4 after exposure to silver-protein solution. Silver granules have accumulated in the central l~men and in the middle part of the canals. Due to the level of sectioning the inner part of the canals is not visible in this particular preparation. >, 46,000. Fig. 6. Large, plug-pore sensillum from the anterior pit of Haller's organ; crosssection, double stained. Numerous dendritic branches occupy the lmnen while the thick cuticle wall is perforated by plug-pores. ×23,000. Inset: Plug-pore at higher magnification. A cuticular lens is centrally suspended in the wide pore opening. The material connecting this plug to the pore wall is of lower electron density than the cuticle and shows short projections toward the inside. :49,000. Fig. 7. Large, plug-pore sensillum after exposure to silver-protein solution: crosssection, unstained. Note accumulation of silver granules in pore necks. -34,000. Inset: Plug-pore at higher magnification with silver granules accumulated in the pore neck region. >:85,000. Figs. 8-12. Thin-walled capsule sensilla with large plug-pores after exposure to silver-protein solution, All sections unstained. Fig. 8. Longitudinal section, All silver granules are retained in the pore necks and none of them contacts the dendritic branches (db) inside the lumen. Arrows indicate extensions of the material that suspends the pore plugs. , 83,000. Fig. 9. Tangential section. Pore plug (p) with surrounding suspending material, seen et1/'ace. Note that silver granule distribution is restricted to the area of the suspending material. "<97,000. Fig. 10. In some cases the material suspending the pore plug (p) extends far into the lumen and those extensions are in close apposition with dendritic branches (db). Silver granules do not penetrate into those extensions. > 108,000. Fig. 1 I. Heavy accumulation of silver granules in the pore neck region and in the material that suspends the plug (p). Apparently the pore is also filled with some extracellular substance (compare Fig. 6, inset). :,, 105,000. Fig. 12. Extended exposure time to silver-protein solution leads to break-down of certain pores (arrows) and silver granules can freely diffuse into the lumen. Note that silver granules remain accumulated in intact pores. >:20,000.
®
,h
/:)h
®
! !ii .,i
,
j
~
..~•
i'~' ......
~
~
~ ,,~.~:~ ~
~
~,~:~i.~,~~ ~ ;~ ~i~,~ ~ ,~ .: ~ ~ ~ :~ ~ ~,.~:7.~~ i~ ~ .... ~
.....
....
~,~::
•
~
: ~ ':,~!~,,,~~~::'~:,~,-'~" ~,
~:~
~~
~' ~
~:,:~,~, ~
~
~i/~
~::~ . . . . . . . .
~:~ .... :~,~,~19~i ~,~' ..... •
~
:!~
~
~,,,.~i~:,.~i:~~ ~ ....... :~ ~;:ii~:.~ ~, ~
~
db
~,: ~ ~ :~ : ~
~ ~ ~ ~ ~:i~,::,,iiii ~' :;.~-~:,.~i~,~::,.~: ~: ~
~
~i~i~iii~'i~:'i ~.'I~QIIQ/~ ~ ~ ~iii~~
•
~i:i~-:,~ • i~:~ ~ , ~ ' ~
~"~i
PERMEABILITY OF TICK SENSILLA Siemens Elmiskop 1A at 80 kV: silver deposits can be localized as solid black granules, which stand out clearly against the pale background of the tissue.
Results
Among the variety of tarsal sensilla only the two types of poroas sensilla were considered: (a) sensilla with small pores of 100-200 A width, which may form convoluted cuticular canals or narrow slit pores, and (b) sensilla with large, plugged pores (about 1200 /~ in diameter). (a) Small pore sensilla Sensilla with small tubular pores occur in the anterior pit of Haller's organ (A3, A5 ; Foelix and Axtell, 1972). Externally they resemble the "grooved pegs' of certain insects (Slifer, 1970), that is, the surface has several ridges and furrows. The cuticle of the bristle shaft forms a spoke-wheel structure; the center ('hub') is occupied by 2-3 dendrites, while canals pass through the 'spokes" to the furrows on the surface, thus establishing a connection between dendrites and the environment (Figs. 1 and 2). Those sensilla, which had been exposed to silver-protein solutions prior to fixation, show silver granules inside the canals, en route to the center (Fig. 3). There, srnall silver granules apparently can aggregate and form large, irregular masses. Another form of small pore sensilla is found proximal to Haller's organ on the dorsal side of the tick's tarsus ('4-group'; Foelix and Axtell, 1971). In this case the pores run as narrow slit openings (100-200 ~ ) along the bristle shaft; they are pentagonally or hexagonally arranged and have a peculiar 'vase'-shape (Fig. 4). One or two unbranched dendrites occupy the central lumen of this receptor. Silver granules can enter through the narrow slit pores and reach the central lumen (Fig. 5), where they can make contact with the dendrites. Although all ticks survived the manipulation of being submerged in a silver-protein solution for 30-105 rain, one must bear in mind that this exposure is quite unphysiological and certainly detrimental to at least some receptors. Indeed, many electron micrographs show severe effects on the
133 cellular parts, i.e. dendrites or processes of enveloping cells may become distorted or completely destroyed, Such sensilla exhibit a heavy accumulation of silver granules in all interspaces (i, Figs. 1 and 2) standing between the ~spokes', but this is thought to be an artifact. Sensilla which have been exposed for longer than 30-45 rain (Exp. 2) to the silver-protein solution under low pressure generally show more of such artifacts. (b) Large phtg-pore sensilla All large pores (1200 /~ in diameter) are provided with a central cuticle plug which is suspended by fine strands at the pore wall (Fig. 6). Plug-pore sensilla can be either thick-walled (0.5 t~; AI and A2 sensiIla of the anterior pit of Haller's organ) or extremely thin-walled (0"1-0.2 t~; capsule sensil la of Haller's organ) ; both types possess numerous branches of several dendrites (3-9) inside the lumen. In sensilla, which have been submerged in silver-protein solution for 25-40 rain (Exp. 1) the silver granules accumulate in the neck region of the pore, just above and around the cuticle plug (Figs. 7 and 9). No granules were ever found to diffuse into the lumen, though they apparently penetrate the material which suspends the pore plugs (Figs. 10 and ll). Exposure for 96105 min (Exp. 2) did not yield further penetration of the silver granules, except in those instances, where the pore plugs were destroyed; in these cases, of course, silver granules could freely diffuse into the sensillar lumen (Fig. 12). A striking difference between olfactory sensilla of insects (Ernst, 1969; Slifer, 1970; Steinbrecht, 1970) and the thin-walled capsule sensilla of ticks was the apparent lack of 'pore tubules' in tick sensilla (Foelix and Axtell, 1972). In insects, silver granules penetrate into the pore tubules and can be traced en route (Ernst, 1969; Chu and Axtell, 1971). At least in some micrographs (Figs. 8 and 10) it now appears as if similar structures are also present in the plug-pore sensillum of the tick. In rare cases extensions (about 140 in diameter) of the material that suspends the pore plug even seem to contact dendritic branches (Fig. 10). However, these structures do not occur consistently, nor are they clearly tubular. Perhaps most important, these extensions were never penetrated by even the smallest silver granules.
FOELIX
134
Discussion In small pore sensilla silver granules can penetrate into the central lumen via long cuticular canals. It can hence be assumed that molecules from the outside could directly interact with dendritic membranes. In contrast, silver granules were not found to enter the lumen of the plug-pore sensilla, but rather were retained in the pore necks: thus a direct contact with the dendrites is nowhere indicated. The silver-protein procedure is, of course, an artificial test situation, using relatively large particles. From previous experiments (Foelix and Axtell, 1972) it is known that the large, plug-pore sensilla are permeable to dyes and it might well be possible that odour molecules can diffuse into the lumen of the sensillum. Ernst (1969) considered the pore tubules as a stimulus transmitting system but did not decide whether odour molecules affect the dendrites directly or whether they diffuse first into the sensillar fluid ('Sensillenliquor'). There is now evidence that the pore tubules are often in direct connection with dendrites (Myers, 1968; Steinbrecht, 1970) and Steinbrecht (1970) has pointed out that the odour molecules do not necessarily have to diffuse into the sensillar fluid. This suggestion is in good agreement with Ernst's experiments (1969) using silver-protein and hemolymph, respectively, none of which entered the lumen but were retained in the pore tubule system. In very recent experiments Borg and Norris (1971b) used a radioactive labelled feeding stimulant (Ha-catechol) as a tracer on olfactory sensilla of a beetle. They claim that the stimulant actually penetrates into the lumen via the pore tubules. However, since the application of Ha-catechol was extremely artificial (beetles were bathed in 80°,; ethanol with Ha-catechol added) it is difficult to draw such explicit conclusions. Also, the limited resolution of autoradiographic techniques (Rogers, 1969) does not allow one to precisely identify the site of stimulation. The pore tubules, formerly believed to be
fine dendritic extensions (Slifer, 1967), have been shown to be an extracellular part of the cuticle wall (Ernst, 1969). More recently, Borg and Norris (1971a) described the pore tubules as invaginations of a continuous membrane which lines the inner side of the sensillum wall. We do not know the chemical nature of this "membrane" but it apparently differs from the normal cuticle. Ernst (1969) describes a swelling effect on the pore tubule material after exposure to distilled water; tick sensilla which had been submerged in an aqueous silver-protein solution for a longer period of time (Exp. 2) often show ruptured pores, due to damage to the material which suspends the pore plugs. It seems reasonable to draw an analogy between the material composing the pore tubules in insects and the substance that suspends the pore plugs in tick sensilla. At least in the thin-walled tick sensilla this material forms a separate layer on the inside of the cuticle wall and appears to be continuous; furthermore, extensions of this material protrude into the lumen, which are similar to the pore tubules in insects with respect to their diameter and relationship to dendrites. In any event, stimulants have to penetrate into this material and the experiments using small silver granules indicate this pathway. Whether stimulation in vivo occurs directly at those dendrites which are closely opposed to the material suspending the pore plug, or whether the molecules have to penetrate further into the lumen ('Sensillenliquor'), cannot be decided at present.
Acknowledgements This work was supported in part by a contract with the Office of Naval Research (R. C. Axtell, principal investigator) and PHS Training Grant ES 00069. Paper no. 3535 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh. The author thanks Drs R. C. Axtell, L. B. Coons and M. A. Roshdy for reviewing the manuscript.
PERMEABILITY
OF TICK
SENSILLA
135 Ret~renees
B~)RC,, T. K. and NORRIS, D. M. 1971a. Ultrastructure of sensory receptors on the antennae of Scolytus multistriatus (Marsh.). Z. ZellJorsch. mikrosk. Anat., 113, 13-28. BoaG, T. K. and NORRJS, D. M. 1971b. Penetration of H~-catecho}, a feeding stimulant, into chemoreceptor sensilla of Scolytus multistriatus (Coleoptera: Scolytidae). Ann. Entomol Soc. Amer., 64, 544-547. Cn~3, I-Wu and AXTHa., R. C. 1971. Fine structure of the dorsal organ of the house fly larva, Musca domestica L. Z. Zel![brsch. mikrosk, Anat., 117, 17 34. ERNST, K. D. 1969. Die Feinstruktur yon Riechsensillen auf der Antenne des Aaskfif~rs Necrophorus (Coleopteral. Z. Zellforsch. mikrosk. Anat., 94, 72-102. FOEL1X,R. F. and AXrELL, R. C. 1971. Fine strtmture of tarsal sensilla in the tick Amblyomma atttericanum (L.). Z. ZellJorsch. mikrosk. Anat., 114, 22 37. FowLlX, R. F. and AXTELL, R. C. 1972. Ultrastructure of Haller's organ in the tick Ambh, omma americattttm (L.I.Z. Zel(/brsch. mikrosk. Anat., 124, 275 292. MYERS, J. 1968. The structure of the antennae of the Florida queen butterfly, Danaus gi[ippe berenice (Cramer). J. Morph., 125, 315-328. ROGERS, A. W. 1969. Techniques o/'autoradiography, p. 62. Elsevier, Amsterdam, London, New York. SLIWR, E. H. 1967. Thin-walled olfactory sense organs on insect antennae. In htsects and Physiology (J. W. L. Beament and J. E. Treherne, editors). Oliver and Boyd, Edinburgh and London. SLIWR, E. H. 1970. The structure of arthropod chemoreceptors. Ann. Re)'. EtltomoL, 15, 121-142. STWINBRECHT, R. A. 1970. Stimulus transferring tubules in insect olfactory receptors. 7idme Congr. Int. Micr. Eh, ctr., Grenoble, 947 948.