Z. Zellforsch. 117, 476484 (1971)
9 by Springer-Verlag 1971
Electron Microscopic Studies on the Palpi
of Cybister fimbriolatus fimbriolatus (Say)*
I. E x a m i n a t i o n o f t h e C u t i c u l a r S u r f a c e s b y M e a n s o f t h e S c a n n i n g
and Transmission Electron Microscopes
FUMIOKI YASUZUMI a n d SAKIMORI YAMAGUCHI
Department of Anatomy, Nara Medical University (Director: Prof. G. u
M. D.)
GERALD H. JOHNSON
Department of Wildlife and Fisheries, School of Natural Resources, University
of Michigan (Director: Prof. John E. Bardach, Ph. D.)
Received March 1, 1971
Summary. The maxillary palpi of the predaceous diving beetle Cybister /imbriolatus
/imbriolatus Say were observed by transmission and scanning electron microscopy.
Scanning electron microscopy shows at least two types of sensilla at the tip of palp,
which are referred to as "circumvallate and naked sensilla". The former are innervated
with outer segments of distal processes of sensory cells, but the latter are provided only
with chitin and cuticular substances.
Key.Words: Maxillary palpi - - Circumvallate sensilla - - Naked sensilla - - Sensory
cells - - Cybister/imbriolatus (beetle).
A large n u m b e r of studies necessary for u n d e r s t a n d i n g insect sense organs
h a v e been m a d e b y electron m i c r o s c o p y (e.g., A d a m s , 1961; Slifer, 1961; Slifer
a n d Sehkon, 1961; Stiirckow, 1962; Larsen, 1962, 1963; T h u r m , 1964; Slifer a n d
Sehkon, 1 9 6 4 a - c ; Slifer et al., 1964; Peters, 1965; Boeekh et al., 1965; I v a n o v ,
1966; T h u r m , 1966; Stfirckow, 1967; E r n s t , 1969; T o m i n a g a et al., 1969; I v a n o v ,
1969; Dallai, 1970; A l t n e r et al., 1970). H o w e v e r , t h e m a x i l l a r y p a l p of t h e predaceous diving beetle Cybister ]imbriolatus/imbriolatus S a y has n e v e r been observ e d in d e t a i l b y electron microscopic procedures, so far as t h e p r e s e n t a u t h o r s are
aware.
A l t h o u g h t h e t r a n s m i s s i o n electron microscope c o n t r i b u t e s to analyzis of t h e
fine s t r u c t u r e of tissue cells, sections used in t r a n s m i s s i o n electron m i c r o s c o p y
are r e m a r k a b l y thin a n d r e p r e s e n t only a small s a m p l e of t h e t o t a l tissue. Therefore, t h e y m a y n o t suffice in themselves to p r o v i d e an u n d e r s t a n d i n g of t h e t h r e e
d i m e n s i o n a l o r g a n i z a t i o n of t h e tissue. Biologically significant features, such as t h e
insect p a l p surfaces, especially t h e i r t i p surfaces, are so c o m p l e x t h a t their orient a t i o n a n d basic s t r u c t u r e s do n o t emerge from s t u d y of sections alone. Our
p r e s e n t i n c o m p l e t e knowledge of the s t r u c t u r e of insect sense organs seems to
* This work was supported by Grant NB 04687 from the National Institutes of Health
of the United States of America.
We wish to express our gratitude to Prof. H. Stanley Bennett, Laboratories for Reproductive Biology, School of Medicine, University of North Carolina, Prof. G. Yasuzumi,
Electron Microscope Research Laboratory, Department of Anatomy, Nara Medical University,
and Prof. John E. Bardach, Department of Wildlife and Fisheries, School of Natural
Resources, University of Michigan, for their valuable advice to the present work.
Palpi of Cybister/imbriolatus
477
derive in p a r t from i n a b i l i t y of sectioning techniques to r e v e a l f e a t u r e s of t h e
whole organ. Scanning electron m i c r o s c o p y p e r m i t s e x a m i n a t i o n of t h e surface
m o r p h o l o g y a n d t h u s reveals i m p o r t a n t aspects of t h e whole organ.
T h e p r e s e n t s t u d y a t t e m p t s to clarify t h e s t r u c t u r a l details of t h e surfaces
of m a x i l l a r y p a l p i of t h e p r e d a c e o u s d i v i n g beetle b y scanning a n d t r a n s m i s s i o n
electron microscopes.
Material and Methods
Techniques for Scanning Electron Microscopy: A pair of palpi of the predaceous diving
beetle Cybister/imbriolatus ]imbriolatus Say were carefully removed from their basal attachment. They were fixed with 6.25% glutaraldehyde for 1 hour at 4 ~ C, and subsequently
with 1% osmium tetroxide for 1 hour at 4 ~ C. Each fixative was adjusted to pH 7.2 with
0.1 M sodium cacodylate buffer. The fixed specimens were for 2 weeks stored in 70%
ethanol solution. After allowing the ethanol to dry in air, the specimens were coated with
a conductive layer of carbon 100-200 A thick followed by a layer of gold of equal thickness
in a vacuum evaporator. The specimens were examined in a Hitachi scanning electron
microscope, model HSM-2, at an accelerating voltage of 25 kV.
Techniques for Transmission Electron Microscopy: The specimens fixed as described were
dehydrated in a series of increasing concentrations of ethanol solutions and embedded
in Epon 812 (Luft, 1961). Sections were cut with an LKB ultrotome using glass knives,
mounted on Formvar-coated specimen grids and stained by the lead citrate method of
Reynolds (1963). These sections were examined in a Hitachi electron microscope, model
HU-11D-S or HU-12 at an accelerating voltage of 75--100 kV.
Observations
The m a x i l l a r y pulp of t h e p r e s e n t m a t e r i a l consists of four segments, in which
t h e distal s e g m e n t m e a s u r i n g a p p r o x i m a t e l y 1.0 m m in length a n d 0.3 m m in
m a x i m a l width, b u t t h e o t h e r ones 0.5 to 0.7 m m in l e n g t h a n d 0.37 m m in
m a x i m a l w i d t h (Fig. 1). I t is covered with the chitinous cuticular elements which
are m a d e u p of a set of h e x a g o n a l p l a t e s interlocking r e g u l a r l y w i t h one a n o t h e r
(Fig. 3). A small n u m b e r of microsetae are found, each being inserted in a punctule.
I t is n o t i c e d t h a t t h e seta is often t r u n c a t e d in several heights. The m i c r o s e t a
is o b s e r v e d i s o l a t e d a t several p o i n t s (Figs. 1, 2, 4), b u t occasionally in a group,
each h a v i n g a n i n d i v i d u a l punctule. I t looks to be a n t e n n a to catch electro-magnetic w a v e s (Fig. 3).
T h e surfaces of p a l p i are r e l a t i v e l y s m o o t h e x c e p t for t h e presence of microsetae, while t h e t i p of t h e d i s t a l s e g m e n t is n o t a l w a y s smooth, i t is r a t h e r
c o n c a v o - c o n v e x (Fig. 4). On t h e surfaces one can discern several t y p e s of sensi]la
basiconica, m o s t of t h e m n e a r t h e t i p of pulp (Fig. 5). One can recognize a t least
t w o t y p e s ; some s u r r o u n d e d b y a t h i c k circular walt, some not. The former are
referred to as " c i r c u m v a l l a t e sensilla", a n d t h e l a t t e r as " n a k e d s e n s i l l a " .
A m o n g s t t h e c i r c u m v a l l a t e sensflla, smaller c o n s t i t u e n t s t r u c t u r e s v a r y in t h e
shape, size a n d n u m b e r . Some sensilla are flame-like in shape, being s u r r o u n d e d
b y a deep, circular furrow, a r o u n d which is a wall. Others d i s p l a y several small
p r o j e c t i o n s s u r r o u n d e d b y a similar wall a n d m o a t (Fig. 6).
I n sections i t is possible to observe t h a t t h e o u t e r segments of distal processes
of sensory cells e x t e n d i n t o t h e central p r o j e c t i o n of t h e c i r c u m v a l l a t e sensillum
(Fig. 8). Such a s t r u c t u r e can n o t be seen in t h e n a k e d sensillum which is
p r o v i d e d only w i t h dense chitin a n d less dense cuticular elements, b u t which do
478
F. u
S. Yamaguchi and G. H. Johnson:
Palpi of
Cybister/imbriolatus
479
Fig. 3. Four microsetae different in length, each having individual punctule. The cuticular
surface is covered with a set of plates showing a hexagonal appearance. X 1400
Fig. 1. Scanning electron micrograph of a pair of the maxillary palpi of the predaceous
diving beetle, each consisting of four segments, x 49
Fig. 2. Enlarged micrograph of one of the distal segments in Fig. 1, showing microsetae
and an uneven surface of the tip of palp. X 140
32 Z. Zellforsch., Bd. 117
480
F. Yasuzumi, S. Yamaguchi and G. H. J o h n s o n :
Fig. 4. The tip surface of palp is markedly uneven. The microsetae truncated at their
apices arc visible in about eight punctules. • 480
Fig. 5. A part of the tip of palp. Numerous papillar structures are visible in a group. X 1400
Palpi of Cybister/imbriolatus
481
Fig. 6. The tip of palp is covered compactly with sensilla basiconica which are differentiated
at least into two varieties: circumvallate and naked, x 4 500
not display any cellular ones (Fig. 7). The wall surrounding the circumvallate
sensillum is approximately 0.7 ix thick and consists of a dense, homogeneous
chitinous material bounded by a less dense, lamellar cuticular layer. A member
of the epidermal cell complex (Larsen, 1963), the trichogen cell, is observed
just beneath the cuticular layer (Fig. 8).
Discussion
The scanning electron microscope provides an excellent opportunity to study
the structure of large pieces of insect sense organs within a wide range of magnification at a good resolution. I t is possible to investigate both natural surfaces
as well as those artificially produced by preparing techniques: for example, some
sensflla are flame-like in shape, being surrounded by a deep, circular furrow around
which is a wall; others display several small projections surrounded by similar
walls and moats. Such multiple features m a y be due to artifacts of preparative
techniques such as fixation, dehydration, air drying and others.
Since the scanning electron microscope provided three-dimensional pictures of
the palpi of the predaceous diving beetle, it was possible to obtain reconstructions
32*
Fig. 7. A longitudinal section through a sensillum without wall. I t is covered with a dense
chitin layer, and composed of chitin and cuticular elements. The lamellar cuticular layer
is situated beneath the sensillum. X 20000
Fig. 8. An oblique longitudinal section through a circumvallate sensillum which is surrounded
by a deep furrow and a dense wall. At least four outer segments of distal processes of the
sensory cells are enclosed in the cuticular sheath. The trichogen cell (TC) containing a large
number of dense mitochondria and vacuoles is encountered under the cuticular layer (CL).
x ll000
F. Yasuzumi, S. u
and G. H. Johnson: Palpi of Cybister/imbriolatus
483
f r o m pictures t a k e n b y t r a n s m i s s i o n electron microscopy. I t is believed t h a t these
two techniques p r e s e n t t o g e t h e r excellent possibilities for s t r u c t u r a l analysis of
biological tissue.
R e c e n t l y , the u l t r a s t r u c t u r a l o r g a n i z a t i o n of t h e c h e m o r e c e p t i v e a n t e n n a l
sensillum of t h e beetle Acilius sulcatus was o b s e r v e d b y t r a n s m i s s i o n m i c r o s c o p y
( I v a n o v , 1969). The u l t r a s t r u c t u r e of the c i r c u m v a l l a t e sensillum o b s e r v e d in t h e
p r e s e n t m a t e r i a l is v e r y similar to t h a t of Acilius sulcatus. More recently, t h e
p o s t a n t e n n a l sensillum of t h e collembola was s t u d i e d b y t r a n s m i s s i o n electron
m i c r o s c o p y b y A l t n e r et al. (1970), a n d t h e cuticular s t r u c t u r e of t h e h e a d a n d
a b d o m e n of such a n insect b y scanning electron m i c r o s c o p y (Dallai, 1970) separately. I t is impossible to pursue a relationship b e t w e e n two k i n d pictures t a k e n
b y different electron microscopic techniques. A characteristic insertion of microsetae o b s e r v e d in t h e collembola (Dallai, 1970) was n e v e r seen in t h e m a x i l l a r y
p a l p of t h e p r e s e n t material.
S c a n n i n g electron m i c r o s c o p y showed for t h e first t i m e a t least two t y p e s of
sensilla a t the tip of t h e m a x i l l a r y palp, c i r c u m v a l l a t e a n d n a k e d sensilla. The
f o r m e r were p r o v i d e d with o u t e r segments of d i s t a l processes of sensory cells, b u t
t h e l a t t e r only w i t h chitin a n d cuticular substances. Accordingly, t h e l a t t e r m a y
function as an a p p a r a t u s for s u p p o r t i n g the i n n e r v a t e d c i r c u m v a l l a t e sensilla.
Combined e x p e r i m e n t s with electron microscopic a n d electrophysiological
procedures m a y reveal m o r e s t r u c t u r a l a n d functional details t h a n could be
recognized b y each p r o c e d u r e respectively. Such results will be p u b l i s h e d elsewhere
in n e a r future.
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Fumioki Yasuzumi
Electron Microscope Research Lab.
Dept. of A n a t o m y
Nara Medical University
Kashihara City, Nara 634
Japan
Gerald H. Johnson
Dept. of Wildlife and Fisheries
School of Natural Resources
University of Michigan
1006 Natural Resources Building
Ann Arbor, Michigan 48104
U.S.A.