Surface Ultrastructure of the Egg Chorion of Eri Silkworm, Samia ricini (Donovan) (Lepidoptera: Saturniidae)
The eggshell of the eri moth, Samia ricini (Donovan) (Lepidoptera: Saturniidae) was investigated by scanning electron microscopy. The surface of the egg chorion of eri moth, revealed the structural elements viz., the micropylar rosette surrounding the micropyle, micropylar canals, shell imprints, aeropyles and the regional differentiation at the different poles. The oval shaped eggs were measured 820-990 (878.72±21.23) μm in length. The highly decorated chorion of S. ricini had a micropylar rosette of 45-51 (48.0±1.02) μm diameter with a centrally located micropylar pit of 2.08-2.31 (2.21±0.036) μm diameter. The micropylar pit consists of seven micropylar openings, each micropylar opening ranged from 0.26-0.28 (0.27±0.003) μm in diameter. Each micropylar opening opens into the seven distinct micropylar canals. The micropylar apparatus encircled by 11-14 petal-shaped primary unequal cells. These cells ranged from 12.0-12.9 (12.48±0.13) μm in length forming an asymmetrical rosette. The secondary petal-shaped cells were short in length and measured 8.4-9.1 (8.72±0.12) μm surrounded the primary petal cells. The entire surface of the chorion had a reticulate pattern of pentagonal and hexagonal cells, each boarded by 4-6 aeropyles of diameter 0.22-0.25 (0.23±0.05) μm. Further, the sunken aeropyles were also observed for the first time in second hemisphere of the egg and measured 0.22-0.24 (0.23±0.003) μm in diameter.
The insect eggs are characterized by an outer shell secreted by the follicular
epithelium during the process of egg formation and provide strong and elastic
mechanical protection to the developing embryo besides allowing air for respiration
at the aeropylar regions. Another specialized area of the egg chorion called
micropyle paves the way for the penetration of sperm (Leuckart, 1855). The morphology
and architectural pattern of the egg chorion in different insects depend on
the imprints of the follicular secretory cells on specialized regions such as
micropyle, aeropylar, stripe and flat regions (Beament, 1948; Sakaguchi et
al., 1973; Kafatos et al., 1977; Regier et al., 1980). The
structures of insect eggshells are usually quite complex and a typical insect
egg capsule consists of the vitelline envelope and chorion (Kumar et al.,
2002a). The sculpturing of the outer part of envelope appears to be specific
and is of considerable interest because of potential taxonomic applications
(Rosciszewska, 1991). Further, the basic knowledge of insect eggshell structure
and function, in particular of Lepidoptera, was provided by Leuckart (1855)
and Korschelt (1887). Light Microscope (LM) and physiological studies of gas
exchange and permeability (Beament, 1948; Wigglesworth and Beament, 1950) preceded
a second phase of basic research that began with the establishment of transmission
(TEM) and Scanning Electron Microscopy (SEM). Since then, the fine structure
of a large number of lepidopteran eggs has been studied with particular emphasis
on surface sculpturing (Matheny and Heinrichs, 1972; Mazzini, 1974; Downey and
Allyn, 1980, 1981; Hill, 1982; Salkeld, 1983, 1984; Fehrenbach et al.,
1987; Arbogast et al., 1989; Kumar et al., 1999; Kumar et al.,
2002b; Kumar et al., 2003). The high resolution and 3-dimensional clarity
of the scanning electron microscope make it an invaluable tool for studying
diagnostic characters of insects. Hinton (1969) used scanning electron micrographs
to illustrate respiratory systems of various insect eggshells. SEM studies revealed
the egg surface to be regionally differentiated (Kafatos et al., 1977;
Margaritis et al., 1980), also allowing a better distinction between
closely related species than by Light Microscope (LM) (Arbogast et al.,
The surface structure of lepidopteran eggs, as revealed by scanning electron microscopy, provides reliable characters for separation of species (Arbogast et al., 1989) and the external morphology of a wide variety of insect eggs has been examined using different techniques (Hinton, 1981). A taxonomy key has been formulated based on the egg chorionic characteristics and its architecture in sod webworms (Matheny and Heinrichs, 1972).
India is the only country, which produces all four kinds of silk viz., mulberry, eri, tasar and muga and the second highest country in the total production of silk, after China. The eri silk is produced by Samia ricini (Donovan) and is an economically important insect for sericulture point of view. Here a study has been taken on the surface ultrastructure on the egg chorion of eri silkworm, Samia ricini (Donovan) using scanning electron microscope. The results of the study reported here describing the surface ultrastructure of the egg chorion of Samia ricini (Donovan) may find use in the study of taxonomy and phylogeny of the insect.
MATERIALS AND METHODS
A laboratory culture of Samia ricini (Donovan) was established from adults that emerged from green pupae. Freshly emerged male and female adults were released in a cage (28x18 cm) and were provided with a 5% sucrose solution for feeding and fresh caster branches for egg layings. After mating, the freshly laid eggs were gently removed from the caster leaves using a fine tipped hair brush.
For scanning electron microscopic study, the eggs were fixed for two hours at room temperature in 2.5% glutaraldehyde prepared in 0.2 M cacodylate buffer (pH = 7.2), dehydrated in a graded alcohol-acetone series and dried in a critical point drier (EMS-850) using CO2 as the transition fluid. The dried samples were mounted onto copper stubs and coated with gold (20 nm thickness) in a Sputter coater (EMS-550) and examined using a JEM 100 CX II electron microscope fitted with ASID 4D attachments (JEOL Ltd., Tokyo, Japan) at 20 kV.
RESULTS AND DISCUSSION
Gravid females of Samia ricini laid eggs on the lower surface of its
food plant leaves. The eggs were found deposited upright serially arranged in
6 to 36 clutches of 317 to 510 eggs. They were attached to the leaf with their
posterior poles and among each other with their lateral sides, by a sticky secretion
(Fig. 1-5). The freshly laid eri eggs were
slight white in colour. As the embryo developed inside the egg, the colour of
the shell changed from whitish to yellowish, yellowish to ashy and finally ashy
to blackish just before hatching (Fig. 2). The eri eggs were
of medium size compared to mulberry or muga egg and have oval shape. Fehrenbach
et al. (1987) have investigated the eggs of three lepidopteran moths
by scanning (SEM) and transmission (TEM) electron microscopy, which revealed
that the eggs of Heliothis virescens were standing type (upright) and
ca 550 μm in diameter and ca 600 μm in length, whereas
the eggs of Spodoptera littoralis was also standing type and almost spheroid
with a diameter of 500-550 μm and slightly less in length.
||A single egg (Eg) of Samia ricini showing anterior
end (Ae), posterior (Pe) and anteriorly located micropyle (Mp) and the shell
imprints (Si) on the surface. (Scale bar = 190 μm)
||A neonate larva (L) emerges out through the anterior end.
Arrows indicate the anterior cut surface of the egg (Eg). (Scale bar = 120
However, the eggs of Cydia pomonella belonged to the lying (flat) type
which measured ca 1350 μm in length and ca 1050 μm
in width. Arbogast et al. (1989) studied the eggs of three tineid pests
under scanning electron microscope and found the eggs of Tinea pallescentella
Stainton as subcylindrical, 0.61-0.67 (0.64±0.02) mm long and 0.31-0.35
(0.33±0.01) mm in diameter at the widest point. But the eggs of Tinea
occidentella Chambers was subcylindrical, 0.61-0.65 (0.62±0.01) mm
long and 0.36-0.39 (0.38±0.01) mm in diameter at the widest point, bluntly
rounded at both ends; the anterior end usually broader than the posterior. Though
the eggs of Niditinea fuscella (L.) were also found subcylindrical their
anterior end was bluntly rounded and the posterior end more acutely rounded,
but occasionally ellipsoid, 0.42-0.50 (0.45±0.02) mm long with 0.26-0.30
(0.28±0.01) mm in diameter at the widest point (Arbogast et al.,
1989). Hinton (1981) emphasized that in most Lepidoptera, the egg is a prolate
or more or less spherical or rarely hemispherical.
||Vacated egg shows the anterior opening (Ao), which was cut
open irregularly by the hatched larvae. Arrow head shows the inner surface
(Is), The detached lateral side (Left sides: single arrow head in Fig. 3)
is also shown with glued substances. (Scale bar = 120 μm for Fig.
3; 60 μm for Fig. 4 and 30 μm for Fig.
He also revealed that the size and shape of the eggs of certain insect like
a tortricid, Rhyaciona duplana (Hub.) may also vary slightly according
to the species of pine on which they are laid. In any insect eggshell there
are three major layers viz., vitelline membrane, the endochorion and the third
one is occasionally absent (Margaritis, 1985). The protection against environmental
hazards during embryogensis is one of the functions of the eggshell.
The eggs of S. ricini measured 820-990 (878.72±21.23) μm in length. Matheny and Heinrichs (1972) have studied the egg chorion of 15 species of Lepidoptera moths and measured 0.598±0.002 μm in Thaumatopsis edonis, 0.425±0.009 mm in Chrysoteuchia topiaria and 0.537±0.009 μm in Pediasia trisecta. Recently, Kumar et al. (1999) observed the eggs of a lepidopteran moth, Spilarctia obliqua using electron microscope and measured 600-650 μm in diameter at the widest point. Further, Kumar et al. (2003) studied the fine structure of the egg of a lepidopterous moth, Amata passalis and measured the eggs 488±0.595 μm in diameter under scanning electron microscope.
The eggshell of S. ricini has a highly decorated chorion and its micropylar
apparatus is located at the anterior pole of the egg (Fig. 1,
7-16). Hinton (1981) stated that the micropyles
are without exception at the anterior pole of the lepidopteran eggs. Sakaguchi
et al. (1973) have described the arrangement of follicular imprints of the
micropylar and aeropylar regions of the egg chorion of Bombyx mori. In
recent past many workers also observed the micropylar apparatus at the anterior
end in the egg chorion of lepidopterous moths (Arbogast et al., 1989;
Fehrenbach et al., 1987; Fehrenbach, 1989; Kumar et al., 1999,
2003). However, an unusual position of the micropylar structure was found on
the lower surface of an egg in a lepidopterous moth Cydia pomonella (Fehrenbach
et al., 1987). A micropylar rosette consisting of micropylar openings
encircled with primary petal-shaped cells, which are followed by the secondary
petal-shaped cells, was observed in S. ricini. The micropylar rosette
of S. ricini ranged 45-51 (48.0±1.02) μm in diameter (Fig.
8). Fehrenbach et al. (1987) have observed a slightly bigger micropylar
rosette ca 65 μm in diameter in Heliothis virescens, 55 μm
in Spodoptera littoralis and about 50 μm in Cydia pomonella.
A rosette of 11-14 petal-shaped primary cells surrounds the micropylar opening
of S. ricini (Fig. 8-10, 13).
The primary cells were again surrounded by a second row of 15-19 petal-shaped
cells of unequal sizes (Fig. 8). The number of primary cells
around the micropylar pit is 15-19 in A. passalis (Kumar et al.,
2003), 10-14 in S. obliqua (Kumar et al., 1999), 5-7 in T.
pallescentella, 5-8 in T. occidentella and 6-9 in N. fuscella
(Arbogast et al., 1989).
||The posterior end of the egg showing its surface detached
form host leaves (arrows). The membranous structure (Ms) which adheres to
the leaf is connected to the egg by short vertical bars (arrow heads). (Scale
bar = 30 μm)
||General view of anterior egg pole revealing the rosette (Mr)
of eleven primary petals follwed
by fifteen secondary petals (*) and centrally located micropyle (Mp). Sometime
a protuberance was also observed on the rosette cells. (Scale bar = 20 μm
for Fig. 7; 12 μm for Fig. 8; 7.5
μm for Fig. 9 and 6 μm for
However, in some eggs of N. fuscella the primary cells were found to
be of subequal length forming symmetrical rosettes. Fehrenbach et al.
(1987) described the fine structure of three lepidopteran moth eggs and explained
that in Heliothis virescens, the micropylar rosette is composed of 13-15
petal-shaped primary cells with 4 or 5 micropylar canals opening into the micropylar
plate, whereas in Spodoptera littoralis the number of the rosette petals
varies between 6 and 11, 7 and 8 being the most frequent and with 3-4 micropylar
openings present in the slightly depressed rosette center. In Cydia pomonella
the micropylar rosette is composed of 8 or 9 rosette petals.
In a few eggs of S. ricini, the primary cells were subequal in length,
arranged in a symmetrical rosette, but more often, some primary cells were longer
than others and formed an asymmetrical rosette (Fig. 8 and
13). Similar asymmetrical rosettes of unequal petal-shaped
cells have been reported in the eggs of six other lepidopteran moths viz.,
Tinea pallescentella, T. occidentella, Niditinea fuscella
(Arbogast et al., 1989), Spilarctia obliqua (Kumar et al.,
1999) and Amata passalis (Kumar et al., 2003).
The length of each primary rosette cells of S. ricini eggs ranged 12-12.9
(12.48±0.13) μm and some of the cells were observed with a protuberance
on the distal end of the cell (Fig. 9-11).
The secondary petal-shaped cells were found short in length and measured 8.4-9.1
(8.72±0.12) μm (Fig. 8 and 9).
||Arrow head shows the petal cell with a single protuberance
(Pb) while the adjacent petal cells were observed without protuberance (arrows).
(Scale bar = 3 μm)
||Top view of egg pole showing micropylar region entrally located
micropyle (Mp) and seven micropylar canals (Mc). (Scale bar = 1 μm)
||A top view of the other egg shows its micropylar rosette (Mr)
with fourteen petal cells surrounding the micropyle (Mp). Scale bar = 7.5
Each micropylar pit was 2.08-2.31 (2.21±0.036) μm in diameter and
had seven distinct micropylar openings ranged from 0.26-0.28 (0.27±0.003)
μm diameters and each micropylar opening radiates down into micropylar
canal in micropylar plate in the eggs of S. ricini (Fig.
14-16). Kumar et al. (1999) studied 50 eggs of
S. obliqua under SEM and found interestingly only a single egg had three
micropylar openings whereas in all other eggs the micropylar apparatus had four
micropylar openings (micropylae). Hinton (1981) described that in Lepidoptera;
eggs have usually four micropyles, but may have more also. In eggs of 15 species
of Notodontidae studied by him, the number of micropyles varied from 4 to 20.
The number sometimes varies considerably in same species, e.g., in a few shells
of Cerura vinula, the number of micropyles varied from 16 to 20. Study
of micropyles has been a favorite subject since early times. Muller (1938) gave
a very good description of the 4 micropyles of the pyralid, Plodia interpunctella
and also described how they develop. Fehrenbach (1989) studied the fine structure
of eggshells of 4 primitive moths and revealed that the egg chorion of Hepialis
hecta and Wiseana umbracula had a different egg surface, with two
micropylar openings at the opposite sides of the longer axis of the oval micropylar
plate. In Mnesarchaea fusitella 2-3 oval micropylar openings were seen
at the base of the micropylar rim. Matheny and Heinrichs (1972) studied the
egg chorion of 15 species of sod webworm moth eggs (Lepidoptera) and developed
a taxonomic key for egg identification.
In S. ricini the rosette of two rows of petal-shaped cells at the top
and anterior pole of eggs is followed by the dense ridged shell imprints which
cover rest of egg surface (Fig. 7-10, 19
and 22) except at the posterior pole where the egg was attached
to the substratum (Fig. 6, 7, 20
and 21). Further, it was also observed that each shell imprint
bears a centrally located blunt protuberance (Fig. 8-10).
Though the posterior poles of egg sometime do not have the shell imprints but
the blunt protuberances were observed in that area (Fig. 17).
Similar to the observation on S. obliqua (Kumar et al., 1999),
the shell imprints were mostly pentagonal or hexagonal in shape and measured
9.0-9.6 (9.24±0.10) μm in diameter in S. ricini. Generally
it was found that 4 to 6 aeropyles of the size of 0.22-0.25 (0.23±0.05)
μm were located on the ridges of each shell imprints, which transverse
the shell down to the trabecular layer, on the anterior and middle region of
the egg (Fig. 18 and 19), however, the
posterior surface, which was observed with or without shell imprints, have the
aeropyles in a pit (sunken aeropyles) (Fig. 20 and 21).
The diameters of sunken aeropyles were measured 0.22-0.24 (0.23±0.003)
μm. In S. ricini the total number of aeropyles on a single egg is
too high to be counted. Matheny and Heinrichs (1972) have investigated the egg
chorion of 15 species of sod webworm moths, using scanning electron microscope
and revealed a smallest aeropyle i.e., 1.46±0.07 μm in Agriphila
ruricollatta whereas the largest aeropyles were 3.03±0.16 μm
in diameter in Crambus lequealellus. Fehrenbach et al. (1987)
have reported about 50 aeropyles per egg measuring ca 1.9 μm in
diameter in Heliothis virescens, ca 400 aeropyles per egg in Spodopters
littoralis measuring about 0.9 μm wide, whereas the number of aeropyles
was 140 per egg and ca 0.6 μm in diameter in the eggs of Cydia
pomonella (Fig. 23 and 24). Arbogast
et al. (1989) reported the aeropyles ranging from 0.67-1.20 (0.94±0.15)
μm in T. pallescentella, 0.55-0.93 (0.77±0.04) μm in
T. occidentella and 0.65-1.50 (1.10±0.36) μm in Niditinea
fuscella. Recently, Kumar et al. (2003) observed the aeropyles of
Amata passalis which measured 0.36±0.08 μm in diameter. However,
the aeropyles were not observed on the first two rows of cells at the micropylar
zone in S. ricini, as in the eggs of S. obliqua (Kumar et al.,
1999) and S. littoralis (Fehrenbach et al., 1987). The panels
of shell imprints adjacent to the micropylar zone in S. ricini have less
number of aeropyles, whereas the shell imprints which are away from micropylar
zone are completely boarded by aeropyles as in S. obliqua (Kumar et
al., 1999). In T. occidentella aeropyles are distributed over the
entire surface of egg whereas in T. pallescentella and N. fuscella
they are restricted towards the anterior and posterior end of the eggs.
(Arbogast et al., 1989). The number of aeropyles in S. obliqua
varies from 18 to 47 on a single shell imprint; however, some large aeropyles
were also observed which may be formed by the fusion of two adjacent small aeropyles
(Kumar et al., 1999).
||The micropyle (Mp) reveals the distinct seven-micropylar openings
(Mo), which further radiates down into the micropylar canal. (Scale bar
= 2 μm for Fig.14; 1.2 μm for Fig.
15 and 0.4 μm for Fig. 16)
||Posterior end (Pe) of an egg revealing a large number of protuberances
(Pb) with out definite boundary of shell imprints. (Scale bar = 30 μm)
||Middle region of an egg revealing the shell imprints (Si)
and centrally located protuberance (Pb). The aeropyle were observed on the
shell imprints (Si). (Scale bar = 20 μm for Fig. 18
and 6 μm for Fig. 19)
||Sunken aeropyles (SAp) were observed on the posterior region
of an egg. The sunken aeropyles were not found located on the shell imprints.
(Scale bar =3 μm for Fig. 20 and 1.2 μm for Fig.
||Extreme posterior end of the egg shows that each protuberance
(Pb) is encircled by a shell imprints (Si). (Scale bar = 7.5 μm)
|| Two normal aeropyles (Ap) situated on the shell imprints
(Si). (Scale bar = 2 μm)
||Magnified view of egg chorion reveals two normal aeropyles
(Ap) on shell imprints (Si). (Scale bar = 0.6 μm)
The main function of the aeropyles is to conduct ambient air into the trabecular
layer (gas containing meshwork), which finally passes on to the oocyte. Very
little is yet known about the permeability of the chorion. Tuft (1950) demonstrated
that when the aeropyles of the eggs of Rhodnius were blocked with shellac,
the egg continued to take up oxygen, though only at about a tenth of the normal
rate. It may be that eggs depend not only upon oxygen entering through the aeropyles
but also on that diffusing through the areas of chorion without aeropyles.
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