Recent Advances in Insect Embryology in Japan
Edited by H. Ando and K. Miya.
ISEBU Co. Ltd., Tsukuba 1985

25

Oosorption In the Lady Beetle,
Henosepilachna vigintioctomaculata(Coleoptera, Coccinellidae)

Morihisa KURIHARA

SynopsisMorphological changes in the egg follicle of Henosepilachna vigintioctomaculata induced by starvation
were observed light and electron microscopically, comparing with those in the normal one. Remarkablestructural changes, enlargement of endobody and crystallized proteinous structure, are seen first in thegerminal vesicle. Subsequently, the following fine structural changes occur in the follicular cells; significant increase of autophagic vesicles in the apical region of the follicular cells, extrusion of cytoplasmic
projections into the oocyte following disappearance of the microvilli, deformation and liquefication of
the proteinous and lipid yolk spheres by lysosome-like bodies, and formation of residual structures suchas myelin figures, vacuoles in various sizes, membranous components and others. According to the electron histochemy, acid phosphatase activity is recognized in the almost all of the lysosome-like bodies,microvilli and intercellular spaces between the follicular cells. These changes in the follicular cells may
indicate that the follicular cells uptake actively the dissolving ooplasm by their phagocytotic activities andacid phosphatase probably acts not only to autolize the microvilli of the follicular cells and oocyte but todissolve the cortical ooplasm.
When the starved females are refed, only the oocytes at the early phase of oosorption in which suchmorphological changes in the follicular cells do not yet occur are able to develop normally and are oviposited. In these oocytes, two kinds of abnormal structures developed in the germinal vesicle disappear
quickly.
From these results, the functional significance of oosorption in H. vigintioctomaculata is discussed
particularly from the viewpoint of the morphological changes in the germinal vesicle.

Introduction
Oosorption is a special case of cell death (Bell and Bohm, 1975) and is often induced byunfavorable conditions for oocyte growth. Its process, however, varies largely among insects,

26

Kurihara, M.

perhaps by different patterns of oogenesis and/or mode of oviposition.
Up to this time, many studies have been done on oosorption, especially for researching of
the internal and external factors inducing oosorption, and for analysis of the endocrine
mechanism concerned to the control of oogenesis. However, cytological and fine structural
approaches to clarify the functional changes of the egg follicle during oosorption are
insufficient. Particularly, there is no study which suggests the causal relation between
abnormality of the germinal vesicle and degeneration of ooplasm.
In Henosepilachna vigintioctomaculata Motschulsky, oosorption occurs during theprediapause period in the adults induced by short photoperiod (Mald et al., 1964; Kono,
1980) and temperature (Mald and Kurihara, 1965). Furthermore, oosorption also inducedby the deficiency of the nutrition as food quality, especially by starvation (Kurihara, 1975;
Kono, 1979). Kono (1980) clarified the endocrine mechanism inducing oosorption in thisinsect, mainly by the histochemical architecture on the changes of secretory activity of theneurosecretory cells of the brain under the short photoperiodic condition. However, he did
not observe the morphological changes of the oocytes.
This review is the summary of the studies on the oosorption of the lady beetle, withspecial reference to the demonstration of the changes of germinal vesicle, including both theresults obtained so far (Kurihara, 1967, '68, '75, '76, '81) and some unpublished data on therecovery of the oocyte development and oviposition in the females refed after varying
periods of starvation.

Materials and Methods
1. Rearing conditions: Insects were reared under 16hr-photophase and 25° C with supplyof potato leaves to observe the normal oogenesis. Thereafter, some of them were transferredto the 8hr-photophase and starving condition to obtain the oosorptive ovaries. Furthermore,
the insects were replaced on the 16hr-photophase with food after varying periods of
starvation and redevelopment of the oocytes was observed.
2. Morphological observation
1) Anatomical observation: The ovaries were fixed with Carnoy's flUid for 2 hr andthen stained with carbol-thionine, dehydrated and mounted in ceder oil.
2) Trypan blue injection: For exact determination of vitellogenic or oosorptive oocytes,
1% trypan blue (buffered with an aquatic solution containing 0.02 M KCI, 2x 1(f4M CaCh ,2 xl 0-4M MgCl 2 , 0.25 M tris malate, at pH 6.2) was injected about 0.025 ml to the female
abdomen (modified from Anderson and Telfer's method, 1970). At an hour after the
injection, the ovaries were dissected and fixed with Bouin's fixative for 3 hr Observation
was done by total preparations which were dehydrated and mounted in ceder oil.
3) Histological and histochemical observation: The almost all of the ovaries were fixedin cold gultalaldehyde solution for 2 hr and then embedded in JB-4 or Acrytron-E, andstained with toluidin blue. Histochemically, the following methods were used: PAS staining(Hotchkiss and McManus method) for polysaccharides and glycogen with salivary treatment,
Feulgen test and MG-P staining (Kurnick method) for DNA and RNA with ribonucleasetreatment control, Sudan B.B or osmium staining for lipids, and Gomori's method for acidphosphatase to observe by electron microscope.
4) Electron microscopic observation: The ovaries removed in ice-cold gultalaldehydeOosorption in the lady beetle

27

solution were fixed with 1% osmium tetroxide buffered with veronal buffer (pH 7.4) for 1.5
hr. Mter usual dehydration by ethanol, the ovaries were embedded in Epon-812. Thin
sections were made by Poter Blum MT- 1 ultramicrotome and double-stained with uranyl
acetate and lead nitrate. Observation was done by Hitachi HU- 125 electron microscope.

Results
I. Normal oogenesis

1. Mode of oocyte development and oviposition
As described in the previous papers (Kurihara, 1967, 1975), each ovary in this insect
consists of 32.1 ± 2.7 ovarioles, each of them attaches to the egg calyx with its short pedicel.
About a half of the ovarioles exist in outer portion of the egg calyx and surround theremaining ovarioles which arrange themselves in ring like layers.
Under 16hr-photophase, OViposition is continued periodically, every or every other day as
an egg mass containing about 30 eggs that is equal to about a half of total ovarioles. In each
oviposition cycle, about 15 chorionated eggs did not be oviposited and remain always in theegg calyx. This oviposition cycle well corresponds to the cyclicity of oocyte development asshown in Text figure 1. Until 6 days, the previtellogenic growth of the basal oocytes can not
be distinguished in the outer or inner ovarioles. Thereafter, almost all of the basal oocytes in
the outer ovarioles begin to deposit the proteinous yolk, while the remaining basal oocytes
in the inner ovarioles are staying in the previtellogenic stage (Text fig. I-A, El). When thelargest basal oocytes in the outer ovarioles reached to maturity and chorionated, no growthof inner basal oocytes can be ever observed as in Text figure I-A (Text fig. I-B, E'I). Text
figure 1-C shows the state which a half of the mature basal oocytes in the outer ovarioles(El) had descended to the egg calyx. The remaining half of them are wholly chorionated.
Some basal oocytes in the inner ovarioles (E'l) begin yolk deposition. Replacing the firstbasal oocytes descended to the egg calyx, the penultimate oocytes situate at the basal
portion of the ovarioles (Text fig. 1-C, E2). As the result of descending of the remaining halfof the basal oocytes (El), the basal portion of all outer ovarioles is filled with thepenultimate oocytes (Text fig. 1-D, E2). All basal oocytes in the inner ovarioles (E'I), on thecontrary, reach to the vitellogenic stage and also the half of them chorionate as same as thestate of the basal oocytes in the outer ovarioles as shown in Text figure 1-B. In the second or
more successive oviposition cycles, the same development of oocytes will be also repeatedperiodically. Therefore normal OViposition may proceed basically as follows: the mature
eggs stored in each egg calyx, number of which correspond to about one fourth of the total
ovarioles, will be pushed out into the common oviduct by the subsequent descending of thebasal oocytes from the vitellarium and deposited as a mass containing the eggs of about halfof the total ovarioles.
2. Fine structure ofnonnal egg follicle
1) Morphology of ovariole
Morphological characteristics of the ovariole in H. vigintioctomaculata were described by
Matsuzaki (1964) and Kurihara (1975). The ovariole of this insect belongs to the telotrophic
type like in other coleopteran insects. Namely, each trophocyte packed tightly in the greaterpart of the germarium has a large spherical nucleus, ca. 26 J.Lm in diameter, and is delimited

28

Kurihara, M.
15·IV+18·111
33Ch

8·p·C+15·111
30

ViI

A
Ch

10

c

~

15·IV+17·111~

15·111 + 9·C;7·IV
31

32

·~1

ViI

.....
..

.c

c

B
024

6

8
o rdel

10

12

14

16

18

~

o

o

D

Text figure 1. Mode of oocyte development in the I oviposition cycle under 16hr
photophase.
Figures A-D show the oocytes in an ovary arranged in order of their longitudinallengths. El, E2 Basal and penultimate oocytes of outer ovarioles (e), E'l Basal
oocytes of inner ovarioles (0), A Viteilogenesis begins in El, B Some El
chorion ate, C About a half of El descent to the egg calyx and E2 replace them,
D Remnant El descend to the egg calyx and basal portion of each ovariole isoccupied with E2.
Formula inserted in each figure represents;
OocyteNumber of oocytes Oocyte + Number of oocytes
In outer ovarioles • stage
in inner ovariole• stage
Total number of basal oocytes+

Ill, IV and C Previtellogenic, viteilogenic and chorionated oocytes,
Oocytes
descend to the egg calyx, CH beginning of chorionation. Vit Beginning ofVitellogenesis.

Oosorption in the lady beetle

29

by a definite cell membrane. The oocytes, on the other hand, are detectable only in the
protoplasmic nutritive chamber. They are relatively small and are situated among many
prefollicular nuclei. The nutritive cords are formed appearently as the elongation ofcytoplasm of both the trophocyte and oocyte (Kurihara, 1975).
2) Fine structure of the oocyte and development of the germinal vesicle
According to the developmental stages of oocytes divided by Kurihara (1975), the finestructure of the oocyte and the germinal vesicle will be described.
Stage L The youngest oocytes restricted at the protoplasmic nutritive chamber: The cellorganelles are very poor. A few slender mitochondria and lipid droplets inflow withnumerous free ribosomes into the oocyte through the nutritive cords. In the germinal vesiclea primary nucleolus and DNA-containing bodies intermingle with each other and form alarge complex. Both structures can be easily detected with their histochemical and finestructural characteristics, because the primary nucleolus is pyronine positive and consists of
only fine fibrilar component with high electron density, while the DNA-containing body is,of course Feulgen or methylgreen positive and is relatively electron-medium compared to
the former (Plate I, Fig.l).
Stage IL The younger oocytes at the anterior zone of the vitellarium: Prefollicular cells
crowd around the oocyte but do not yet form a single epithelial layer. Ooplasm more or lessincreases. Well developed Golgi aparates, some lysosome-like bodies and sometimes one large
electron-medium and compact sphere, probably an aggregate of the nuclear emission
granules, are particular in the ooplasm of this stage. In the germinal vesicle, the primary
nucleolus and DNA-containing body begin to get untied. The primary nucleolus aggregates
to form an electron dense mass. Attaching to it, the DNA-containing body also grows to thelarge mass which consists of fine fibrous materials and has usually a single cavity in the center(Plate I, Fig.2). At the late stage 11, the primary nucleolus begins to separate to small
granules, ca. 900 A, and disperse throughout the nucleoplasm, following the subsequent
granulation and migration of the DNA-containing bodies (Plate I, Figs.3 and 4).
Stage Ill. The previtellogenic oocytes at the middle of the vitellarium: Ooplasm largely
increases. The follicular cells form a single·layered epithelium, but their microvilli do not yet
develop. Until the beginning of this stage, disintegration of the DNA-containing body andthe primary nucleolus has already finished. Thereafter, at the middle of this stage, formationof the karyosphere begins; the long thread-like chromosomes accumulate loosely around thepyronine negative body which increases rapidly in size, ca. 4 ~m in diameter. Electron
microscopically this body consists of only fibrous component (Plate 11, Fig.6). At the sametime, a number of small spheres of three types produce actively around the surface of the
karyosphere. The first type of the spheres has ca. 2 ~m in diameter and shows high electron
density with fine granules on its surface (BNl). The 2nd, the same size of the 1st type,shows a ring-shaped sphere having an electron-dense thin cortex (BN2), and the last is anelectron-medium, compact sphere consisting of only fibrous component and closely
resemble to the endobody first described by Bier et al. (1967) (BN3) (Plate 11, Fig.5).
Stage IV. Vitellogenic oocytes: The proteinous yolk spheres begin to deposit from theperiphery of the oocyte by pinocytosis and are filled in the ooplasm at the end of this stage.
The BNl, BN2 and BN3 spheres increase considerably in number through the early stage ofvitellogenesis. Finally, they become undetectable, when the vitellogenesis almost finished.
Stage V. The completed eggs with chorion: Fine structure of the vitelline membrane andthe chorion formation was described by Matsuzaki (1965). The follicular cells degenerate.

30

Kurlhara, M.

The germinal vesicle becomes crescent and the nuclear membrane breaks and finallydisintegrates.
11. Oosorption
1. Cessat ion of oviposition and changes of the mature eggs induced by starvation
Immediately after ovipositon, the female beetles were transferred to the 8hr-photophase
under starving condition, and the mature eggs produced daily (oviposited eggs plus
chorionated eggs in the egg calyx) were examined for 8 days.
Normally, the starved females deposit a few eggs discontinously during the first 4 days of
starvation, while the chorionated eggs in the egg calyx increase progressively, perhaps, as theresult of chorion formation of the late vitellogenic oocytes in the basal portion of thevitellarium and subsequent descending to the egg calyx. After 6 to 8 days of starvation,
however, the deposited eggs more or less increase but the chorionated eggs in the egg calyx
decrease conversely. Consequently the same number of the mature eggs were obtained in all
starving periods investigated, although the number of eggs produced were slightly increasedin the longer starvation. Beyond the 8 day-starvation, subsequent oviposition is notobserved and no remaining eggs can not be ever seen in the egg calyx. This may be indicatedthat some of the nearly matured oocytes and the chorionated eggs stored in the egg calyx do
not degenerate but are deposited, while no further development of the vitellogenic oocytes
occurs in the vitellarium.
2. Changes of immature oocytes during starvation

When the females are starved, all of the immature oocytes in the vitellarium continue todevelop slowly for several days and then oosorption starts. The relation between thedevelopmental stages of the oocyte and occurrence of oosorption is summarized in Text
figure 2.
At stage I and 11, the oocytes develop slowly until formation of karyosphere (stage Ill).
Dispersion of the primary nucleolus and DNA-containing body in these oocyte nuclei seemto be normal. During such slow growth, the BN3 (endobody) begins to develop enormously,ca. 40 tL1ll in a maximum diameter (Plate V, Fig.12). At stage III and IV, however, theenlargement of the endobody completes within 24 hours. After such slow growth of theoocytes the morphological abnormalities become detectable in the egg follicle; decreasing
stainabilities of the trophocytes for nucleic acid-staining, folding and irregular arrangementof the follicular cells, coagulation of ooplasm, fusion and liquification of the proteinous yolk
spheres with decreasing PAS-stainability, etc., are typical changes detected by lightmicroscope (Kurihara, 1967, '68).
3. Fine structural changes of the egg follicle during oosorption with special reference to thechange of germinal vesicle

Fine structural changes of the follicular cells and oocytes during oosorption were
described by Kurihara (1976, '81).
At the slow growth period of oosorption, any particular changes can not be observed in
the egg follicle, except enlargement of the endobody. Subsequently. first visible changes

Oosorption in the lady beetle

31

occur in the apical region of the follicular cells and the cortical ooplasm. Namely, somelysosome-like vesicles which contain acid phosphatase appear significantly, while in the basal
region of the cells just beneath the tunica propria, well-developed rER, many mitochondria
and Golgi aparates distribute as in the normal (Plate Ill, Fig.8). According to thehistochemistry with electron microscope, free acid phosphatase granules distribute widely
throughout the cytoplasm of the follicular cells and are also detected in their microvilli. With
progress of the oosorption, the microvilli of the follicular cells disappear rapidly, perhaps
because of resolution by acid phosphatase (Plate Ill, Figs.7 and 8). Substituting for the
microvilli well elongated cytoplasmic projections of the follicular cells extrude more deeply
and more irregularly into the liquefied ooplasm (Plate IV, Fig. 11). Frequently, at the midto late oosorption large lysosome like structures including various residual bodies, such as
deformed proteinous yolk spheres, lipid droplets fused each other in a large mass, myelin
figures and some membranous components etc., are included in the follicular cells. In thesestructures the acid phosphatase can also be detected (Plate IV, Fig. 9). Finally, at the lateperiod of oosorption the oocytes become almost empty, and the nuclei of follicular cells
inflow into the liquefied oocyte and autolyze. These processes of destruction of thefollicular epithelium can be easily detected by trypan blue injection. Namely, as shown infigure 10a-d (Plate IV), the degenerating follicular cells uptake the dye spottedly in theearlier oosorption (b) and massively in the later oosorption (c), respectively.
Changes in the germinal vesicle: During the early phase of oosorption, prior to thecytoplasmic changes of the egg follicle, two particular structures are formed in the germinal
vesicle; crystallized proteinous structure and an enlarged endobody. The fine structure, theorigin and formative process of them are clarified electron microscopically (Kurihara, 1976).
The crystallized proteinous structures are formed by accumulation and crystallization of
the small granules that are derived from the primary nucleolus and scattered evenly
throughout the nucleoplasm. Namely, at first, the granules aggregate on the surface of the
BN3 and/or on the concave portion of the inner surface of the nuclear envelope.
Subsequently, the accumulated granules develop to the short threads and finally they form adefinite structures (Plate V, Fig.l4).
The enlarged endobody is finally formed only one in the germinal vesicle by abnormal
development and fusion of the BN3 distributed throughout the nucleoplasm. The enlarged
endobody has a thin fibrous envelope consisting ofphospholipoid material in nature which isone of the components in the BN3. The contents, on the other hand, do not containphospholipids and nucleic acids, but are rich in proteins (Plate V, Figs. 12 and 14).
Ill. Recovery of oocyte development and oviposition in the refed females after varying periods of starvation

1. Ozanges afimmature aacytes in the vitellarium af the refed females
To confirm whether the immature oocytes in different degrees of oosorption are able toresume normally their growth or not, the 3 days-starved females were refed and therelationship between redevelopment of the oocytes and changes of the enlarged endobody
were examined. In the 3 days-starved females, oosorption proceeds in different extent
corresponding to the oocyte development, as follows:32

Kurihara, M.
Inner ovariole

Outer ovariole

60

50

..

.

~ 40
.r=

'"

~ 30

20

E3
10

o~~__~~__~~~~~__~_-~--~~o23refed- 0

4
1

5
2

6
3

7
4

8
5

9(~ays)6

Text figure 2. Development of oocyte in the refed females under l6hr-photophase 25°C,
after 3 days-starvation. CE Chorionated eggs,' Endobody begins to enlarge, {}Anatomically, oosorption becomes visible in the oocyte and follicular cells,_. _. Oosorption curve, - - Redeveloping curve, - - - - - - Oosorption
curve under continuous starvation, El, E2, E3 Basal, penultimate, and 3rd
oocytes in outer ovarioles, E'2, E'3, E'4 Penultimate, 3rd and 4th oocytes in
inner ovarioles.

1) Oosorptive stage I (D I). Early previtellogenic oocytes: Anatomically, any abnormality can not be observed in the egg follicle. Probably, oosorption does not initiate.
2) Oosorptive stage 11 (D 11). Mid- to late previtellogenic oocytes with an enlarged
endobody in the germinal vesicle: Other abnormalities are not recognized.
3) Oosorptive stage III (D Ill). Early to mid-vitellogenic oocyte in which oosorption
proceeds: Hematoxylin-positive ooplasm begins to coagulate partially and arrangement of
the follicular cell becomes irregular. Proteinous yolk spheres are deformed. In the germinal
vesicle, an enlarged en do body reaches to the maximum size.
The results are divided conveniently into the outer and inner ovarioles, as shown in Text
figure 2
Outer ovarioles: In the outer ovarioles of the 3 days-starved females, the basal oocytes
are in D Ill. Even if the females were refed, the oocytes can not develop and oosorption
continues (El in Text Fig. 2, broken line) as in the case of continuous starvation (dottedline). The penultimate oocytes (E2) in D 11, on the contrary, begin to grow normally assoon as the starved females are refed.
Inner ovarioles: The ovarioles contain the oocytes in D I and D 11. The mature oocytes atthe basal portion of the ovarioles descend already to the egg calyx during 3 days of
starvation and only corpus luteum remains. Therefore, in the basal part of the vitellarium,Oosorption in the lady beetle

33

the penultimate oocytes in the late previtellogenic stage (E'2) exist and reach at D n. Whenthe starved females are refed these oocytes begin to grow rapidly, and come to maturity withthe chorion after 3 days. The 3rd oocytes (E'3) were in D I, and they also resume the normal
development without oosorption.

2. Changes of the enlarged endobody in the germinal vesicle of the refed females
Enlargement of the endobody occurs only in the germinal vesicle of the oosorptive
oocytes after the mid-previtellogenic stage (Stage Ill) and is a typical syndrome of the earlyphase of oosorption.
To observe the change of the enlarged endobody in the redeveloping oocytes, the24hr-starved females were transferred to the 16hr-photophase with food. The females having
the oocytes with enlarged endobody rapidly decrease although they increase temporarily 16
to 20 hours after. Lastly, the enlarged endobody can not be found at 24 hours after
re feeding. In the germinal vesicle of the redeveloping oocyte, many fine residual particles(Plate V, Fig.15a) and/or discontinuous envelope of the body (Plate V, Figs.l5b, c) are
scarecely observed. This may be due to rapid breaking of the envelope and an instantaneous
fusion of contents of the body with the surrounding nucleoplasm.

3. Hatching ability of the eggs deposited by the refed females
Hatching ability of the eggs deposited in the 1st to 4th oviposition cycles of the refedfemales after 24hr starvation were investigated. The number of eggs per one egg mass does
not differ in each oviposition cycle. Extreme low hatching ability is obtained only in the
2nd oviposition cycle (Table 1). Unhatched eggs are dissected and developmental stages ofthe embryos are observed anatomically. Many of them (about 60%) had reached at the fullgrown stage and then die just before or after emergence from the egg shell. Early dead andabnormal embryos and unfertilized or undeveloped eggs are excluded. Almost all of the
newly hatched larvae die as soon as they hatch but the survivals grow and pupate normally.
Text figure 3 is illustrated the state of oocyte development in the 24hr-starved females. It isclear that the only oocytes corresponding to the 2nd oviposition cycle (E2) are in D 11, andabout a half of them can not hatch, although they are able to resume development and arelaid by refeeding.
Table 1. Hatching ability of eggs oviposited by the refed females after 24hr-starvation.
Oviposition cycles
after refeeding

Number of
femalesNumber of eggs
per egg mass

Hatching
ratio

EO
El
E2
E3
E4

10
10
9
7

44.5 ± 6.3

95.7 ± 3.8
89.4 ± 10.6
564. ± 31.4
97.2 ± 5.8
97.3 ± 2.1

6

38.6 ± 7.3
42.2 ± 9.0
31.3 ± 10.8
32.7 ± 5.7

EO, eggs oviposited before starvation.

34

Kurihara, M.

PenultimateOC--- --- -. --Basal OC

MV- LPVEl
Oviposited
EO

Text figure 3. Oocyte development of the 24hr-starved females and mode of oviposition
after re feeding.
EO Eggs oviposited before starvation, El- E5 Eggs oviposited at the first to the
5th oviposition cycles after re feeding At 24hr-starvation, E2 reaches oosorptive
stage II and all of others are in normal (El, E3-E5). CE Chorionated (Stage V),
IV Late vitellogenic, MV Mid-vitellogenic, MPV Mid-previtellogenic, EPV Early
previtellogenic oocytes.

Oosorption in the lady beetle

35

Plate I.
Fig. 1. Oocyte nucleus at stage I. A large complex consisted of DNA-containing body (D)and primary nucleolus mingled with each other. X 8,300.
Fig. 2 Oocyte nucleus at stage II. DNA-containing body with a single cavity in its center(D) and primary nucleolus (PN). X7,200.
Fig. 3. Oocyte nucleus at early stage III. Primary nucleolus migrates to the one side of thenucleus and there breaks into small granules (SG). X 9,200.
Fig. 4. Oocyte nucleus at mid-stage Ill. DNA-containing body also breaks into several
fascicles and soon disappears. X3,SOO.

36

Kurihara, M.

Plate It
Fig. 5. A part of karyosphere and electron-light sphere (BN3) at stage Ill. CH condensed
chromosome, BNI electron-dense budding nucleolus. X 9,900. Inset shows total
aspect of karyosphere. X 760.
Fig. 6. Karyosphere at late stage Ill. FB endobody-like fibrous body, BNI and BN2electron dense and ringshaped budding nucleolus. X 4,500. Inset shows total
aspect of karyosphere. X760.

Oosorption in the lady beetle

37

Plate III
Fig. 7. Follicular cells and oocyte at the early phase of oosorption. Microvilli of thefollicular cells become unclear and cytoplasmic projection elongate into the
oocyte (arrows). The groups of many small vesicles (V) appear at the corticalcytoplasm of the oocyte. X 8,600. Inset shows acid phosphatase in microvilli(MV) and in small vesicle in the oocyte (V). X 36,000.
Fig. 8. Follicular cells and oocyte at the early phase of oosorption. Microvilli disappear. In
the basal region of the follicular cells many lysosome like vesicles (L Y) appear.
Cortical cytoplasm of the oocyte is filled with electron dense liquified ooplasm. X
4,300. Inset shows acid phosphatase in lysosome like vesicles. X 18,000.

38

Kurihara, M.

Plate IV.
Fig. 9. Apical region of the follicular cells at the late phase of oosorption. Myelin figures(MY), vacuoles of various sizes (V) and deformed lipid droplets (L) in thecytoplasm. X 7,500. Inset shows acid phosphatase in large lysosome-like structure.
X6,000.
Fig. 10. Trypan blue uptake in oocytes. a: normal, b-d early: mid-, and late oosorption.
Fig. 11. Basal region of the follicular cells. Well-developed cytoplasmic projections extrude
into the liquefied ooplasm. X 9,000.

Oosorption in the lady beetle

39

Plate V.
Fig. 12. Oocyte nucleus at the early phase of oosorption. Ka diminished karyosphere, RBring shaped body (enlarged endobody) with thin envelope, CPS crystallized
proteinous structure, NM nuclear membrane. X950.
Fig. 13. Diminished karyosphere at the early phase of oosorption. X 5,700.
Fig. 14. Fine structure of ring shaped body (RB), and crystallized proteinous structure(CPS). NM nuclear membrane. X3,700.
Fig. 15. Light micrograph showing disintegration of ring··shaped body. a Fragments of theenvelope band c Discontinous portion of the envelope (arrows). X 450.

40Kurihara, M.

Discussion
Peculiarity of oosorption process

In H vigintioctomaculata, oosorption process can be divided mainly into two significant
phases; the preoosorptive and the oosorptive. During the former phase, oocytes slightly
grow and sustain the reversible ability for growth, although a typical abnormality,
enlargement of the endobody, appears in the germinal vesicle. In the latter phase following
the former one, however, the oocyte growth stops and then oosorption progresses
irreversibly even if the starved females would be returned to the adequate conditions for
oocyte growth, such as sufficient food supply under the long photophase. This slow growthof the oocyte is indicated well by the low synthetic activity of RNA and proteins by thetrophocytes as shown by the decreasing stainability for methylgreen-pyronine in H
vigintioctomaculata (Kurihara, 1968) and for Ponsau S or acridine orange in Rhodnius
prolixus (Pratt and Davey, 1972a-c ), and decreasing incorporation of H3 -uridine in Blatta
orientalis (Sams, 1975). Such transient growth seems to be in general; previtellogenic growth
proceeds slowly and then oosorption occurs just before proteinous yolk deposition. For
example, in the starved mild weed bug, Oncopeltus fasciatus, the previtellogenic oocytes
develop until i3 and 'Y stages (probably nearly late previtellogenic) before degeneration(Johansson, 1958), and in the allatectomized or the starved R. prolixus, the oocytes
continue to develop until disappearance of the nutritive cord jointing the oocyte and thetrophocytes (Vanderberg, 1963).
Timing for onset of oosorption

As to the timing for onset of oosorption in vitellogenic oocytes, the more delicate
differences have been observed not only in the species belonging to the different orders but
among the closely relating species. In some starved cockroaches, resorption of the oocytes ischaracterized by different patterns; it occurs simultaneously with beginning of vitellogenesis
in Brysotria, while vitellogenic growth does not initiate in Leucophaea (Roth and Stay,
1962). Otherwise vitellogenesis continues within 10 days of starvation and then resorption
occurs in Periplaneta (Bell, 1971), while in Blatta vitellogenic arrest continues during 15
days of starvation and then oosorption initiates (Sams, 1975). Therefore, Sams (1975)concluded that, in general, different insects evolve different strategies to cope with period of
starvation, the strategy of Blatta is to arrest vitellogenesis and to maintain viable oocytes forlongest as far as possible without initiating oosorption.
In H vigintioctomaculata, the longer starvation is continued, the less oocytes are able toresume vitellogenesis. At least, within the 2 d·ays-starvation, all of the basal and penultimate
oocytes can switch to redevelop, while in all oocytes of the 8 days-starved females
oosorption always proceeds. The days required for preoosorptive phase in the basal oocytes
seem to be about 2 days in the older oocytes and 3 days in younger ones, respectively.
During this phase! the oocytes in the starved females may initiate or resume thevitellogenesis according to the age of oocytes as shown in Text figure 2.

Oosorption in the lady beetle

41

Function of the follfcular cens at OOSOlrptioo
As to the mechanism of breakdown of yolk sphere during oosorption, two possibilities
have been discussed; one is phagocytic activity by the follicular cells, and the other lysis by
enzymes contained in lysosome-like bodies within the oocytes (Bell and Bohm, 1975).
In nmmaJ vitellogenesis, the follicular cells may secret an important protein relating
uptake of vitellogenin. Anderson and Telfer (I970) clarified that in cecropia moth, thefollicular cells produce H3 -histidine labelled secretion product into the intercellular spaces.
In follicles treated with trypan blue, the dye is bound in large amounts in the extracellularspaces and as the result, vitellogenesis stops. Removing the bound dye yolk deposition canbe reversed. Therefore they suggested that this protein seems to be a necessary agent in thepinocytotic uptake of blood proteins. Such protein synthesis of the follicular cells may
cease at oosorption process. In B. orientalis, first sign of the functional changes of the follicular cells is the termination of protein synthesis (Sams, 1975).
Based on electron microscopy of the allatectomized colorado beetle Leptinotarsa
descemlineata, De Loof and Lagasse (I 970) showed that the first sign of oosorption is theaccumulation of large quantities of electron-dense materials, lysosome-like bodies, between
the microvilli of the follicular cells and the oocyte, and then these materials migrate into the
oocyte. Thereby, they suggested that supplying the precursor oflysosomes into the oocyte isa role of the follicular cells in the oosorption of this insect.
The first step of oosorption in H. vigintioctomaculata is characterized by thedisappearance of the microvilli of the follicular cells and the oocyte like in L. descemlineata.
Prior to the morphological changes, large amount of acid phosphatase begins to accumulate
rapidly in the microvilli, as soon as oosorption initiates. This enzyme may relate ·to bothbreakdown of the microvilli and rapid dissolution of the cortical ooplasm. The increment of
the same enzyme has been observed in the periphery of the oocytes in Nasonia vitripennis
taken out from the host (Hopkins and King, 1964) and in Schistocerca gregaria (Lusis,
1963).
After the disappearance of the microvilli, the cytoplasmic projections begin to extrude
into the oocyte. Finally, the cytoplasm of the follicular cells is filled with large amount ofresidual structures, such as deformed proteinous yolk spheres and lipid droplets attacked bylysosome-like bodies, myelin figures, large vacuoles in various sizes and membranous
structures etc, (Kurihara, 1981). These phenomena may indicate that the follicular cells not
only autolyze themselves but also supply of the enzyme for dissolution of the corticalooplasm and yolk spheres and thereafter, they uptake and resorb actively the dissolving
ooplasm and yolk spheres by their phagocytic activitieso

Rmction of the endobody and significance of its enlargement at oosorption

The endobody has been first described in several insects by Bier et al. (I 967). Similar
structures were also found in Drosophila (Mahowald and Tiefert, 1970; Kinderman andKing, 1973), Ozrysopa (Gruzova et al., 1972; Matsuzaki, 1978). Up to this time, however,the fUnction of the endobody in the normal oogenesis remains unsolved. Normally, theendobody does not synthesize the nucleic acids (Bier et aL 1967, Mahowald and Tiefert,
1970). Bier et al. (I967) suggested that the endobody may be a storage site of42

Kurihara, M.

nucleoproteins because of its active incorporation of H3 -leucin. However, affirmative results
could not be obtained in other insects studied thereafter.
In H vigintioctomaculata, the endobody contains no nucleic acids but is rich inaCidophilic proteins and phospholipids (Kurihara, 1975). The fine structure closely
resembles to that in carabid beetles; it consists of only fibrous component (Kurihara, 1976).
Now, the greatest care must be taken about the difference of growth and disintegration of
the endobody among the insect species. During mid- to late previtellogenesis in H
vigintioctomaculata, a number of electron light spheres (BN3) bud off from the surface ofthe endobody-like fibrous body constructing the karyosphere and disperse throughout thenucleoplasm. illtimately, they diminish in number and disappear at the late vitellogenesis,
while in Drosophila melanogaster and D. immigrans, as studied by Mahowald and Tiefert(1970), the endobody disappears prior to vitellogenesis. Besides, even in the genus
Drosophila, the endobody of D. virilis remains until the beginning of metaphase I in stage 12(Kinderman and King, 1973). In the panoistic ovary, on the contrary, it continues toincrease in size until the end of oogenesis (Bier et al., 1967). The changes of the endobody in
H vigintioctomaculata which has the telotrophic ovary are similar to the changes in thepanoistic ovary rather than those in the polytrophic one. Although the origin and functionalsignificance of the endobody are not yet quite clear either in H vigintioctomaculata or inother insects such difference in development of the endobody must be an interesting andimportant problem relating to the functional difference of RNA synthesis of the germinal
vesicle between the panoistic and meroistic ovaries. So, as pointed out by Mahowald andTiefert (1970), the function of the endobody may be not always the same in different
insects, even if the fine structure is resemblant.
In H. vigin tioctomacu lata, when the females were starved, enlargement of the endobody
in the germinal vesicle is a drastic and first visible sign of the initiation of oosorption. It is areally hollow sphere with a homogeneous thin envelope lipoid in nature and is lastly formed
only one in the germinal vesicle. Since the function of the endobody is not known also in
any insects, the physiological significance of the enlargement of the endobody in the earlyphase of oosorption can not be discussed exactly here. However, it is pointed out that suchabnormality in the germinal vesicle does not affect the fertilization and the further
embryogenesis, because almost all of the redeveloped eggs complete their embryonicdevelopment, although many of them can not hatch. Therefore the enlargement of theendobody may indicate that a temporary storage of the nucleoproteins for reversible usage
when conditions will become again favorable for Vitellogenesis.

Acknowledgements
The author wishes to express his hearty thanks to Dr. K. Miya, Iwate University, for his
constant guidance in the course of the work and for reading this manuscript. The author is
grateful to Drs. N. Yoshitake and H. Watanabe, University of Tokyo, for their valuableadvice and encouragement.

Oosorption in the lady beetle

43

References
Anderson, L. M., and W. H. Telfer, 1970. Trypan blue inhibition of yolk deposition - aclue to follicle cell function in Cecropia moth. J. Embryol. exptl. Morph. 23: 35-52.
Bell, W. J., 1971. Starvation induced oocyte resorption and yolk protein salvage in
Periplaneta americana. J. Insect Physiol., 17: 1099-1111.
& M.K. Bohm, 1975. Oosorption in insects. Bioi. Rev., 50:373-396.
Bier, K., W. Kunz and D. Ribbert, 1967. Strukture und Funktion der Oocytenchromosomen und Nukleolen sowie der Extra-DNS wUhrend der Oogenese panoistischer undmeroistischer Insekten. Chromosoma (Berl.), 23: 214-254.
Gruzova, M. N., Z. P. Zaichikova, and I. I. Sokolov, 1972. Functional organization of thenucleus in the oogenesis of Chrysopa perla L. (Insecta, Neuroptera). Chromosoma(Berl.j, 37: 353-386.
Hopkins, C. R., and P. E. King, 1964. Egg resorption in Nasonia vitripennis (Walker)(Hymenoptera; Pteromalidae). Proc. R. ent. Soc. London (Aj, 39: 101-107.
Johansson, A. S., 1958. Relation of nutrition to endocrinereproductive function in the
milkweed bug, Oncopeltus fasciatus (Dallas). Nytt. Mag. Zool., 7: 1-132.
Kinderman, N. B., and R. C. King, 1973. Oogenesis in Drosophila virilis L. Interactions
between the ring canal rims and the nucleus of the oocyte Bioi. Bull., 144: 331-354.
Kono, Y., 1979. Abnormal photoperiodic and phototactic reactions of the beetle, Epilachna
vigintioctomaculata, reared on sliced potatoes. Appl. Ent. Zool., 14: 185-192.
- - - , 1980. Endocrine activities and photoperiodic sensitivity during prediapause
period in the phytophagous lady beetle Epilachna vigintioctomaculata. Appl. Ent.
Zool. 15: 73-80.
.
Kurihara, M., 1967. Studies on oogenesis of the lady beetle I. Anatomical and histologicalobservation on the reversible development of ovary in the lady beetle, Epilachna
vigintioctomaculata Motschu1sky, induced by the change of photoperiods., J. Fac Agr.,
Iwate Univ., 8: 223-233.
- - - , 1968. Ditto n. Histological and histochemical observation on periodic acidschiff positive substances and nucleic acids in the normal and degenerated ovary of thelady beetle, Epilachna vigintioctomaculata Motschulsky. Ibid., 9: I-IS.
_ _ _ , 1975. Ditto Ill. Anatomical and histological studies on the germinal vesicle indegenerating oocyte of starved females of the lady beetle Epilachna vigintioctomaculata
Motschulsky (Coleoptera, Coccinellidae). Kontyu, Tokyo, 43: 91-105.
_ _ _ , 1976. Ditto IV. Origin and formative process of two nuclear structures in degenerating oocyte nucleus of the starved Epilachna vigintioctomaculata Motschulsky(Coleoptera: Coccinellidae)., J. Fac. Agr.,Iwate Univ., 13: 145-159.
_ _ _ , 1981. Ditto V. Fine structural changes of the follicu1ar cells during oosorption in
the starved lady Beetle, Epilachna vigintioctomaculata Motschulsky, with specialreference to the demonstration of acid phosphatase. Kontyu, Tokyo, 49: 65-80.
De Loof, A., and A. Lagasse, 1970. Resorption of the terminal oocyte in the allatectomized
Colorado beetle, Leptinotarsa descemlineata Say. Proc. Koninkl. Nederl. Akad. Westenschappen, 73: 284-307.
Lusis, 0., 1963. The histology and histochemistry of development and resorption in theterminal oocytes of the desert locust, Schistocerca gregaria. Q.J. microsc. Sci.. 104.
57-68.
Mahowald, A. P., and M. Tiefert, 1970. Fine structural changes in the Drosophila oocyte
nucleus during a short period of RNA synthesis. Wilhelm Roux Arch. EntwMech. Org.,
165: 8-25.
Maki, T., and M. Kurihara, 1965. Effect of temperatures on reversibility of ovariandevelopment in the lady beetle, Epilachna vigintioctomaculata Motschulsky (inJapanese with English summary). J. Fac. Agr., Iwate Univ., 7: 167-172.
, and Y. Ando, 1964. On the reversibility of the ovarian development- __ ,of the lady beetle, Epilachna vigintioctomaculata Motschulsky, by the photoperiodic

44

Kurihara, M.

effects (In Japanese with English summary). l. Fac. Agr., Iwate Univ., 7: 7-17.
Matsuzaki, M., 1964. The electron microscopic studies on the early development of various
insect eggs I. The observation on the ovary of the large 28 spotted lady beetle(Dpilachna vigintioctomacu!ata Motschu1sky) (in Japanese with English summary) Sci.
Rep. Fukushima Univ., 13: 35-40.
_ _ _ , 1965. Ditto Ill. Chorion formation and changes of follicle cell components of the
Epilachna egg (In Japanese with English summary) Sci. Rep. Fukushima Univ., 15:
30-34.
____ , 1978. Developing ovarian follicles during early oogenesis in the adult green
lace wing Chrysopa septempunctata Wesmael (Neuroptera, Chrysopidae) Annot. Zoo!.
lap., 51: 222-235.
Pratt G. E., & K. G. Davey, 1972a. The corpus allatum and oogenesis in Rhodnius prolixus(SW) I. Effects of allatectomy. l. exptl. Bio!., 56: 201-214.
_ _ _ ,and ___ , 1972b. Ditto 11. The effect of starvation. Ibid" 56: 215-221.
_ _ _ ,and ___ , 1972c. Ditto Ill. The effect of mating. Ibid., 56: 223-237.
Roth, L. M., and B. Stay, 1962. Comparative study of oocyte development in false
ovoviviparous cockroaches. Psyche, 69: 165-208.
Sams, G. R, 1975. Vitellogenic arrest in the cockroach, Blatta orientalis. l. Insect Physiol.,
21: 1203-1210
Vanderberg, J. P., 1963. The role of the gonadotrophic hormone in the synthesis of protein
and RNA in Rhodniusprolixus., Bio!. Bull., 125: 576-581.

Author's address:

Dr. M. Kurihara,
Laboratory of Applied Entomology,
Faculty of Agriculture, Iwate University,
Morioka, Iwate 020, Japan