Table Of ContentReference: Biol. Hull. 203: 28l)-30f>. (December 2002)
Structure, Formation, Mechanical Properties, and
Disposal of the Embryo Attachment System of an
Estuarine Crab, Sesarma haematocheir
M. SAIGUSA1'*, M. TERAJIMA1, AND M. YAMAMOTO2
^Laboratory ofAnimal Behavior anil Evolution. Graduate School of Natural Science and Technology,
Oka\ama University. Tsushima 3-1-I, Okayama 700-8530, Japan: and 'Ushimado Marine Laboratory.
Okavama Universit\. Oku-Ushimado, Okayama 701-43. Japan
Abstract. In most decapod crustaceans, fertilized eggs (Ela and Elb). and the adhesiveness rapidly decreases. The
extruded from the gonopore attach to ovigerous hairs within plasticity of the envelope remains, and the funiculus is
the incubation chamber of the female. The attachment is formed, accompanied by kneading of the eggs (40-90 min
effected by an "embryo attachment system." The three after egg-laying). The embryos hatch one month after incu-
continuous components ofthis system are the egg envelope, bation, and the attachment systems all slip offtheir oviger-
the funiculus, and the investment coat, which wraps around ous hairs by the actions of the ovigerous-hair slipping
an ovigerous hair. Transmission electron microscopy substance (OHSS). This substance appears to act specifi-
(TEM) revealed that the embryo of Sesarma haematocheir cally atthe attachment sites on the hair, lysingthebond with
is enfolded by three distinct envelopes (El. E2. and E3), layer Ela. and thereby disposing of the embryonic attach-
whereas the embryo attachment system is composed ofonly ment system and preparing the hairs for the next clutch of
the outermost, singleenvelope (El ) with two sublayers (Ela embryos.
and Elb). Thisenvelope (El) originates from the outerlayer
ofthe vitelline membrane (envelope ofthe ovum) with two
Introduction
sublayers (Ela' and Elb'). The sequence and timing of
events in the formation of the embryo attachment system The oocytes of some decapod crustaceans are fertilized
was determined on the basis of observations of female when they pass through the seminal receptacles where
behavior, ultrastructure, and mechanical properties of the sperms are stored (Hartnoll. 1968). Fertilization is followed
membranes. The egg envelope (Ela' + Elb') is not adhe- by egg-laying; that is, the eggs are extruded through two
sive immediately after extrusion from the gonopore; but 5 gonopores on the ventral side of the thorax of the female.
min after egg-laying, it becomes adhesive a change asso- The number of eggs extruded at once varies with species
ciated with "fusion" of the two sublayers (El) and at- and the duration of incubation; for example, an estuarine
taches the eggs to the ovigerous hairs from 5 to 30 min after semi-terrestrial crab. Sesarma haematocheir. laid 10.000-
egg-laying. The layer Ela' always binds to an ovigerous 50,000 eggs in one batch. After laying, the eggs attach to
hair at specific, electron-dense attachment sites that are ovigerous hairs borne on the maternal ovigerous seta; the
distributed longitudinally on the surface of each hair. Plas- attachment is through a stalk called the funiculus (Herrick.
ticity of the egg envelope changes, and the female kneads 1895; Yonge, 1937. 1946, 1955; Cheung, 1966; Fisher and
her eggs by the movement of ovigerous setae; this move- Clark, 1983; Goudeau and Lachaise, 1983). The mechanism
ment forms the investment coat on the ovigerous hair by which the funiculus forms and attaches to the ovigerous
(10-40 min after egg-laying). Thirty minutes after egg- hairs, as well as the process by which the embryonic enve-
laying, the egg envelope again divides into two sublayers lope forms, have been subjects of controversy for many
years.
Received 5 June 1998; accepted 12 September 2002. Yonge (1937) reported that many secretory glands occur
*To whom correspondence should be sent. on the pleopods of the female of the lobster Homanis
289
290 M. SAIGUSA ET AL.
nilgaris. These glands are identical in structure to the teg- hours after larval release. This process prepares the hairs for
umental glands that occur everywhere beneath the epithe- the attachmentofthe next clutch ofthe embryos (forfurther
lium, and Yonge speculated that the fertilized eggs are details, see Saigusa, 1994. 1995. 1996). But the mechanism
attached by a "cement" secreted by these glands at the time by which the investment coat slips off the hairs is not yet
of egg-laying. Observations by Andrews (1906). Stephens known. OHSS might cause morphological changes to the
(1952). and Aiken and Waddy (1982) supported Yonge's coat. Thus, we have examined ultrastructural changes to
hypothesis. Moreover, FisherandClark ( 1983) reportedthat investment coats that were treated with OHSS solution and
the fertilized eggs of an estuarine shrimp, Palaenum nuic- were subsequently stripped off their hairs.
rodactylus, are attached to each other, or to the ovigerous
hairs of the maternal pleopods, by materials that are pro- Materials and Methods
duced by the pleopods and form the outer layer of the
Animals and strategy for experiment
embryonic envelope.
In contrast, a study ofembryonic envelope formation and The animal used for this study was Sesarma haemato-
egg attachment in the shore crab, Curcinits inaenas, indi- clicir (red-handed crab; akate-gani) inhabiting thickets
cated that the funiculus forms from the outermost layer of along estuaries in western Japan. The females ofthis species
the "trichromatic membranes," all ofwhich are producedby incubate two to three successive clutches of developing
mature eggs after fertilization (Cheung. 1966). Cheung embryos between June and September. After one month of
found no "cement glands" on the pleopod of this crab. incubation, the embryos hatch, and the zoea larvae are
Cheung's hypothesis was also supported by the ultrastruc- released in the water (Saigusa. 1982). The number of fe-
tural studies of Goudeau and coworkers, who showed that males releasing larvae increases at about the time offull and
the funiculus ofCarcimis maenas is an extension ofthe two new moons and decreases at the half moons a semilunar
vitelline layers of the mature egg. These two layers are rhythm.
altered structurally and stretched by the vigorous beating of The most important procedure for this study was to
the maternal pleopods; by this action the material in the collect females that had just begun to lay their eggs. Egg-
layers wraps around the ovigerous hairs without involving laying occurs primarily in the morning, from 0600 to 1200,
any adhesive substance (Goudeau and Lachaise, 1980, but it begins with no visual sign, so in the field we cannot
1983; Goudeau et al., 1987; reviewed by Hinsch, 1990). specify precisely any particular female that has just started
The embryo attachment system of many intertidal and laving eggs. Therefore, these investigations were carried out
estuarine crabs includes, not only the embryonic envelope in the laboratory. We maintained a few hundred females in
and the funiculus. but also a clear "coat" that invests the the laboratory, which enhanced the chance that we could
whole hair (Saigusa, 1994). Ifthe newly extruded eggs are observe females that were starting egg-laying. We also took
attached to the ovigerous hairs by a "cement" or similar advantage ofthe fact that, in S. haematocheir, most females
substance that is secreted from the female, we should ob- incubate their next clutch within a few days after a larval
serve two kinds of material on the ovigerous hair: that is, a release. Therefore if females that had released zoea larvae
layer secreted by the maternal pleopods, and another layer during any night were placed in different containers, we
originating from the egg envelope. On the otherhand, ifthe could expect that many of them would start egg-laying
egg envelope has only been stretched and wrapped around again a few days later. Thus, this species was well suited for
the ovigerous hairs, the coat should consist only of layers the present study.
originating from the egg envelope. To solve this problem,
the modification of the egg envelope of Sesurma haemato- Maintenance ofcrabs and obsen'ations ofegg-laying
cheir an estuarine terrestrial crab was investigated mor-
phologically, and physiological changes in the properties of Females used in this study were collected at Kasaoka,
the envelope are described in terms of adhesion and plas- Okayama prefecture, Japan (Saigusa, 1982). Ovigerous fe-
ticity. On the basis of these results, we present a schedule males on the way to water would cross a road along the
for an egg attachment for this crab. seacoast just after sunset, so each evening at this spot,
Another purpose of this study was to investigate the 50-200 females were collected and transported to the lab-
disassociation of the embryo attachment system from the oratory. After the females released their larvae, they were
ovigerous hair. When embryonic development is complete, washed repeatedly with a large quantity of tap water to
hatching occurs in synchrony with the times of high tide remove adherent hatch water and any zoeas still attached.
(Saigusa, 1992. 1993; Saigusa and Terajima. 2000). An The females were then kept in plastic containers (70 cm
active factor that we call OHSS (ovigerous-hair stripping long. 50 cm wide, and 30 cm in depth), each containing a
substance) is released by the embryos at the time of hatch- small quantity of seawater diluted less than 59r. Females
ing, causing the investment coat and the rest ofthe embryo that had released larvae each night were moved to different
attachment system to slip off the ovigerous hairs several containers. The containers were exposed to the natural day-
EGG ATTACHMENT SYSTEM IN A CRAB 291
night cycle in the laboratory (sunset at 1830-1920 and from the embryo at the time ofhatching. This factor, which
sunrise at 0450-0530 in July-August). Within a lew days, we call OHSS (ovigerous-hair stripping substance), is con-
mostfemales began to incubate theirnew clutch offertilized tained in the hatch water of crabs (Saigusa, 1994, 1995,
eggs. A board in the container, providing shelter for the 1996). In this study, ovigerous seta with premature embryos
crabs (see Saigusa. 1980), was gently removed at times, and attached were separated from a few ovigerous females and
any female that had just started egg-laying was very care- cut into three pieces each. The tip ofthe seta was discarded,
fully isolated to a transparent vessel so that egg-laying and and each clusterofembryos was placed in awell ofaplastic
subsequent behaviors could be observed, and the eggs were culturedishand incubated with0.5 ml ofconcentrated hatch
retrieved. water (see Saigusa and Iwasaki, 1999). Control embryo
mM
clusters were immersed in 0.5 ml of20 Tris-HCl buffer
(pH 8.5). Each segment of embryo cluster was removed
from the hatch water, or the control buffer, after 2 min, 4
Fertilized eggs tor microscopy were obtained from the min, 6 min. 8 min, 10 min. 20 min. 60 min, and 120 min,
abdomen after egg-laying (Fig. 1A and IB), whereas ma- and each was fixed for transmission electron microscopy.
ture. but unfertilized (unextmded) eggs were obtained di- The fixation and ultrathin sectioning were carried out as
rectly from the ovary. These eggs and embryos were fixed described above.
for 1.5 h at room temperature in 2.5% glutaraldehyde dis-
mM mM
solved in a cacodylate buffer (400 NaCl. 100
Na-cacodylate. pH 7.4). and then washed. These egg sam- Results
ples were further post-fixed for 1 h. at 4 C. with [7c OsO4
dissolved in the cacodylate buffer. After post-fixation, the Ultrastntcture ofthe einhryo attachment system in the
specimens were dehydrated through a graded series ofeth- incubatingfemale
anol solutions and embedded in a low-viscosity epoxy resin
(TAAB). Ultrathin sections were cut with a Porter-Blum Egg envelope. The female of Sesarma haematocheir has
MT2-B ultramicrotome and collected on copper grids. four pairs of abdominal appendages; each bears one plu-
These sections were further stained with 1% uranyl acetate mose and one non-plumose ovigerous seta (see Saigusa,
dissolved in 50% ethanol. and with 0.1% lead citrate, and 1994). Embryos attach to the ovigerous hairs by a stalk, the
were then examined with a Hitachi H500H electron micro- funiculus, and rarely to each other (Fig. 2A). The funiculus
scope operated at 75 kV. never is attached directly to an ovigerous hair, but rather to
a coat that invests the hair. Figure 2B shows the fine
Assa\ing the adherence andplasticity ofthe egg envelope structureofthe eggenvelope.The envelopeconsistsofthree
With a fine forceps, 5-20 fertilized eggs were removed layers: the outermost (El), the thick middle (E2). and the
Tevheersye 1e0ggmsinwefrreomplaacfeedmainleajpuostlyasfttyerreengeg-pletaryiindgis(hFi(gF.alIcBo)n. cionnnseirsmtosstofthtiwno(dEi3s)tinlgayueirssh.ablTehesuobultaeyerrmsostdesliagyneartefdurtEhlear
and Elb.
1007; Becton Dickinson Labware, NJ) with seawater di-
luted to one-third (109). Characteristically, when an egg Funiculus. Figure 3A shows the base of the funiculus at
eadvheenriefdthteodtihsehbwoetrteomsloowfltyheshpaekterin bdiyshhaintdw(oFuilg.d 1nCo)t. rTohlle, istisngjluencltaiyeorn,w0i.t3h-0t.h4ejeammbtrhyiock., Twhhiechfuinsiccounlutsincuoonussiswtisthofthea
strength of envelope adherence was estimated as the per- outermost layer (El) investing the embryo. Moreover, the
caednhteargeentofegeggsgswtehraet agdenhtelryedrotloltehdewbiotthtoamfoorfcaepdsi,sht.heIfetnhvees-e utlwuoss(uFbilg.ay3eBr)s.(IEnlacoantnrdasEt,lbn)oacreomaplosonepnrtesoefntE2inotrheE3funwiacs-
lope elongated (Fig. ID). While one end of the elongated ever found in the funiculus. The proximal end of the funic-
envelope () remained attached to the bottom of the dish, ulus is folded onto and wrapped around the ovigerous hair
ttrbhoraeleklaeeokndp.paoAsssutfiafttirehcioiseuenngptdholiyne(,tsa,t1eii)tmthashetteielrlleotnefhgnetcihtlseoonspflveaedtslhtteoihcpeieetnyev.goegrlA.otpInhfeeetw(heaegp-geeatg'rwig)odwwuiaalsssdh tfuiosnrucopalmrluoldodyenudiecneivnhetasthithersestotwonrraltatyhipefpoiinenndeegxi.htna;viTrehf,soetrbmuteethnxiatacmfkcpneoleawests,h(aFoiiifrngs.tFahi3regCeu)cr.soeaoTtmh3edCeti.ifcfmoteeahrstes
was used for each test of eggs removed from a female. coat is only 2-3 jam at its thinnest, but 25 jam at its thickest.
In every part, the investment coat, like the funiculus. in-
Action ofOHSS on the cinhryo attachment svatem: cludes only the El layer and its components (Ela and Elb).
Thus, the investment coat wrapping the ovigerous hair con-
fine structure sists morphologically ofthe same materials as are present in
The embryo attachment system slips offofthe ovigerous the funiculus and the outermost layer of the egg envelope.
hair after hatching. This is due to an active factor released The investment coat is tihtly connected to the ovigerous
292
Figure 1. Egg-layinginScsannahaematocheirandamethodofmeasuring[headhesivenessandplasticityofthe
eggenvelope.(A)Morphologyofthethorax(th)andabdomen(ah)ofafemale.Eggsjustextrudedfromthegonopore
(go)attachtomanyovigeroushairs(seeFig.6A)arrangedontheovigerousseta(os).Noeggsadheretotheplumose
setae(ps).m: monthparts,vvl: walkingleg.an:anus.(B)Theincubationchamber,andaneggclusternewlyextruded
fromthegonopore.Theextrudedeggsliebetweenthoraxandabdomen (i.e., incubationchamber),protectedby the
foldedabdomen,andareincubatedfornearlyamonth.Thewholeeggclusterisimmersedinatransparentincubation
fluid.Abbreviationsarethesameasin (A).(C)Eggs(eg) within5 minafteregg-laying.Theeggenvelope isnotyet
adhesive. (D)Theeggs20-30minafteregg-laying.Theeggenvelope(en) isadheringtothebottomofapetridish.
Atthistime, ifanegg isgently rolled withafineforceps, it movesalittle, then it is slipped(orcrushed)outofthe
envelope,a:oneendoftheenvelope,whereitadherestothebottomofthepetridish(attachmentpoint).</': theother
endoftheenvelopewheretheeggwasrolledorpushedwithaforceps.Theplasticityoftheenvelopewasmeasured
asthelengthofa-u' atthistime. (E)Theegg40minafteregg-laying.Theenvelope(en)showedbothadhesiveness
andplasticity.(F)Theeggs90minafteregg-laying.Adhesivenessoftheenvelope(enIwaslost,sotheenvelopedid
not adhere tothe petri dish. But theenvelope attainedmaximum plasticitv.
hairs (Fig. 3C); when clusters ofembryos were pulled away Investment cunt. When cross and oblique sections of
from the ovigerous seta with fine forceps, most ofthe hairs the attachment were examined (Fig. 4A), it was always
broke away from the seta (not shown: see Saigusa, 1995). sublayer Ela that was seen to adhere directly to the
EGG ATTACHMENT SYSTEM IN A CRAB 293
mm
1
B
Figure 2. Embryo attachment system ofSesarma haematocheir: mature embryos that are within hours of
hatching. (A)Connectionoftheembryo(em)toanovigeroushair(oh),co: investmentcoatwrappedaroundthe
hair, f: funiculus. h: heart ofeach embryo. Note that the funiculus does notattach at aparticularposition with
respecttotheembryo. (B)Finestructureoftheembryonicenvelope. El: theoutermostlayer, whichconsistsof
twofurthersublayers (ElaandElb). E2: the middle layer. E3: the innermostlayer. Theembryo (em), close to
hatching, is wrapped further in two very thin layers: i.e.. exuviae (exl and ex2) which are deposited by the
embryo, zc: zoeal cuticle. Just after hatching, the zoeas cast offthese exuviae and then begin to swim.
ovigerous hair (Fig. 4B, C). A cross section (Fig. 4B) Fig. 4B: much the same magnification). In the oblique
showed the presence of a thin layer 40 nm thick (al) on section, furthermore, the al layer is seen to be underlain by
the surface of the ovigerous hair. This layer contacts a comparatively electron-dense layer (a2). Many tubular
ridges in Ela that are arranged parallel to the hair at structures (25-70 nm in diameter) were observed in this
intervals of 130-160 nm. Tubular structures, 50-70 nm layer andjust below it. The remainder ofthe cuticular wall
in diameter, appear at random in the ovigerous hair ofthehairhasthe usual stratifiedstructures. Ovigerous hairs
(small arrows in Figs. 4B, 5A, B). taken from female crabs before egg-laying were also exam-
In oblique, nearly longitudinal section (Figs. 4C, 5A). the ined (Fig. 5B). Note that iterated electron-dense structures
ridges on layer Ela in contact with the al layer appear as are present on the surface of these hairs, and at the same
slender electron-dense structures arranged at the same in- intervals, although the investment coat, and thus Ela, are
terval as the ridges (130-160 nm) (compare Fig. 5A with absent.
294 M. SAIGUSA ET AL.
OUT
OUT
Ela
100
Figure3. Thecomponentsoftheembryoattachmentsystem: matureembryosthatwillhatchinafewdays.
(A)The funiculus(f) iscomposedoftwoopposedportionsofeggenvelope(El). The funiculusextendstoward
the coat (col wrapping one of the female's ovigerous hairs; the ovigerous hair is not shown. IN: inside the
funiculus. OUT: outside the funiculus. E2 and E3: middle and innermost layers, em: embryo, exI and ex2:
exuviaedepositedbytheembryo. (B)Componentsofthefuniculus. ElaandElb:twodistinguishablesublayers
oftheouterlayerEl. Asubstanceoflowelectrondensityisseeninsidethefuniculus.(C)Acontinuouscoat(co)
investing two ovigerous hairs (oh). Symbols are the same as in (A).
EGG ATTACHMENT SYSTEM IN A CRAB 295
100nm
Figure 4. Attachment of the investment coal to the ovigerous hair; observed in mature embryos. (A)
Sections of an ovigerous hair (oh) invested by the coat (co). CS: cross section. OS: oblique section, nearly
longitudinal, f: funiculus. (B) Inacross section,ridge-likeextensionsofElaareattachedtoalayer(al)onthe
surface ofthe ovigerous hair. al: alayered structure underlying al. Elaand Elbare the two sublayers ofEl.
Smallarrowsindicatetubulesinthea2layer,oh:ovigeroushair.(C)Inanobliquesection(lowermagnification),
theridgesappearasslender,evenly spacedstructuresattachedtosublayerEla. Manytubularstructuresareseen
in the a2 layerandjust inside it (some ofthem are shown by small arrows). Compare with (B).
296 M. SAIGUSA ET AL.
the eggs are laid, the female leaves the water and stands
motionless, with her walking legs stretched out and her
abdomen half folded. At the same time, however, the pleo-
pods the ovigerous setae (Fig. 6A) steadily knead the
clutch. The eggs were never adhesive just after laying.
Within 5 min. however, the volume of incubation fluid (a
transparent liquid appearing in the incubation chamber)
increases, and the egg cluster is immersed in this fluid (Fig.
IB). At this time, the egg envelope becomes strongly ad-
herent to the ovigerous hairs (Fig. 6B). The eggs never
oh attach to the plumose hairs (Fig. 6A).
I <X2 Between 10 and 20 min after egg-laying, the female is
still motionless with her abdomen half open, and the egg
cluster is still being kneaded by the ovigerous seta. But the
number ofeggs adhering to the ovigerous hairs is markedly
B increased. While the eggs are attaching to the hairs, the egg
envelope is expanding; its volume becomes 1.5-1.6 times
larger than that of the egg itself (Fig. 6C), and it is still
T T T T T Y strongly adhesive (Fig. 6D). Although the investmentcoatis
complete between 10 and 40 min after egg-laying, the
funiculus is not yet formed at this time.
Forty minutes after egg-laying, adherence of the egg
envelope seems, actually, to decrease; that is, the eggs still
oh adhere to the bottom of the petri dish (see Fig. 9), but the
attachment point (a in Fig. IE) shifts slightly when the egg
is gently pushed with a forceps. The incubation fluid im-
mersing the egg cluster is also decreased 40 min after
egg-laying. The funiculus begins to form at about this time
anFoivgiugreero5u.s hSatirr.uc(tAur)eAonftohveiagtetraocuhsmehnatirb(eothw)eewnratphepeidnvienstamnenitnvceosattmaenndt (Fig. 6E). and is completed between 60 and 120 min after
coat (oblique section, magnified from Fig. 4C; the scale is the same as in egg-laying. With the funiculus formed, the abdomen of the
Fig.4B);observedinmatureembryos, al:thethin,electron-denselayerto female begins to move up and down, but this movement
which theslenderextensionsfrom layerElaare regularly arranged. Small stops 2 h after egg-laying. With this behavior, all the eggs
arrows show tubules. 2: a layered structure underlying al. The thin, would be well folded by the abdomen. This brief period of
oelveicgterroonu-sdehnasierl(aoyhe)r (f*r)omis aalsfoemoablseertvheadt oins ntohet siunrcfuabcaetionfgEalac.lu(tBc)hAonf abdominal fanning is not likely to aerate the eggs; possibly,
embryos.Thus, thishair isnotinvestedbyacoat. Notetheelectron-dense it serves to fold the clutch into the incubation chamber. At
structures(downwardsolidarrow heads: T)aboutthesameintervalasthe this time, the female becomes mobile again, and can easily
extensionsfromEla(comparewithA).Smallarrows: tubulesseen inboth escape in response to external stimuli.
a1 and a2. Figure 6F shows eggs that were kept in 20 mMTris-HCl
buffer in a petri dish for one night afteregg-laying. At first,
the eggs adhered to the bottom of the dish, or to the other
Formation ofthe embryo attachment s\stem
eggs. In the morning, the diameter of the envelope had
after egg-laving
expanded 3-fold. These eggscouldbe detachedwith forceps
Scheduleofevents. An examination ofbehaviorin several from the dish or from the other eggs without envelope
females revealed the time course ofegg-laying, egg attach- deformation, but such eggs were never again attached to
ment, and funiculus formation in Sesarma. No visible event either a dish or to the other eggs. The plasticity of the
signals the start of egg-laying, and it proceeds rapidly, so envelope was also lost.
determining a precise schedule ofthis process was difficult. Egg envelope: origin oftlie outermost layer (El) and its
In addition, an exact point oftransition between successive morphological change after egg-laving. The fine structure
events could sometimes not be determined. Thus, the timing ofamature, unfertilizedegg is shown inFigure 7A. The egg
of each component behavior summarized in Table 1 in- is enclosed by a follicle cell (about 1 /u,m thick), beneath
volves some speculation. which is an envelope (vitelline membrane)consisting oftwo
The eggs are extruded from the gonopore and into the sublayers (Ela' and Elb'). The sublayer Elb' is about 0.5
incubation chamber (Fig. IB). Between 10 and 15 niin ju,m thick in the ovary (Fig. 7A); but within 5-10 min after
seems to be required for all the eggs to be extruded. When egg-laying, Elb' becomes 5-fold thicker (2.5 ju.m; Fig. 7B).
EGG ATTACHMENT SYSTEM IN A CRAB 297
Table 1
Time course <>/Vi^i; attachment itiulformation of the investment CIHII and(nniiiiliis
Time (min) after egg-laying
10 1? 20 3(1 60 90 120
A. Eggs O
Incubation fluid O
Modification ofihe egg envelope (El)
<F.!a' + Elh'i (El: Fusion) (Ela + Elb)
Adhesiveness ol the egg envelope o
Egg attachment to ovigerous hairs
Expansion ofthe egg envelope
Formation ofthe investment coat and funieulus
(Investment coat) (Funieulus)
B. Females o
Abdomen halfopen
Kneading ofthe egg cluster o-
Up and down movements of the abdomen
Locomotion
*Zero minutes indicates the estimated end ofegg-laying.
O > -
Symbols: start. end. continuing. uncertain limits ofsequential events.
The sublayerElb' is furthermodified, as its structure comes complete 12-24 h after egg-laying (not shown). This layer
to resemble that of sublayer Ela' (Fig. 7C). Soon the may be formed by the secretion from the embryo (see
distinction between the two sublayers is completely lost, arrows in Fig. 7D, E). The third (E3) layer appears a few
and they fuse to form a homologous envelope (1.2 /urn days later, possibly secreted by the egg like the middle layer
thick); we designate this layer El (Fig. 7D). The interme- (not shown).
diate stage in the formation of El (Fig. 7C) was rarely The egg envelope attaches to ovigerous hairs. As men-
observed, indicating that the process of fusion progressed tioned earlier, the egg envelope expandsjust after attaching
very rapidly, possibly within 5 min after the thickening of to the ovigerous hairs (Figs. 6C, 8A). The interaction be-
Elb'. The cause of the rapid modification of sublayer Elb' tween the envelope and the ovigerous hairwas examined by
is not known, but it could be related to the appearance of transmission electron microscope. Attachment of the enve-
clusters ofelectron-dense granules that seem to be secreted lope onto the ovigerous hair occurred about 10 min after
fromtheegg intothe spaces below Elb' (arrows in Fig. 7B). egg-laying, when two sublayers (Ela' and Elb') began to
After the fast modification of Elb'. the homogenous fuse (Fig. 8B). At higher magnification (Fig. 8D). the egg
structure of layer El is retained for 10-30 min after egg- envelope can clearly be seen to be a homologous layer (El)
laying. By 30-60 min after egg-laying. El again divides (see also Fig. 7D).
into two sublayers, Ela and Elb (Fig. 7E); but whether The egg envelope at this time (10-15 min after egg-
these new sublayers correspond to the original Ela' and laying: Fig. 8A-D) is strongly adhesive, so it adheres, not
Elb' (Fig. 7B) is not known. In any event, our morpholog- only to the hair, but also to other envelopes. For example.
ical findings suggest that the outermost layerenveloping the Figure 8B shows an envelope (El) folded on the upper half
egg (El) is produced by the egg itself, rather than being ofthe hair. While this envelope is adhering to the ovigerous
formed by externally secreted materials. Also, formation of hairfor 14-15 jam (from points al to a2). it is also adhering
the middle layer (E2) begins at 10-15 min after egg-laying to itself for 20 jiim (bl-b2). Note that the envelope in
(Fig. 7D). Figure 8B is not attached to the lower part of the hair: this
Formation ofsubsequent layers ofthe egg envelope. In may be due to a weak attachment that was separated during
addition to the outermost layer (El), a thick middle layer fixation or embedding. The large space between El and E2
(E2) and an innermost thin layer (E3) invest the embryo suggests that the envelope (El) had started to expand or
(Fig. 2B). These two layers are included in neither the elongate.
funieulus nor the investment coat. The middle layer (E2) The connection between the newly attached homogenous
appeared at about the time that El was becoming homoge- layer (El) and the ovigerous hair (Fig. SC and 8D) is
neous in structure possibly 10-15 min after egg-laying similar to that in a mature coat (Fig. 5A). In Figure 5A,
(Fig. 7D). The middle layer forms very slowly, and is however, the surface of Ela appears as an electron-dense
298 M. SAIGUSA ET AL.
Kifjure 6. Egg attachment and formation of the funiculus after egg-laying: observed with a stereomicro-
scope. Observations of the eggs that were placed in a petri dish just after laying. (A) A pair of abdominal
appendages of a female: ovigerous seta (os) and plumose seta (ps). Ovigerous hairs (oh) are arranged on the
ovigerous seta. (B) An egg (eg) adhering to an ovigerous hair(oh): 5-10 min afteregg-laying. (C) Expansion
oftheenvelope(en): 10-15 minafteregg-laying;theenvelopeshowsstrongadhesiveness.(D)Formationofthe
iin-c\tniciitcoalfollowingattachment tothe hair: 15-30minafteregg-laying. (E)Formationoffimiculus(f): 60
mm
afteregg-laying. Althoughtheadhesivenessoftheenvelopeislost,theplasticity increases. (F) Eggsplaced
in a petridishcontaining 20mA/Tris-HCl bufferovernight. Eggenvelope swells in allembryos. Adhesiveness
o! the envelope has already been lost.
thin layerdistinguishable from anothersublayer(El; 40-50 with time. Adherence was estimated with 10-20 eggs, and
jitm thick in oblique section). Such a layer-like structure was plasticity was measured with 5-10 eggs (Fig. 9). The egg
never observed in the newly attached envelope. Figure 8E envelope was not adhesive immediately after egg-laying
shows the egg envelope about 80 min after egg-laying. The (not shown), but after 5 min, the envelope became strongly
funiculus has already been formed. Moreover, the envelope attached to the bottom of the dish, and it did elongate as
and funiculus are again divided into two sublayers (Elaand soon as it was gently rolled with a fine forceps.
Elb), and the inner layer (E2) has thickened. In Figure 9, adherence of the egg envelope was 100%
Temporal changes in egg envelope adhesiveness and between 10-50 min after egg-laying. But 40-60 min after
plasticity. Newly extruded eggs were removed from the egg-laying, the fixed attachment point(a in Fig. IE) became
female every 10 min afteregg-laying. The eggs were placed mobile when the eggs were rolled with a forceps. Thus,
in a polystyrene petri dish, and temporal changes in the envelope adhesiveness begins to decrease markedly about
adherence and plasticity of the envelope were examined 40 min after egg-laying. In contrast, envelope plasticity
