A Substance Inducing the Loss of Premature Embryos From Ovigerous Crabs

9 Pages·1994·3.4 MB·English

by  M Saigusa

Reference: Bin/. Bull 186: 81-84. (February. 14941
A
Substance Inducing the Loss of Premature Embryos
From Crabs
Ovigerous
MASAYUKI SAIGUSA
Okayama University, CollegeofLiberalArts& Sciences, Tsushima 2-1-1, Okayanui 700, Japan
Abstract. The embryos ofan estuarine terrestrial crab transparent outer membrane and clustered on pleopod
(akate-gani: Sesarma haematocheir) are attached bya fu- setae beneath the folded abdomen ofthe female(Herrick,
niculusto ovigerous hairs on the maternal pleopods, and 1895; Yonge, 1937, 1946, 1955; Cheung, 1966; Fisher
are ventilated by the female until hatching occurs. When and Clark, 1983; Goudeau and Lachaise. 1983). Theem-
females were kept in a small quantity ofdiluted seawater bryos are attached to the pleopod setae by a stalk: the
(about 10%), hatching and larval release occurred in all funiculus. While attached to the ovigerous hairs by this
cases. Incontrast, whenthe medium washatch water(i.e., stalk, the embryos are ventilated by the movement of
the filtered medium intowhich larvae had been released), pleopods ofthe female until the thick outer membrane
most ovigerous females liberated their embryos prema- breaks, indicatingthe beginning ofhatching. The present
turely, but hatchingdid not occur. Theegg masses(cluster study is concerned, in general, with hatching in an estu-
ofembryos) carried by these females were not released as arine terrestrial crab; it is focused on an active substance
usual, but were gradually extruded from the brooding that is released outside ofthe egg membrane at the time
chamber, and within a few days all had dropped to the ofhatching.
bottom of the beaker. No morphological changes were Hatching enzyme is among the important substances
found on the outer egg membrane, the funiculus, or the that are discharged upon hatching in many groups ofan-
coatinvestingtheovigeroushairsoffemaleskept in hatch imals. In the teleost Oryzias latipes, the enzyme, which
water. But the ovigerous hairs did slip easily out of the is secreted by the embryo, digests the egg membrane, al-
coat, and thiscaused theextrusion oftheegg masses. The lowing the embryo to emerge (e.g., Ishida, 1944; Yama-
active factor called incubation disrupting substance gami, 1972). This teleost enzyme is actually a mixture of
wasstablewith freezing, but heat-labile. In normal females two kinds ofproteases that act cooperatively on the inner
(i.e., those nottreated with hatch water), broken eggcases layeroftheeggcase(Yasumasu etai, 1989a, b). Embryos
and funiculi remain for a time after hatching with the ofthe toad Xenopus laevis also secrete a proteolytic en-
coat on the ovigerous hairs, but they are gone by the zyme(from thefrontal region oftheembryos)thatdigests
morningafter hatching. So thesecretion ofthis incubation- the fertilization membrane (Carroll and Hedrick, 1974).
disruptingsubstance may participateincleaningtheovig- Katagiri (1975) also demonstrated a hatching enzyme of
erous hairs ofold investing coatsand funiculi after larval 55-60 kDa thatdigeststhewholeeggcasein thefrogRana
release, thus preparing for the attachment of the next chensinensis. Similarly, sea urchin embryosat the blastula
clutch ofembryos. In addition, this substance may play stage (about 10 h after fertilization) secrete a proteolytic
a role in hatching. enzyme that dissolves the fertilization membrane. Re-
cently, Lapage and Cache (1989) purified the enzyme, a
Introduction glycoprotein of 51, 52, or 57 kDa that autolyzes to an
inactive form of30 kDa.
The newly oviposited eggs ofdecapod crustaceans, ex- Among crustaceans, adult barnacles (Balanus) release
cept nonbroodingpeneid shrimps, arewrapped in a thick a substance that stimulatesthe hatchingofnauplius larvae
(Crisp and Spencer, 1958). Ifthe barnacles were fed, the
Received4January 1992:accepted 22 November 1993. factor was secreted within 1 or 2 days and the embryos
81
82 M. SAIGUSA
hatched; ifthe barnacles were starved, hatching did not larval release by females occurs about the time of noc-
occur. Because the factor could be extracted from the tis- turnal high tides in the field.
sues ofstarved animals in the laboratory, aswell as those
fed under natural conditions, it may be produced by the Preparation oj hatch water
barnacle'sown metabolism, and not related toaparticular
food source (Crisp and Spencer, 1958). An active factor Eggs of S. haenuitocheir are dark brown at the early
extractedand purified from adultbarnacletissuescontains stagesofembryonicdevelopment, but becomea brownish
a prostaglandin-like compound that stimulatesembryonic green (possibly becauseofyolkconsumption)ashatching
muscle (Clare et al., 1982). approaches. Females carrying brownish-green eggs (i.e.,
In the brine shrimp Anemia salina, glycerol is highly embryos that should hatch within a few days) were se-
concentrated in dormant cysts, and then rapidly disap- lected, removed from the plastic containers, and placed
pears(Clegg, 1962). When theembryosemerge from their individually inglassorplasticbeakers(8.5 cm indiameter,
egg cases, glycerol is released into the medium (Clegg, 12 cm in height) containing 30 ml of 10%o seawater. This
1964). medium isprepared from naturalseawater,which isboiled
Embryos ofthe terrestrial crab Sesanna haematocheir for5-10 min,cooled,andthendilutedwithdistilledwater;
hatch on land around the time ofnocturnal high water. the solution isaerated forat least oneday before use. The
Hatching occurs in a very short time, and the timing is medium wasrenewedat intervalsof1-2days. Underthese
highly synchronized among embryos (Saigusa, 1992, conditions, all ofthe femalescontinued tocarryeggsuntil
1993). As soon as hatching is complete, the crabs enter hatching (Table I, top).
the water and release the hatched larvae by making vig- Hatching of S. haematocheir is highly synchronized;
orous fanning movements of the abdomen (Saigusa, all ofthe embryos may hatch within about 5-30 min in
1982). The embryos ofovigerous females that were col- the laboratory (e.g.. see Saigusa, 1993). When hatching
lected from the field and kept in the laboratory did hatch was completed, and the female had released all of her
simultaneously. This paper first demonstrates that the fil- larvae into the medium by vigorous abdominal fanning,
tered water intowhich zoea-larvae had hatched (i.e., hatch she was removed, and the medium was filtered through
water) contains a substance that disrupts the incubation nylon mesh or filter paper (filtrate I in Fig. 1) which
ofother females. Next, microscopic studies are described removed the zoeas. As shown in Table I, 23 samples of
that indicate thecause ofthe liberation ofpremature em- tillrale 1 were prepared immediatelyafterthe larval release
bryos from the brooding chamber.
Materials and Methods
medium and a female
Animal handling 30ml(10"/..SW)
Experimental animals were ovigerous females of the
hatching
terrestrial red-handed crab (akate-gani), Scsarma hae-
malocheir. collected from the thicketalongasmall estuary removal of thefemale
at Kasaoka, Okayama Prefecture, Japan. (For details of
filtration
the habitat and the chronology oflarval release, see Sai-
gusa, 1982.) Collections were made on 19 August and 2, I
4, 9, 14, 15, and 16 September 1991. The crabs were filtrate 1 zoeas
quicklybrought into theexperimental rooms, where they newmedium to rinse zoeas
were kept in plastic containers (70 cm long, 40 cm wide,
and25 cm high(containingshallowwater(ca. 1 cmdeep) filtration
and hiding spaces. The waterwas renewed whenever one I
or more females released their larvae in the container, filtrate 2 zoeas
and otherwiseat intervalsof3-5 days. The crabswere fed new medium torinse zoeas
every few days.
Light and temperature were controlled in the experi- filtration
mental rooms. A 15-h light and 9-h dark photoperiod, a I
phase that is much the same as that in the field (light-on filtrate 3 zoeas
at0500and light-offat2000), wasemployed. Temperature Figure 1. Collection ofhatchwater. Each medium contained 30ml
washeld constant at 23 1 C. Underthis light condition. of 10%o seawater. Seethe text fordetails.
INCUBATION-DISRUPTING SUBSTANCE IN A CRAB 83
(i.e.. within 30 min); the rest (22 samples) were prepared Anothersample ofhatch water(filtrate I), obtained simi-
aftera while (i.e., more than 30 min afterthe release, but larly from seven specimens, was frozen at 80C for 8
before 10:00 on the next morning following release). days, and then thawed at room temperature. Thissample
The filtered zoeas were immediately immersed in new was divided among seven beakers, and an ovigerous fe-
medium, and this medium was also filtered (filtrate 2). male was again placed in each.
AmongJiltrale 2, nine samples were generated immedi-
atelyafterthe preparation offillrate I. and then thezoeas Results
were rinsed and filtered once more (filtrate 3) (Fig. 1). Eggattachment topleopodsetae
The rest offiltrate 2 (32 samples) was generated within a
fewhoursafterthepreparation offiltrate I. Becauselarval A female ofSesarma haematocheir has four pairs of
releaseoccursat night, these procedureswere usuallycar- abdominal appendages, each ofwhich bearsone plumose
ried out under illumination provided by a small hand- and one non-plumose seta (Fig. 2A). Many fine hairs
held light. (ovigerous hairs) are arranged in whorls along the non-
plumose seta(Fig. 2B). Fertilizedeggsareattachedtothese
Assay oftheactivity ofincubation-disrupting substance hairsbyafuniculm'(Fig. 2C, D). When theovigeroushair
was removed with fine forceps, the coat that had been
Ovigerous females, that had not been used to collect wrapped around the hair was broken but remained at-
the hatch water, were selected randomly from the stock tached to several funiculi (Fig. 2E). Thisobservation sug-
containers and placed individually in beakers containing gests that the investment coat is formed ofthe same ma-
hatchwater(filtrates 1. 2. or3).Theactivityofincubation- terial as the funiculus (Fig. 2E, F). The morphological
disruptingsubstance ineach filtratewasevaluated interms features shown in Figure 2D, E, and F suggest that this
of the following three grades: (+), a portion of the egg material also constitutes the outer egg membrane, or at
mass protrudes from the brooding space; (++), the egg- least covers the surface ofthis membrane.
mass protrusion progresses further, and some embryos
are scattered at the bottom ofthe beaker, (+++), all, or Protrusion and droppingofegg masses from an
almostall, oftheeggmasshasslipped out ofthebrooding incubatingfemale
space. Observations were finished 4 or 5 days after the As shown in Figure 3A, an incubating female carries
female had been placed in each beaker. To prevent con- her clutch in the brooding space between the thorax and
tamination ofthe medium by fecesand foods, thefemales the flexed abdomen. Premature embryos never slipped
were not fedduringtheexperiment. With time, however, out ofthe brooding space when the females were in 10%o
they often ate theirown embryos, eitherthose released in seawater, and hatching occurred in all cases (Table I). At
thebeakerorthosesqueezed outofthebroodingchamber. the completion of hatching, these females released the
But females nevereat theirembryos in normal brooding. hatched larvae into the medium with vigorous fanning
For these reasons, the effect oftest solutions wasjudged movements ofthe abdomen.
within 4-5 days. Soon after ovigerous females were placed in hatch wa-
ter, a mass of premature embryos protruded from the
Observations with a scanning electron microscope brooding chamber, and then dropped into the beaker.
Embryosattachedtothepleopod setae, aswell asthose This process is shown in Figure 3B-E'. Figure 3B shows
released in the hatch water, were collected and fixed with a portion ofthe incubating egg mass starting to protrude
5% formalin in distilled water for 2 or 3 days. They were from the brooding space (+). In Figure 3C and D, the
then washedafewtimesin 50% ethanol, then dehydrated embryo-protrusion has proceeded further (++). These
through a graded series of ethanol concentrations and premature embryos were often released by the females in
dried in a critical point apparatus (Hitachi HCP-1). The association with abdominal fanning behavior and were
samples were mounted on metallic stubs with a piece of scattered at the bottom ofthe beaker. In Figures 3E and
double-sided stick tape, plated with gold, and observed 3E', the embryos have all (or almost all) dropped from
with a scanning electron microscope (JEOL JSM-T300). the brooding chamber (+++). A remarkable feature of
this phenomenon is that, from the beginning ofembryo-
Boiling andfree-ing ofthe test samples protrusion to the time that all of the egg masses have
slipped out ofthe female takesonlya fewdays. Asshown
A sample offiltrate 1. obtained from 15 females that in Table I, 37 of 45 females (82%) treated with hatch
hadjust released their larvae, was boiled for 5 min. After water(filtrate 1) releasedtheireggmassesintothebeaker.
it had cooled, the solution wasdivided among 15 beakers Clearly, the active substance that disrupts incubation is
(30 mleach), andan incubatingfemalewasput intoeach. contained in hatch water.
84 M. SAIGUSA
ga
Figure 2. Thepleopodsetaeandattachmentofeggs.(A left)Thorax(T)andabdomen(A)ofaSi'Mirnm
haematocheir female, wl: walking leg: an: anus; ga: genital aperture. (A right) The second abdominal
appendageshowingplumoseandnon-plumosesetae,ps:plumoseseta:os(nps):non-plumoseseta;oh:ovigerous
hair. (B)Ovigeroushairsonthenon-plumoseseta. Scale = 100Mm. (C)Theoutereggmembrane(om)and
fumculus(fu). Scale = 10^m. (D) Embryosattached toan ovigeroushairby funiculus. Thisspecimen was
fixed with 5% formalin and observed in 50% ethanol. em: embryo; fu: funiculus; co: thecoat investingan
ovigerous hair; oh: ovigerous hair; im: inner(or lining) membrane; om: outer thick membrane. In living
INCUBATION-DISRUPTING SUBSTANCE IN A CRAB 85
The activity ofany sample ofhatch water (filtrate 1) eggs attached to intact females. The embryos were ob-
seems to vary with the number of zoea-larvae released viously alive (Fig. 4A), and the funiculus remained at-
into the medium. When the hatch waterwasderived from tached to the coat investing each ovigerous hair. But a
medium into which a large number of zoea had been cleardifference wasobserved in thecoat investing oviger-
released, almost all ofthe females dropped theirembryos oushairs. Eggmasses incubated byuntreated femaleswere
within 1 dayaftercontact. But hatchwaterprepared from attached tightly to the ovigerous hairs (Fig. 2F and F'),
a medium containing only a small number oflarvae, in- whereas those dropped in hatch water always lacked an
duced this phenomenon 1-3 days later. ovigerous hair in the twig of the investment coat (Fig.
Fourfemales, though treated with hatch water(filtrate 4B). Premature embryo release is thus due to the invest-
]), showed no sign ofegg mass protrusion (Table I). This ment coat separating from the ovigerous hairs, and not
negative result, however, could have been due to an in- the funiculus detaching from the coat.
sufficient concentration of active substance. When the When hatch-water-induced protrusion of egg masses
unresponsive females were isolated after the experiment was in progress (+ or ++; Fig. 5A), groups ofembryos
ina new, unoccupied container, they finallydropped their wouldeasilyslipofftheovigeroushairswhengently pulled
egg masses some days later (unpub. obs.). In four other with a fine forceps (Fig. 5B). These embryos were always
females (Table I), normal hatching and larval release oc- accompanied by the funiculus and coat. In contrast, when
curred before egg mass protrusion could begin. Presum- theeggmassofacontrol femalewaspulled, theovigerous
ably, theseovigerousfemaleshadbeencollectedjustprior hairs were broken away from the seta (not shown).
to hatching, which simply occurred on schedule, uninflu-
enced by the incubation-disrupting substance reported
Discussion
here.
The 15 samples ofboiled hatch water had no effect on The embryos of most decapod crustaceans are incu-
incubation: no egg masses protruded from the abdomen bated by the females until hatching occurs. So it may be
(Table I), sothe incubation ofthe test femaleswasclearly natural that a portion ofthe egg cluster is lost from the
maintained. In contrast, the egg masses protruded and female during embryonic development. For example,
dropped as usual when seven females were exposed to some embryos may be dropped when the female cleans
hatch water that had been frozen and thawed (Table I). the clutch using her chelae. Laboratory-maintained fe-
Incubation-disruptingsubstance isthusstabletofreezing, males ofthe lobsters Homarus americanus and H. gam-
but is heat-labile. mants often lose a large percentage ofembryos because
To determine whether the active substance is released the funiculus has been improperly formed (Talbot and
in association with larval hatching or is generated by the Harper, 1984). In Sesarma haematocheir, predation on
lcaormvpaearaeftderwtihtehretlheaatseo,ftfhielterfafteect1so(fTafibllterat1e).s2Thaendac3twiveirtey emgaginmaesxsterisnsbiyc tfhacetomragfogrottsheolfosaspoafraesmitbircyoflsy icnanthbeefitehled
was markedly decreased in filtrate 2, and no effect was (unpub. obs.). But the phenomenon reported in the pres-
detected in filtrate 3. Because filtrate I contains most of ent study is quite different from such types ofegg loss.
sthpeonisnicbulbeatfiaoctno-rdiissraulpltirnegleaacsteidvittyo,tIhespemceudlaituemthwathetnhetrhee- noDmiesnruopntifoonr oovfiignecruobuastfieomnaliessc;erbtuatintlhyisapnhuennuosmuealnopnhei-s
egg membrane opens at the time ofhatching. obviously adaptive for females that have released their
larvae. Observationsofthe femaleabdomenjust afterlar-
Microscopic observations ofthe egg masses liberated val release (Saigusa, 1992, 1993) demonstrated that the
prematurelyfrom thefemale broken eggcase(outereggmembrane), funiculus,andthe
Clusters ofembryos shed into the incubation medium coat still remain attached to the ovigerous hairs (see also
were observed under a stereomicroscope (Fig. 4A). The Fig. 6A). A similar event occurs in the lobsters H. amer-
surface of the embryos (i.e., outer egg membrane) and icanus and H. gammams: egg cases and stalks remain
thefuniculuswerenotdifferentmorphologically from the attached on the ovigerous hairs, and they seem to be cast
Figure 2. (Cimliniicil) specimens, the embryocompletely fills, and is pressed against the eggcase, but
in the fixed samples, theouter membrane swells, revealingan inner membrane ora membrane liningthe
eggcase(toregg membranes, seeSaigusa, 1992). (E) Thecoat from which an ovigerous hairwas removed
withafineforceps.Whitearrowheadsshowtheregionwherethefuniculus(fu)branchesoutoftheinvestment
coat.Scale= 100jam.(F)Thecoatwrappinganovigeroushair.Scale = 100^m.Upperleft(F'l: Magnification
ol theportion where this hairpenetratesthecoat. Scale = 10/JITI.
86 M. SAIGUSA
3C
Figure3. Prematureegg-releasefromafemale.(A)A normalclutch incubatedbyafemale, rb:arubber
band to fix the female's body and legs. (B) A female in which egg-release from the brooding chamber is
beginning(+).(CandD)Femalesinwhichprotrusionofeggmasshasproceededfurther(++).(E)Afemale
from which almost all eggs were released (+++). (E') The inside ofthe brooding chamber in this female.
Although some eggs still remained in the chamber, they have already been detached from the ovigerous
hairs.
Figure 4. Morphology ofpremature eggs liberated from the female. (A) Embryos (eml extruded from
the broodingchamber and dropped to the bottom ofthe beaker. These embryos (at early developmental
stage)areallalive. Thecoatsthat investedtheovigeroushairsareseen tobein place. (B) An ovigeroushair
isabsent from its investment coat(co). An ovigeroushairitselfisalso hollow, but itsappearance isclearly
different from that ofthecoat (comparewith Fig. 2F').
INCUBATION-DISRUPTING SUBSTANCE IN A CRAB 87
Table I
Liberationofprematureembryosinducedbythehatch water
Activity offiltrate
Maintenanceof Hatchingand
Medium Number incubation larval release
Freshmedium(10%o SW) 82 (0) 82
Filtrate 1
Generated within 30 min after
hatching 23 21
Generated morethan 30 min
afterhatching 22 16 3
Boiling 15 14
Freezingandthawing 7 6
Filtrate2
Generated within 1 h after
hatching 9 8
Generated for3-1 h after
hatching 32 23
Filtrate3
Generated within 1 h after
hatching
offfrom the ovigerous hairs at the next molt (Goudeau tachment to the ovigerous hairs and the source of the
et al., 1987). attachment stalk have been controversial for a number
Incontrasttothelobster, theremnantsontheovigerous ofyears(Yonge, 1937, 1946, 1955; Cheung, 1966; Fisher
hairsof5. haematocheircompletely disappearduringthe and Clark, 1983). Goudeau and Lachaise (1983) showed
nightofthelarval release, andthe nextclutch isproduced that the funiculusofthe American lobsterHomarus folds
withinafewdays. Afterlarval releaseiscomplete, females around an ovigerous hair, formingseveral turns, and that
unfold theirabdomen and pluck the empty eggcasesand for the innermost coiling turn, the stalk wall adheres
funiculitoeat;indeed, someoftheremnantsareprobably tightlytothe hairsurface, although noadhesivesubstance
disposed ofin this way. But it is not conceivable that a is detectable. In 5. haematocheir, the funiculus extends
purelymechanical pluckingbehaviorcancompletely clear to the coat wrapping an ovigerous hair, which suggests
ovigerous hairsand setae. I conclude that the incubation- that the same material forms both the stalk and the coat
disruptingsubstance, as reported in this paper, causesthe (Fig. 2E, F). Furthermore, this material might make up
investment coat to slip offthe ovigerous hair, facilitating the outeregg membrane (at least the surface ofthe mem-
post-release remnant-eating ofthe female, and releasing brane), as well (Fig. 2C). Because the coat dropped from
the remnants that are not eaten. Because the ovigerous the femaleshowsnoobvious morphological changes(Figs.
hairscompletelycleanoffbythemorningfollowinglarval 4A, B), then the egg masses must be liberated, either by
release (compare Fig. 6A and B), incubation-disrupting a deformation ofthe ovigerous hairs or by a separation
substance may also make it easier for the female crab to ofthecoat in some unknown way from the hairs. But the
get the debris off her ovigerous hairs without damaging deformation ofthe ovigerous hairs must be detrimental
or breaking the hairs which, after all, must be in good to the hairs because a new clutch has to be attached to
condition to receive the new clutch ofeggs. In brief, the them.
incubation-disrupting substance clearsthe ovigerous hairs EmbryosoftheestuarinecrabRhithropanopeusharrisii
for the next clutch ofeggs. can be detached by bovine or porcine trypsin, and are
What mechanisms cause the investment coat to slip released into the medium by the pumping movements of
from ovigerous hairs? The exact mechanisms ofegg at- the females. Some ofthem prematurely hatched as im-
Figure5. Detachmentoftheeggmassesfromtheovigeroushairs(livingspecimen).(A)Twoovigerous
hairs(seethe blackarrowheads)andthe investingcoat thatbeginstoslipoutofthesehairs. Awhitearrow
head indicatestheedgeofthe investmentcoat, em: embryo;os: ovigerousseta. (B)Theend(awhitearrow
head)ofasection ofcoat that had slippedoffitsenclosed ovigeroushairs.
M. SAIGUSA
Figure6. Ovigeroushairsarecompletelycleanedoffbythemorningafterlar\alrelease.(A)Anovigerous
seta just after larval release. The empty egg cases, funiculi, and investing coats are still attached to the
ovigeroushairs. (B) An ovigerousseta(os|on the next morning(about 10h)afterthe larval release.
mobile larvae(RittschoftV al.. 1990). In .V haematocheir, Literature Cited
ovigerous females treated with hatch water certainly lib-
erated premature embryos (Fig. 3B-E'); but in no case CarrOo/lIlR,SKA./ci.1v.s,Jrm.o,rpahnodl.oJg.iIc..alIleevdernitcks.an1d97e4v.ideHncaetcfhoirnaghaintcthhiengtoeandzy\mcen.-
was the funiculus dissolved and detached from the in- /Vi Bid 38: 1-13.
vestment coat (see Fig. 4A). As shown in Figures 4B, 5A. Cheung, I. S. 1466. Thedevelopment ofegg-membranesand eggat-
and 5B, the coal did slip offthe ovigerous hairs. So what tachment in the shore crab. Cairinnx maeiiax. and some related
is liberated from 5. Inicinatoclieir females is masses of decapods../ Mar. Bid. .-l.v.vur. C'.A 46: 373-400.
premature embryos, and no individual free embryos. Clareeg,gAh.aSt.c,hGin.g:Waalkneorv,elD.roLl.e1folrolalapndr,osatnadglDa.ndJi.n-Clriikspe.c1o9m8p2.oundBa.rnMaaclre
The next question concerns the other functions ofthe Bin/. 1.ell 3: I 13-120.
incubation-disruptingsubstance. Becausethisfactorisre- C'legg..). S. 1962. Freeglycerol in dormant cystsofthe brine shrimp
leased from theeggcaseat thetime ofhatching, the factor Ark'ima \alina. and itsdisappearanceduringdevelopment. llml. Bull
mustalsobe moredirectly involved in hatching. Recently. 123: 295-301.
aDcetiVvirtiyesisarnedleFaosredwaorudts(i1d9e9o1f)tihnediecgagtemdetmhbatraanperotienolaystsioc- ClegAogsn,meJo.mtiSi.ac1pM9r6i-em1s.asu.reT./h.aenA'd.cv/oitn.hterBio,rl>o!olef4e1om:fef8rr7ge9ee-ng8cl9ey2ca.enrodlmientadbeovleilsopmibnygecxytsetrsnaolf
ciation with thehatchingofestuarinecrabs; theassaywas Crisp, D. J., and C. P. Spencer. 1958. The control ofthe hatching
based on the proteolysis ofcasein. But how, exactly, such processin barnacles. Proc. Roy Soc L.md B 148: 27X-244,
an enzyme contributes to hatching remains obscure. As De Vries, M. C., and R. B. Forward, Jr. 1991. Mechanisms ofcrus-
shown in Figures 4A, 5A, and 5B, the incubation-dis- tMaacreanBmelg.g h11a0t:chi2n8g1:-2e9v1i.dence forenzyme release by crabembryos.
rupting substance dissolved neither the outer egg mem- Fisher, \V. S., and \V. II. Clark, Jr. 1983. Eggsof1'alaem.ni macrn-
brane nor the funiculi. Neither was the investment coat ilaeniux: I. Attachment tothe pleopodsand formation oftheouter
itselfdissolved (Fig. 4B). But these negative results might investmentcoat. Biol. Bull 164: 189-200
ohfavtehebeeegngdmueemtbortahneer.elaItnivealnyycoeavresnet,sctahnenipnosgsmibiiclriotysctohpayt Goudaenadud,epMo.s,itainodn oFf.eLmabcrhyaiosnei.c1e9n8v3e.lopeSstriunctaurceraob.ftThiexxeugcg&fuCneilclul1u5s:
47-62.
the incubation-disrupting substance also functions as a Goudeau, M., P.Talbot, and R. Harper. 1987. Mechanism ofeggat-
hatching enzyme remains to be explored. tachmentstalk formation inthelobster, Hmnarux. dameleRex 18:
279-289.
Acknowledgments derrick, F. II. 1895. TheAmerican lobster astudyofitshabitsand
development. Bulletin tilic L'nilcdStale* Fish Cinniix\ni 1895:
This study followed from stimulative discussions with 1-252 plus 54 plate pages.
Drs. Masamichi Yamamoto and Hiroko Shirai at Ushi- Ishida.J. 1944. Hatchingenzyme in the fresh-water fish. Oryiias la-
mado Marine Biological Laboratory. Okayama Univer- ///>o Annul tool .//"(. 22: 137-154.
seiatryl.yDmra.nYusacmraipmtotwaoskrienaddlyanhdelepdeidtemdebywiDtrh.tHhierSokEoM.ShTirhaei lKa.taap.ga/.igreIi-,:,.\CpI...,/1<a9<n7>d/5..C.19G3P:raocp1he0er9.t-i1e11s988o.9f.thePhuarticfhiicnagtieonnzaynmdecfhraormacftreorgiezmatbiroynoso.f
and by Dr. Katsuhiko Endo, Faculty ofScience, Yama- theseaurchin embryo hatchingenzyme.J. Biol. Client 264: 4787-
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INCUBATION-DISRUPTING SUBSTANCE IN A CRAB 89
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