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By Dieter Adelung
All CHIVES
FISHERIES RESEARCii BOARD OF CANADA
Translation Series No. 108 I
Release and function.of the moulting hormone during an intermoult interval in the cOmmon shore crab '
'(Ca:Èdiriuà'Illà.èrias L.).
Original titles Die Ausschuttung und Funktion vàn .liâutungshormon w'ghrend eines Zwischenh.âutungs-
' - Intervalls:bei der Stràndkrabbe Carcinus maenas L..
•• From: Zeitschrift fur Naturforschung -(Journal for Natural
History Research), 24 - (11): 1447-1455 1969.
• Translated by the Translation Bureau(PFB) Foreign Languages Division
Department of the Secretary of State of Canada
Fisheries Research Board of Canada Biological Station St. Andrews, N.B.
1970
21 pages typescript
!.;-£) V . SECRÉTARIAT D'ÉTAT
BUREAU DES TRADUCTIONS DIVISION DES LANGUES ÉTRANGÈRES
DEPARTMENTOFTHESECRETARYOFSTA1E
TRANSLATION BUREAU
FOREIGN LANGUAGES DIVISION
CANADA
YOUR NO. DEP AR TMEN T DI VISION/RANCH • CITY
VOTRE N° MINISTRE DIVISION/DIRECTION VILLE '
L
-7 69-1 8-1 4 Fisheries and Forestry Fisheries Research Board St.. Andrews, N.B.:
. OUR NO. LANGUAGE - TRANSLATOR (INITIALS), , , DATE
NOTRE Nb LANGUE TRADUCTEUR(INITIALES)
1937 German P.F.B.
THE RELEASE, AND FUNCTION OF
NIOULTING HORMONE DURING AN
INTERMOULT INTERVAL IN
THE COMMON SHORE CRAB
CARCINUS MAENAS L.
By D. Adelung
II. Zoological Inatitute of the Ju .à.tus Liebig-University, Giessen and
Physiological-Chemidal Institute 9f the Philipps-University,'Marburg.
.SUMMARY
The moulting cycle of the shore crab has been divided into 21
different stages on the basis of new criteria. Besides the moephologiéal
characteristics'that have been used hitherto as criteria for the individual
' stages, the state of development of regeneratéd appendages and the speed
with which the crabs complete their moulting cycle have also been used
for this purpose.
The content of moulting hormone has been determined in several •
505..200.. 10..31
animals for each stage and a curve for the titre of the hormone during
UNEDITED DRAFT TRANSLATION Only for information
TRADUCTION NON REVISÉE - Information seulement
- 2 -
a moulting cycle han been constructed. The function of the moulting hor-
mone in inferred from the changes in the hormone coritent and from the
morphological and physiological changes that occur siMultaneously. Two
releases of hormone occurred before the, moult, one six'to eight day, the
other One to two days before ecdysià. The first releaSe of hormone initiates
,probably the apolysis, the second causes an increase in the osmotic value
of the haemolymph and thus leads te the initiation of the moult in the
more restricted sense. Immediately after the moult there is another release
of hormone that Presumably regulates the hardening process in the cuticle.
After all processes connected with the preceding moult have been completed,
a further brief release of hormone initiates the regenerative development
and perhaps the next moulting cycle.
A comparison of the content of moulting hormone in the shore
crab during a moulting cycle with that in insects during the pupal moult
shows a few agreements, for example, in the amount of the hormone content
and there are also a few differences that can be explained by the dif-
ferences in the mode of living.
The growth in size in decapod crustaceans is connected with
moults. The moment of moulting depends on many external and.internal fac-
tors. Thus temperature, continuous light, the presence of others of the
same species or lack of food can delay a moult that may otherwise be
due 1 e 2 . Under constant conditions, however, the animals do moult at definite, 1448 , 1
predictable intervals3. One of these endogenous factors is body size.
The larger an animal, the longer is the time interval between moults. How-
ever, it is not only the size, but also the potential increase in size that
the animal will experience at a moult, that plays a role in the initiation
- 3 -
in some species: a certain increase in size must be warranted - before each
moult, in order that a Moult is to take place.
The exogenous and endogenous factors influence the rhythm of -
.moulting in the crustaceans by way of the central ,nervous-systeM.,This s.
determines through a hormonal mechanism when a moult is going to take
place. When a moult is tolpe prevented, thè central nervous System causes
the release of a moult inhibiting hormone. This is produced in the neuro-
secretory cells of the complex'of the sinus gland-X organ that belongs
to the tritOcerebrum. The moult inhibiting hormone presumable apts in an
inhibitOry manner on the moulting gland of the crustaceans4. If the effect
of the mould inhibiting hormone is lacking, the moulting gland, the Y 'or-
gan, produces the moulting hormone orustecdysone. This is a steroid.hor-
mono and is only slightly different from the insect moulting hormone éc-
dysone 6 . Recent investigations have shoWn that ecdysone can be transformed
into crustecdysone in crustaceans as veil, as in insects7 . The crustecdysone
initiates the moulting in crustaceans' 9. Several findings indieate that
the hormone is not alone in initiating moulting1 0 .
However, when is a moult being initiated by the hormone? What
is the amount necessary and which other processes are being regulated by
the hormone? A first answer is made possible by measuring the level of
hormone during an intermoult interval and comparing it with the changes
that aPPear in the crustaceans during this time. In addition, such measure- [P. 1448 , r.c.]
ments allow inferences about the speed of the release and of the inac-
tivation of the hormone. Since the moulting processes in insects and
crustaceans show many agreements, the comparison with curves of the titre
,1 of hormones in inseets112 is of some interest.
A substantial prerequisite for'this work is the exact analysis
and division of the intermoult intervaLinto different,-easily recognizable
stages. The fundamental investigations aboutthe moulting cycle in decapod
crustaceans were carried out b3r - Drach 1 3 in 1939. He ddvided the entire
moulting cycle into 12-different sections on the bais of—differences in •
the formation of the cuticle, the condition of the epidermis and the
behaviour of the animals. HoweVer,-it is sometimes difficult to recognize
in the living animal kme of these,âharacteristics for the division into
stages of .the brachyurous decapods. Skinner14 therefore propored as an
additional, easily recognizable criterion the development of regenerated
aPPendages. Investigations of the land'crab Gecarcinus lateralisl showed
that the regenerated parts that develop in place of a severed appendage
stand in a definite relation to the size of the animal.
In order to be able •o take into aceount also rapid changes in
.the level - of hormone we had to divide the moulting interval into many
more stages than had been done hitherto and we had also to pay attention
to the duration of the individual stages. - For Carcinus maenas we have
therefore divided anew the intermoult interval into 21 different stages
before we embarked on tÉe hormone determinationr.
M - ATERIAL AND METHODS
The Keening of the ExPerimental Animals
We used as eyperimental animals the common shore crab, Carcinus
maenas. PreviouFly we had already investigated in detail its moulting LP. 1449, 1 .c.]
rhythm under laboratory conditions. The crabs were collected at the North
Sea, shore of Schleswig-Holstein when they were 10 to 15 mm wide. They were
kept individually in compartmented 200-1 sea-water aquariums in the labor-
atory under constant environmental conditions (water temperature 25°C,
-5-
8 hours light and 16 hours darkness). In order to obtain a unifOrm nutrition ,
the animals were fed daily sufficient amounts of shellfish meat to make '
them Tully satiated.
The sea-water is circulated at a rate of about 40 1/min and
cleaned •by passing it through a filter of activated charcoal. The filtered
Water is injeCted . through a.nozzle as a shàrp jet into the aquariums. The
air carried along by the jet guarantees an optimum aeration of the,water:
Under these conditions the crabs Moult., ecording to size, at intervals of ...
14 to 24 days. Only animals with a carapace width.of 12 to 22 mm are used
in the experiments. The crabs are used in the experiments Only after they
have moulted at least once under laboratory conditions.
For reasons that.will be discussed later, on the animal all legs
are amputated, with exception of one posterior ambulatory leg. This is done .
at the latest one-half day after the moult. The amputation is parried out .
in a simple fashion by holding the animals by the Pertinent legs, which
are consequently severed by autotomy. A few days after amputation, regener-
ates grow from the leg stumps that remained behind'. The regeneratea - become
fully developed Until the next moult. The speed of growth of the regenerates
allows to make inferences about the physiologiOal condition of the animals.
For this purpose the regenerates are measured every other day and the values
are recorded. When the growth of the regenerates ceases, it is a sign for
sosie disturbance in the animals. Such animals are excluded from the eXPer6.-
iments.
The Extraction of the Moulting Hormone
The crustecdysone can be obtained by the same method that has
been developed by Karlson and Shaaya15 'for the extraction of the insect
moulting hormone ecdysone. This method has been modified by us only slightly
and has already been descrfbed earlier3. The extraction is based in prin-
ciple on the remoVal of the hormone from the aqueous crab homogenizate by
n-butanol. The extract obtained is brought'to dryness and used for the
quantitive determination of the hormone after it has been dissolved in
a small amount of. water. .SAnce with this extraction method it is possible
to obtain amounts of hormone as , small as 0.03eg, a single animal vas
sufficient for,each extraction.
The Quantitative Determination of the Moulting Hormone
The amount of hormone in the extracts was determined by a biàlogical [P. 1449, r.c.]
test. For this we,have specially.developed a test procedure that is being
described in-detail elsewhere 16 . This is five times as sensitive as is the
Calliphora test 15 that has hitherto been used for the determination of ec-
dysone and crustecdysone. In carrying out the test, 5 el each per animal
of the solution to be tested are injected into the ligated posterior ends
of maggots of the housefly, Musca domestica, which posterior ends have re-
tained their larval character. Twelve to twenty-five maggots are used for
each test. The solution to be tested is diluted so far that the amount of
fluid injected causes the pupation (formation of a puparium) of about 50
per cent. The average degree of pupation of all animals subjected to the
test is ascertained 20 hours after injection. From a calibration curve
can be read off the hormone concentration that corresponds to this degree
of pupation. The calibration curve has been obtained through the injection
of pure hormone solutions. From the hormone concentration can be calculated
the hormone content of the crab by ,taking into accoUnt the dilution of the
test solution.
RESULTS
. The Division of . the Intermoult Interval
' The division.of the intermoult interval that is based exclus-
ively on morphological charâcteristics, as, e.g.,.the size of the réne7,
gerates, is not suitable for physiological investigations. It is easy.to
understand that a crab, which remains for any reason whatever twice as
long in one and the same stage as does a:nether one of the same,size,. must
differ from it in its physiologicaa state. That is the reason why we. have
taken into consideration in the new partition into stages, as a substan-
tial factor the time that the crabs require for the different stages.
This, however, is possible only when the crabs pass continuously through
the individual stages. However, it can happen frequently, especially when
the environmental conditions are net quite constant, that the crabs spend
a longer time in one stage (C 4 according to Drach). In order to eliminate
such delays we amPutated the ambulatory legs of the crabs, as has been
described above. The loss of many ambulatory legs results in a shortening
of .- he entire intermoult interval 17 and in mUch greater uniformity in the
'moulting of the crabs 18 . A further advantage.is the poSeibility of being •
able to judge the developmental state of the crabs on hand of several . -
regenerates substantially better than with only one.
LP• 1450, 1 . c ] Table 1. The duration of the intermoult interval of leg-less crabs at 25 ° C under short-day illumination
LCarapaxbreite = width of carapace, Hautungsintervalldauer_in.Tagen = duration of intermoult interval in days, Tage = days, Zahl der Einzel- •
werte = number of individual values] • •
Carapnxbreito 12 13 14 15 16 17 • 18 19 20 21 • 22 23 24 25 , 26
[m n ]
IlitutungElintervall, . . . . dauer in 'fawn 11,7 12,8 13,4 13,7 14,1 15,2 16,7 ' 17,5 18,6 19,3 20,2 21,5 ' 20,5 23,0 24.3
± [Togo] 0,3 0,3 0,4 0,3 0,3 0,3 0,4 - 0.3 0,4 0,4 0,4 . 0,7 .0.5 1,3 1,1
Zahl der Einzel-• wort° 22 39 37 39 44 . 34 . 25 29 • 86 23 36 ' 23. 37 15 12
The amputation of legs has the effect that animals of the same
size moult at practically the same tinte intervals, so that the individual
scatter is greatly reduced and that the differences in size have substan-
tially less effect on the duration of the moulting interval than they have
in normal - animals. Thus leg-less animals that are twice the size of others
(cf. Table 1) require - only eight daya ionger for their moult; corresponding
. animals with legs reqUire, - however, an extra 14 days or more.
In order to ascertain how much time the animals require for the
individual moulting stages, we recorded, to begin with, the duration of the
entire ,intermoult interval in almost 500 animals. The result is shown in
Table 1. This shows that the'duration of the moulting interval is practical-
ly the same for animals that differ in size only slightly. It increases
slowly with the. size -of the animal.
To begin with, we have divided the moulting interval arbitrarily
into 10 periods, of which 7 have about the ,same length. We have designated
these periods with the roman numerals I to X. We have deviated from the
division into sections of equal duration only at the beginning (stages -1
and II) and at the end (stage X), because . during these phases there occur
draatic physiological 'changes in the crabs in a very short time. In order
to increase still further the accuracy of the evaluation of the stages,
we recorded the measurements of the groWth of the regenerates in 70 crabs.
The formation of regenerates begins four to five days after a moult, that
in, after about one quarter of the moulting interval has already pàssed.
It begins with the aPPearance of small humps on the fracture face of. the
remainingstumps of the legs. The regenerates grow rapidly during the time
from stage Vb to IXa. With about 80 per cent they have reached at that
time approximately their final size and,begin to become pigmented.
- 9 -
At first there aPPear only isolated green chromatophores, whdch, hoWeveri
increase'in numbers rapidly from then on. The regenerates that were celour-
less to begin with, take on a greenish aspect. Afterwards, black chromato- - [P. 1450, r.c.
phorps are formed that give the regenerate a dark-green to blaCk , colovi, .
Inunediately afterwards, about one day before the -moult, the colour changes
again, 'that is, to yellowish-brown. This is caused by the haemolymph, which
was colourless to milky white, sudden],y becoming yellowish-red. We do not
yet know the cause of this colour change. However, it must be considered
as an indication of intensive-metabolic processes. Since the haemolymph
shines through the very thin skin of the regenerates it aleo changes their
appearance.
During the phase of pigmentation the growth of the regenerates -
progresses only slowly and it is completed only in stage Xa. Under our
constant environmental conditions the final size of the regenerates stands
in a firm ratio to the size of theanimals. Our measurements have shown
that a definite size of regenerate corresponds to each time interval, that
is, the regenerates have attained a certain percentage of their size in,
each stage. The growth of the regenerates and the differentiation are thur
very suitable as criteria for the division into different phases of the
intermoult interval also in Carcinun maenas.
Fiirther criteria are provided by the changes in the structure
of the cuticle that have already been described by Drach (cf. Table 2) 13 .
On the basis of the division that has been carried out according'
to these three principles for the partition (time, growth of regenerates,
condition-of the cuticle) we have succeeded in dividing each moulting in-
terval into 21 stages that can easily be distinguished from one another.
This constitutes a gubstantial prerequisite for the construction of a
size and with known moulting data. LP. 14510',‘ 1.
•••■
■••■
M.»
Xc 2.0
Xa XI)
4.0 3.0
- 10 -
curve for the hormone titre.
The different stages' and their charadteristic criteria have •been
asasmbled in Table 2. These criteria make it possible to recognize after
some practice at once the physiological conditionof à crab of a known-'
Table 2. Partition of the intermoult interval of the shore crab .Carcinus maenaa L. into 21 stages. Further explanations in the text.
Duration Length Duration of of
of inter- regener- ,,Colour Stage Stages moult ates as, of Condition Water Food
Stage after as per- interval percen- régen- of up- in-
Drach centage as Per- tage Of erates cuticle take . take . of total centage final duration of total -size
duration '
_ , fully soft 44. - parchm , like + - ful.pliable - - - , - , caraP.dorsal.- ( 4')
› plia. in spots _ caraP. fief'. - + _ It It ..., ++
and thickened .. - - _ ++
22:0 0.5 colorless . - -
23.0 2 , fg - . ++
29.0 - 3 : eg . - .., +4-
34. 0 4 /I ... -1-1-
39. 0 12, li _ ++
44.0 25 • II _. ++
51.0 28 •1 _ ++
63. 0 53 li _ ii.
.68.0 85 light green _ ++
73. 0 , 91 green . resorptions - .+
85.0 96 dark green II _ ( 4')
90.0 - black
94.0 100 yellow-brown o. _
94.0 100 do. parting of +
97.0 97..0 . pleural seams
100.0 100 do. og " ++ wide open
Ina - 02 4.0
nib C3 7.0 • .
ine c 3 3. 0 .
1Va D 9 1.5 o '
IVb 5.0
Va 5.0
Vb 5.0
Via 5.0
Vib . 7.0
VII D D1 11.0
VIII 7.0
IXa 3 6.0
in 12.0
IXe , 5.0
Ia A1 0.5 ' 0.5
Ib A2 1.0 0.5
lia B.1 .4. 2 3.0 2,0
IIb C l 4.0 5.0 8.0 12.0 12.0 19.0
'MO
- 1 1 -
. .
. _ . [P. 1451,* Ï. Table 3. The content of moulting The Level of Hormone during an
hormone in the shore Crab,
Carcinus maenas L., during - - Intermoult Interval '. • a moulting interval.. . .
On hand of. the methods
Stage Crustecdy- Min- Max- Test Error described. eready, we havé deter-, : soneactiv- im- im- No. ity ng/g um um fresh weight
_ - of the crabs. At least ten determin- I ' 12.1 1 3.1 2.5 35,0 13 25.5 ' ' II a, ' 24,0 ± 12. 9 0.0 140,0 11 51.0
16.1 ± 60,0 ations with one crab each were car-
Il b 41.1 0.0 13 30.5 111a 15.8 ± 7.0 0,0 64.4 10 44.2 II 1 b 14.6 :1: 5.6 0.0 48.5 1° 38.3 ried out for each stage. Altogether 111e 4,9 ± 1,5 0.0 22.0 14 • 30,1 IV a 31,0 ± 10,4 0,0 136,6 .14 33.6 IV') 8,4 4- 3.3' 0,0 30,0 10 39.3 '210 individual determinations Were
.
Va 18,3 I 5,0 OM 42.0 11 27.6 • V b .23.5 4- 7,2 0.0 60.0 11 30.7
Via 27,5 ± 6,2 6.0 60.6 H 22 .6 made. In order to obtain comparable-. VII) 40.4 ± 18,8 0.0 226.6 12 46.3 V I I • 35,1 -_,L. 9.8 ' 0,0 115,0 14 27.9 VIII 79.51:16. 9 2 .) ,8• "").2 H , ..20 ,3,: data, the content of hormone hara 1 x a 63.3 -i : 14.4 9.8 200,0 12 22.7. 1X1) 29 q ...4_ 8 7 0.0 114 0 . 15 ; 29 8
■ - ..._ • • ' . been related to the fresh weight of •I X e Xe 70.7 • 8.0 7.7 250.0 27 127 1 X b the animals. For each stage has been X1)--c. 109,9 :1 : 41.6 39.0 540.0 12 40.9 Xe 30:8 1 5.4 2.7 77.1 22 14.2 •
• . calculated the average of the indiv-
idual determinations and the mean
error of the averages. The result is-
shown in Table 3. This reveals that the hormone content changes continuously
during the moulting interval. Here it is striking that the mean er/40r of
the average is very large in some ratages and is small in others. The small
errors prove that the systematic error in the hormone determinations is.
relatively small. It is therefore not possible to explain the large errcirs
by errors in the determinations of the hormone. They must rather be caused LP. 14529 1.c.
by the fact that the partition of the moulting interval into 22 different
stages is not fine enough to cover exactly all changes in the physiological [P. 1452, r.c.]
condition of the animals. Large errors are therefore a . sign that very rapid
changes in the level of the hormone took place in the corresponding stages.
mined the .content of hormone for 19
cent of the stages of the intermodtphase
120
100
86'
60
. 40
20
o
Table 4: The water content of Carcinus maenas L. at different
stages of the intérmoult interval.
Water content Stage as percentage n
of fresh weight
- 12 -
• Hoeing ilaulung
F S 1 I -: i
/-It _I / I ile,`
d 1 / .% ■ -1 I I
/e/
/ _I ‘î
I I I % /' 1 - i %
i I /
% / i 2. . t / / 1 % ,p--- ..... 4 t ,
. . 'Ili/ '!.i.
e■ tl, ' 1 ...o• ' K, - 1151 ‘,_ **4 1 ■ d ' I Nr,
t( ■,...o.......,,,:, , 1 ..• _ •:;,./ b,- 8
t1
III 11 1 III 1 1 1 1 I I 1 1 1 1 1 1 lita b fila b clVab Va b Via b
Fig. 1. The titre of moulting hormone of the shore crab, Carcinue maenas L., during an intermoult interval. The stages are marked on the abscissa in such a way that
they correspond to their actual duration in the inter-moult interval..[Ordinate = mot,lting . hdrmone/g - frésh
weight, abscissa = stages of the intermoult interval, eiltung =
17111 11e b c ra b
In order to obtain a review
of the speeds of the changes in the
hormone content, we have represented the
méasured values graphically in the form
of a curve for the titre of the hormone.
In its construction we have taken into 1 al-b 8 5,0 J1- 1,0 3
HI a , 77,5 ± 0,5 •2 account the duration of the individual I II b 71,0 ± 2,0 2 - In 71,0 I Vb 63M --t- 3,0 3' stages of-the intermoult (cf. Fig. 1). VI b 67,7 ± 1,0 3 VIII OM -I- 1M 3 I X a—c 64.3 -1-- 1 ,'2 8 Moulting hormone is found in our ex- X a—b 64M ± 1,7) - 3
, X e 65,8 -I- 2,3 4 perimente animals during almost the entire
intermoult interval, during which the
animals are in diecdysis, that is, in a period of consecutive moulting
cycles. Only during the stages IIIc and IVb there is 'practically no hor-
mone present. In contrast, one finds marked maxima in stages IVa, VII and
- 13 -
Xa to Xc. In spite of their large - mean errors the maxima are, with P
0.01 in • a T- test highly significant when compared with the values•of.their
neighbouring stages. . _ .
illhere is a furthe small maximum in stage. IIa that is-not-sig- - :
nificant statistically. However, if ones takes into' account the effect. of
dilution that is caused by the large amounts of water that the crabS take
up at each moult in order to increase their volume, this maximum in. stage
.11a 'stands out much more markedly than when one relates the hormone con- .
tent to the fresh weight of the aniMals. As.can be seen in Table 4p the .
water content of the crabs increases from about 65. per cent before-the [p. 1452, r.c.]
moult.to 85 per cent in stage I. During the subsequent stages, the water •
taken up is gradualy, being replaced by new tissue. The.crabs have regained'
their normal water content in stage IV. The water that has been taken up
during the moult only dillites the substances present and when one sub-
tracts this from the fresh weight and relatés the. hormone. content to the -
corrected weight, one obtains the curve•that is shown dotted in Fig. 1.
It deviates from the other cu:me only during stages I to IIIe and shows
a distinct maximum'in stage Ilb.
DISCUSSION
Now the following question presents itself: - .what is the function
of the moulting hormone? It seems clear that it has to fulfill not only ,
one, but several tasks, because it is being released at quite different L I) , : 1453, 1.c.]
moments during the intermoult interval and in different strengths. In
order to obtain an indication for the different effects of the hormone, we
have investigated which.morphological and physiological changes corres-
pond to the changes ifn the level of the hormone. Since the level of the
- 1 4-
hormone,drops steeply already before the moult proper until stage I after
the moult, the hormone is 6bviously. no longer required during the actual
moulting procese. Since, the level of hormone does not subside completely
and does even rise again teMporarelY in stage II, the hormone is obviously
necessary for the regulation of metabolic processes even after_the moult.
Here the assumption presents itself that the hormone affects the hardening
processes in the new cuticle: Hardening of the cuticle that is still com-
pletely soft in stage I takes place above all in stage II. This hardening
is caused by the formation of new layers of chitin and the deposition of
protein and in this connectien of Calcium carbonate into the already exis-
ting layers of the cuticle. According-to reCent findings 19 , the déposition
of calcium does not take place through the action of enzymes, but it pre-
supposes the presence of layers of protein in the new cuticle. The for-
mation of layers of both protein and of chitin requires a high activity of
enzymes. Since it has been possible to demonstrate that the 'insect moulting
• hormone ecdysone stimulates the synthesis of protein in vitro , . the assump-
tion presents itself that the moulting hormone of crustaceans in stage I
and stage 1I stimulates the synthesis of enzymes that are required for
the hardening of the cuticle. This is supported also by the fact that the
level of hormone has dropped practically to zero after the process of har-
dening in ..the stage III-has been completed.
After stage IIIc follows either a period of anecdysis, that is,
a, period in which moulting rests and in which no hormone can be demonstrated
in the animals, or a new cycle of moulting. This begins with stage 1Va,
which lasts only one day and which is characterized by the start of the
development of regenerates. During this time takes place a relatively 1.13 . 1453, r.c.]
copious release of hormone. Since no other physiological changes can be
- 15 7
Seen in the crabs, eXcept the, formation of 'igeneratea, we surmise that .
the hormone triggers the formation of regenerates. , .
, The primary ?process during the formation of regenerates, is cell . , .
divisions, , theaame as during. the'process of mdulting,The effect or the •
moulting hormone in this Case . eould'therefore be tià trigger cell divisions.
However, it . has to remain undecided whether cell divisions . occur only
in the buds of the.regenerates or whether they take place simultaneously
alÉo in the:other tissuesias seems to be indicated . by initial findings.
In this case one would have to consider the stage IVa as the. beginning . of
the new moulting proceFs anà.one would have to equate it with the stage lb
of Drach.
The question whether animais in possession of - all legs, that is,
thoFe that do not-have to form regenerates, show a corresponding release
of hormone, cannot be answered, because it is difficult and :subject to a •
great probability of errors to determine in these animais a phase that is -
comparable to stage
After a renewed drop to zero value, the level of hormone rises
again cOntinuously, beginning in Stage IVb until it reaches again a maximum
in stage VIII. During this time the only visible changes are the rapf.d
growth of the regenerates. It cannot yet be dedided in how far thè hor-
mone is involved in this or whether it fulfills other tasks also. Our his-
tological investigations give a hint in regard to the function of the hormone
in stage VIII: they show that the apolysi s 21 takes place illimediately
after stage VIII. It is thus very probable that thiaimportant atep in
moulting that corresponds to phase D1 of Drach is being triggered by this
copious release of hormone.
During the stages IKa and.IM - the level of hormone drops again,
-e
but then shows a renewed ripe and attains its greatest maximum in stages
iXo to Xb to c. During the drop of the;Ourve of the hormone (stage iXa to
1Xb)-, the regenerates are growing substantially more slowly than before
.and. start to become pigmented. However, the'curva of • the hormone des not
furnish any clue in regard of an intimate connection between pigmentation
and •hormone.
SimultaneOusly with the rise of the level of the hormone in [p. 1454, l.c.]
stages IXcto Xb take place the changes in the haemolymph of the animals
that have already been mentioned. The most visible change is the change in
the colour of the haemolymph. Newly formed chromophorous substances give
the previously colourless to slightly opalescent blob(' a yellow to red
colour. In addition, the level of blood calcium rises by about 80 per cent 22 .
The content of blood protein also rises. It is not yet clear how far this
concerns material that haS beenresorbed from the old cuticle". In any
case, these changes result in an increase in the osmotic value of the
blood. The haemolymph that Was . previously isotonic to slightly hypotonic
in regard to sea-water, now becomes hypertonic. Drach 1 3 was able to show
that now the sea-water swallowed by the crabs flows through the thin walls
of`the stomach into the interior of the body. This raises the internal
pressure so much that the old cuticle ruptures at pre-formed places and
that the animal can free itself from the old carapace through the continuing
increase in volume that is caused by the constant uptake of water.
The present findings and the coalp“.15 just mc,Lcie show
that the moulting hormone triggers the entire process of moulting not
through a single release of hormone, but that it is rather interfering
with the moulting process both before and after the moult in a directing
capacity. This raises the question: how is it possible that a hormone can
- 1 7
trigger such divers processes as, for example, oeil divisions and hargenirig
processes? ln principle this is possible in two different ways:
--(1) the height of the hormone titre.provides the decision about whether
this procesn or another process is - to belnitiated;
(2) the physiological age of the animals providep the decision which -
process is to be initiated by, the hormone.
The first possibility is sUggested by the fact that thé fOur
maxima of thel.evel of the hormone that have been demonstrated by our
. investigations differ distinctly 'in regard 'to their heighte.
.The second route is indicated by the injection experiments that
have been carried out by us without any succese. We did not succeed in •
triggering a moult or one of - the processes that are typical for mOulting -
by single or multiple (in two-day intervals) injections of Various.amounts 113 . 1454, r.b.
(50 tb 2,000 C units per injection) 'of ecdysone. This makes it obvious
. that the timing of the release of hormone is of substantial importance.
This is not invalidated by the successl'ul injection experimente.of Carlisle 9 .
He was able to release moulting in crabs through multiple injections of .
hormone extracts. It is true that in his animals the moult inhibiting hor-
mone had been removed beforehand. Exactly the moult inhibiting horMone .
can play a decisive role in this case. In addition, it concerned large
animals that had not had a' moult for a considerable time and who reacted
to the hormone perhaps for this reason.
It is probable that in the régulation of the moulting process
and in the triggering of the moult both the physiological age and the
height of the level of hormone are of importance for the decision about
which prOcess is triggered at what time.
A problem that is not unimportant in this connection is the .
• ..
- 18-
Speed of release and the speed of inactivation of the hormone. As can
be seen from an inspection of the curve of the hormone titre in the
stages 1Xa and Xb to c, the content of hormone rises very rapidly and
drops again at least just as ràpidly. However, what causes the rapid in-
crease and the sudden drop? Our inviistigations do not provide an answer
for this question.
The rapid increase in the hormone content in the animals could
be caused by the hormone being transformed from an inactive storage form
into an , active working form or that it ds being synthetized very rapidly'
de novo..So far there are no 'indications to show which route is actually
being followed. On the other hand, experiments on the 'inactivation of
the insect moulting hormone allow well-supported comparisons.
Theoretically the inactivation of the moulting hormone can
take place in two ways: through the elimination of the hormone in its
active form or through the transformation of the hormone in the metabolism
into inactive degradation produCts. The curve for the hormone titre shows
(cf. Fig. 1, stage X) that the crustacean moulting hormone is being in,-
activated or secreted within a few houra. This finding corresponds to the
investigations in insects, which showed that the moulting hormone ecdysone LP. 1455, 1.c.]
has a half-life of 1 to 2.5 hours 23-25 . In addition, it could be shown in
insects that the inactivation of the moulting hormone takes place by an
enzymatic route 23 . Since more recent findings have shown that ecdysone .
can be transformed to crustecdysone in insects as well as in crustaceans,
it appears probable that crustecdysone is being inactivated in the same
manner as ecdysone, namely by enzymatic action. It is true, it has been
possible to show that the hormone has been excreted in active form, but
this appears to be of small importance only 26 .
t . en
- 19-
ComParison of the Release of Hormone 11.1 Carcinus maenae with the Release
of Hormone in other'CrustaCeans 2:22.4 in Insects
• Since no data were availablè hitherto for thecomParison of.
•the release of moui.Éing hormone during an intermoult interval in other
crustaceans, we have started corresponding investigations in the river
crayfish Orconectes affinis. The first results of these investigations 27
show that the content of crustecdybone in the river crayfish before and
after the moult is several times lower than in the shore crab and that
before the moult no,significant fluctuations in-the level of the hormone
are to be found in the river crayfish. What significance is to be ascribed
to these differences cannot be said at the moment.•
There are available two detailed investigations of the budget
of the moulting hormone during development in insects, namely on the ec-.
,dysone content of Ca4iphora erythrocePhala and of Bombyx mori ll ' 12. The
comParison of the curve of the hormone titre of CalliPhora and Bombyx
during the pupal moult with that of thé shore crab Carcinus maenas shows
a few substantial agreements. This is:interesting, since it concerns.two
animals who, although they belong to two closely related classes, haV:e
nevertheless very different modes of life.
As in the shore crab, the hormone titre of pupating fly larvae
shows characteristic fluctuations. Thè hormone content also is-the same
as far as the order of magnitude is .concerned. Calliphora it attains LA. 1455, r.c.
a height of 70 to 80 ng of ecdysone/g fresh weight, in the shore crab the .•
figure is 110 Pg. -
In the same manner as in the shore crab, the level of hormone
inCl....,a.hor_a rises steeply before the moult. However, it shows only one
peak and not two as in the shore crab. The maximum persists during the
formation of the puparium, but it drops prior to the pupal moult p±oper
. and it thua corresponds to the conditions in the shore crab, where the .
leVel of the hormone also drops steeply shortly before the moult. The
fact that there are two maxima in the shore crab, but only one in the
blowfly can be explained by the difference in the mode of Moulting in the
two animals. Whereas the fly larvae remain in the old larval skin that has ,
been consolidated into the Puparium,, thé shore crab has to divest itself
of its old- cuticle. For this are required, as has already been mentioned,
fundamental changes in the composition of the haemolymph, which are probably
initiated and governed by the second release of hormone befàre the moult. -
Since Salell changes in the haemolymph are not required in the land dwelling
fly larvae, the release of hormone that woilld be required for such a change
can be dispensed with.
In Bombyx one finds, as in the shore crab, two maxima before the
moult. However, here the first release of hormone is held responsible for
the initiation of the spinning process, which is lacking in the blowfly,
and the second release of hormone only is responsible for the initiation
of the Moult.
As in the shore crab, in . Calliphora the level of hormone drops
steeply before the mcult, but it also remains at a lower level after the
moult. In the pupae the level of the hormone even rises substantially in
the interim. However, it is.probable that the hormone released after the
moult serves different purposes in insedts and crustaceans. Whereas it
presumably regulates the process of hardening in the 'crustaceans, in the
insects it might be responsible'for the zeorganization.of the pupa into
the imago. •
The work haa been carried out with support by the Deutsche
Forschungsgemeinschaft.
•-• ,
- 21 —
R E'FERENC.E S
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3 D. ADELUNG, Verh. dtsdt. zool. Ges. Glittingen 30, Supple. mentbd., 264 [1966].
4 L. M. PASSANO, in : The Physiology of Crustacea 1, 473 Academic Press, New York 1960.
5 F. HAMPSHIRE U. D. S. H. HORN, Chem. Commun. 2, 37 [1966].
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22 D. ADELUNG, Verb: dtsch. zool. Ges. Würzburg, im Drucic.
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27 11. KELLER u. D. ADELUNG, Roux Ardtiv Entwicklungs-medutn. Organismen, in Vorbereitung.
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