CHARACTERIZATION OF POSTEMBRYONIC DEVELOPMENTAL STAGES
OF THE FEMALE CASTES OF THE HONEY BEE,
APIS MELLIFERA L.
Heinz
REMBOLD,
Jean-Pierre KREMER’Gabriele M. ULRICH
Max-Planck-Institutfür Biochemie,
D-8033 MarlinsriedSUMMARY
Queens
and workers of thehoney bee, Apis mellifera.
have been reared as larvae and pupae under definedconditions,
and thedevelopmental
stages have been associated withunequivocal
features. The larvalinstars
are characterizedby
the head diameter andweight
of the larvae. The stages ofpupal development. are
definedby
the colours shownby
thecompound
eyes and the thorax. Thetemporal
durationof the
successivedevelopmental
stages has also been determined. With these criteria a larva or apupa can be
assigned
to a definiteposition
on thedevelopmental
time axis.INTRODUCTION
The formation by Apis mellifera of female
castesfrom larvae with the
samegenotype offers
afascinating model for studies on differentiation. During
a
comparatively short space of growth and metamorphosis, the originally bipotent female
larva develops into either
aqueen
or aworker. The direction of development is determined during the first 3 1/2 days of larval life by external factors such
asnutrition
(Z A N
DER and B E CK ER , 1925; WEAVER, 1957; R EMBOLD and H ANSER , 1964). The resulting differences in size, function, behaviour, morphology and metabolism have been investigated by S TABE (1930), 1VI ELAMPY and W ILLIS (1939), M YSER (1954), J AY (1963), O SANAI and R EMBOLD (1968).
Such comparative studies with the
castesof honey bee larvae and pupae require
anexact
determination of the developmental stages. Several authors have described the
growth
ratesof honey bee larvae (S TABE , 1930; W ANG , 1965). Previous studies from
* In
part
from a doctoral work to be submittedby
J. P. Kremer toUniversity
of Miinchen, 1980.this laboratory used age and position of the larvae in their cells for characterization
(C
ZOPPELT and R EMBOLD , 1970; O SANAI and R EMBOLD , 1970). A more precise staging of the larva is required sometimes, particularly for investigation of morphological, physiological and biochemical aspects of honey bee differentiation.
The present study describes
asimple method of determining the developmental stage of
aworker
or aqueen larva and pupa by head diameter, body weight and pigmentation. These criteria
arethen used
to measurethe duration of the different stages.
MATERIALS AND METHODS
The larvae and pupae used were collected from
Apis mellif t ra
carnica colonies held near the instituteor near
Saulgrub (Bavaria).
The correlation of head diameter andbody weight
was established with worker larvae and pupae from strong field hives.Queens
were rearedby general
methods as describedby
Z ANDER
and BECKER
(1925)
and WEiss(1971);
the larvae selected for this purpose(0-24
hoursold, weight
0.1-0.3
mg)
weregrafted
into cell cupsplaced
inqueenless
nurse hives. Head diameter andbody weight
of 501 worker and 464 queen larvae with a distribution all over theweight
range were measured.Duration of the different larval instars was determined as follows. The queen of a
breeding colony
was restricted to one comb for 4-6 hours. After 3
days, only
the larvaeemerged
within an interval of4 hours were retained for further
rearing.
Worker larvae were left in thebreeding colony
and theprospective
queens weregrafted
as mentioned above. Head diameters andbody weights
of queen and worker larvae collected at different times afterhatching
were determined.Disturbing
the larvae too oftenduring
anexperiment
was avoidedby rearing
9 distinctseries,
each with a differentcollecting interval,
togive
differences in age of 4-6 hours between the larval groups.The
pigmentation
and duration of the differentpupal
stages of queens and workers was studied with animals that grew up undi-sturbed in the colonies until their cells were sealed or untilpupation.
The tops of the cells were then firstopened
farenough
to allow observation of thecompound
eyes and thethorax,
and then closed with a coverslide. The cells werekept
in an incubator at 34 &dquo;C with a relativehumidity
of 70-80 ’Yo and were examined every 6 hours until adult
ecdysis
occured. Finalanalysis
wasperformed
with 370 data from 22 queens, and 1860 data from 55 workers.
Eye pigmentation
was estimatedaccording
to the colour chart RAL-F 2(RAL, 1976).
RESULTS
Characterization of larval and pupal stages
In 1890 D YAR showed that head width in larval lepidoptera is nearly
constantduring
adefined larval stage, and increases according
to aregular geometric progression with the successive ecdyses of a larva. This observation
was confirmed for the honey bee by B ERTHOLF (1925) and his developmental data have been used by
a
number of research groups, but statistical data concerning the material
asexamined by
B
ERTHOLF
were notpublished.
We have compared the head diameters and weights of 501 worker and 464 queen
larvae. The resulting correlations (Fig. 1) demonstrate the existence of five groups of
paired data for both queen and worker larvae. A comparison of the average head diameters reveals a highly significant difference between the corresponding groups
(2 p
<0.001). This clearly demonstrates that head diameter can be used
to
characterize each of the 5 larval instars. The average head diameter and
corresponding minimum and maximum weights for each larval instar
aregiven in
Table 1.
The fifth larval instar is longer than the preceding ones (see Fig. 2) and
can be subdivided into
an open-cell phase (L 5) and
a spinning phase after the sealing of the cell (LS). According
to our observations,
a worker cell is sealed
at a larval weight of
150-165 mg and
aqueen cell
at aweight of about 190 mg.
After spinning, but without ecdysis, the larvae
enterthe prepupal stage (PP), during which they appear straightened and motionless in their cells, while the larval cuticle progressively loosens from the pupal
oneunderneath, beginning in the head region. This stage ends with the pupal ecdysis.
Pupal development was followed by noting the pigmentation of the compound
eyes and the thorax (Tab. 2). The observed colour stages of the eyes
werecoordinated
with the chromaticity values of the Rn L -colour chart (R AL , 1976), and divided into
larger groups (pink, red brown, dark brown) for easier application.
Duration of larval and pupal stages
Queens and workers, characterized
asdescribed above,
wereused
tocalculate the duration of the different developmental stages.
Although carefully dated, the larvae did
notall grow
atthe
samerate,
so aminimum and
amaximum duration is given for each larval instar. The temporal durations indicated for pupal development correspond
totime intervals comprising
75 % of the observations within each colour group (Tab. 3 and Fig. 2).
DISCUSSION
Analysis of
castedifferentiation during the postembryonic development of Apis mellifera and investigation of the related problems of morphology, histology and metabolism require an exact characterization of the animals studied. In the present
study
wehave established simple external criteria that permit
aclassification of female larvae and pupae in the successive developmental stages.
Different starting points have been proposed for this purpose. S TABE (1930) and
W
ANG (1965) correlated the weight and age of larvae, but external factors such
astemperature (MtLUn-t, 1930), humidity, food quality and quantity, etc., obviously
influence the duration of developmental phases,
soage is
not areliable basis for
characterizing larvae reared under different conditions and it would probably be still
less good if the larvae
wereof different
races.A ninety hours old larva, for example,
may be in either the fourth
orthe fifth larval instar (see Fig. 2).
The instar,
onthe other hand, is
afundamental step in larval development.
B
ERTHOLF (1925) used the head size of larvae
todetermine stage and ecdysis and the
same
procedure
wasapplied by WEAVER (1974). Our data on head diameter (Tab. 1)
are
in substantial agreement with the findings of B ERTHOLF . Figure 1 and Table 1, which correlate larval head diameter and weight, allow
alarva collected
atrandom from
acomb
tobe assigned to a defined position on the development axis. For routine
the development axis. For routine
characterization of larval instars
wehave used « ecdysis » rather than
«apolysis
»(apolysis
=detachment of the epidermis from the old cuticle; J ENKIN and H INTON , 1966). Apolysis usually can not be observed without histological investigation, except in the prepupa.
The different stages of pupal development are described in
term of the pigmentation
of compound eyes and thorax (Tab. 2). Several authors have described the
pigmentation of the honey bee, e.g. S TRAUS (1911), Z ANDER , L6 S C HEL and M EIER (1916), v. R HEIN (1933), J AY (1962). As judgment of colour gradations is well known
to be subjective,
we have tried to objectify our observations of Apis mellifera carnica
pupae by using a colour chart,
as T HOMPSON (1978) did for A. mellifera adansonii.
A further characterization of the postembryonic development is obtained by determining the duration of the different stages in larvae and pupae
(Tab. 3). Corresponding descriptions from K OEHLER (1923), B ERTHOLF (1925) and
WEAVER (1974), e.g., show partially differing results when compared with each other
and with
our owndata. This is
notsurprising since, as indicated by the overlapping of
time intervals in Figure 2, certain undetermined factors either delay
oraccelerate growth of the animals. The external criteria of head diameter, weight and pigmentation are appropriate
to allot
a larva
or pupa randomly taken
out of
a comb
to
a
defined place
onthe developmental axis.
Receired Jbr publication
in August 1979ACKNOWLEDGEMENT
We are
grateful
to GerhardS CHMIDT , Saulgrub,
for queenrearing.
ZUSAMMENFASSUNG
CFiARAKTERISIERUNG DER POSTEMBRYONALEN ENTWICKLUNGSS&dquo;1’ADIL-N BEI DEN WEIBLICHEN KASTEN DER HONIGBIENE,
APIS MELLIh’ERA L.
Die
Entstehung
weiblicher Kasten bei derHonigbiene
ausursprünglich bipotenten
Larven bietetneben
praktischen Gesichtspunkten
auch ein faszinierendes Modell zum Studium vonDifferenzierungs vorgängen,
sowohl ausmorphologischer
undphysiologischer,
als auch aus biochemischer Sicht.Entsprechende Untersuchungen
setzenjedoch
einemöglichst
genaue Bestimmbarkeit des Entwick-lungsstadiums
der Larven undPuppen
voraus. In dervorliegenden Untersuchung
werden einfach zubeobachtende äussere Merkmale beschrieben, die es gestatten, die Arbeiterin- und
Königinlarvcn
bzw. -puppen der
Honigbiene
auf derEntwicklungsachse
in eine definierte Position einzuordnen. AlsUntersuchungsmaterial
dienten Bienen der Carnica-Rasse.Zur
Charakterisierung
der Larvenstadien verwandten wir dieMessung
desKoplkapsel d urchmessers.
- Nach βt:aTHOt.t!
(1925)
bleibt dieser bei der Bienenlarve innerhalb eines bestimmten Larvenstadiums nahezugleich
und nimmt mit den einzelnenHäutungen sprunghaft
zu. Um statistischsignifikante Aussagen
machen zu
können, verglichen
wirKopfdurchmesser
und Gewicht von 501 Arbeiterin- und 464Königinlarven.
Diegraphische Darstellung (Abb. 1)
liefertjeweils
5Gruppen,
die den Larvenstadien derHonigbiene entsprechen.
In Tabelle 1 sind die hochsignifikant
voneinander verschicdenen Mittelwerte desKopfdurchmessers (2
p <0.001)
sowie diedazugehörigen
Gewichtsbercichc fürjedes
Larvenstadiumangegeben (L
1-L 4 = 1.-4.Larvenstadium:
L 5 - 5. Larvenstadium in der offenen Zelle; LSSpinnmade
im 5. Larvenstadium in der verdeckelten
Zelle).
Die Arbciterinzcllen wurden bei einemLarvengcwicht
von150-165 mg, die
Königinzellen
bei etwa 190 mg verdeckelt. NachBeendigung
desSpinncns geht
die Larvc in dasVorpuppenstadium (PP)
über, das durch diegestreckte
undunbewegliche Lagc
der Larve in der Zellegekennzeichnet
ist.Gleichzeitig
wird die zunehmendeAblÖsung
der larvalen Kutikula von der darunterliegenden Puppenkutikula
sichtbar.Die anschliessende
Puppenentwicklung
wurde anKöniginnen
und Arbeiterinnenderen
Zcllcnnach dem Verdeckeln oder nach der
Verpuppung
an derSpitze
etwasgeöffnet
und mit cincmDeckglits
wieder verschlossen wurden. Die
Puppen
wurden im Brutschrankgehalten
und alle 6 Stundcn auf diePigmcntierung
derKomplexaugen
und des Thorax untersucht(Tab. 2).
DieAugcnpigmenlicrung
wurdemit llilfe der Farbtabelle RAI.-F2
2 (Rm&dquo; 1976)
beschrieben.Gleichzeitig
wurde die Dauer derPuppenentwicklung registriert.
Die Dauer der einzelnen Larvenstadien wurde mit Larven bestimmt, die innerhalb von 4 Stunden
geschlüpft
and zu verschiedenenZeitpunkten
in einem 4-bis6-Stundenrhythmus gesammelt
waren. Da beiden Larven und
Puppen
einegewisse Streuung
in derEntwicklungsgeschwindigkeit festgestellt
wurde, sindfür die Larvenstadien
jeweils
eine minimale und eine maximale Dauer und für diePuppen Zcitintervallc,
die jeweils
75 °!> derBeobachtungen
beinhalten.angegeben (Tab. 3). Abbildung
2gibt
eineÜbersicht
über dieGesamtentwicklung
und das Alter, zu dem die einzelnenEntwicklungsphascn
zu crwarten sind. Diesenkrechten Balken deuten die sich überschneidenden Zeitintervalle an.
RÉSUMÉ
CARACTÉRISATION DES STADES DE DEVELOPPEMENT POST EMBRYONNAIRE DES CASTES FEMELLES CHEZ L’ABliiLLli. /lI’is Aii.i.i.fi !R.-1 1..
Le
développement
des castes femelles de l’Abeille àpartir
de larvesbipotcnticllcs représente,
à côtédes aspects
pratiques impliqués,
un modèle intéressant en vue d’une étudemorphologique, physiologique
ou
biochimique
des processus de différentiation. Toutefois, lesanalyses
de ce genreprésupposent
la déter-mination la
plus
exactepossible
du stade dedéveloppement
danslequel
se trouvent les larves et les nym-phes
examinées. Les auteurs de laprésente
étude décrivent descaractéristiques
extérieuressimples
à obser-ver, permettant
d’assigner
uneposition
définie sur l’axe dudéveloppement
à des larves et desnymphes
d’ouvrière ou de reine. L’étude a été effectuée avec des abeilles de la race carfii<a.Les différents stades larvaires ont été caractérisés à l’aide de la mesure du diamètre de la tête.
D’après
BeaTHO!F
(1925),
le diamètre enquestion
demeure presqueinchangé
chez les larves d’Abeille durant un stade larvaire défini et il augmente fortement avecchaque
mue. Afin d’obtenir des donnéesstatistiquement significatives,
les auteurs ontcomparé
le diamètre de la tête avec lepoids
de 501 larves d’ouvrières et de 464 larvesroyales.
Lareprésentation graphique
faitapparaître
5 groupescorrespondant
aux stades larvai-res de l’Abeille
(figure 1).
Le tableau 1indique
les valeurs moyennes du diamètre de la tête pourchaque
sta- des les différences entre les stades consécutifs sont hautementsignificativcs (2
p <0.001).
En outre, le tableau donne lespoids
larvaires minima et maximacorrespondants (L
1 L 4 )&dquo;4’ stadelarvaire;
L 5 -- 5’ stade larvaire dans la cellule non
operculée;
LS = 5‘ stade larvaire dans la celluleoperculée
lorsdu
filage
ducocon).
Les cellules d’ouvrière ont étéoperculées
avec unpoids
larvaire de 150 à 165 mg, les cellulesroyales
avec 190 mg environ. Le cocon étantfilé,
les larves entrent dans le stadeprénymphal (PP) qui
se caractérise par laposition allongée
et immobile des larves dans leur cellule. Pendant ce temps l’on peut observer le détachementprogressif de
la cuticule larvaire de celle de lanymphe.
Le
développement nymphal
a été observé avec des reines et des ouvrières dont les cellules ont été ouvertes à lapointe après l’operculation
ou lapupation,
et refermées à l’aide d’uncouvre-objet
en verre.Les cellules furent maintenues dans un
incubateur,
ledegré
de lapigmentation
des yeux et du thorax desnymphes
a été examiné toutes les 6 heures(tableau 2).
Lapigmentation
des yeux a été décrite au moyen du tableau de couleurs RAt 2(RAL, 1976).
La mêmeexpérience
a servi à la mesure de la durée dudévclop-
pement
nymphal.
La durée des différents stades larvaires a été déterminée à l’aide de larves écloses dans une
période
de4 heures et
prélevées
à desâges
différents avec unrythme
de 4 à 6 heures.Étant
donné une certaine varia- tion dans la vitesse dudéveloppement
observée tant chez les larves que chez lesnymphes,
le tableau 3indique
pour les stades larvaires une durée minimum et maximum, et pour lesnymphes
un intervalle detemps
comprenantchaque
fois 75 % des observations faites. Lafigure
2 montre un aperçu dudéveloppe-
ment
post-embryonnaire
et del’âge
aveclequel
les différents stades de cedéveloppement
peuvent être atten- dus. Les barres verticalesindiquent
les intervalles de tempsrespectifs
ainsi que leur recoupemcnt.REFERENCES
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AY
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YSER
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ANG
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