Effects of Supernumerary Corpora Allata and Farnesol Compounds on Ovary Development in the Worker Honeybee

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Journal of Apicultural Research
ISSN: 0021-8839 (Print) 2078-6913 (Online) Journal homepage: https://www.tandfonline.com/loi/tjar20
Effects of Supernumerary Corpora Allata and
Farnesol Compounds on Ovary Development in
the Worker Honeybee
Ber-Lin Chai & R. W. Shuel
To cite this article: Ber-Lin Chai & R. W. Shuel (1970) Effects of Supernumerary Corpora Allata
and Farnesol Compounds on Ovary Development in the Worker Honeybee, Journal of Apicultural
Research, 9:1, 19-27, DOI: 10.1080/00218839.1970.11100240
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Journal of Apicultural Research 9(1) : 19-27 (1970) 
EFFECTS OF SUPERNUMERARY CORPORA ALLATA AND 
FARNESOL COMPOUNDS ON OVARY DEVELOPMENT IN 
THE WORKER HONEYBEE 
BER-LIN CHAI* AND R. w. SHUEL 
Department of Apiculture, University of Guelph, Guelph, Canada 
Manuscript received for publication 16 December 1969 
Summary 
Implantation into a 4-day worker larva of corpora allata from a queen larva 3 or 4 days old was 
followed by a reduction in the extent of ovary regression in the prepupal and pupal stages. The 
average number of ovarioles in adults developing from treated larvae was increased by several times, 
but no oocytes developed. Corpora allata from a worker larva had no effect on ovary regression. 
Injection of farnesol or farnesyl methyl ether did not affect the number of ovarioles but did result 
in the development of oocytes. The prepupal period was lengthened both by supernumerary 
corpora allata from queen larvae and by farnesol compounds. Results were consistent with a 
hypothesis of endocrine control of retrogressive changes in the worker reproductive system. 
Introduction 
Queens and workers of the honeybee (Apis mel/ifera) differ strikingly in the develop-
ment of their reproductive organs. Zander et al. (1916) reported appreciable differ-
ences in ovary size as early as 48 hours. Wang and Shuel (1965), however, in an 
examination of large numbers of larvae whose ages had been estimated to within 
±4 hours, found very little difference between queen and worker larvae with respect 
to ovary volumes or numbers of egg tubules before the completion of caste deter-
mination (around 84 hours). Thereafter development is very rapid in the queen. 
At maturity each of her large pear-shaped ovaries contains some 160-180 egg tubules 
or ovarioles (Oertel, 1930). Reproductive development in the worker is arrested 
early in the fifth instar, and the ovaries regress during the prepupal and pupal stages 
(Zander et al., 1916). Egg tubules are reduced in number from more than 100 to 
3 or 4 in each ovary, and the ovaries themselves are thin and shrunken (Oertel, 1916). 
The rest of the reproductive system appears equally rudimentary. Despite its 
rudimentary appearance, however, the reproductive system of the adult worker can 
become functional to a limited degree. When the supply of queen substance in the 
colony is lacking or deficient, oocytes may develop in the ovarioles of the worker, 
and she may lay a few unfertilized eggs (Ribbands, 1953). 
Although the retrogressive changes in the reproductive system of the worker 
honeybee can be ascribed to diet, the physiology of the changes has received little 
attention. Ligation experiments by l'Helias (1950, 1952), Schaller (1952) and 
Lukoschus (1955) have confirmed for the honeybee the classic pattern of endocrine 
control of metamorphosis. From the analogy between metamorphosis and poly-
morphism (Wigglesworth, 1954) one might expect the latter, like the former, to be 
under endocrine control. Shuel and Dixon (1959, 1960) suggested that in the honey-
bee the endocrine system mediates between diet and form, in the initiation of caste 
differences during the first 3 or 3-! days of larval life, and in the completion of these 
* Present Address: Department of Zoology, University of Toronto 
20 
differences in the post-determination period. During the period of caste determi-
nation the likely candidate organ for the role of intermediary is the corpus allatum, 
the only endocrine organ in which conspicuous caste differences are visible (Canetti 
et al., 1964). Differences in other endocrine organs are apparent at 96 hours (Ritcey, 
1969). Canetti et al. interpreted caste differences in size and appearance as indicative 
of greater activity in the corpora allata of the queen larva. In addition to their role 
in maintaining juvenile characters throughout insect larval moults, the corpora allata 
produce a secretion, believed to be identical with the juvenile hormone (Wigglesworth, 
1964) which induces yolk formation in the adult female of Rhodnius prolixus (Wiggles-
worth, 1936) and is involved in lipid and protein metabolism during ovary development 
in many insects (Pfeiffer, 1945; Strangways-Dixon, 1961; Highnam et al., 1963; 
Thomas & Nation, 1966). Juvenile hormone activity has been found in a number 
of chemicals, notably farnesol and its methyl ether derivative (Wigglesworth, 1963). 
Recently the juvenile hormone of Hyalophora cecropia has been identified as methyl 
1 O-epoxy-7ethyl-3, ll-dimethyl-2, 6-tridecadienoate (Roller et al., 1967). 
To test the possibility that the retrogressive changes in worker ovaries are under 
endocrine control, we attempted to change the natural hormonal balance through 
implantation of supernumerary corpora allata or the injection of farnesol and its 
methyl ether into larvae 4 days old. As criteria of experimental effects we recorded 
changes in the appearance of the reproductive system and increases in numbers of 
ovarioles. We also recorded certain secondary data related to characteristic patterns 
of queen and worker development, i.e. patterns of cocoon spinning and larval 
orientation in the cell, and the duration of larval, prepupal, and pupal stages. 
Materials and Methods 
Collection of larvae and larval food 
Larvae were taken from colonies in the apiary during the summer months and from 
indoor flight rooms during cold weather. Queen larvae were obtained by transferring 
larvae 6-12 hours old from worker cells to artificial queen cups in nurse colonies. 
Fresh royal jelly and worker jelly were collected by aspiration from 3-day queen 
cells and 1- to 3-day worker cells respectively. 
Implantation of corpora a/lata 
The donor larvae were placed in a dissecting dish with the ventral side up, and the 
head was pushed forward by placing a pin across the neck. A drop of sterile saline* 
was placed on the head. The neck was cut open with a fine scalpel, exposing the 
corpora allata under the tentorium, and the corpora allata were cut loose with 
scissors and sucked up into a capillary tube. The host larva was cut at the second 
thoracic segment, the tip of the capillary inserted into the incision and the corpora 
allata discharged. The incision was sealed with agar heated to 60°C. Instruments 
used in the operation were sterilized with alcohol. 
The earliest age at which larvae could be operated was about 4 days, when theyweighed around 60 mg. Younger larvae died from injuries or bleeding. Sham-
operated controls and untreated controls were included in each experiment. 
Injection of farnesol compounds 
Farnesyl methyl ether was prepared from 99·1 % commercial farnesol by the William-
son synthesis (Hill & Kelly, 1944). Purified peanut oil was used as a carrier for 
* 0·9% saline containing 0·1 % penicillin, 0·1 % erythromycin and 0·15% phenolphthalein 
21 
both farnesol and its methyl ether. Injections were made into the intersegmental 
suture on either side of the mid-ventral line of the thorax, using a 50 jlg micrometer-
driven syringe. Half-microlitre volumes containing 2·2 or 4·4 jlg of the farnesol 
compound were used. Larger doses of farnesol killed the larvae within a few days. 
Peanut oil alone was injected into the sham-operated controls. 
Post-operative care of larvae 
After surgical treatment larvae were placed in artificial beeswax cells in desiccators 
at 35°C and 97% relative humidity. They were fed either royal jelly or an amended 
worker jelly containing 120 mg per gram of glucose and fructose in equal parts. 
The composition of the latter diet is close to that of modified worker jelly, the diet 
of older worker larvae (Shuel & Dixon, 1959, 1968). We were not able to obtain 
enough modified worker jelly from 4-day worker cells. 
At the beginning of defaecation, larvae were washed with warm water, dried, and 
placed horizontally in 1 x 3-mm pupation cells capped with tissue paper. The 
relative humidity was reduced to 80% for pupation and to 60% thereafter. 
Examination of ovaries 
The fixative of Weaver and Thomas (1956) was used to preserve the material for 
dissection. A median longitudinal incision was made in the first four abdominal 
segments with a scalpel and fine tweezers, under a x 20 binocular dissecting micro-
scope. The eight dorsal plates were removed to expose the adipose tissue, digestive 
organs and ovaries. The method described by Hess (1942) was followed for ovariole 
counts. 
Statistical methods 
As ovariole counts deviated from the normal distribution, the range being propor-
tional to the mean, a logarithmic transformation (Steel & Torrie, 1960) was applied 
to the data before analysing them. Data on the relative duration of each 
developmental stage were changed to percentages and compared by the x2 test. 
Analysis of variance was used to evaluate treatment differences in total times to 
emergence. 
Results 
General appearance of the reproductive system 
Because of the difficulty of obtaining clear photographs, the ovaries of an adult bee 
developing from a worker larva fed on the worker jelly + sugar diet and implanted 
with a pair of corpora allata from a queen larva 3 days old are shown diagrammatically 
(Fig. 1 ), with ovaries of a normal untreated worker adult and a queen for comparison. 
There was a partial development of the ovaries in the treated individuals, though it 
was limited in comparison with development in a queen. No oocytes could be seen 
in the ovarioles. The spermatheca was rudimentary though somewhat larger than 
in the control groups. Development in individuals receiving corpora allata from 
queen larvae 4 days old was very similar. Ovaries in the unoperated and sham-
operated bees were much reduced in size and hardly traceable among the surrounding 
tracheae. 
The ovarioles of larvae fed on royal jelly and implanted with queen corpora allata 
were longer and more closely packed than those fed on worker jelly and sugar. 
Like the latter, they contained no oocytes. 
22 
The ovaries of bees developing from larvae injected with farnesol or farnesyl 
methyl ether were much smaller than those of bees that had been given corpora allata 
implants, but-in contrast to the ovaries of the implanted bees--some of the ovarioles 
contained oocytes (Fig. 2), and were thus atypical of either caste. 
w ow Q 
FIG. 1. (left) Diagrams showing comparative ovary development in a normal adult worker (W), 
in an adult developed from a worker implanted with corpora allata from a 3-day queen larva 
(OW), and in an adult queen with fully matured ovaries (Q). 
FIG. 2. (right) Oocytes in ovarioles of worker honeybees developed from larvae injected with 4·4 Jlg 
of famesyl methyl ether. 
Ovariole counts 
Quantitative data on survival and ovariole development are recorded in Tables 1-4. 
Survival of operated bees was very poor. Operated individuals were especially 
sensitive to jarring or to slight changes in temperature at the time of the prepupal 
moult. Surgery without implantation was somewhat less injurious. Injection with 
farnesol also reduced survival, though not as drastically as surgery. Bees injected 
with the peanut oil carrier alone survived better than those receiving farnesol 
compounds. 
Numbers of ovarioles varied greatly in both treated and control groups. Despite 
this variability, it can be seen from Tables I and 2 that the average number of 
ovarioles was substantially higher in the groups receiving supernumerary corpora 
allata from queen larvae than in the untreated and sham-operated groups. Results 
with queen donors 3 and 4 days old were similar, but treatment with corpora allata 
from 3-day larvae produced more ovarioles. Implantation with corpora allata 
from 3-day worker larvae had little or no effect on the ovariole count (Table 3), nor 
did injection with farnesol or farnesyl methyl ether (Table 4). Control groups fed 
on royal jelly had more ovarioles than controls on the worker diet (means 18·8 and 
9·5 respectively, P<0·005). 
Effects on other aspects of development 
Larvae receiving queen corpora allata discharged their faeces earlier than those in 
the control groups. The silk was light yellow, resembling the silk of queens more 
than that of workers. 
Control larvae lay on their backs facing the cell capping in the normal fashion 
23 
of workers (Jay, 1963), but treated larvae varied in their orientation. Some lay on 
their sides facing the capped end, whereas others faced the cell base. Similar 
variability was seen in larvae injected with farnesol compounds. 
Duration of development 
Bees receiving corpora allata from 3- or 4-day queen larvae entered the prepupal 
stage earlier than the controls, but they remained as prepupae longer, with the result 
TABLE l. Implantation of corpora allata from queen larvae into 4-day worker 
larvae fed on worker jelly with added sugar. 
No. ovarioles in adults 
Age No. No. Statistical 
of donor larvae surviving Mean Range significance 
3 days 0: 815 8 71·9 ± 13-21 19-141 
O>S,C 
S: 504 8 9·0 ± 2·89 1-28 
P<0·005 
C: 129 13 6·3 ± 1·39 2-13 
4 days 0:224 5 35·2 ± 3·24 26-46 
O>S,C 
S: 61 5 8·2 ± 1·90 4-16 
P<0·005 
C: 20 5 9·6 ± 1·97 2-15 
0: operated; S: sham-operated control; C: unoperated control 
TABLE 2. Implantation of corpora allata from queen larvae into 4-day worker 
larvae fed on royal jelly. 
No. ovarioles in adults 
Age No. No. Statistical 
of donor larvae surviving Mean Range significance 
3 days 0:229 4 72-5± 1·95 33-113 
O>S,C 
S: 117 4 18·0 ± 8·95 2-48 
P<0·025 
C: 32 6 41·3 ± 20·40 3-92 
4 days 0: 128 4 61·5 ± 16·07 19-120 
O>S,C 
S: 33 4 10·8 ± 2-36 3-15 
P<0·025 
C: 12 5 13-4 ± 4·78 1-27 
0: operated; S: sham-operated control; C: unoperated control 
24 
that the total time until emergence ofthe adult was longer than in the controls (Fig. 3). 
Implantation of corpora allata from worker larvae had no effect on the duration of 
development. The extension of the prepupal stage, and the total time to emergence, 
were especially pronounced in the injected bees, being about two days. In no 
instance was the length of the pupal stadium changed by the experimental treatment. 
In both operated and injected groups total developmental time was shorter on the 
royal jelly than on the worker jelly + sugar diet. 
TABLE 3. Implantation of corpora allata from 3-day worker larvae into 4-day 
worker larvae. 
No. ovarioles in adults 
No. No. StatisticalDiet larvae surviving Mean Range significance 
Worker jelly 0: 163 5 5·0 ± 1·03 1-10 
with added S: 42 5 5·6 ± 1·05 2-11 P>0·10 
sugar C: 25 5 4·1 ± 0·89 1-9 
Royal jelly 0: 77 4 11·0 ± 1·06 7-16 
S: 39 4 13·8 ± 1·42 6-21 P>O·IO 
C: 14 4 14·0 ± 0·78 10-17 
0: operated; S: sham-operated control; C: unoperated control 
TABLE 4. Injection of farnesol and farnesyl methyl ether into 4-day worker larvae. 
No. ovarioles in adults 
No. No. Statistical 
Diet Treatment larvae surviving Mean Range significance 
Worker jelly 4·4 )lg 0: 30 4 6·3 ± 1·64 2-11 
with added farnesol S: 15 4 10·0 ± 0·93 5-20 P>0·1 
sugar C: lO 6 7·0 ± 1·38 2-11 
Worker jelly 4·4 Jlg 0: 120 6 14·8 ± 3·10 6-26 
with added farnesyl S: 35 6 6·2 ± 1·71 3-15 P>0·1 
sugar methyl C: 10 6 5·8 ± 0·87 3-10 
ether 
Royal jelly 4·4 )lg 0: 30 4 10·7 ± 3-80 1-9 
farnesol S: 15 4 7·7 ± 2·11 3-14 P>0·1 
C: 10 4 22·7 ± 8·33 3-48 
Royal jelly 4·4 Jlg 0: 30 4 54·2 ± 14-40 29-103 
farnesyl S: 15 4 47·5 ± 16·10 16-94 P>0·1 
methyl C: 10 4 26·8 ± 13·70 4-73 
ether 
... 
z 
Ill 
~ 15 ... 
ct 
Ill 
Ill: 
t-
., 
>-
ct 5 
Q 
QUEEN CORP. ALL . WORKER CORP. ALL. 
' i 111111 ... 
0 5 c 
Cl 
0 5 c 
b 
0 5 c 
c 
0 5 c 
d 
25 
FARNESYL M. E. 
II 
0 5 c 
• 
II 
0 5 c 
f 
FIG. 3. Effect on duration of development stages in worker bees (L=larval, PP=prepupal, 
P=pupal) of the following: 
a, b - implanting 3-day queen corpora allata 
c, d - implanting 3-day worker corpora allata 
e, f - injecting 4·4 Jlg famesyl methyl ether 
O=operated; S=sham-operated; C=control 
WJS=worker jelly and sugar; RJ =royal jelly 
Statistical significances are: 
for total time to emergence of adults in a, e, f: O>S,C (P<O·OI) 
for relative duration of developmental stages in a, b, e, f: P<0·05 
inc, d: P>O·IO 
Discussion and Conclusions 
Retrogression of the reproductive system of the worker honeybee normally begins 
about the fifth day of larval life, after the caste has been determined. Implantation 
of a pair of corpora allata from a queen larva 3 or 4 days old into a worker larva 
4 days old reduced the degree of retrogressive development. Implantation of corpora 
allata from 3-day worker larvae failed to produce a like effect. One may infer from 
these observations (I) that the corpora allata of the queen are involved in the post-
determination development of her reproductive system, and (2) that the corpora 
allata of the worker are less active in promoting reproductive development than 
26 
those of the queen. The histological evidence of Canetti et al. (1964) is thereby 
substantiated. 
The reduction in the usual retrogressive process in the workers was limited, 
however, to a partial development of only about one-quarter of the number of egg 
tubules normally found in the queen. Three possible reasons for the limited success 
of the experimental treatment come to mind: (I) the transplanted corpora allata 
had less than their normal in situ activity; (2) the ovaries of the host larvae had lost 
most of their capacity for progressive development at 4 days of age; (3) other en-
docrine organs in the host larvae (whose activation may be essential for complete 
ovary development) may no longer have been competent at 4 days of age. Lukoschus 
(1952) observed a rapid development of the prothoracic glands in the queen around 
4 days, and Ritcey (1969) observed a marked reduction in size of the neurosecretory 
cells of queen larvae-which she interpreted as indicating a massive release of neuro-
secretion-around 96 hours. Possibly our operation came too late to precipitate a 
similar event in the host larvae. It is unfortunate that technical difficulties prevented 
an earlier operation. 
There is abundant evidence (reviewed by Shuel and Dixon, 1960) that the diet 
supplied to the queen or the worker cell during early larval life sets the stage for post-
determination events. A comparison of ovary development in control groups fed 
on worker jelly - sugar and on royal jelly diets in both the corpora allata and the 
farnesol experiments indicates that some dietary influence persists into the post-
determination period. The occurrence of ovary regression soon after the worker 
diet is modified by a large addition of honey is probably a coincidence rather than 
evidence of a cause-effect relationship. 
The qualitative difference between corpora allata and farnesol effects on ovary 
development-the first increasing ovariole number, the second stimulating oocyte 
maturation-is surprising, and it suggests that more than one hormone may be 
involved in the regulation of ovary development. 
Corpora allata implantation and farnesol injection had similar effects on the timing 
of the late developmental stages. Fukaya and Mitsuhashi (1961) found that farnesol 
prolonged the developmental period of Periplaneta americana from 17 to 30 days. 
The experimental effects on ovariole numbers are consistent with a hypothesis of 
endocrine control over retrogressive changes in the reproductive system of the 
worker honeybee. The fact that our treatments made worker larvae more queen-
like in one respect (reproductive development) and less queen-like in another (length 
of the developmental period) underlines the complexity of the endocrine influence on 
caste development. 
Acknowledgements 
We are indebted to Dr. S. E. Dixon, Department of Zoology, University of Guelph, 
for the corpora allata implantation technique, and to Dr. M. V. Smith and Mr. A. 
Adie, Department of Apiculture, for help in procuring material. This work, which 
formed part of a thesis by the senior author for the M.Sc. degree, was supported by 
the Ontario Department of Food and Agriculture and the National Research Council 
of Canada. 
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