/ . Embryo!, exp. Morph. Vol. 40, pp. 245-251, 1977
Printed in Great Britain © Company of Biologists Limited 1977
245
Variation in Habrobracon juglandis ovariole
number
I. Ovariole number increase induced by extended cold
sliock of fourth-instar larvae
By DANIEL S. GROSCH, 1 ROBERT G. KRATSAS
AND ROBERT M. PETTERS
From the Department of Genetics, North Carolina State University
SUMMARY
Variation from zero to five ovarioles in the braconid wasp, Habrobracon juglandis, is
reported for dissections of females from laboratory stocks. Four ovarioles (two per ovary) is
the standard phenotype for the species. Storage of fourth-instar larvae in a refrigerator
resulted in a large proportion of females with an increased number of ovarioles (5-11). A
1- to 2-week cold exposure during the period of ovarian differentiation is required for the
maximum effect.
INTRODUCTION
While great variation in ovariole number occurs between insect species, the
number of ovarioles per ovary remains constant within most species (Chapman,
1969). Some exceptions to this rule are known, as in Drosophila selection experiments (Robertson, 1957) and in certain wasps of the family Ichneumonidae
(Iwata, 1960). Perhaps there are many more cases occurring in nature which
escape detection especially when there are dozens of ovarioles. In ovaries comprised of bundles of ovarioles, one tube more or one tube less is neither morphologically obvious, nor particularly significant to fecundity.
The parasitic wasp Habrobracon juglandis (Ashmead) = Bracon hebetor Say
has been the subject of intensive laboratory investigation since 1918 (P. W.
Whiting, 1918), and the number of ovarioles has been repeatedly reported to be
four (two per ovary) (Genieys, 1925; Speicher, 1936; A. R. Whiting, 1940;
Bender, 1943; Grosch & Sullivan, 1954; Erdman, 1961; King &Cassidy, 1973).
The present paper documents both natural and experimentally-induced variation in the number of ovarioles per ovary of H. juglandis. The experiments
were performed in an attempt to elucidate morphological variability which came
to our attention during other kinds of investigations.
1
Author's address: Department of Genetics, North Carolina State University, Raleigh,
North Carolina 27607, U.S.A.
246
D. S. GROSCH, R. G. KRATSAS AND R. M. PETTERS
MATERIALS AND METHODS
The braconid wasp, known to geneticists as Habrobracon, is ectoparasitic
upon the larvae of the Mediterranean flour moth, Ephestia kuehniella Zeller
( = Anagasta kuehniella Zeller). Both the host and parasite are easily reared in
the laboratory. Two females, two males and four host caterpillars are placed in
shell vials for routine stock maintenance. The optimum rearing temperature of
29 ± 1 °C is maintained in standard incubators. At this temperature the lifecycle is 9-10 days. Synchronized progeny can be obtained by allowing pre-mated
females to oviposit on paralysed caterpillars for 1 h. Offspring age is calculated
from the time at which the mothers were placed with host larvae. Thus, a group
of 96 h individuals contain wasps from 95 to 96 h of age. A large cabinet refrigerator (5 ± 1 °C) was used for all cold treatments. Prior to and following the
cold treatment the wasps were incubated at 29 ± 1 °C. After eclosion the female
wasps were collected, allowed to oviposit for a few days and then dissected.
Dissections of individual wasps were performed with sharp needles in a drop
of saline (1 % NaCl) under a binocular microscope (7-30 x).
Unlike the Diptera and the generalized textbook representations for insects,
there is no sheath enclosing the ovary. Each braconid ovary normally comprises a pair of ovarioles attached anteriorly by the union of a terminal
filament from each tube (Genieys, 1925). In dissection, this as well as a copious
tracheal net must be severed from the body wall. The tensile strength of these
suspensory structures typically exceeds that of the ovariole sheath so that
attempts to drag the ovaries out of the abdomen by pulling on the last abdominal
segments results in torn ovarioles. This can lead to errors both of omission and
of commission in scoring ovariole number. In experiments where many females
must be examined for oocyte damage, it may not be convenient to check for the
presumed missing ovariole among the various fragments.
RESULTS
Over 500 control females reared at 29 ± 1 °C were dissected for these and
other related experiments in fecundity (to be published elsewhere). Females
with four ovarioles were in the majority (77 %) and should be considered the
normal situation. Variations above and below four ovarioles are shown in
Table 1, which compares results for a standard laboratory stock no. 33 with
two more recently obtained stocks. Additional observations on females from
no. 33 are shown as controls in Tables 2 and 3. Individuals with none or one
ovariole were seen. Few untreated individuals showed more than four ovarioles.
Exposure to cold temperature for long periods of time can increase the number of ovarioles if the cold treatment is applied during the fourth instar (Table 2).
In expt. 1 the females which were 96 h old when treated (group 1) showed a
dramatic increase in the frequency of individuals with more than four ovarioles -
247
Variation in Habrobracon ovariole number
Table 1. Variation in ovariole numbers found in dissection of Habrobracon
females reared at normal temperature (29 ± 1 °C)
No. of females with ovarioles numbering:
Stock
designation*
No.
dissected
No. 33
LUM
eA
59
48
40
1
1
1
56
46
32
1
1
1
* Stock no. 33 in the laboratory for over 35 years, originally from California. LUM was
recently collected in Lumberton, NC. eA designates an ebony body stock from A. M. Clark,
University of Delaware.
Table 2. Variation in ovariole numbers found in dissections of Habrobracon
females stored at low temperature (5 ±1 °C)for 14 days
Age when
>laced in cold
F
(h)
No.
females
dissected
—
96 h
120 h*
144 h*
168 h*
Females with listed ovariole numbers (%)
A
f
2
3
4
5
6
7
8
9
10
31
20
16
20
14
— — —
— —
— — —
— — —
— — —
3
90
50
100
95
93
7
20
—
—
—
—
15
—
—
—
—
10
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
84 h
96 h
108 h
21
4 — — 5 91 — — — — —
8
7 — —
54 23
8 — — —
— — 3 18 75 4 — — — —
— — — — 100 — — — — —
—
—
—
—
1
2
3
4
5
76 h
78 h
80 h
82 h
84 h
16
14
10
5
7
— — —
— — 6
— — —
40 — —
— — —
Expt. 4
Controls
1
2
3
—
76 h
78 h
80 h
23
13
7
7
—
—
14
—
Expt. 1
Controls
1
2
3
4
Expt. 2
Controls
1
2
3
Expt. 3
13
28
4
0
1
—
—
—
—
c
—
5
7
—.
—
—
20
—
— —
— —
— 29
— —
44
29
40
—
72
37
29
30
—
14
6
29
—
40
14
13
7
20
—
—
—
—
10
—
—
— —
— —
— —
— —
— —
96 4 — — — —
30 15 31 8 8 —
43 — — — 14 —
29 — — 57 — 14
* Prepupae. All other groups were 4th-instar larvae when placed in the cold.
—
8
—
—
248
D. S. GROSCH, R. G. KRATSAS AND R. M. PETTERS
Table 3. Variation in ovariole numbers found in dissections of Habrobracon
females stored at low temperature (5 ±1 °C)for different periods of time
Group
Duration
of
treatment
(days)
No.
females
dissected
Controls
—
Treatment begun at 76 h
1
1
2
3
5
3
4
7
184
24
19
44
31
26
5
14
Treatment begun at 80 h
6
1
68
7
3
105
8
5
79
9
9
81
10
14
50
Females with listed ovariole numbers (%)
0
1
9
2
5 10 28 55 —
—
8 17
5 5 11
2 11 12
3 3 7
7 — —
—
—
—
—
—
2
7
2
1
2
29
21
36
19
8
46
58
39
55
19
—
—
—
13
23
16 82
1182
22 75
11 63
6 8
—
—
1
16
20
8 12
—
—
—
9
32
—
—
—
—
18
—
—
—
—
8
4
—
—
—
—
4
10 11
4
—
—
—
—
—
—
—
—
—
—
2
45 % of the sample. No increase was seen in those groups older than 96 h at
treatment. Experiment 2 (Table 2) revealed a cold treatment induction of extra
ovarioles in the 84 h group but few in the 96 h group. In the third experiment
the controls were omitted because the frequency of the standard ovariole
number was established. All five groups in expt. 3 exhibit an increase in ovariole
number per female. Furthermore, the 84 h groups in expts. 2 and 3 have similar
values for the percent of individuals with more than four ovarioles (expt. 2,
group 1 = 31 %; expt. 3, group 5 = 28 %).
A comparison of the results of expt. 3 with those from expt. 4 shows a consistent increase in supernumerary ovarioles between groups treated at 76 and
80 h. 'Seventy-six hour' individuals (group 1 in both experiments) yielded 56 %
and 69 % with more than four ovarioles. Similarly, the 80 h groups (group 3 in
both experiments) yielded 60% and 71 %. The frequency in the 78 h groups
dropped from 65 % in expt. 3 to 14 % in expt. 4, but the latter group included
an exceptionally large proportion of females with three ovarioles.
Groups of females exhibiting an increase in the percentage of individuals with
more than four ovarioles usually have a mean number of ovarioles per female
that is greater than four. Due to the variability in the composition of the samples, the skewed distribution, and the small size of the samples, the mean
number of ovarioles per female appears to be a less suitable indicator of the
cold shock effect on ovariole number than the percentage of individuals with an
ovariole number greater than normal.
The duration of cold temperature necessary to produce an effect on ovariole
number was examined in the experiment shown in Table 3. With the 76 h
Variation in Habrobracon ovariole number
249
samples, 1 week of cold temperature was sufficient to increase the frequency of
extra ovarioles from 0 to 13 % but 2 weeks of cold storage was even more
effective, raising the frequency to 66 %. The 80 h samples were even more sensitive. Eighty-four % of the wasps stored in the cold for 2 weeks showed supernumerary ovarioles. The results in Table 3 for the 2-week exposures compare
well with the values obtained in the experiments shown in Table 2. Exposures
shorter than 7 days did not increase the number of ovarioles.
DISCUSSION
The occurrence of individuals with less than four ovarioles in H. juglandis
stocks reared at standard conditions was unexpected. All previous literature
cited unequivocally two ovarioles per ovary except for the isolated case of no
ovarioles mentioned by Grosch, 1971. Here we report for the first time that
females with less than four ovarioles are present in at least three separate stocks
of H. juglandis. Perhaps investigators anticipating the normal number of ovarioles assumed that dissections revealing less than four may have been due to the
'loss' of an ovariole or two. Even experienced technicians can 'lose' a small
detached egg tube, hidden under and attached to a segment of exoskeleton.
Recent gene mutation can probably be ruled out as an explanation because of
the observation of variation in a number of different stocks. Possibly a decrease
in ovariole number is caused by an undetected parasite as several are known
that castrate host insects (Wheeler, 1910). Investigations are in progress to
elucidate the origin of this spontaneous variation in ovariole number.
In every braconid laboratory, refrigeration of pupal and adult wasps has been
a standard storage procedure. Some morphological consequences of this practice came to our attention during studies of anti-vitellogenic agents in which all
wasps were dissected. Adults refrigerated as early pupae tended to have short
ovarioles with attached germaria. Genieys (1925, p. 200) reported that Habrobracon collected during a cold period (14 °C) possessed short, convoluted
ovarioles. However, this could result from oocyte resorption in the absence of
the food supply. In 1973, females with up to six ovarioles were discovered in a
sub-line in the no. 33 stock, but inbreeding to derive a pure breeding strain was
unsuccessful (Gary J. Smith, personal communication). This was a period of
extreme excursions in temperature due to faulty air conditioning. The experiments reported above were a systematic attempt to ascertain the effects of cold
storage during the late larval stages of Habrobracon.
The low-temperature effects on insect development include changes in the
rates of development and survival (Howe, 1967; Bursell, 1974). Cold shock of
eggs can lead to an increased number of gynandromorphs both in Apis (Drescher
& Rothenbuhler, 1963) and in Habrobracon (Petters & Grosch, 1976). Young
larvae of Tenebrio molitor placed in a cool cellar showed teratisms in the adults
(Lengerken, 1925). Cold shock has also been used to induce phenocopies of
250
D. S. GROSCH, R. G. KRATSAS AND R. M. PETTERS
certain genes in Drosophila (Gloor, 1944, 1945, cited in Hadorn, 1961, p. 240).
However, the usual approach involves only a brief exposure at a specific developmental stage. The results presented here differ from classical phenocopy data
in that a lengthy period of exposure is required.
Ovariole number in H. juglandis can be increased by extended cold shock if
the treatment is applied before the end of the fourth larval instar. The optimum
age for treatment appears to be between 76 and 96 h. Larvae older than 96 h
do not respond to the cold treatment. Presumably, the critical developmental
event is the division of the ovary anlagen which occurs between 70 and 90 h
(Henschen, 1928; Erdman, 1961). It appears that the cold temperature must be
present prior to and during this critical stage. The experiment shown in Table 3
demonstrated that at least a week of exposure to the cold was necessary for a
recognizable effect. The proportion of individuals with an increased number of
ovarioles is consistent between experiments performed over a period of one
year.
The mechanism by which cold temperature leads to an increase in the number
of egg tubes is open to speculation. Cold temperature is used to prevent the
larval to pupal molt in the Anagasta host caterpillars. The temperature does not
stop the growth of these caterpillars, and individuals become much larger than
normal. Perhaps a similar pattern of growth without differentiation is taking
place in the ovary anlagen during the cold treatment. A larger anlage at the
time of ovariole establishment may be divided by investing sheath cells into an
ovary with more than the normal number of tubes. Alternatively, perhaps the
cold treatment affects the partitioning of the normal-sized anlagen in such a
way that 'extra' ovarioles are established which subsequently develop to equal
size. An inhibition of cell division could be postulated here. A literature search
yielded few references on the subject of cold temperature effects on gross insect
morphogenesis. As this paper suggests, cold storage of insects, while quite
convenient, should be considered as an experimental variable especially when
dealing with immature stages.
The work presented here was supported in part by grant no. ES 00044 from the National
Institutes of Health.
Paper no. 5130 of the Journal Series of the North Carolina Agricultural Experiment
Station, Raleigh, North Carolina.
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{Received 5 January 1977, revised 2 February 1977)