878 K.G. Davey Insect Biochemistry and Molecular Biology 30 2000 877–884
pounds such as fenoxycarb mimic these effects. We have shown that both T4 and particularly 3
9,3,5-triiodothyron- ine T3, when applied to follicle cells of locusts in vitro,
induce the same rapid reduction in cell volume as JH III, and this effect is inhibited by ouabain, implicating
Na
+
K
+
ATPase Davey and Gordon, 1996. More recently, we have shown that T3 also mimics the action
of JH I in causing both a reduction in volume and an increase in patency in the follicle cells of Rhodnius pro-
lixus. Moreover, the effects of T3 on the follicle cells of L. migratoria appear to be mediated by the same recep-
tor as JH III. The effect on follicle cell volume is blocked by incubation in an antibody raised against the 35 kDa
binding protein for JH III, T3 shows specific and satu- rable binding to membrane preparations of follicle cells,
and JH III will compete for these binding sites Kim et al., 1999.
In this paper, I further explore the effects of deriva- tives of thyroxine on follicle cells, and examine the
occurrence and probable origin in locusts of immunore- active thyroxine derivatives.
2. Materials and methods
2.1. Insects Locusts were obtained from a large colony maintained
on a 12:12 light:dark cycle with the temperature at 36 °
C during the day and at 22
° C during the night.
2.2. Chemicals 3
9,59,3-Triiodothyronine, 3,39-diiodothyronine and 3
9,59-diiodothyronine were purchased from Henning Berlin. All other chemicals were from Sigma St.
Louis, MO, USA. Rhodamine conjugated T3 rho-T3 was prepared and
purified according to Cheng et al. 1980. This material has been shown in mammals to bind only to those sites
to which T3 binds Cheng et al., 1980.
2.3. Changes in cell volume Suspensions of follicle cells of ovaries in mid to late
vitellogenesis were prepared as previously described Davey and Gordon, 1996. Changes in cell volume were
detected by measuring the optical path difference OPD by quantitative interference microscopy as described in
Davey and Gordon 1996. Changes in the OPD under the conditions used in this study are inversely pro-
portional to changes in cell volume, so that an increase in OPD indicates a decrease in cell volume. In practice,
measurements for any particular hormone were perfor- med on 25 cells selected at random. The results
presented in each graph were performed on a suspension prepared from a single ovary. Each graph thus represents
results from a single experiment: all experiments have been replicated at least three times with qualitatively
identical results in terms of the differences in OPD among treatments, although the control values varied
among individual locusts. Unless otherwise indicated, the cells were exposed to the various test solutions for
30 min before the OPD was measured.
2.4. Uptake of rho-T3 Uptake of rho-T3 by the follicular epithelium was
measured by fluorescence microscopy. Individual ovari- oles were dissected from locust ovaries in a medium con-
sisting of 40 Eagle’s Medium in Schneider’s Droso- phila Medium Gibco, Grand Island, NY, USA, and
their connective tissue sheaths removed. For any one experiment, ovarioles from a single female were used.
In a typical experiment, six to seven ovarioles were immersed in 3 ml of medium containing 1.0
µ M rho-T3
at room temperature for 60 min. They were then rinsed quickly in medium, and mounted in medium on a micro-
scope slide under a coverslip for viewing and measure- ment by epifluorescence. In some experiments, follicles
were pre-incubated in inhibitors or an antibody raised against the JH III membrane receptor Kim et al., 1999
for 30 min before exposure to rho-T3.
Fluorescence was measured using a Zeiss Photomicro- scope III with an attached photometer model 01 K with
photomultiplier R446. Illumination was from a mercury lamp HBO 100 W2 driven by a stabilised DC power
supply, and the light path was chopped by a slotted disk at 60 Hz. The epi-illumination dichroic mirrorfilter set
was 31002 D 54025 mirror, 565 DCLP filter from Chroma Technology Corporation Brattleboro, Vermont,
USA. The photometer was adjusted so as to read 0 when no specimen was in the field, and a follicle which had
not been exposed to the rho-T3 also yielded a reading of 0. For measurement, a field of follicle cells on the
terminal follicle of an ovariole was centred and focused under tungsten illumination, and then switched to fluor-
escence, and the measurement taken immediately in order to avoid the rapid photo bleaching of the specimen.
This was repeated for five fields on each of at least five follicles, using a 40
× Neofluar objective. The readings
for each ovariole were typically within 10 of one another and the average was taken as the reading for a
follicle. The data are reported as means of at least five follicles. The photometer is very sensitive, detecting the
very weak fluorescence produced by this procedure.
2.5. Radioimmunoassay RIA Thyroid hormone immnunoreactive substances were
initially extracted from tissues and food by homogenis- ing them in phosphate buffer at pH 7.8 containing 1 mM
879 K.G. Davey Insect Biochemistry and Molecular Biology 30 2000 877–884
6-n-propyl-2-thiouracil PTU to inhibit deiodinases. The supernatant remaining after centrifuging at 1500g
was used for RIA. For more quantitative determinations, tissues or food were homogenised in 3 ml of phosphate
buffer containing PTU and 2.5 mg pronase. The homo- genate was incubated at 37
° C for 12 h, and then 1.5 ml
of NH
4
OH were added. The supernatant from centrifug-
ation at 1500g was evaporated to dryness and the residue taken up in 1 ml of distilled water for processing by
RIA. Haemolymph was not extracted prior to assay. RIA was performed using commercial kits Inter-med-
ico, Markham, Ont. for total T4 and T3. The standards supplied in the kit were used for most samples. Dilution
curves of gut contents and food were linear and parallel to the standard curves. For quantitation of haemolymph
T3, a standard was prepared based on locust haemo- lymph. Twenty millilitres of haemolymph were extracted
from locusts through a small incision in the cervical membrane. A small quantity a few crystals of phenyl
thiourea to inhibit darkening, and 0.1 sodium azide to inhibit bacterial growth were added. The haemolymph
was subjected to extraction with activated charcoal fol- lowed by centrifugation at 1550g to remove any T3, and
the resulting supernatant used as a solvent in preparing the standards of T3.
3. Results
