237 M.-P. Tu et al. Journal of Insect Physiology 47 2001 233–244
activity 200 mCimmol]. We used CAs from sugar-fed flies because they showed less background activity than
liver-fed flies. Before the CAs were introduced into the medium, 2, 20, or 200 pmol of synthetic Mas AT were
added. The CAs were incubated for 4 h before JH biosynthesis was estimated using a scintillation counter
to monitor the incorporation of [H
3
]-methionine into the JHs.
3. Results
3.1. Generation of the antisera Each rabbit produced over 100 ml of high titer anti-
serum designated as CY16 and CY17. CY16 and CY17 at different dilution of 1:500 and 1:1000, respectively
yielded comparable results when used immunochem- ically in our whole-mount preparations of insect neural
tissues. This suggests that antisera might have some slight differences in affinity andor antibody titer towards
tissue-bound Mas AT or Mas AT-like substance. Anti- sera from both rabbits were also used to develop com-
petitive enzyme-linked immunosorbent assays cELISA for testing their cross-reactivity to Mas AT, Lom MT,
and
a deduced
mosquito AT-like
peptide at
nanogramtest level. Under our cELISA conditions, comparable results were obtained if antisera CY16 and
CY17 were used at 400,000- and 200,000-fold dilutions, respectively. Again, this suggests that there were slight
differences in our antisera in terms of their affinity andor titer towards Mas AT.
3.2. Characterization of anti-Mas AT antisera 3.2.1. Localization of Mas AT-immunoreactive cells in
M. sexta larvae When probed with the CY17 antiserum, immunoposi-
tive neurons were identified from day-2, last 5th instar larval M. sexta in their frontal ganglion [Fig. 1A], brain
[Fig. 1B], and terminal abdominal ganglion [Fig. 1C]. The number and the location of these neurons were
identical to those reported by Taylor et al. 1996. As they have reported, there are two neurons each in the
frontal and the terminal abdominal ganglia, as well as numerous immunoreactive neurons in the brain. CY16
antiserum yielded the same results.
3.2.2. Lack of cross-reactivity to other Mas AT-like peptides
The cELISA for Mas AT was used to check possible cross-reactivity
to other
peptides that
somewhat resemble Mas AT. Two peptides were synthesized and
tested. The first is the [Ala
6
, Leu
7
, Ser
8
]-allatotropin, which is originally discovered from the male accessory
gland of L. migratoria. The second is a peptide deduced from the DNA of A. aegypti showing a high degree of
homology to Mas AT. Results from experiments with CY16 Fig. 2 showed that under the stated conditions,
the cELISA had a linear detection range of 25–2500 ng of synthetic Mas AT. It took ca. 110 ng of Mas AT, or
greater than 2000 ng of [Ala
6
, Leu
7
, Ser
8
]-allatotropin, to reach the point of 50 inhibition. The difference was
about 20-fold. Interestingly, the affinity of CY16 was so low towards the deduced mosquito AT-like peptide,
competitive inhibition never exceeded 20 in this assay. CY17 antiserum yielded similar results.
3.2.3. Lack of reactivity to MylATr MAP Our results showed practically no reactivity between
our antisera and the MylATr MAP. Regardless of the concentration of antisera tested, the reactivity to Mas AT
MAP was at least 17 times ranged from 17 to 46 times higher than that to the MylATr MAP. It appeared that
our antisera contained little or no antibodies that recog- nize the heptalysine core, which is the only feature
shared by both MylATr MAP and Mas AT MAP. Other- wise, we would see considerable reactivity of the anti-
sera towards both MylATr MAP and Mas AT MAP. We conclude that our antisera were specific to the Mas AT
portion of the MAP.
3.3. Localization of Mas AT-immunoreactive cells in P. regina and other insects
When probed with CY17 antiserum, immunopositive neurons were identified in the cephalic nervous tissues
of P. regina [Fig. 1D–G] but not in the thoracico- abdominal ganglion. In each brain hemisphere of sugar-
and liver-fed adult P. regina females, two immunoposi- tive neurons were found in the region where lateral neu-
rosecretory cells would be expected [Fig. 1D,F]. Also, 9–11 immunopositive cells were found in a region
located in between where median neurosecretory cells would be expected and the pharyngeal aperture [Fig.
1D,E]. This pattern of distribution of Mas AT immuno- positive neurons differed significantly to the distribution
pattern of such cells in the larval brain of M. sexta [Fig. 1B]. It was interesting to note that immunoreactive
material was present in the CC of liver-fed P. regina 6 h after the onset of the feeding. The CA of both sugar-
and liver-fed flies, as well as the CC of sugar-fed flies, was void of Mas AT-like substance. In addition, no Mas
AT immunoreactivity was observed in the anterior midgut of this fly. CY16 antiserum yielded similar
results. When CY17 antiserum was used against the brain andor frontal ganglion of other insects, immunore-
active materials were identified in: 1 the adult brain of a hemipteran, O. fasciatus [Fig. 3A]. In this bug,
immunopositive cells were found scattered all over the brain; 2 the adult brain of the oriental fruit fly, D. dor-
salis [Fig. 3B], where immunopositive materials
238 M.-P. Tu et al. Journal of Insect Physiology 47 2001 233–244
Fig. 1. Manduca sexta allatotropin Mas AT immunoreactivity in the central nervous system of 2-day old, 5th instar larva of M. sexta. A
Dorsal view of frontal ganglion of larval M. sexta. Two distinct neurons contained strong immunoreactivity in the perikaryons and axons descending into the recurrent nerve not shown. Scale bar
= 105
µ m. B Dorsal view of the brain. Many immunoreactive neurons are present in the entire
brain. Scale bar =
105 µ
m. C Dorsal view of the terminal abdominal ganglion. Two distinct neurons containing large quantities of immunoreactive materials. In B and C, no Mas AT immunoreactivity was observed in any nerve fibers. Scale
= 105
µ m. D–E Mas AT-like immunoreactivity
in the central nervous system of liver-fed, adult female, P. regina. D Posterior view of the brain showing the left optical lobe OL, position of the median furrow of the protocerebrum double-headed arrow pointing to each hemisphere, trachea T, two clusters of immunoreactive neurons
located in the region in between where the median neurosecretory cells may be expected and the foramen for the pharynx or pharyngeal aperture thin arrow points to either left or right cluster of neurons, and two distinctively reactive neurons thick arrow in the left hemisphere in the region
where lateral neurosecretory cells may be expected. Scale bar =
140 µ
m. E Immunoreactive neurons at the median region. It was clear that the nuclei are not immunopositive. Scale bar
= 50
µ m. F Two immunoreactive neurons at the lateral region. Again, nuclei are not immunoreactive.
Scale bar =
50 µ
m. G Immunoreactivity in the CC–CA complex. Strong immunopositive responses were restricted to the CC portion of the complex. The overall appearance of the immunoresponses in the CC were quite different from that found in the brain neurons. The immunoreactivity did
not appear to be associated with the somata of innate CC neurons. Scale bar =
50 µ
m. All micrographs, except D, were obtained using a confocal microscope, D was obtained by using a fluorescent microscope.
showed up clearly in three pairs of neurons; and 3 the brain and frontal ganglion from the last larval instar lor-
eyi leafworm, M. loreyi. Two immunopositive cells were identified in the frontal ganglion [Fig. 3C], with dozens
of cells found scattered around the entire brain [Fig. 3C]; and 4 brains of both adult female [Fig. 3D]
and male results not shown M. loreyi contained immu- nopositive cells, which were scattered around the two
hemispheres. Results not shown, from two more larval lepidopterans, B. mori and A. bipunctata, were very
similar to those observed for the larval loreyi leafworm.
No immunopositive Mas AT materials were found in the adult brain neurons of A. mellifera honey bees,
Camponotus sp. carpenter ants, L. decemlineata Colorado potato beetles, and M. tsudai walking
sticks. Also, no immunopositive Mas AT neurons were
found in the female adult brains of sugar- and liver- fed P. regina if the brains were probed with antisera
that had been pretreated with synthetic Mas AT. This indicated that at the three doses 20, 40, and 80
µ g
per pretreatment used here, synthetic Mas AT could effectively abolish the recognition of Mas AT or
Mas AT-like substance in brain neurons. This result also attested to the specificity of our antisera to
Mas AT.
239 M.-P. Tu et al. Journal of Insect Physiology 47 2001 233–244
Fig. 2. Relative affinities of Mas AT, Lom MT, and deduced Aee
AT-like peptide toward our anti-Mas AT antiserum. Results of a com- petitive enzyme-linked immunosorbent assay cELISA for Mas AT
showed that the affinity of Lom MT was 20-fold less than that of Mas AT towards the antiserum. Very little inhibition was detected for the
Aee AT-like peptide up to 2000 ng level suggesting very low affinity to the antiserum.
3.4. Stimulation of JH biosynthesis by the CA of P. regina with synthetic Mas AT
Three replicates were conducted to examine the effect of three doses of Mas AT on the in vitro JH biosynthesis
of the CA of P. regina. In each replication, 3–5 incu- bations were conducted. Results Fig. 4 showed that the
synthetic Mas AT stimulated JH biosynthesis by the CA from sugar-fed females in a dose-dependent manner at
2, 20, and 200 pmol of Mas AT per 25
µ l of TC199 or
80, 800, and 8000 nM. Stimulated CAs produced 1.96, 2.64, and 2.00 times more JHs than the respective con-
trols.
4. Discussion
