Enhanced haemolymph circulation by insect ventral nerve cord: hormonal control by Pseudaletia unipuncta allatotropin and serotonin
1 Department of Zoology, University of Toronto, Toronto, Ontario, M5S 3G5
Canada
2 Department of Biology, Laval University, Sainte-Foy, Quebec, G1K 7P4
Canada
* Author for correspondence (e-mail: stephen.tobe{at}utoronto.ca)
Accepted 17 July 2002
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Summary |
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Key words: allatotropin, haemolymph circulation, Manduca sexta, Pseudaletia unipuncta, serotonin, ventral diaphragm, ventral nerve cord
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Introduction |
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In most Lepidoptera, the VD has developed into a unique structure,
originally named the Cord of Leydig after the researcher who made the original
observations (Leydig, 1862; cited in
Brocher, 1920). Instead of a
continuous sheet lying dorsally to, yet separated from, the ventral nerve cord
(VNC), the lepidopteram VD consists of a discontinuous arrangement of fine
aliform muscle fibres originating ventrolaterally on the abdominal sternites,
and inserting onto a connective tissue matrix on the abdominal midline
(Richards, 1963
;
Ashhurst and Richards, 1964a
;
Hessel, 1969
;
Chapman, 1971
;
Kristensen and Nielsen, 1980
).
This tissue matrix, comprising primarily collagen (Ashhurst and Richards,
1964a
,
b
;
Ashhurst, 1968
), is fused to
the top of and/or partially surrounds the VNC. Consequently in Lepidoptera,
contractions of the VD musculature can cause lateral oscillations of the VNC
itself, and not just the movement of the VD as seen in other insects. In the
moths Bombyx mori and Arctias luna, the VNC was reported to
oscillate at frequencies of approximately 80 and 120 cycles per minute
(Gerould, 1938
;
Richards, 1963
). In addition,
data indicate that these contractions were able to specifically move
haemolymph from the anterior to the posterior portion of the abdomen
(Brocher, 1920
;
Hessel, 1969
), suggesting that
the function of the collagen matrix (and the associated musculature) was more
than just providing a protective sheath for the VNC. Furthermore, Brocher
(1920
) noted that the frequency
of these lateral movements changed, being low when the insect was at rest and
high when the insect was `excited'. Since these initial observations, however,
we are not aware of any research that has been undertaken to elucidate any
physiological mechanisms underlying these changes in frequency in the
Lepidoptera.
The true armyworm Pseudaletia unipuncta has been used as a model
system to study the role of juvenile hormone (JH) in the pheromone-mediated
reproductive biology of moths undertaking seasonal migrations in response to
habitat deterioration (Delisle et al., 1987;
McNeil, 1987; Cusson et al.,
1990
,
1993
,
1994a
,
b
; McNeil et al.,
1994
,
1995
,
1996
,
2000
). We have cloned the gene
for the neuropeptide, Manduca sexta allatotropin (Manse-AT), in
P. unipuncta (Truesdell et al.,
2000
); the gene was originally identified and characterized in the
moth M. sexta based on its ability to stimulate in vitro JH
biosynthesis (Kataoka et al.,
1989
). Both the expression of this gene, and Manse-AT-like
immunoreactivity, were shown to be distributed throughout the brain and
abdominal ganglia of the armyworm
(Truesdell et al., 2000
). The
pleiotropic nature of this peptide has been reported for several moth species
and non-lepidopterans; it is not only capable of stimulating in vitro
JH biosynthesis (Audsley et al.,
1999
,
2000
;
Oeh et al., 2000
;
Rachinsky et al., 2000
;
Tu et al., 2001
), but also
acts as a cardioacceleratory agent
(Veenstra et al., 1994
;
Rudwall et al., 2000
) as well
as inhibiting midgut ion transport (Lee et
al., 1998
). A general model, including the possible pleiotropic
effects of Manse-AT on the reproduction and migration of P.
unipuncta, was proposed (McNeil and
Tobe, 2001
) and subsequent work demonstrated that Manse-AT affects
both JH biosynthesis and cardioacceleration in true armyworm adults
(Koladich et al., 2002
).
Furthermore, it was shown that serotonin (5-hydroxytryptamine, 5-HT) is a
potent stimulator of heart rate in adult P. unipuncta
(Koladich et al., 2002
), as
seen in M. sexta (Prier et al.,
1994
) and several other invertebrates
(Chiang et al., 1992
;
Fox and Lloyd, 1997
;
Saver et al., 1999
;
Zornik et al., 1999
). Although
5-HT affected the intensity and duration of the cardioacceleration
significantly more than Manse-AT, a clear synergistic interaction was seen
when the dorsal vessel was stimulated simultaneously with both compounds
(Koladich et al., 2002
).
The true armyworm P. unipuncta is a powerful flying insect, and has an efficient circulatory system that is essential for long-distance migrations in search of suitable habitats. In the present paper, we extend our investigation of the roles of both Manse-AT and 5-HT in haemolymph circulation in P. unipuncta by analyzing their effects on the VD. We suggest that this structure plays a more active role in the circulatory system than previously thought, and is essential to the animal in terms of carbohydrate and lipid mobilization, fight energetics and thermoregulation.
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Materials and methods |
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Reagents
All chemicals were supplied by Sigma (St Louis, MO, USA), except for goat
anti-mouse secondary antisera (Jackson ImmunoResearch Laboratories Inc., West
Grove, PA, USA); Manse-AT was custom-synthesized and purified by high
performance liquid chromatography (to >90% purity) by Research Genetics
(Huntsville, AL, USA).
Anatomy and ventral nerve cord function
To obtain still images of the VNC and VD structures, entire male and female
abdomens were fixed with 2.5% glutaraldehyde in 0.1 mol l-1
Sorenson's phosphate buffer, pH 7.2, for 12-14 days. Approximately 500µm of
the extreme anterior and posterior ends of the abdomen (primarily cuticle)
were removed prior to fixation to facilitate penetration of the
glutaraldehyde. For cross-sectional images, multiple transverse sections were
made with a scalpel, and the sections stained for 30s with 0.1% Methylene Blue
and 7.5 mmol l-1 ascorbic acid in ddH2O to enhance
contrast. Alternatively, intact VNC and associated VD musculature were
carefully dissected out of fixed abdomens and stained for 30s with 0.1%
Methylene Blue and 7.5 mmol l-1 ascorbic acid in ddH2O
followed by staining for 30s with 0.1% Neutral Red in ddH2O. After
staining, both transverse sections and intact VNC/VD tissues were thoroughly
rinsed in phosphate-buffered saline (PBS; 10 mmol l-1 phosphate
buffer in 0.9% NaCl, pH 7.2). All still images were immediately captured on an
Olympus C4040 Zoom 4.1 Megapixel digital camera mounted on an Olympus BX60
System light microscope.
To acquire images of the oscillating VNC, live animals were pinned dorsal
side up with their head, legs, wings and removed. The dorsal cuticle was
removed along with the dorsal vessel. The fat body, gut and reproductive
organs were carefully dissected away under a modified Weever's insect saline
(7.0 mmol l-1 NaCl, 34.0 mmol l-1 KCl, 16.0 mmol
l-1 MgCl2, 4.0 mmol l-1 CaCl2,
184.0 mmol l-1 glucose, pH 6.8)
(Lehman et al., 1993), thus
exposing the oscillating VNC. Tissues were then flooded with 0.1% Methylene
Blue and 7.5 mmol l-1 ascorbic acid in ddH2O for 20s,
followed by thorough rinsing in modified Weever's saline. The movement of the
VNC was then recorded in the form of digital video using a Panasonic GP-KR412
charge-coupled device mounted on a Zeiss dissecting light microscope. Still
images were then captured on a computer from sequential frames of video.
To acquire images of the circulatory abilities of the oscillating VNC, live animals were dissected as described above. However, the fat body, gut and reproductive organs were left intact. Dye was then introduced with a fine syringe into the intact thorax, or into the exposed fat body of either the anterior or posterior abdomen. Sequential images of the resultant movement of dye were generated as described above.
Whole-mount immunocytochemistry
All steps were performed at room temperature unless otherwise stated.
Animals were anaesthetized by chilling on ice. Brains and VNC were dissected
under ice-cold modified Weever's saline, and placed immediately in ice-cold
fixative (2% paraformaldehyde in Millonig's buffer). Tissues were fixed
overnight at 4°C and rinsed thoroughly in PBS. Tissues were then incubated
for 2 h in PBS containing 4% Triton X-100, either 10% normal goat serum (NGS;
for Manse-AT assays) or 10% normal sheep serum (NSS; for 5-HT assays) and 2%
protease-free bovine serum albumin (BSA), followed by 30 min in PBS containing
3% skimmed milk powder. The 4% Triton X-100/10% NGS/NSS, and skimmed milk
incubations, were performed to increase perineurium permeability, enhance
antibody penetration and reduce non-specific binding of antibodies. Primary
antibodies were also pre-incubated for 24 h at 4°C in PBS containing 0.4%
Triton X-100, 2% BSA and 2% NGS (Manse-AT) or 2% NSS (5-HT) to occupy
non-specific antigenic sites.
To identify Manse-AT-like immunoreactivity, tissues were incubated in a murine monoclonal antibody diluted 1:800 in PBS containing 0.4% Triton X-100, 2% NGS and 2% BSA for approximately 5 days at 4°C. To identify 5-HT-like immunoreactivity, tissues were incubated in a rabbit polyclonal antibody diluted 1:800 in PBS containing 0.4% Triton X-100, 2% NSS and 2% BSA for approximately 2 days at 4°C before being washed overnight in PBS containing 0.2% Triton X-100 at 4°C. Manse-AT-like immunoreactivity was visualized by incubating the tissues for 18 h at 4°C in fluoroscein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antiserum diluted 1:200 in PBS containing 0.4% Triton X-100, 10% NGS and 2% BSA. 5-HT-like immunoreactivity was visualized in the same manner using Cy-3-conjugated sheep anti-rabbit secondary antiserum in 10% NSS. All tissues were then rinsed overnight at 4°C in PBS containing 0.2% Triton X-100, cleared and mounted on slides in 5% n-propyl gallate in 80% glycerol (pH 7.3). Images were captured using a Zeiss LSM 510 laser scanning confocal microscope employing an argon laser/FITC filter set (Manse-AT) and heliumneon laser/Cy-3 filter set (5-HT).
Liquid-phase pre-adsorption of the Manse-AT primary antiserum with 10 µmoll-1 synthetic Manse-AT, or the 5-HT primary antiserum with 10 µmoll-1 5-HT, abolished signals in all immunopositive tissues. Furthermore, no immunoreactivity was detected in either assay when either the primary or the secondary antibody incubation was omitted from the procedure.
Ventral nerve cord assays
Live animals were pinned dorsal side up into a Sylgard dish with their
head, legs and wings removed. Dorsal midline incisions in the abdomen were
made, and the heart, gut and reproductive organs were removed. The VNC was
exposed and kept flooded with modified Weever's saline. The electrodes from an
impedance converter (UFI Instruments model 2991; Morro Bay, CA, USA) were
positioned approximately 3 mm apart with the VNC centered between them. The
resultant signal was captured on a chart-recorder (Linear Instruments Corp.
model 1200; Reno, NV, USA). All electrophysiology equipment was appropriately
grounded and shielded in a Faraday cage. These semi-isolated VNC preparations
were bathed in 0.5 ml of modified Weever's saline for a minimum of 20 min
(with changes every 5 min), and only those preparations that maintained a
stable rate and did not respond to the changes in saline, were used. Different
concentrations of synthetic Manse-AT, 5-HT or both chemicals, dissolved in
modified Weever's saline, were then manually applied with 3x0.5 ml
washes. Changes in the rate of VNC oscillation were quantified by comparing
the average rate (beats min-1) for the 2 min immediately prior to
application of a compound with the rate during the 1 min immediately after
application for doseresponse assays, and for every subsequent minute
for an overall period of 20 min in the case of time-course experiments.
Because of the nature of the signal detected by the impedance converter, only
changes in frequency, not amplitude, could be quantified accurately. A minimum
of five animals was used per data point.
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Results |
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The abdomens of adult virgin P. unipuncta were densely packed with fat body. However, surrounding the VNC, there was a perineural sinus approximately 300-500 µm wide, essentially forming a hollow tube that runs longitudinally along the entire abdomen and inside which the VNC oscillates (Fig. 1B). Although the VNC is only approximately 80 µm wide, it was occasionally observed to oscillate with an amplitude of approximately 200 µm from the ventral midline (Fig. 2).
|
When dye was introduced into either the thorax or anterior abdomen of live animals from which the dorsal cuticle had been removed, it reappeared through the fat body in the region dorsal to the terminal abdominal ganglion (TAG; Fig. 3) within approximately 0.5 s, which was a shorter time period than would be possible by diffusion alone. No dye was ever observed moving laterally through the fat body or elsewhere along the length of the VNC. In addition, following the introduction of dye to the posterior end of the abdomen, it remained at the site of introduction and was not detected in either the anterior abdomen or thorax.
|
Manduca sexta allatotropin-like immunocytochemistry
Immunocytochemistry of the VNC of day-3 adult male and female P.
unipuncta indicated the presence of three pairs of soma arranged medially
within the abdominal ganglia, and axons within the VNC connectives that
demonstrated Manse-AT-like immunoreactivity. Furthermore, there was an
extensive network of Manse-AT-like immunoreactive fibres running the entire
length of the abdominal nerve cord, but not in the TAG
(Fig. 4A). These immunoreactive
axons exit the abdominal ganglia laterally and immediately branch dorsally,
sending both contra- and ipsilateral projections anterior and posterior along
the dorsal surface of the tissue matrix fused to the VNC. These axons appear
to possess extensive sites of release. No differences were noted between the
sexes.
|
Serotonin-like immunocytochemistry
2-3 ventrolateral pairs of 5-HT-like immunoreactive cells were located
posterior in each of the abdominal ganglia, which contributed to the
immunoreactivity detected in axons running within the VNC connectives, as well
as those exiting laterally from the VNC. An extensive network of 5-HT-like
immunoreactive axons was detected along the entire length of the tissue matrix
associated with the VNC, but was absent from the TAG, similar to the pattern
seen with Manse-AT-like immunoreactivity. These axons, which also appeared to
possess numerous release sites, were also situated dorsally and frequently ran
both within and along the surface of the matrix
(Fig. 4B). Positive 5-HT-like
soma were also detected in several sites in the brain, corpora cardiaca and
TAG of day-3 adult males and females (data not shown). No differences were
noted between the sexes.
Stimulatory effects of allatotropin
The VNC of both sexes of P. unipuncta oscillate laterally at rate
of approximately 135 cycles min-1 when resting. Treatment of the
VNC of day-3 adult male and female armyworms with Manse-AT at
10-9-10-5 mol 1-1 resulted in positive
dose-dependent responses in the rate of VNC oscillations in both sexes
(Fig. 5). The EC50
values in males and females were comparable at 1.2x10-7 mol
1-1 and 2.2x10-7 mol 1-1, respectively.
Concentrations at or below 10-9 mol 1-1 had essentially
no significant stimulatory effect, whereas both sexes showed significant
increases from the resting rate at 10-6 mol 1-1 and
above (t-test; P<0.05). Maximum levels of stimulation
(approximately 25% above resting) reached a plateau at approximately
10-6 mol 1-1.
|
Stimulatory effects of serotonin
Serotonin also proved to be a potent stimulator of the rate of VNC
oscillations in P. unipuncta, with day-3 males and females showing
similar dose-responses at concentrations from 10-10 mol
1-1 to 10-5 mol 1-1
(Fig. 6). However,
EC50 values for 5-HT at 6.8x10-9 mol
1-1 and 2.0x10-9 mol 1-1 for males and
females, respectively, were several orders of magnitude lower than the
EC50 values seen for Manse-AT. Accordingly, significant increases
over resting rates are seen at 10-8 mol 1-1
(t-test; P<0.05). In addition, 5-HT treatment resulted in
maximum rates of oscillation >55% above the resting rate, which was more
than twofold higher than the increase of approximately 25% seen with
Manse-AT.
|
Allatotropin and serotonin time courses
Manse-AT and 5-HT, applied at concentrations close to their EC50
values (1x10-6 mol 1-1 for Manse-AT;
1x10-8 mol 1-1 for 5-HT), both evoked a rapid
acceleration of the rate of VNC oscillations in day-3 adult females, but
resulted in dramatically different temporal patterns
(Fig. 7). Manse-AT treatment
alone resulted in a significant increase in rate of oscillations within the
first minute of application (t-test, P<0.05) but rates
gradually returned to pre-exposure levels within 5 min. Subsequent
applications of 10-6 mol 1-1 Manse-AT at 2, 4 and 6 min
after initial stimulation (t=0) resulted in a sustained significant
level of stimulation for 7 min (t-test, P<0.05) but rates
nonetheless returned to approximately pre-exposure levels in less than 3 min
following the last treatment (Fig.
8).
|
|
Treatment with 5-HT alone also resulted in a dramatic, significant increase within the first minute. However, in contrast to the results from exposure to Manse-AT, rates remained significantly elevated above resting levels for the duration of the experiment (Fig. 7; t-test, P<0.05). Furthermore, 5-HT stimulation consistently resulted in levels of stimulation that were significantly higher than the Manse-AT treatment over the entire time course of the experiment (t-test; P<0.05).
Simultaneous treatment of day-3 adult females with both Manse-AT and 5-HT at approximately their EC50 values displayed a temporal pattern similar to, but slightly reduced from treatment with 5-HT alone, and did not show any additive or synergistic effects (Fig. 7). However, whereas the slope of the line for 5-HT-treated VD from t=2 to 20 min showed a gradual decline that was significantly different from zero (slope=-0.3926; F=49.81, P<0.001), the slope of the line for Manse-AT+5-HT treated VD maintained a constant, accelerated rate that was not different from zero (slope=-0.0541; F=0.2824, P=0.6179), indicating that there was no decline over the period examined. In addition, the slopes of these two lines are significantly different from each other (ANCOVA; F=8.510, P=0.015).
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Discussion |
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These findings certainly suggest that, at least in P. unipuncta,
the VD may play a much more active role in the circulation of haemolymph than
previously thought. There are several ways in which this would be of
particular importance for species such as the true armyworm, which has very
large fat reserves and uses powered flight during long-distance migration.
Orchard et al. (1991) have
shown that in the true armyworm, haemolymph lipid levels, under the control of
adipokinetic hormone, increased >150% above resting after 1 h of sustained
flight. Thus, there would be considerable physiological benefit to an
effective mobilization of energy sources from the abdomen to the flight
muscles. Similarly, the efficient removal of metabolic waste from the thorax
would ensure that their accumulation did not adversely affect muscle function.
These two processes would be facilitated by the complimentary action of the
dorsal vessel and the VD.
Another very important function of active haemolymph movement from the
thorax to the abdomen is thermoregulation, as shown in a number of insects
including dragonflies, bees and Lepidoptera (Heinrich,
1970a,
1974
,
1976
;
Heinrich and Casey, 1978
).
Sustained flight generates considerable heat, and without an effective means
of dissipating heat from the thorax, temperatures would soon rise to critical
levels. Heinrich (1971
) has
clearly shown that, as thoracic temperature rises in the tobacco hornworm
M. sexta, there is an increase in abdominal temperatures. In
addition, it occurs sooner and reaches higher levels beneath the VD than in
the dorsal section of the abdomen. Furthermore, heart rate increases with
increasing thoracic temperatures and is regulated by neural mechanisms
(Heinrich, 1970b
). Thus, it is
possible that a similar neural feedback exists with respect to the VD.
The presence of extensive immunoreactivity and numerous release sites for both Manse-AT and 5-HT along the length of the VNC suggests that secretion of either or both of these neurohormones into the haemolymph would occur quickly and efficiently. This would not only expedite the rapid movement of cardioactive compounds to the dorsal vessel, but the corelease of other neurohormones with either Manse-AT or 5-HT would ensure very rapid and efficient circulation to other parts of the body an elegant solution for a moth with an abdomen densely packed with fat body.
Interestingly, the effects of Manse-AT on both the VD and dorsal vessel are
very short-lived, even with repeated exposure
(Koladich et al., 2002). In
our time-course experiments, the VD only showed a significant response to
Manse-AT with the initial exposure, and reapplication of peptide resulted in
only minor increases in rates of VD oscillation
(Fig. 8). This suggests rapid
desensitization of Manse-AT receptors, although it does not rule out the
possibility that Manse-AT is rapidly degraded by peptidases released in
response to the initial application of peptide. This may nevertheless have
some bearing on the pleiotropic nature of the Manse-AT. Manduca
sexta-AT is capable of stimulating JH biosynthesis in the corpora allata
of adult P. unipuncta (Koladich
et al., 2002
), so it may be desirable to have only small
quantities of this peptide released from the VNC in vivo to `prime'
the VD and/or dorsal vessel for exposure to 5-HT during migratory flight by
sexually immature individuals. If larger amounts of Manse-AT were required for
maintaining stimulated levels of the dorsal vessel and/or the VD, then
untimely effects on JH biosynthesis might be result. This is significant, for
while low levels of JH are required during migration, higher levels would
induce premature sexual maturation and reproductive behaviour in adult male
and female P. unipuncta (Rankin
and Riddiford, 1978
; Delisle et al., 1987;
McNeil 1987
; Cusson et al.,
1990
,
1994a
,b
;
McNeil et al., 1994
,
1996
,
2000
).
In conclusion, our data suggest that the VD plays an important role in fluid circulation of adult P. unipuncta. Both Manse-AT and 5-HT are critical hormonal elements that augment circulation by modulating the VD as well as the dorsal vessel, allowing enhanced transport of haemolymph, lipids, and metabolic and thermal wastes, very necessary for this long-distance migrant.
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Acknowledgments |
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