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Department of Developmental Neurobiology, Faculty of Biology, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands;
* Queen's University, Belfast, School of Biology and Biochemistry, Medical Biology Centre, N. Ireland; and
Department of Dermatology, Amsterdam Leiden Institute for Immunology (ALIFI) Academisch Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands
1Correspondence: Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands. E-mail: mdejong{at}bio.vu.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: host interaction • LyNPY • reproduction • food intake • glycogen storage
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies using the well-validated model system, the schistosome
Trichobilharzia ocellata and its intermediate snail host
Lymnaea stagnalis, have demonstrated that what occurs is not
a competition for nutrients, with the parasite as the winner, but
rather that the parasite selectively interferes with neuroendocrine
mechanisms regulating the main determinants of the energy budget in the
host's reproduction and growth (1)
. Recently,
differential cDNA screening was used to identify parasite-induced
alterations in gene expression within the host's central nervous
system (CNS). A significant up- or down-regulation of several
neuropeptide-encoding genes was observed. These changes were closely
related to the developmental stage of the parasite (2)
.
One of the genes that were up-regulated appeared to encode a
Lymnaea neuropeptide Y homologue (LyNPY). This up-regulation
of the LyNPY gene coincided with the energy requiring developmental
stage of the parasite, i.e., the onset of a continuous and high
production and release of parasites (cercariae), known as the
`shedding stage'. In this stage, characteristic physiological effects
also occur in the host: inhibition of reproduction and an increase in
growth (3
, 4)
. As NPY is known to play a central role in
regulation of energy budgeting with food intake, reproduction, and
growth as the main determinants in vertebrates (e.g., refs 5
, 6
), we investigated whether a similar physiological role can be
ascribed to this neuropeptide in an invertebrate host. This might
explain why the host's NPY gene is an important target for parasitic
action.
Here we describe the cloning of a novel cDNA encoding the
Lymnaea NPY precursor protein and the sites of gene
expression in the CNS. The functional significance of up-regulation of
this gene in parasitized snails was studied in the physiological
experiments we performed with synthetic LyNPY. As these animals do not
have a blood–brain barrier, molecules administered to the open blood
system have easy access to neurons and even to synapses in the brain
(7)
. Synthetic LyNPY was administered to nonparasitized
snails by implanting a slow-release pellet (long-term effect) or by a
single bolus injection (short-time effect). It caused a profound
inhibition of egg mass production and brought about suppression of
growth. LyNPY had no effect on food consumption, which is a continuous
activity in this animal unless it is inhibited under certain
environmental conditions or during other activities such as egg laying
and copulation (8)
. When the LyNPY titer had returned to
normal and reproduction and growth were resumed, a significant increase
in food consumption was observed. In this short hyperphagic period,
glycogen stores were replenished.
These physiological data showing that LyNPY plays an important role in
the central regulation of metabolic and endocrine processes are
supported by morphological observations on whole mount CNS
preparations. An association was found between LyNPY-positive axons,
which constitute a broad bundle running through all ganglia and their
connectives, and the neuroendocrine centers thought to be involved in
the regulation of reproduction, ovulation and egg laying, and growth
(9
10
11)
. The figures do not show synaptic(-like) contacts
between LyNPY axons and these two neuroendocrine systems.
LyNPY-positive axons also pass the buccal ganglia, where the
motoneurons innervating the feeding apparatus (buccal mass) are
localized, but do not approach these motoneurons.
In sum, elevation of the titer of LyNPY inhibits reproductive activity and reduces growth in a dose- and time-dependent manner without affecting food consumption. This study helps to explain why endoparasites cause up-regulation of the NPY encoding gene: it benefits their own energy budget.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Molecular aspects of LyNPY and its precursor
Cloning mRNA-encoding precursor LyNPY: isolation of a cDNA clone
encoding preproLyNPY
Standard molecular procedures were performed as described by
Sambrook et al. (13)
. Restriction enzymes were purchased
from Boehringer Mannheim (Almere, The Netherlands).
Total RNA isolated from Lymnaea CNS was converted into first
strand cDNA using oligo(dT) and reverse transcriptase as described
previously (14)
. Two degenerate primers were synthesized
based on the amino acid sequence of LyNPY—sense [S1], residues
38–43 in Fig. 1
: 5'-CCNAA(T/C)GA(G/A)(C/T)TN(A/C)GNAA(A/G)TA-3'; antisense [AS1],
residues 55–60: 5'-AANC(T/G)NGGNC(T/G)NCCNAC-3'
(15)
—and used in a polymerase chain reaction
(PCR): 94°C for 20 s, 50°C for 20 s, and 72°C for 1 min
for 32 cycles. PCR products were directly cloned in the pGEM-T vector
(Promega, Madison, Wis.) and sequenced. Two specific oligonucleotides
based on one of the obtained sequences apparently to encode
LyNPY—sense [S2]: 5'-CAAGGCTTTGAATGAGTACTACGC-3'; antisense [AS2]:
5'-AATGGCGTAGTACTCATTCAAAGC-3')—were synthesized and used in a PCR
with cDNA isolated from a ZAP II cDNA library of the Lymnaea
CNS as template. cDNA was amplified using either primer in combination
with a reverse primer (RP) complementary to a pBluescript sequence
(RP/S1 or RP/AS1) for 40 cycles: 94 0°C for 20 s, 58°C for
20 s, and 72°C for 1 min. The longest PCR products from both
PCRs were cloned in pGEM-T and sequenced. Two LyNPY-specific primers
encompassing the coding region could be developed on these sequences:
sense, S3: 5'-TTTTATCTGACTGTTTCCGACCTG-3'; and antisense [AS3]
5'-ATCGCATGGCTGCGTGGTCAGTGG-3'. These primers were used in three
independent PCR reactions in combination with cDNA from
Lymnaea CNS as template and Pfu polymerase (Stratagene,
Cambridge, U.K.) containing proofreading activity (94°C for 20 s, 65°C for 20 s, and 72°C for 1 min for 32 cycles). A single
PCR product of ~400 bp was obtained, cloned into EcoRV
digested pZErO (Invitrogen, San Diego, Calif.), and four independent
clones from each PCR reaction were sequenced. Automated sequencing was
performed with an ABI 373 automated DNA sequencer (Applied Biosystems,
Foster City, Calif.).
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In situ hybridization (ISH)
In situ hybridization using oligonucleotide primers
was performed as described by De Lange et al. (16)
.
LyNPY pellet formulation and pharmacodynamics
Pellet formulations were constructed of neuropeptide
solution:cholesterol (75 µl, at an appropriate concentration: 56 mg)
and the control pellet was composed of Ringer's buffer:cholesterol (75
µl:56 mg). Pellets were dried (35°C, 30 min) and the resulting
powder was mixed with melted cocoa butter (18.5 mg). Implantation
pellets (2.5 mm x 1 mm) were prepared using a stainless steel
tube and rod and left to solidify at 4°C (unpublished results).
Pellets of three concentrations (0, 300, and 3000 µM LyNPY) were placed in 1 ml Ringer's buffer for 1, 3, 5, and 7 days and shaken continuously using a Thermolyne (AROS160) Adjustable Reciprocating Orbital Shaker, at speed 022.
The pellet was discarded and the remaining solution was applied to RP-high-performance liquid chromatography (RP-HPLC) using a Narrow Bore C18 column. The detector of the HPLC was set at 214 m. The solvent system consisted of 7.5 mM TFA (solvent A) and solvent A/70% acetonitrile (solvent B). After washing, a linear gradient of 0 to 100% solvent B was used over 35 min at a flow rate of 300 p1/min. LyNPY elutes at 67% B in this system. Integration values were expressed as peak height (mAU), and peptide concentrations in the fluid were calculated according to a standard curve.
Diffusion of LyNPY from the pellet is concentration dependent, the
lower concentration pellet (300 µM) reaching a maximal concentration
at day 3; in the case of the higher concentration pellet, the maximal
value occurred at day 5 (see Fig. 1
).
Application methods of LyNPY
Implantations
Snails were anesthetized by the injection of 500 µl
MgCl2 (50 mM) and the cholesterol pellet was
implanted into the head sinus of the animal from a small incision on
head skin. Animals had recovered within 4 h after surgery.
Injections
Snails were injected in the foot region with 30 µl 15.3
µg/ml LyNPY dissolved in Ringer's buffer (17)
containing NaCI (25 mM), sodium methylsulfate (6 mM), KCl (1.7 mM),
MgCl2·6 H2O (1.5 mM),
CaCl2·2H20 (4 mM),
Na2HPO4 (0.5 mM), and
NaHCO3 (25 mM).
Measurement of physiological parameters
Reproduction
Egg masses were collected daily and their occurrence was scored.
The number of eggs per egg mass was counted, yielding total egg
production.
Growth
Using permanent colored paint, three dots were made near the
edge of the shell at the widest point of the last whorl
(18)
. The average distance from the dots to the edge of
the shell was measured using a micrometer placed in a binocular (2 x 20), producing a scale with 50 µM increments. Growth was measured
daily at the same time.
Food intake
Each snail was fed daily with two circular discs of
lettuce with a total surface area of 40 cm2, an
amount exceeding the actual consumption level. The lettuce was a broad
leaf variety; only the flat parts of the outer green leaves were used.
Remaining lettuce was collected on each day and the surface area was
calculated using an Area Meter (model 3100). The difference between the
surface area supplied and the remaining surface area was used as a
measure of food consumption. The jars were provided with a floor of
pins to prevent coprophagy (19)
.
Weight determinations
Wet and dry weights were recorded after implantation and
injection of LyNPY. After the shell was removed, the wet weight of soft
body parts (total body, mantle tissue, or albumen gland, one of the
female accessory sex glands) was determined and then the dry weight
after overnight freeze drying in preweighed vials. Values were
expressed as dry weight density, a measure for the amount of stored
energy (20)
.
Enzymatic glycogen determinations
Glycogen was determined (at the same intervals as body weight)
according to Joosse and van Elk (3)
. Briefly, the
freeze-dried mantle regions were digested in sodium hydroxide (2 M) for
~1 h and neutralized with hydrochloric acid (1 M). From portions of
these digests and of standard glycogen preparations, glycogen was
hydrolyzed to glucose by incubation (30°C; 30 min) with 2 U of
-amyloglucosidase (EC 3.2.1.3; Boehringer) in sodium acetate buffer
(0.2 M, pH 4.5), final volume 200 µl. Glucose was determined
enzymatically, as shown below. Tissue free glucose was negligible.
Values were expressed as mg glycogen/mg mantle dry weight.
Enzymatic glucose determinations
Glucose was measured on a microscale using the glucose
dehydrogenase method. The reaction mixture (final volume, 250 µl)
containing potassium phosphate buffer (0.25 M; pH 7.6), NAD (1 mM) and
sodium chloride (15 mM) was put into a disposable semi-microcuvette.
The initial extinction (E0) was measured at 340
nm using a Vitatron Reaction Rate Spectrophotometer fitted with a
special microcuvette adaptor. Glucose dehydrogenase (24.4 mU, EC
1.1.1.47; Merck, Rahway, N.J.) in ammonium sulfate (3 M) was added.
After 10 min at room temperature, the extinction
(E1) was recorded. The glucose concentration was
calculated from E1-E0.
Effect of `reproductive status' on LyNPY-induced physiological
changes
Animals were divided into two groups according to their
reproductive or ovipository status (those that produced an egg mass on
the day of the experiment and those that had produced an egg mass the
day before the experiment). Egg mass production was assessed before
noon. Injections were carried out using the previous concentration of
LyNPY (30 µ1; 15.3 µg/ml) dissolved in snail Ringer. Effects of
injected LyNPY on growth, reproduction, and food intake of the two
groups were measured as before.
Immunocytochemistry
Antisera used
The synthetic peptide was synthesized (ID-DLO, Lelystad, The
Netherlands) and a polyclonal antiserum was raised in mice against the
entire peptide, as described (16)
. The polyclonal antisera
raised against synthetic LyNPY, caudo-dorsal cell hormone (CDCH; ref 21
) and molluscan insulin-like peptides (MIPc; ref 22
), whose
specificity has been described previously, were used on whole mount
preparations of the CNS.
Whole mount preparations
The CNS were dissected and treated with 0.5% protease
(type 14, Sigma, St. Louis, Mo.) in Aquadest for 20–30 min and
subsequently fixed in 1% paraformaldehyde (PF; 1 h, room
temperature), 2% PF (1 h, room temperature), and 4% PF (overnight at
4°C). After being rinsed in 2% Triton X 100 in Tris-NaCl (six times
for 10 min), the tissue was incubated with the first primary antiserum
(anti-CDCH), which was diluted 1:100 with Supermix (TBS-gelatin:
6.06 g Tris, 8.77 g NaCl, 2.50 g gelatin, and 5 ml
Triton X 100 per liter; pH 7.4) for 3 x 24 h at 4°C,
rinsed in Tris-NaCl buffer (6 x 10 min), and incubated (overnight
at 4°C in the dark) in the second antiserum (SwaR/FITC, Dako) diluted
(1:50) in Supermix. From then on, all handling was performed in the
dark. After rinsing in the Tris buffer (six times for 10 min), the
tissue was transferred to the second primary antiserum (anti-LyNPY
1:125 diluted with Supermix) and incubated for 3 x 24 h at
4°C. After rinsing in Tris buffer (6 x 10 min), the tissue was
incubated (overnight at 4°C) in the second antiserum (Go-a-M/Cy5;
Amersham Life Science, Arlington Heights, Ill.) 500x diluted in
Supermix. The tissue was rinsed again (six times for 10 min), embedded
in a few drops of a glycerol mixture (3 ml glycerol and 1 ml
phosphate-buffered saline with 1% ethylene-diamine (anti-bleach)
between two coverslips/glasses, and stored in the dark at 4°C.
The immunostained whole mount preparations were studied with a confocal laser scanning microscope (Zeiss LSM 410). The 488 nm line of the Ar laser was applied to excite the FITC fluorophore, using a dichromic beam splitter FT 510 and an emission band-pass filter BP 515–565. The 633 nm laser line (He-Ne laser) was applied to excite the Cy5 fluorophore, using a dichroic beam splitter FT 655 and an emission long-pass filter RG665.
Statistics
To analyze the physiological data, repeated-measures
analysis of variance was used with time points (days) as repeats. This
was followed by post hoc Bonferroni protected tests between
experimental groups at the specific time points. Where applicable,
means and standard deviations are given. Probability values are
Bonferroni corrected.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This precursor protein has an organization similar to other NPY
precursors; this similarity includes the presence of a 21 aa long
signal peptide, followed by LyNPY and a carboxyl-terminal peptide. The
signal peptide is cleaved off at residue Cys 21 according to the von
Heijne algorithm for signal peptide cleavages. The predicted sequence
for the mature LyNPY is fully consistent with our protein purification
and sequencing data (15)
and continues after the Arg at
position 38, with a Phe residue (followed by the sequence Gly-Lys-Arg)
being the common signal for processing and subsequent amidation
(23)
. Finally, the precursor contains a 26 aa long peptide
at the carboxyl terminus.
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A peptide was synthesized on the basis of this LyNPY sequence and our
purification and sequencing data for native LyNPY (15)
and
used for physiological assays and antibody production. For in
situ hybridization, we used the A3 oligonucleotide primer and
conditions as described by De Lange et al. (16)
.
Effect of LyNPY on reproduction, growth, and food
consumption
Implanted LyNPY pellet
To study long-term effects of LyNPY, cocoa pellets
containing different amounts of LyNPY were implanted into mature
snails. Egg laying activity was almost completely suppressed for up to
7 days (Fig. 3
A). The suppression during the first 5 days was dose
dependent with a Kd of ~65 µg per gram of body
weight in the pellet (Fig. 4
A). The number of eggs per egg mass was unchanged for the
duration of the experiment (21 days; data not shown). After initial
suppression in all groups, egg laying was significantly suppressed from
day 2 until day 7 (P<0.01; Fig. 3A
). The onset
of the effect occurred sooner with higher doses, but the return to
control level occurred at ~8 days regardless of dose (Fig. 3B
). During the 21 days of the experiment, the LyNPY-treated
animals did not compensate for the reduced reproductive output either
in terms of number of eggs or number of egg masses.
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In the first 5 days after implantation, the growth rate was also less
with increasing doses (Fig. 4B
). This effect was already
apparent on the first day after implantation, when growth rate (at
doses of 500 µM and higher) was lower than that of controls
(P<0.01) and persisted until about day 7 (Fig. 3C
). On the first 2 days of the experiment, growth rates
were relatively low in all groups, possibly due to handling. The
decreased growth rates resulted in smaller overall shell lengths at
higher doses of LyNPY. This holds true for lengths measured after 7
days and lasted until the end of the experiment at day 21. The wet
weight of the soft body parts showed a decrease only at the highest
concentrations of LyNPY. This did not result in significant changes in
dry weight densities (P<0.01).
There was no change in food consumption during the first 5 days of the
experiment (Fig. 4C
). However, during the period from 5 to
10 days after implantation, consumption was elevated in a
dose-dependent manner (Fig. 4D
). At the highest dose (1000
µM), increased consumption occurred on days 9 and 10
(P<0.01; Fig. 3D
). The increased consumption
occurred later at lower doses. At 100 µM, it occurred on days 11 and
12 (P<0.01; Fig. 3D
).
In summary, female reproductive effort was decreased early on, but not immediately, at intermediate doses. This suggests that some animals were ready to lay an egg mass and did so regardless of the presence of LyNPY. Growth was suppressed almost immediately. These effects were transient and lasted between 5 and 7 days. By contrast, food consumption was unchanged at first, but increased later, when the effects on growth and reproduction had worn off.
Injections with LyNPY
Effect of LyNPY on egg laying depends on reproductive status
The results of the implantation experiments described above
suggest that the onset of the effect of LyNPY on reproduction depends
on the ovipository status of the animals: when animals are due to lay
an egg mass, LyNPY has no effect, in animals that are not, LyNPY
prevents egg laying. This suggests differences in the reproductive
status of the recipients; administration of LyNPY to animals that have
recently laid eggs would suppress egg laying more than application to
animals that have gone longer without eggs. To obtain a better temporal
resolution of the effects of LyNPY than can be achieved with slow
release pellets, LyNPY was injected at a final concentration of 100
nmol/g. Since the pond snail lays an egg mass once every 2 days under
long day-length conditions, we compared animals that laid eggs during
the 24 h before injection (day of eggs) with animals that laid
eggs between 24 and 48 h before injection (day after eggs).
Clearly, egg laying was suppressed in both NPY-injected groups
(Fig. 5
A). However, in the group that laid eggs during the 24 h
prior to injection, suppression lasted for 2 days, whereas in the group
that had not laid eggs for at least 24 h, egg laying was
suppressed for only 1 day.
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Growth was significantly suppressed on day 1 in the animals that laid
eggs 24 h before the injection (0.237±0.060 vs. 0.125±0.063 µM
controls vs. NPY; P<0.01) as well as in the animals
injected more than 24 h after egg laying (0.234±0.056 vs.
0.124±0.055 µM, controls vs. NPY; P<0.01). Food
consumption had significantly increased on day 4 both in the animals
that were injected soon after egg laying and those injected the day
after egg laying (Fig. 5B
).
From these data, we conclude that 1) LyNPY suppresses egg laying longer when it is applied sooner after egg laying and 2) LyNPY elevates food consumption, but that this effect is probably indirect.
Effects of LyNPY on energy storage
Glycogen is the main energy source of Lymnaea
(24)
. Storage of glycogen takes place in specialized
cells, the vesicular connective tissue cell, which are the main
components of mantle tissue (25)
. To study the effect of
LyNPY on energy storage, we injected animals with LyNPY (100 nmol/g)
and measured dry weight density of the mantle as well as its glycogen
content. Growth, food intake, and reproductive activity were also
determined. Administration of LyNPY again caused an initial suppression
of growth and egg laying (significant at day 2), whereas food
consumption showed a significant increase at the same time. At day 2,
glycogen storage had increased significantly over control levels
(P<0.01; Fig. 5D
); the mantle showed an increase
in dry weight density (P<0.01; Fig. 5C
), which
probably reflects increased storage. By contrast, both dry weight
density and glycogen content had decreased significantly
(P<0.01) by day 5, indicating loss of stored materials. At
day 7, measures were not significantly different between controls and
LyNPY injected animals. The decreased glycogen and dry weight density
occurred at about the time food consumption was heightened (compare
Fig. 5B
).
Morphological studies
Whole mount immunocytochemistry
Combined laser scan pictures of whole mount preparations of the
ganglia of the Lymnaea CNS (see Fig. 6A
for gross anatomy CNS) stained with anti-LyNPY show that
LyNPY-positive axons form a thick bundle that runs as a circular band
through all ganglia passing the commissures connecting the ganglia.
Although LyNPY-positive cell bodies can be found in all ganglia except
for the two buccal ganglia, the majority of these neurons can be found
in the paired cerebral and pedal ganglia and in the unpaired visceral
ganglion (see Fig. 6B
).
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Double-immunostained CNS preparations revealed that projections of
LyNPY-positive neurons located in the central part of the cerebral
ganglia make contact with CDCH-positive axons from the caudo-dorsal
cells (CDCs) in a specialized area, called the `loop area' (Fig. 6C
), which is composed of CDC axons before they run into the
cerebral commissure and make contact with the CDC cluster in the contra
lateral cerebral ganglion. Axons derived from neurons in the pedal and
pleural ganglia closely pass this group of LyNPY-positive neurons in
the cerebral ganglia. Besides CDCH-positive axons, LyNPY-positive axons
(up to six) have also been found to cross the central part of the
cerebral commissure (Fig. 6C
). A (smaller) bundle of
LyNPY-positive axons running through the cerebral ganglia appeared to
pass the cerebral-buccal commissures and form a network around
CDCH-positive neurons (1 or 2) located in each of the paired buccal
ganglia (Fig. 6D
). In the unpaired visceral ganglion, a very
characteristic LyNPY-positive neuron projects its axon together with
CDCH-positive axons from neurons from other parts of the brain into the
nervus intestinalis (Fig. 6E
).
No obvious differences were found between whole mounts of the CNS from parasitized and nonparasitized snails immunostained with anti-LyNPY.
LyNPY-positive axons can also be found in the area where the
axons of one cluster of MIP-producing light green cells (LGCs; two
clusters in each cerebral ganglion) form a bundle (Fig. 6F
). They do not contact the cell bodies of these
clusters of LGCs. No LyNPY-positive axons run into the lateral lobes,
which are attached to the cerebral ganglia and are involved in growth
regulation. A giant neuron is present in these lobes that also stains
with anti-MIPc (22)
.
In situ hybridization
The ISH studies showed neurons expressing the LyNPY gene in all
ganglia of which the CNS is composed, except for the two buccal ganglia
innervating the buccal mass. Comparison of the ISH pictures with those
of whole mount preparations of CNS from nonparasitized animals confirms
that corresponding neurons express the gene and contain the peptide
(see Fig. 6
E,
inset).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 26
27
28
29
30)
, but
also its well-known function in regulating energy budgeting (31
, 32)
. This became evident by the fact that schistosome parasites
caused up-regulation of the NPY gene in their snail host. Cloning of
the cDNA encoding a Lymnaea NPY precursor was achieved by a
PCR-based approach. The coding region is only 270 bp long and encodes a
simple precursor molecule containing a signal peptide, followed by a
dipeptide prohormone containing LyNPY, which is separated by a
classical dibasic proteolytic site from a carboxyl-terminal peptide.
This latter carboxyl-terminal peptide may serve in maintaining a
3-dimensional structure within the prohormone for correct folding
and/or proper processing of the precursor.
We have tried to mimic the parasitized condition by elevating the LyNPY
titer in nonparasitized snails. The data on the diffusion pattern of
LyNPY from pellets kept in snail's Ringer showed that the release was
concentration dependent, with a maximal release at day 5. This
corresponds with the period of ~7 days during which the physiological
effects of LyNPY administered by means of an implanted pellet were
obvious. The effect of injected LyNPY, on the other hand, lasted only
1–2 days. LyNPY administered in pellets appeared to inhibit both egg
mass production and growth in a dose- and time-dependent manner.
Histological observations support the idea that these physiological
changes result from an effect of LyNPY on central neuroendocrine cells
involved in regulation of reproduction and growth. However, this has to
be confirmed by future LyNPY receptor localization studies. In the
cerebral ganglia of the CNS, LyNPY-positive axons approach axons
derived from the neuroendocrine CDCs regulating ovulation and egg
laying (33)
and from the LGCs thought to regulate growth
(11)
. However, these LyNPY axons do not make
synaptic(-like) contacts with either cell bodies or axons of these two
neuroendocrine systems, suggesting that the effect of LyNPY is exerted
nonsynaptically (7)
. LyNPY probably does not directly
inhibit the release of the neuropeptides regulating ovulation, egg
laying, and accompanying behavior since application of LyNPY did not
change
theelectrical properties and hence the release of these neuropeptides from
the CDCs in situ (P. M. Hermann, A. Ter Maat, and
R. F. Jansen, unpublished results). These data differ from
observations on the Aplysia bag cells, neurosecretory cells
comparable to CDCs in Lymnaea that also regulate ovulation
and egg laying. These bag cells were immunopositive for
Aplysia NPY, whereas this NPY homologue also had a prolonged
inhibitory effect on these cells (34)
. Other than this
effect in Aplysia, data on the physiological role of NPY
homologues in vertebrates are lacking. Recently, the transmitter role
of LyNPY, also a NPF, has been described in the innervation of muscle
cells serving the male copulation organ in Lymnaea
(35)
. The current study is the first that not only
identifies the peptide structure and mRNA sequence of a NPY, but also
establishes its physiological role in the regulation of energy flows in
an invertebrate, confirming the best known role of NPY in vertebrates
(31
, 32)
.
In vertebrates, inhibition of reproduction by NPY is ascribed to its
inhibiting effect on release of reproductive hormones from the
hypothalamus and the pituitary (31)
. This makes it
interesting to study whether and how LyNPY affects synthetic activity
in these CDCs and how such an effect depends on the secretory status of
the CDCs, namely, the reproductive status of the animal. The fact that
in our experiments not all animals immediately stop laying eggs on the
first day of chronic/long-term LyNPY administration suggests that other
regulatory factors play a role as well. We hypothesize that the female
reproductive tract plays an important role. In Lymnaea, egg
masses are large and consist mainly of materials secreted by the
accessory glands associated with the female reproductive tract.
Immediately after egg mass production, these glands have released large
amounts of material. The restoration period of the glands almost covers
the egg laying interval, under long-day conditions of 1 to 2 days
(36)
. The response to environmental stimuli that trigger
egg laying is lower in animals that have recently laid an egg mass
(33)
, suggesting that activation of egg laying depends at
least in part on the state of the accessory female glands. In the
current experiments, the suppression of egg laying by LyNPY in such
animals lasts longer, in keeping with this suggestion. In vertebrates,
the effect of NPY on neuroendocrine cells regulating reproduction has
appeared to depend on circulating estrogens derived from such
peripheral organs as the gonads (37
, 38)
.
Our observation that both a LyNPY-positive neuron and a
CDCH-positive neuron in the visceral ganglion project their axon into
the nervus intestinalis, which also innervates the reproductive tract
(including the accessory sex glands), provides additional evidence that
LyNPY plays a role in regulating activity of the accessory sex glands.
In vertebrates it has also been found that NPY plays a role as a
modulator of neuroendocrine functions not only at the central, but also
at the peripheral level, as demonstrated by its role in the innervation
of the epididymis in the male (39)
. Recently, published
data have shown that the reproductive status of female rats also
influences the level and number of neurons expressing galanin (GAL)mRNA
in the nucleus paraventricularis of the brain, whereas it does not
affect the hypothalamic NPY neurons (40)
. This supports
that GAL and NPY, though with similar functions in the control of
reproduction and feeding, are part of different neuronal networks
(31)
.
The clear inhibiting effect of an elevated titer of LyNPY on snail
growth is a persisting effect: even after 21 days, the shell height of
the animals that had received a LyNPY pellet remained smaller. We have
obtained morphological support for such an effect of LyNPY on growth in
that axonal branches of the LyNPY system follow those of the LGC
clusters, which are known to be involved in regulation of growth
(11)
. However, we cannot exclude the possibility that the
effect of LyNPY on growth is mediated by other neurons as well. Other
possible targets of LyNPY are the paired lateral lobes attached to the
cerebral ganglia. Extirpation of these lobes induces an increase of
growth as occurs in parasitized animals, an increase of the wet weight
not paralleled by an increase of the dry weight (11)
.
However, we have no indication that LyNPY-positive axons contact
neurons in the lateral lobe. In vertebrates, hypothalamic NPY has an
inhibitory action on the release of growth hormone (GH) by modulating
the release of growth hormone-releasing hormone and somatostatin (41).
The inhibitory action of NPY on pulsatile GH secretion can be prevented
by leptin (42)
. The inhibitory effect of LyNPY on growth
and the fact that this is not counterbalanced by a change in food
consumption seem different from the situation in vertebrates, where NPY
is known to play a stimulatory role in the regulation of food intake.
However, in rats parasitized with the intestinal helminth
Nippostrongylus, in situ hybridization
studies have shown that the NPY gene was up-regulated but did not
result in an increase of the amount of food consumed by these anorectic
rats (43
, 44)
. In the current study, food intake of
Lymnaea appeared to increase as soon as glycogen-storing
cells (comparable to vertebrate fat cells) in the connective tissue
become depleted. In vertebrates, leptin produced by the fat-storing
cells links storage with the feeding system (45
46
47)
. This
suggests that in Lymnaea an analogous factor from the
storage cells plays a dominant role in the regulation of feeding.
The increase of both dry weight density and glycogen content of mantle tissue observed in snails 2 days from being injected with LyNPY may reflect an excess of energy resulting from the coinciding inhibition of reproduction and growth. The decrease of the parameters for storage observed 5 days after LyNPY injection may point to a disturbed balance between blood glucose concentrations and the amount of glycogen stored when reproduction and growth are resumed.
The feeding motor program of Lymnaea is localized mainly in
the buccal ganglia innervating the musculature of the feeding
apparatus, the buccal mass (48)
. The LyNPY fibers were
only found in close contact with the neuron(s; one or two) in each of
these ganglia, which appeared to be immunopositive with anti-CDCH,
the egg laying hormone (49)
. This indicates that LyNPY
does not directly influence activity of neurons of the feeding motor
program, but may play a role in egg laying behavior. Preceding
deposition of an egg mass the snail shows a typical behavior: she
prepares the substrate, the surface of a lettuce leaf, for egg mass
deposition. At this time the buccal mass starts making high-frequency
movements, called `rasps', which do not lead to normal ingestion of
food. The animal merely scrapes the surface of the leaf exactly where
the egg mass is to be deposited (50)
. Thus, CDC-derived
peptides as well as LyNPY might be involved in regulating this type of
behavior.
The question now is whether elevated titers of LyNPY in the parasitized
host can explain the effects on the host's reproduction, growth, and
feeding. Inhibition of reproduction in this stage of infection can be
explained by the fact that parasites cause up-regulation of the LyNPY
gene, resulting in a permanently high titer of LyNPY. However, one
should realize that during parasitic infection, the internal defense
system (IDS) of the host is activated. In snails with an open
circulatory system, this IDS consists of not just one type of blood
cell—hemocytes—but also of cells residing in the connective tissue
that produce humoral factors as molluscan defense molecule, a member of
the immunoglobulin G superfamily (51)
, and schistosomin
(see ref 52
). The identified cytokine-like factor
schistosomin, whose synthesis and release is induced by these parasites
at the same time that the LyNPY gene is up-regulated, is derived from
tiny connective tissue cells around axons. It plays a role in this
sterilizing effect: it interferes with the action of gonadotropic
hormones on their targets and affects electrical activity of CDCs; it
inhibits electrical activity of CDCs and hence the release of CDC
neuropeptides (see ref 1
). Apparently these two effects induced by the
parasite cooperate to establish an immediate and lasting effect on
reproduction. The observation in rats that the cytokines
Interleukin-1ß and the ciliary neurotrophic factor inhibit the effect
of NPY on feeding, resulting in anorexia and weight loss, is not
only interesting but also links the status of the immune system to the
neuroendocrine system (53
54
55)
.
Although the enhanced growth of snails in this stage of infection can
be ascribed primarily to an increase of the wet weight, our data do not
suggest that this effect on water balance is caused by LyNPY. LyNPY
only had an effect on the body wet weight at high concentrations, but
did not affect the dry weight density. It has been demonstrated in
vertebrates that NPY plays a role in salt and water homeostasis,
namely, salt loading has an effect on the density of neurohypophysial
NPY binding sites (56
, 57)
. Supposedly more/other key
factors play a dominant role in establishing the peculiar effects
parasites have on the growth of their snail host. It has been
demonstrated that the cytokine-like factor schistosomin not only
interferes with neuroendocrine regulation of reproduction, but also
with that of growth; it stimulates electrical activity of the
growth-controlling LGCs (58)
. Another example affecting
growth is the surface coat factor secreted by plerocercoids of
Spirometra mansonoides into their rodent host that is both a
growth hormone agonist and a cysteine proteinase (59)
.
The observation that LyNPY does not affect food intake corresponds with
previous data that feeding is not affected in patently infected
L. stagnalis (60)
even though the blood glucose
level and the amount of glycogen stored had reached levels below the
controls in this stage of infection. However, parasitic interference
with energy budgets in the host can yield several effects on
reproduction, growth, and feeding even within other, closely related
parasite-host combinations (1)
. These effects on energy
flows, however, have to be considered in relation to changes in
processes associated with metabolic rate such as oxygen consumption,
rate of energy conversion, food assimilation, heart beat, locomotory
activity, and heat production. These effects depend as well on the
developmental stage of both parasite and host and on the parasitic
burden.
The data presented here demonstrate that the (Ly)NPY encoding gene is
likely an important target that parasites can switch on to direct the
energy flow in the host for their own benefit. The fact that increased
levels of NPY mRNA were also observed in the hypothalamus of rats
infected with the intestinal helminth Nippostrongylus
(43
, 44)
indicates that it might be a general and
lucrative strategy used by endoparasites to affect the NPY gene
expression in the host. Our model system lends itself well to
investigations of how parasites or their excretory/secretory products
affect gene expression in the host. The supposition that the conserved
NPY gene in the host is an important target for parasitic action opens
interesting possibilities to interrupt the life cycle of medically
and/or economically important parasites.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-) or 3000 µM (-
-) LyNPY into snail's Ringer (1
pellet/ml) during 1, 3, 5, or 7 days. The diffusion is concentration
dependent. Diffusion from the pellet with the lower concentration
reached a maximal concentration at day 3, the pellet with the highest
concentration at day 5.
-), 300 (-•-), or 1000 (-
-) µmol LyNPY
were implanted on day 0. n=20 in all groups.
A) Production of egg masses (number/snail/day). Egg mass
production was significantly lower in the (1000 µmol) LyNPY-treated
group on days 2–7. B) Cumulative plot of egg mass
production (per 20 animals) showing a complete return to control
productivity after day 7 in both groups of LyNPY-treated snails.
C) Growth (µM/day) was transiently suppressed for the
first 7 days in the 1000 µmol group. D) Food
consumption (cm2 lettuce/day per animal) was not affected
until after 9 days after implantation, when it increased significantly
in the 1000 µmol LyNPY group. Consumption in the other experimental
group increased later, but also significantly (P<0.01).
Asterisks refer to the comparison between controls and the group
implanted with 1000 µmol of LyNPY and denote P<0.01.
The data shown are taken from the same experiment from which the
dose-response curves shown in Fig. 4
