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Insights into Embryo Defenses of the Invasive Apple
Snail
Pomacea canaliculata
: Egg Mass Ingestion Affects
Rat Intestine Morphology and Growth
Marcos S. Dreon
1,2.
, Patricia E. Ferna
´ndez
3.
, Eduardo J. Gimeno
3
, Horacio Heras
1,4
*
1Instituto de Investigaciones Bioquı
´micas de La Plata (INIBIOLP), Universidad Nacional de La Plata (UNLP) – CONICET CCT, La Plata, Argentina, 2Facultad de Ciencias
Me
´dicas, Universidad Nacional de La Plata, La Plata, Argentina, 3Instituto de Patologı
´a B. Epstein, Ca
´tedra de Patologı
´a General Veterinaria, Facultad de Ciencias
Veterinarias, Universidad Nacional de La Plata, La Plata, Argentina, 4Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata, Argentina
Abstract
Background:
The spread of the invasive snail Pomacea canaliculata is expanding the rat lungworm disease beyond its native
range. Their toxic eggs have virtually no predators and unusual defenses including a neurotoxic lectin and a proteinase
inhibitor, presumably advertised by a warning coloration. We explored the effect of egg perivitellin fluid (PVF) ingestion on
the rat small intestine morphology and physiology.
Methodology/Principal Findings:
Through a combination of biochemical, histochemical, histopathological, scanning
electron microscopy, cell culture and feeding experiments, we analyzed intestinal morphology, growth rate,
hemaglutinating activity, cytotoxicity and cell proliferation after oral administration of PVF to rats. PVF adversely affects
small intestine metabolism and morphology and consequently the standard growth rate, presumably by lectin-like proteins,
as suggested by PVF hemaglutinating activity and its cytotoxic effect on Caco-2 cell culture. Short-term effects of ingested
PVF were studied in growing rats. PVF-supplemented diet induced the appearance of shorter and wider villi as well as fused
villi. This was associated with changes in glycoconjugate expression, increased cell proliferation at crypt base, and
hypertrophic mucosal growth. This resulted in a decreased absorptive surface after 3 days of treatment and a diminished rat
growth rate that reverted to normal after the fourth day of treatment. Longer exposure to PVF induced a time-dependent
lengthening of the small intestine while switching to a control diet restored intestine length and morphology after 4 days.
Conclusions/Significance:
Ingestion of PVF rapidly limits the ability of potential predators to absorb nutrients by inducing
large, reversible changes in intestinal morphology and growth rate. The occurrence of toxins that affect intestinal
morphology and absorption is a strategy against predation not recognized among animals before. Remarkably, this defense
is rather similar to the toxic effect of plant antipredator strategies. This defense mechanism may explain the near absence of
predators of apple snail eggs.
Citation: Dreon MS, Ferna
´ndez PE, Gimeno EJ, Heras H (2014) Insights into Embryo Defenses of the Invasive Apple Snail Pomacea canaliculata: Egg Mass
Ingestion Affects Rat Intestine Morphology and Growth. PLoS Negl Trop Dis 8(6): e2961. doi:10.1371/journal.pntd.0002961
Editor: Matty Knight, George Washington University School of Medicine and Health Sciences, United States of America
Received November 18, 2013; Accepted May 9, 2014; Published June 19, 2014
Copyright: ß2014 Dreon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Comisio
´n de Investigaciones CientI
´ficas de la Provincia de Buenos Aires (CICBA) to MSD and Agencia
Nacional de Promocio
´n CientI
´fica y Tecnolo
´gica (PICT #1865), Argentina to HH. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: h-heras@med.unlp.edu.ar
.These authors contributed equally to this work.
Introduction
The invasive apple snail Pomacea canaliculata (Lamarck, 1822)
(Architaenioglossa, Ampullariidae) has become a serious aquatic
crop pest in Asia and a vector of the rat lungworm Angiostrongylus
cantonensis that causes human eosinophilic meningitis, a potentially
fatal disease considered an emerging infectious disease. Unfortu-
nately angiostrongyliasis (rat lungworm disease) continues to be
reported in new regions beyond its native range which has been
associated with the expansion of this snail [1;2]. P. canaliculata is the
only freshwater snail listed among the 100 worst invasive species
worldwide [3]. Their successful establishment in invaded areas
may be related, among other factors, to their high fecundity, and
the unusual characteristics of their eggs that increase the risk of the
expansion of the disease. Females of P. canaliculata deposit
hundreds of bright pink-reddish egg masses, each containing 30–
300 eggs [4;5]. These egg clutches are remarkable in three
respects: they are cemented outside the water, they are brightly
colored and have virtually no predators, presumably because they
have unusual defenses against predation [5–8]. Though filled with
a perivitellin fluid (PVF) containing large amounts of carbohy-
drates and storage proteins (called perivitellins), these toxic eggs
have no predators reported in their original South American range
and only one in the newly colonized habitats in SE Asia: the fire
ant Solenopsis geminata (Fabricius, 1804). The presence of these egg
defenses [6;8;9] would explain the behavior of the snail kite
Rostrhamus sociabilis (Vieillot, 1817) and Norway rat Rattus norvegicus
(Berkenhout, 1769) that invariably discard the gland that
PLOS Neglected Tropical Diseases | www.plosntds.org 1 June 2014 | Volume 8 | Issue 6 | e2961
synthesizes the egg defenses when predating on adult female P.
canaliculata [10–14].
Work in the last two decades identified the perivitellins PcOvo
and PcPV2 in the egg defenses against predation [6;8;13;15–17].
These are the most abundant perivitellins stored in large quantities
in the PVF (57.0% and 7.5% of egg total protein for PcOvo and
PcPV2, respectively) [15]. Both are resistant to proteolysis
reaching the intestine in a biologically active conformation [6;8].
PcPV2 is a neurotoxic storage lectin with a strong lethal effect on
selected neurons within the spinal cord of mice [9;18]. It is a novel
combination of a tachylectin-like subunit with a membrane attack
complex/perforin (MACPF)-like subunit, not reported in animals
before [8]; PcOvo, on the other hand, is a storage carotenoprotein
that provides the conspicuously reddish coloration of the clutches
which presumably advertises to visual-hunting predators the
presence of egg defenses (aposematic warning) [19]. In addition,
PcOvo is a proteinase inhibitor limiting the ability of predators to
digest egg nutrients. In fact, oral administration of purified PcOvo
to rats significantly diminished rat growth rate presumably by a
dual mechanism: the inhibition of trypsin activity (antidigestive
role) and the resistance of the inhibitor to digestion by gut enzymes
(antinutritive) [6;20–22]. A recent proteomic analysis of P.
canaliculata PVF identified a small amount of over 50 other
proteins, including two F-type lectins, many proteins involved in
innate immunity in other mollusks and some with potential roles
against insects and fungi [23].
As the epithelial cells along the digestive tract of animals are
fully exposed to food contents, they are possible target sites for
defense proteins. In this regard, plants have evolved a wide array
of toxic dietary lectins that interact with the membrane
glycoproteins of the luminal side of the gut of higher animals
having an important role in plant defenses against predation [24].
There are, however, no reports in animals of such a defense
mechanism [25].
With the aim to further understand the role of egg defenses of a
host of the lungworm disease, in the present work we studied the
effect of P. canaliculata PVF on the small intestine of rats. Through
a combination of biochemical, histopathological, cell culture and
feeding experiments, we provide evidence that oral administration
of apple snail PVF adversely affects rat small intestine metabolism
and morphology and consequently rat growth rate, presumably by
proteins displaying lectin-like activity. This overall effect has not
been found in other animals, but it is remarkably similar to that for
plant seed lectins on the gastrointestinal tract of rats and other
vertebrates.
Materials and Methods
Ethics Statement
All studies performed with animals were carried out in
accordance with the Guide for the Care and Use of Laboratory
Animals [26] and were approved by the ‘‘Comite´ Institucional de
Cuidado y Uso de Animales de Experimentacio´n’’ of the School of
Medicine, UNLP (Assurance No. P08-01-2013).
Eggs
Egg masses of P. canaliculata were collected either from females
raised in our laboratory or taken from the wild in streams or
ponds near La Plata city, Province of Buenos Aires, Argentina,
between November and March of consecutive reproductive
seasons. Only egg masses with embryos developed to no more
than the morula stage were employed. Embryo development was
checked microscopically in each egg mass as described elsewhere
[16].
Rats
All experiments with rats were performed using male Wistar rats
from the Animal Facility of the School of Medicine of the National
University of La Plata (UNLP), Argentina. Rats came from a
colony started with the strain WKAHlHok (Hokkaido University,
Japan). Six-week-old animals weighing 18062 g at the start of the
experiments were housed in cages with 12 h day-night cycle,
temperature of 2261uC and relative humidity of 45–60%.
Preparation of PVF
Fertilized eggs were repeatedly rinsed with ice cold 20 mM
Tris-HCl, pH 6.8, containing a protease inhibitor cocktail (Sigma
Chemicals, St. Louis) and homogenised in a Potter type
homogeniser (Thomas Sci., Swedesvoro, NJ). Ratio of buffer:
sample was kept 5:1 v/w. The crude homogenates were then
sonicated for 15 sec and centrifuged sequentially at 10,0006g for
30 min and at 100,0006g for 60 min. The pellet was discarded
and the supernatant comprising the egg PVF was equilibrated in
50 mM phosphate buffer pH 7.4 using a centrifugal filter device of
50 kDa molecular weight cut off (Millipore Corporation, MA) to
eliminate potentially interfering compounds. Total protein con-
centration of the PVF (13.3 g/L) was measured by the method of
Lowry et al. [27].
Isolation and purification of PcOvo. PcOvo was purified
from the PVF of the eggs by high performance liquid
chromatography (HPLC) (Hitachi Ltd., Tokyo, Japan). First,
the sample was analyzed in a Mono Q HR 10/10 (Amersham-
Pharmacia, Uppsala, Sweden) using a gradient 0 to1 M NaCl in
a 20 mM Tris buffer. The PcOvo peak was then further purified
by size exclusion chromatography (Superdex 200 HR 10/20,
Amersham-Pharmacia, Uppsala, Sweden) using an isocratic
gradient of sodium phosphate buffer 50 mM, 150 mM NaCl,
pH 7.6. Purity of the single peak obtained was checked by native
PAGE performed in a Mini-Protean III System (Bio Rad
Laboratories, Inc.). Protein content was determined by the
method of Lowry et al. [27].
Author Summary
Filled with nutritious substances to nourish the embryos,
eggs of most animals are often the targets of pathogens
and predators. An exception are the eggs of Pomacea
canaliculata –known as the apple snail– which have hardly
any predators. This freshwater snail is a serious aquatic
crop pest in several continents, listed among the 100 worst
invasive species. It is the host of a roundworm responsible
for the rat lungworm disease causing human eosinophilic
meningitis. The spread of this emerging infectious disease
has been associated with the expansion of apple snails.
They lay eggs above water level in bright pink-reddish
masses, presumably a warning coloration. Indeed, eggs
have chemical defenses, including neurotoxic and anti-
nutritive proteins. The authors found that the ingestion of
egg extracts adversely affects rat small intestine inducing
large, reversible changes in the intestinal wall that limits
the ability to absorb egg nutrients causing a diminished
growth rate. Apple snail eggs are the first animal known to
deter predators by this mechanism, but remarkably this
defense is rather similar to the toxic effect of plant seeds
proteins. These overlapping egg defenses that predators
have not managed to overcome yet may partially explain
the reproductive success of P. canaliculata.
Snail Egg Defenses against Predation
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Experimental Protocols
Experiment 1. Effect of egg PVF-supplemented diet
on rat growth. Groups of control and treated rats, 10 animals
each, were fed ad libitum with a commercial diet for up to 7 days.
Treated animals were administered PVF fraction (4 mg protein) in
the water on a daily basis, while the control group received the
equivalent volume of buffer. Food consumption as well as body
weight were determined on a daily basis for each animal. The
standard growth rate (SGR) was calculated as follows:
SGR~Wto=Wt
ðÞ
1=t{1
|100
Where W
to
is the initial weight, W
t
is the final weight, and tis the
time in days. [28].
Experiment 2. Effect of egg PVF-supplemented diets
on rat intestine size and morphology. To establish the time-
dependent changes induced by PVF on intestine, rats (groups of 5
rats each) were given either the commercial diet or the diet+PVF
feed which contained 4 mg PVF protein rat
21
day
21
and
sacrificed after, 4 or 10 days.
A similar setting was employed using PcOvo-supplemented diet
at 4 mg PcOvo protein rat
21
day
21
. Animals were sacrificed after
4 and 8 days and the absorptive surface of the intestine measured
as described below (next section).
Rats were euthanized by CO
2
inhalation in a closed chamber
[26]. Care was taken to kill all animals at comparable times of the
day (late morning). Intestines were removed and their length
measured from pylorus to ileocaecal junction.
Sections of the first part of the duodenum were cut, washed
several times with PBS to remove food and fixed in 4% formalin in
phosphate buffered (pH = 7) for histological examination. In rats
from day-4 treatment, sections for SEM analysis were also
obtained (see scanning electron microscopy section).
To establish the time needed to recover after the changes
induced by egg PVF on intestine, rats (groups of 5) were given
either the commercial diet or a diet-and-egg PVF, which
contained 4 mg protein rat
21
day
21
. Animals were fed with the
extracts for 10 days and then switched to a control diet and
sacrificed 4 days after switching treated animals to control feed
and processed as above.
Histology and Morphological Measurements
Cylindrical tissue samples of the small intestine were post fixed
in 10% neutral formaldehyde for 24 h at room temperature and
then embedded in paraffin wax. Representative 5–7 mm sections
were stained with haematoxylin and eosin for histological
examination of general morphology. In addition, periodic acid
Schiff (PAS) staining was performed to highlight carbohydrate
distribution and goblet cells.
Fifty properly oriented villi and crypts from duodenum were
selected at random from each animal and their length and width
measured to calculate mucosal absorptive surface area following
the method of Kisielinsky [29] whose results have no significant
differences compared with the Harris method, widely used in
rats [30]. The method considers a geometric mucosal unit of a
cylindrical villous with rounded tip surrounded by cylindrical
crypts. It assumes that the whole mucosa is an iteration of this
unit, and the surface area can be calculated with mean values of
structures that define the mucosal unit: villus length, villus
width, and crypt width. Thus, the mucosal-to-serosal amplifi-
cation ratio Mwas calculated considering these 3 variables, as
follows:
M~
(villous width .villous length)z(villous width
2zcrypt width)2
2(villous wi dth)2
2
(villous width
2zcrypt width)2
2
Immunohistochemistry
Small intestine sections were assayed by immunohistochemistry
(IHC) to evaluate cellular proliferation using a primary monoclo-
nal mouse against the proliferating cellular nuclear antigen
(PCNA) as a proliferation marker (Dako, Clon PC10). The
antibody was diluted in 0.1% BSA in phosphate buffer and
incubated overnight at 4uC. PCNA is a nuclear acid protein which
functions as dDNA polymerase helper. In the presence of PCNA
and a replication C factor, dDNA polymerase starts the synthesis
of DNA and the progression of the cellular cycle.
Samples were incubated overnight at 4uC as mentioned above,
and visualized using the LSAB kit (Dako Cytomation Lab,
Carpinteria, USA) detection system which is based on a modified
labeled avidin-biotin (LAB) technique in which a biotinylated
secondary antibody forms a complex with peroxidase-conjugated
streptavidin molecules. In short, after incubation with the
appropriate primary antibody, a sequential 10 min incubation
with an anti-mouse biotinylated antibody and peroxidase-labelled
streptavidin is performed. Then staining is completed by
incubation with 3,39diaminobenzidine tetrahydrochloride (DAB)
and H
2
O
2
. Positively stained cells showed a golden, dark-brown
color. All sections were counterstained with Maeyer haematoxilyn
before analysis. Primary antibody was replaced by normal mouse
antiserum in control sections.
Lectin Histochemistry
Small intestine sections were assayed with seven lectins (Table 1)
(Lectin Biotinylated BK 1000 Kit, Vector Laboratories Inc.,
Carpinteria, CA, USA) namely: Con A (Concanavalia ensiformis),
DBA (Dolichos biflorus), SBA (Glycine max), PNA (Arachis hypogaea),
RCA-I (Ricinus communis-I), UEA-I (Ulex europaeus-I) and WGA
(Triticum vulgaris) to reveal possible changes of the glycosylation
pattern.
In short, paraffin sections were deparaffinized with xylene
dehydrated with 100% alcohol twice, 10 min each, and then
endogenous peroxidase activity was quenched by incubating
5 min with hydrogen peroxide in methanol 0.3–3.0%.
They were then hydrated, washed in phosphate-buffered saline,
and incubated with biotinylated lectins overnight. Then sections
were washed with PBS, followed by 10-min incubation with
streptavidin-HRP (streptavidin conjugated to horseradish perox-
idase in PBS containing stabilizing protein and anti-microbial
agents (Vector Laboratories Inc., USA). Finally the bound lectins
were visualized by incubation during 4–10 min with a buffered
Tris-HCl solution (0.05 M, pH = 6.0) containing 0.02% 3,39-
diamino-benzidine tetrahydrochloride (DAB) and 0.05% H
2
O
2
(DAB; Dako, Carpinteria, USA). Positively-stained cells were
demonstrated by a dark golden brown coloration. The sections
were counterstained with Maeyer haematoxilyn.
Scanning Electron Microscopy (SEM)
After 2-hour fixation in 2% (v/v) glutharaldehyde, samples were
dehydrated in graded series of ethanol. Then ethanol was replaced
by liquid carbon dioxide and samples were dried by critical point
in a CP-30 (Balzers). Samples were gold metalized in a JEOL Fine
Ion Sputter, JCF-1100. Observations and photomicrographs were
Snail Egg Defenses against Predation
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obtained with a JEOL JSM 6360 LV SEM (Jeol Technics Ltd.,
Tokyo, Japan) at the Service of Electron Microscopy, Facultad de
Ciencias Naturales y Museo, Universidad Nacional de La Plata,
Argentina.
Hemagglutinating Activity
Horse, goat, rabbit and rat erythrocytes were obtained from the
animal facilities at the University of La Plata (UNLP). Blood
samples were obtained by venous puncture and collected in sterile
Elsever’s solution (100 mM glucose, 20 mM NaCl, and 30 mM
sodium citrate, pH 7.2) (Sigma Chemicals, St. Louis). Prior to use,
erythrocytes were washed by centrifugation at 1500 g for 10 min
in TBS buffer (20 mM Tris, 150 mM NaCl, pH 7.4). This
procedure was repeated several times until the supernatant
remained clear. Hemagglutinating activity was assayed in micro-
titer U plates (Greiner Bio One, Germany) by incubating a two-
fold serial dilution of PVF (6 mg/mL) in TBS with 2% erythrocyte
suspension in TBS at 37uC for 2 h. Results were expressed as the
inverse of the last dilution showing visible hemagglutinating
activity by naked eye.
Cytotoxicity
Human colorectal adenocarcinoma cells (Caco-2) were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) (4.5 g/liter D-
glucose) supplemented with 10% newborn calf serum, penicillin
(10 U/mL), streptomycin (10 mg/mL), amino acids and vitamins
(Life Technologies-Invitrogen). Cells were cultured at 37uCina
humidified atmosphere of 5% CO
2.
Culture medium was replaced
every 2 days and subcultured by trypsinization when 95%
confluent. Passages 60 through 65 were used for the experiments.
Prior to each experiment, the viability of the cells was determined
by trypan blue exclusion. Viability of every cell preparation
exceeded 90% as determined by counting the stained cells.
The cytotoxic effect of the PVF on Caco-2 cells was evaluated
using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay [31]. Cells were seeded in 200 mLof
culture medium on 48-well plates at densities that ensured
approximately 90% confluency after 24 h. Once cell cultures
reached the desired confluence, 50 ml/well of a serial dilution of
PVF (6 mg/mL) in PBS were added and incubated at 37uC for
24 h. Control wells were prepared with 50 mL/well of PBS. After
treatments, culture medium was removed and cells were incubated
with fresh medium containing 0.5 g/L of MTT at 37uC for 1 h.
Plates were then centrifuged, the supernatant discarded and the
cells were washed three times with PBS. Finally the cell
monolayers were extracted with 200 mL/well of DMSO and the
absorbance of each well recorded at 540 nm with background
substraction at 640 nm in a microplate reader Multimode
Detector DTX-880 (Beckman Coulter, Inc., CA, USA). Cell
viability was expressed as control percentage [31].
%Viability = (OD treated cells/OD control cells) 6100
Statistical Analysis
Data collected from all experiments were analyzed individually
by either t test (histology) or ANOVA (bioassays) using Instat
v.3.05 (Graphpad Software Inc.). Where significant differences
between samples occurred, a post-hoc Tukey’s HSD test was
performed to identify the differing means. Results were considered
significant at the 5% level.
Accession Numbers
GenBank accession numbers for PcOvo subunits: JQ818215,
JQ818216 and JQ818217; GenBank accession numbers for
PcPV2 subunits: JX155861 and JX155862.
Results
Effect of Snail Egg Supplemented Diet on Rat Growth
Rate
During the first 3 days of treatment with PVF, treated rats
showed a significantly lower standard growth rate than the control
ones (Fig. 1). This effect on growth rate disappeared after the
fourth day of treatment and animals began to grow at the same
rate as control groups. Daily food ingestion was similar in control
and treated rats along the experimental period (results not shown).
Effect of PVF on Rat Gastrointestinal Tract
Oral administration of PVF for 10 days increased the mean
intestinal length of the rats though a tendency was already evident
after a 4-day treatment (Fig. 2). Within four days of switching the
10-day treated animals to a control diet, the total length of the
small intestine returned to control values (Fig. 2). Crypt
dimensions and general morphology of intestine were virtually
restored to normal.
At day 4, samples from control animals showed the character-
istic tall, finger-like villi, whereas villi from treated animals showed
significantly less height and were wider with some proliferation in
the basal zone of the epithelia. In certain areas of the epithelium of
treated animals, altered villi with a double, fused or ‘‘tongue’’
shape, displaying a bridge pattern were observed by SEM and
light microscopy (Fig. 3). PAS staining was moderate on the
glycocalyx of villi and crypt enterocytes while the mucin of goblet
Table 1. Lectins used in this study and their major specificities.
Acronym Lectin Specificity Concentration (mg/ml)
UEA-I Ulex europaeus-I a-L-Fuc 30
DBA Dolichos biflorus, a-D-GalNAc 30
PNA Arachis hypogaea b-D-Gal (b1-3) D-GalNAc 10
SBA Glycine maximus a–D-GalNAc; b–D-GalNAc 30
WGA Triticum vulgaris, b-D-GlcNAc; NeuNAc 30
RCA-I Ricinus communis-I b-Gal 30
Con-A Concanavalina ensiformis a-D-Man; a-D-Glc 30
Specificities according to Goldstein and Hayes [50]. Fuc: Fucose; Gal: Galactose; GalNAc: N-Acetyl galactosamine: Glc: Glucose; GlcNAc: N-Acetyl glucosamine; Man:
mannose; NeuNAc: Acetyl neuraminic acid (sialic acid).
doi:10.1371/journal.pntd.0002961.t001
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Figure 1. Effect of egg PVF-supplemented diets on Wistar rats’ standard growth rate during the first 7 days. Control (black square),
treated (red circle).Values represent the mean 61 SD (n = 10); **p,0.01.
doi:10.1371/journal.pntd.0002961.g001
Figure 2. The effect of egg PVF on the length of the rat small intestine. Blue: Control; grey: PVF supplemented diet; 4 d: 4 day feeding; 10 d:
10-day feeding; 10 d+4d recovery: 10-day PVF feeding +4-day diet switched to control conditions. *p,0.05. Bars represent the mean 61 SD of 5 rats
per group.
doi:10.1371/journal.pntd.0002961.g002
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cells showed a strong stain in both control and treated samples.
Mucose epithelia from treated animals showed an increased
number of goblet cells (Fig. 4 A,B).
Scanning Electron Microscopy
SEM analysis of control and treated animals confirmed the
remarkable differences on the length and width of the villi of
treated animals (Fig. 3). The increased amount of mucus on the
mucosa in the treated animals was also observed with this
technique, as well as areas displaying conical, dome-shaped
mucosal elevations which seem to connect two villi in a bridge-
epithelial pattern (Fig. 3).
Lectin and Immunohistochemistry
PCNA labeling showed moderate immunostaining in the basal
areas of the epithelium from controls, while a stronger staining was
evident on the treated animals. (Fig. 4 C,D arrows).
Intestinal epithelial cells were studied using a set of 7 lectins, of
which PNA and SBA produced the most remarkable results
(Fig. 4). PNA was strongly positive on the supranuclear region of
enterocytes of control animals, while in treated animals its binding
was observed not only in this region (strong staining) but also in the
whole enterocyte (light staining) (Fig. 4, E,F arrows). Besides, SBA
lectin binding was strong on the glycocalyx of the apical zone of
the enterocytes of treated rats in comparison to the moderate
staining in control group, indicating SBA-binding glycans were
more expressed on enterocytes exposed to snail egg PVF (Fig. 4
G,H arrows).
Absorptive Surface
When the effect of PVF on rat small intestine absorptive
surface was quantified on histological sections, a significant
decrease of the 4-day treated animals was observed while if the
ingestion is continued for 8 days, the absorptive surface reverted
to normal (Table 2). When rats were exposed to PcOvo, the
small intestine did not show significant changes in absorptive
surface for up to 8 days (Table 2) and villi morphology was
normal.
Hemagglutinating Activity
The egg PVF of P. canaliculata showed hemagglutinating activity
against horse red blood cells up to a protein dilution of 0.15 mg/
mL, indicating the presence of active lectins. Moreover, a
moderate agglutinating activity against rabbit and rat red blood
cells was also observed at 0.6 mg/mL of PVF protein concentra-
tion (Fig. 5).
Cytotoxic Effect of PVF on Caco-2 Cell
The MTT assay showed that PVF displays cytotoxic activity on
Caco-2 cell monolayers in a dose-dependent manner. A very
significant reduction of cell viability to only 6.660.6% in PVF-
treated monolayers as compared to control ones was observed at a
PVF protein concentration of 0.6 mg/mL (Fig. 6).
Figure 3. Changes in small intestine morphology after 4-day treatment with a diet supplemented with snail egg PVF. A,D control;
B,C,E treated. Villi from treated animals are shorter and wider and displayed dome-shaped mucosal elevations which seem to connect two villi in a
bridge-epithelial pattern (C, arrowhead) and are covered with more mucus (C, arrow). There is an increase in the proliferation of the basal epithelia in
treated animals (B, arrows). A,B HE 10x; C,D,E SEM; bar = 100 mm.
doi:10.1371/journal.pntd.0002961.g003
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Discussion
The Effect of Apple Snail Egg PVF on Rat Intestine
Resembles That of Dietary Plant Lectins
The ingestion of apple snail PVF severely affects the gastroin-
testinal tract rapidly causing a decrease in growth rate. Shortly
after feeding a diet containing PVF the rat intestinal morphology
undergoes a dramatic change. This included shorter and wider
villi and fusion of villi by epithelial bridging, which might be
related to the ability of the epithelial cells to stretch in order to
cover denuded areas [32]. The observed enlargement of both
villous and crypt thickness in treated animals was associated with
Figure 4. Effect of PVF administration on rat small intestine glucids, cell proliferation and glycosylation pattern. Rats were fed for 4
days on a diet without (A,C,E,G) or with (B,D,F,H) PVF containing 4 mg protein. A,B. PAS stain highlighting the goblet cells (arrowheads). C,D. PCNA
Immunohistochemistry showing moderate and strong staining in control and treated samples, respectively. Arrows indicate the proliferation of the
basal zone of the epithelium. E,F. PNA lectin histochemistry. Arrows: supranuclear zone, arrowhead: enterocyte; G,H. SBA lectin histochemistry in
control and treated samples, respectively. Arrows indicate glycocalyx. The nuclei were counterstained with haematoxylin. A,B,E,F,G,H: Bar 100 mm; C,
D: Bar 45 mm.
doi:10.1371/journal.pntd.0002961.g004
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the presence of hyperplasic crypts and hypertrophic mucosal
growth changes. The notable increase in enterocyte proliferation
and the presence of immature enterocytes in the crypts suggest
increased mitotic activity in treated animals. This in vivo effect was
further supported by the analysis of PVF cytotoxicity toward
differentiated intestinal cells which indicates the presence of toxins
somehow damaging these enterocytes. This damage in turn would
induce the proliferative response observed at the crypt. Thus, the
ingestion of PVF seems to interfere with gut and systemic
metabolism, inducing hyperplasia and hypertrophy of the small
intestine and alterations in organ function. Despite this effect on
the gut being well established for plant toxic lectins [33] it has not
yet been reported for the ingestion of animal proteins.
The enterocyte proliferation was also associated with changes of
the glycosylation pattern revealed by the differential binding of the
plant lectins PNA and SBA. PNA binds to the supranuclear
portion of enterocytes where Golgi apparatus is located. It has
been reported in humans orally intoxicated with PNA that the
perturbation of cell kinetics and the more rapid cell migration and
turnover of enterocytes may be reflected as synthesis of incomplete
nascent glycoproteins, and expressed by altered PNA binding
patterns [34]. This is also a well-known effect caused in rats
intoxicated by other plant lectins that, as metabolic signals, can
radically alter the pattern of glycosylation of the gut epithelium
and thus further amplify their potent physiological effects [33;35].
These similarities between the effects of PVF and plant lectins lead
us to look for lectin hemagglutinating activity in the PVF, which
was found positive for some mammalian erythrocytes. This agrees
with the recent identification of two putative lectins in a proteomic
study of P. canaliculata PVF [23]. As mentioned before, one of these
lectins, PcPV2, is the second most abundant egg protein. A
functional study performed after its ingestion by rats showed that
PcPV2 has the ability to withstand protease digestion, displaying
structural stability within the pH range of the gastrointestinal tract
of rats. Moreover, this toxic lectin binds to the glycocalyx of rat
enterocytes in vivo and to Caco-2 cells in culture [8]. This
interaction is also in agreement with the high cytotoxic effect of the
snail PVF on Caco-2 cells observed in this study.
These properties are concurrent with those of many plant lectins
which are resistant to mammalian gastrointestinal digestion and
their toxicity is mainly attributed to the binding to the glycan
surface of the small intestine epithelial cells, which leads to
interferences with the digestion and anatomical abnormalities [35–
38].
Besides lectins, PVF also contains the proteinase inhibitor
PcOvo. When the effect of a PVF-containing diet on rat growth
rate (Fig. 1) is compared with that of a PcOvo-containing diet [6],
a larger decrease of rat growth rate was observed with PVF,
indicating there are more defensive compounds acting synergis-
tically. In addition, a PVF-supplemented diet, unlike a PcOvo-
supplemented one, diminished intestinal absorptive surface. A
literature survey reveals no information on animals in this regard
but again, a reduction of the absorptive surface area was reported
after the administration of diets containing plant lectins to rats,
causing malabsorption of nutrients [33;39]. As a whole, the
decrease on rat growth rate and changes in intestine morphology
and absorptive surface caused by PVF ingestion together with the
reported ability of PcPV2 toxic lectin to bind intestinal cells were
rather similar to the effect observed on rodents fed with diets
containing plant lectins strongly suggesting that PVF lectins may
be involved in the observed effect of snail toxic eggs on the gut of
the rat.
Rats Gut Adapt to Prolonged Exposure to PVF
If the ingestion of PVF is continued, the rat growth rate
becomes indistinguishable from that in control rats indicating an
adaptation overcoming the antinutritional effect. Changes in the
length of small intestine are often related with the difficulty in
digesting the food. Greater length increases the transit time, thus
maximizing digestion [40]. The adaptation to the PVF involved a
time-dependent increase of the small intestine length, clearly
observed after 10-day treatment. Similar effects were also observed
in rats 3 days after administering diets containing phytohemaglut-
tinin (PHA) from red kidney beans and other plant lectins [41;42].
However, those studies have shown that PHA-treatment of rats
Table 2. Small intestine mucosal absorptive surface (M) of rats ingesting control, PVF-supplemented diet or PcOvo-supplemented
diets after 4 and 8 days.
4 days 8 days
Control diet 7.1361.28 7.3861.78
PVF-supplemented diet 4.7461.07* 7.1962.49
PcOvo-supplemented diet 7.1261.49 7.2562.37
Values represent the mean 6SD of 5 rats (n = 50 cross-section/animal).
*p,0.05%; M: Mucosal-to-serosal amplification ratio.
doi:10.1371/journal.pntd.0002961.t002
Figure 5. Hemagglutinating and hemolytic activity of
P.
canaliculata
egg PVF against different types of mammalian
erythrocytes. Well 1 to 5: 1.6, 0.8, 0.4, 0.2, and 0.1 mg/ml PVF protein.
doi:10.1371/journal.pntd.0002961.g005
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resulted in pancreas growth [42;43]. No such effect was observed
in the current study (results not shown). In addition, the increase in
mucous secretion suggests another adaptation allowing the
isolation and protection of the intestinal surface from the toxic
proteins. The change in length was virtually reverted 4 days after
the elimination of the toxins from the diet along with the recovery
of the normal tissue morphology. It is worth recalling that the
mucosa of the small intestine is lined with epithelium that has the
shortest turnover rate of any tissue in the body and in about 3
days’ time the entire surface is covered with new cells [44].
Although there is no report of other animal lectins causing this
effect, a fast remodeling of intestine by reversible effects on
anatomy and morphology are known in rats and pigs administered
diets containing plant lectins [41;45].
Ecological Implications
Resting eggs are particularly vulnerable, since they are most
attractive to potential parasites and predators and may lack an
active defense system (because of their inactive metabolic state).
Apple snails seem to have evolved passive defense systems to
protect their developing embryos; the preferential accumulation of
large quantities of lectins, and protease inhibitors is certainly
indicative of that strategy. Moreover, it is believed that the main
antinutrients responsible for reducing the nutritional value of
many plant seeds are a combination of lectin and trypsin inhibitors
[46]. Similarly, in apple snail eggs these two types of proteins may
be also the main factors responsible for this effect. This further
highlights the previously reported similarities between apple snail
egg and plant seed embryo defenses [6;8]. In a broader view, the
overall effect of P. canaliculata PVF on rats bears many similarities
with the effect of plant dietary lectins not only against mammals
but also birds, insects and nematodes, preventing these predators
from digesting and incorporating nutrients from the tissues
consumed [47–49]. Unlike plants, P. canaliculata advertises its
defenses by a conspicuous coloration of the egg masses. Eggs
indeed seem to have a large number of defensive proteins against
predation, such as other protease inhibitors, chitinases, glycanases,
lectins and antifungal proteins, as the analysis of the PVF
proteome revealed [23]. Interestingly, all of these defensive
proteins are also present in many plant seeds. It is possible that
the combined effect of these defensive perivitellins -some targeting
the digestive system while others aiming at other organs- may be
an evolutionary adaptation. Although these defenses may not
completely protect an egg from consumption, they may very well
confer an advantage that increases its fitness helping to explain the
virtual absence of egg predators. With more than 80,000 species,
gastropods are the second largest class of animals after insects. It is
therefore not surprising that a better understanding of gastropod
egg biochemical defenses, little studied to date, is unveiling novel
strategies not previously recognized among animals. In this regard,
this study provides insights on the unique defenses against
predators of a snail egg that are advertised by conspicuous
coloration, and suggests that the acquisition of this protection may
have conferred a survival advantage. This places apple snail eggs
in the ‘‘winning side’’ of the predator-prey arms race.
Conclusions
In this work we demonstrate that the oral administration of
apple snail egg PVF promotes alterations in rat growth rate and
small intestine morphophysiology for short periods, whereas
prolonged exposure to the toxic PVF induces an adaptation
overcoming the antinutritonal effects. This defense has not been
reported in animals before, but resembles those well established for
plant seeds.
The severe effects of PVF on digestive tract adds another line of
defense to the previously reported suite of biochemical defenses of
apple snail eggs. This study helps to explain the near absence of
predators and their successful establishment in invaded areas.
Acknowledgments
MSD is member of Carrera del Investigador CICBA, Argentina. HH and
EJG are members of Carrera del Investigador CONICET, Argentina. We
Figure 6. Cytotoxic effect of PVF on Caco-2 cells monolayers evaluated using MTT assay. Values are the mean 6SD (n = 6).
doi:10.1371/journal.pntd.0002961.g006
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thank Patricia Sarmiento from the SEM service of Facultad de Ciencias
Naturales y Museo, UNLP for her invaluable help optimizing intestine
sample preparation and M. Yanina Pasquevich for her help in the cell
culture experiments.
Author Contributions
Conceived and designed the experiments: MSD HH. Performed the
experiments: MSD PEF. Analyzed the data: MSD PEF EJG HH.
Contributed reagents/materials/analysis tools: HH. Wrote the paper:
MSD PEF EJG HH.
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