Journal of
The Malacological Society of London
Molluscan Studies
Journal of Molluscan Studies (2014) 80: 62–66. doi:10.1093/mollus/eyt042
TOLERANCE TO LOW TEMPERATURE AND DESICCATION IN TWO
INVASIVE APPLE SNAILS, POMACEA CANALICULATA AND
P. MACULATA (CAENOGASTROPODA: AMPULLARIIDAE),
COLLECTED IN THEIR ORIGINAL DISTRIBUTION AREA
(NORTHERN AND CENTRAL ARGENTINA)
KAZUHIRO YOSHIDA 1, KEIICHIRO MATSUKURA 1,
NESTOR J. CAZZANIGA 2 AND TAKASHI WADA 1
2
1
NARO Kyushu Okinawa Agricultural Research Center, Suya 2421, Koshi, Kumamoto 861-1192, Japan; and
Departamento de Biologia, Bioquimica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahia Blanca, Argentina
Correspondence: T. Wada; e-mail: [email protected]
(Received 15 March 2013; accepted 8 October 2013)
ABSTRACT
We collected the invasive apple snails, Pomacea canaliculata and P. maculata (formerly, P. insularum), in
their native endemic regions of Argentina and investigated tolerances to cold and desiccation, in order
to ascertain any difference between the species and to compare these traits with those reported for apple
snails in invaded areas of Asia. All of the four studied populations of P. canaliculata and P. maculata
showed enhanced cold tolerance after cold acclimation, as reported for P. canaliculata in invaded areas.
Two populations of P. canaliculata and one population of P. maculata that had been suggested to be
hybrid based on nuclear gene structures were found to have a tolerance to cold temperature approximately similar to that reported for P. canaliculata in Japan. However, the remaining population of
P. maculata, which had been suggested to be the pure line, was less tolerant to cold. The same population
of P. maculata, collected in a permanent lake, was much less tolerant to desiccation than the other three
Pomacea populations, which were collected from ephemeral ponds. Two probable hybrid populations of
P. canaliculata and P. maculata showed desiccation tolerance that was intermediate between the pure lines
of both species. On the basis of these results, in addition to probable hybridization between the two
species, we suggest that P. canaliculata snails might have a better ability to colonize Asian paddy ecosystems than P. maculata.
INTRODUCTION
South American apple snails of the genus Pomacea Perry, 1810,
were introduced in the 1980s into Asia for human food and the
aquarium trade (Mochida, 1991; Litsinger & Estano, 1993).
They subsequently became major pests of rice after invasion into
the environment (Halwart, 1994; Wada, 2004; Hayes et al., 2008).
As it is difficult to identify apple snails on the basis of morphological characteristics, their taxonomy has been rather confused
for a long period, not only in the countries they have invaded but
also in their original South American habitats (Wada, 1997;
Cowie, Hayes & Thiengo, 2006). Molecular biology has made it
possible to identify apple snails more precisely (Rawlings et al.,
2007; Hayes et al., 2008; Hayes, Cowie & Thiengo, 2009a).
Mitochondrial DNA analysis of samples collected in various
countries in Asia and South America have revealed four species of
South American apple snails in Asia: P. canaliculata, P. maculata
(formerly, P. insularum; Hayes et al., 2012), P. scalaris and P. diffusa
(Hayes et al., 2008). Among them, P. canaliculata and P. maculata
are widely distributed in rice ecosystems in Southeast and East
Asia. However, P. canaliculata has become distributed more widely
than P. maculata. These two species, with morphologically very
similar characteristics, have become a serious threat to rice production and ecosystems in Asia (Carlsson, Bronmark & Hansson,
2004; Hayes et al., 2008). They are distinct species according to
mitochondrial DNA (COI) analysis (Rawlings et al., 2007; Hayes
et al., 2008; Matsukura et al., 2008a) and shell, anatomy and egg
morphology (Hayes et al., 2012). Recently, Matsukura et al.
(2013) investigated nucleotide sequences of nuclear elongation
factor 1-alpha and suggested genetic exchanges (hybridization)
between the two species in invaded Asia and in their native
endemic region of Argentina.
Both P. canaliculata and P. maculata occur in temperate Japan,
one of the northernmost countries invaded by Pomacea species in
# The Author 2014. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved
COLD AND DESICCATION TOLERANCE OF POMACEA SPP.
Asia (Matsukura et al., 2008a). However, P. canaliculata accounts
for the majority of the apple snails, while P. maculata is found
only in the southern islands of Okinawa and some areas of
Honshu. Cold weather in winter apparently limits the expansion
of P. canaliculata in Japan (Ito, 2002; Wada & Matsukura, 2007;
Yoshida et al., 2009). Pomacea canaliculata in Japan shows seasonal
adaptation; both cold acclimation and dry conditions enhance its
cold tolerance during winter (Matsukura & Wada, 2007; Wada
& Matsukura, 2007). Snails whose cold tolerance has been
enhanced through cold acclimation also show increased desiccation tolerance, and both forms of tolerance are physiologically
linked (Wada & Matsukura, 2011). Physiologically cold-tolerant
snails have increased body glycerol content and decreased glycogen content (Matsukura et al., 2008b). Essentially, the snails are
freezing-intolerant but, more importantly from an ecological
viewpoint, they are killed in fields by exposure for considerable
periods to mild cold above freezing temperatures (Matsukura
et al., 2009).
Among Pomacea species living in South America, the distribution of P. canaliculata extends furthest south (Martin, Estebenet
& Cazzaniga, 2001) and few studies have reported the influence
of cold on its distribution. Low temperature has been reported
to affect various snail activities, including feeding and crawling
(Seuffert, Burela & Martı́n, 2009). However, the distribution of
the southernmost population of P. canaliculata is reportedly constrained by salty, alkaline, poorly-vegetated habitats with high
desiccation risk, rather than by low temperatures (Martin et al.,
2001). A recently introduced population in northern Patagonia
was reported by Darrigran, Damborenea & Tambussi (2011).
We collected samples from four populations of Pomacea snails
in their native range of northern and central Argentina in 2010.
Based on mitochondrial molecular analysis they were P. canaliculata or P. maculata; their genetic backgrounds (including a
nuclear gene) were described by Matsukura et al. (2013). In the
present study we report tolerances of these snails to environmental stress, in particular to cold temperature and desiccation. In
the experiments we used juveniles with intermediate shell size,
because this is the most common stage during winter in temperate regions (Watanabe et al., 2000). We compared these data
between the species and with those already reported for populations in invaded areas (Wada & Matsukura, 2007, 2011) and
discuss the implications of our findings for invasive snail biology.
rearing room under a 16L:8D photoperiod at 258C, which is
considered to be the optimal water temperature for juvenile
growth and survival of P. canaliculata (Seuffert & Martin, 2013).
The snails were provided eggplant ad libitum as basic food, a few
grains of carp diet (0.5 –1.2 g depending on snail sizes) and a
small amount of oyster shell powder (c. 1 g) as a source of
calcium, approximately twice a week. When the majority of
juveniles had reached a shell height of 7.5 –15.0 mm (about a
month after hatching), only the snails that had attained this
shell height were selected for experiments. Pomacea canaliculata
snails of this size class exhibited the highest cold tolerance
(Wada & Matsukura, 2007).
Cold tolerance test
We examined snails for two types of cold tolerance: those
without cold acclimation and those after cold acclimation. Cold
tolerance in each type of snails from the four populations was
examined at the same time. During the cold-acclimation phase,
snails wrapped with moist paper were confined in a plastic container (31 26 cm, 10 cm deep). The temperature was reduced
by 58C per week for 3 weeks, from 258C to 108C, and then maintained at 108C for 25 d. Before and after this cold acclimation,
we examined the survivorship of snails exposed to 08C for
various durations in the following way. Forty snails from each
population, wrapped with moist paper, were confined in a
plastic container (11 9 cm, 4 cm deep). They were kept at 08C
for 2, 5 and 10 d and then tested for mortality as described
below.
Desiccation tolerance
Snails without cold acclimation were used in this experiment,
which was also carried out at the same time for all four populations. About 280 snails randomly selected from each population
were divided into seven groups. About 40 (39–41) snails in each
group were confined in round plastic containers (10 cm in diameter, 4 cm deep) without a lid, in the bottom of which was
placed a dry filter paper. The plastic containers with the test
snails were kept in a rearing room at 26.48C (+1.08C) under a
16L:8D photoperiod. Relative humidity in the room varied
daily between 38% and 78%. Although we could not fix the humidity at a constant level, this fluctuation did not influence comparison of desiccation tolerance among populations, because all
populations were exposed to the same humidity conditions. One
out of the seven groups was taken at random from the rearing
room on day 1, 3, 6, 9, 12, 24 and 48 after the start of the experiment, and examined for mortality.
MATERIAL AND METHODS
Snails used
We collected Pomacea snails in late November, 2010, at four
locations in Argentina: a pond in Catalinas Park, San Miguel
de Tucuman (26.8329558S, 65.1906398W; referred to as the
‘Tucuman’ population), a pond in Juan de Garay Park, Santa Fe
(31.6359688S, 60.7211468W; ‘Santa Fe’), the shore of Setubal
Lake, Santa Fe (31.74258S, 60.675288W; ‘Setubal Lake’) which
is located some 8 km distant from Juan de Garay Park, and a city
pond at Paseo del Bosque, La Plata (34.9103498S, 57.9372068W;
‘La Plata’).
According to mitochondrial COI gene sequences, the snails collected in Tucuman and La Plata were identified as P. canaliculata,
whereas the snails in Santa Fe and Setubal Lake were P. maculata
(Matsukura et al., 2013).
Mortality test
Survivorship of the snails was tested using the method described
previously by Wada & Matsukura (2007). In brief, snails were
immersed in tap water in a glass container and left at room temperature (21–288C) for 1 d. Snails that extruded the foot and/or
tentacles from the shell within 24 h were considered alive.
RESULTS
Cold tolerance
None of the snails in any of the populations without cold acclimation survived exposure to cold treatment at 08C for 5 d, and
only 3% of the snails on average survived exposure to 08C for
2 d (Table 1). After cold acclimation, all of the populations
showed enhanced cold tolerance. Survivorship was significantly
higher after exposure to 08C for both 2 d and 5 d in all the populations, except for the case of the Setubal Lake population
Rearing and tolerance experiments
Snails from each population were reared in a plastic tank
(c. 200-l), provided a cabbage and carp diet (Hikari Co.,
Japan) and egg masses were obtained from field-collected snails.
Hatched juveniles from four to six egg masses from each population were reared in an aquarium (72 l) with aeration in a
63
K. YOSHIDA ET AL.
Table 1. Survivorships of juvenile progeny of Pomacea species collected in Argentina, after various cold treatments.
Collection site
Species
Cold acclimation
0oC2d treatment
No. used
Live snails
0oC5d treatment
%
No. used
Live snails
0oC10d treatment
%
No. used
Live snails
%
0.0 a
Snails before cold acclimation
Tucuman
canaliculata
No
40
2
5.0 a
40
0
0.0 a
La Plat a
canaliculata
No
40
1
2.5 a
40
0
0.0 a
Santa Fe
maculata
No
40
2
5.0 a
40
0
0.0 a
Setubal Lake
maculata
No
40
0
0.0 a
40
0
0.0 a
Snails after cold acclimation
Tucuman
canaliculata
Yes
40
36
90.0 ab*
40
38
95.0 c*
40
0
La Plat a
canaliculata
Yes
40
31
77.5 a*
40
28
70.0 b*
40
0
0.0 a
Santa Fe
maculata
Yes
40
40
100.0 b*
40
40
100.0 c*
40
1
2.5 a
Setubal Lake
maculata
Yes
40
40
0
40
0
0.0 a
o
37
o
92.5 ab*
0.0 a
o
Snails were exposed to 08C for 2 d (0 C2d), 08C for 5 d (0 C5d) and 08C for 10 d (0 C10d), respectively. Asterisks (*) indicate that the survival rate was significantly
different from that of the partner snails before cold acclimation, according to Fisher’s exact probability test. Percentages followed by the same letter are not
significantly different at the 5% probability level, according to analysis of multiple comparisons of proportions (Zar, 1996).
Table 2. Survivorship (%) of the progeny of Pomacea species collected from Argentina, after exposure to desiccation.
Collection site
Species
Days after exposure to desiccation
1
3
6
9
12
24
48
17.9 a
Tucuman
canaliculata
97.5 a
97.5 ab
87.5 a
82.9 a
77.5 a
45.0 a
La Plat a
canaliculata
95.0 a
85.0 b
33.3 b
15.4 b
5.0 b
0c
0b
Santa Fe
maculata
100 a
100 a
82.5 a
68.3 a
65.0 a
17.5 b
0b
Setubal Lake
maculata
100 a
13 c
0c
0c
0b
0c
0b
Juvenile snails were tested without cold acclimation. Percentages followed by the same letter are not significantly different at the 5% probability level, according to
analysis of multiple comparisons of proportions (Zar, 1996).
exposed to 08C for 5 d, whose survivorship was zero. Pomacea
maculata from the Setubal Lake strain were less tolerant than the
other three populations, according to the result of exposure to
08C for 5 d. The survivorship of P. canaliculata from La Plata was
also significantly lower than that of snails collected at Tucuman
and Santa Fe. No snails, except for one acclimated snail collected at Santa Fe, survived exposure to 08C for 10 d.
the sequences of two clades (C and M), which represent
P. canaliculata and P. maculata, respectively, as reported by Hayes
et al. (2009a). However, some snails had both clade C and clade
M or the opposite clade sequences, suggesting genetic exchange
between snails of the two clades. They also tried experimental
mating between two species in the laboratory and succeeded in
producing fertile hybrid populations. With regard to the present
study, P. canaliculata collected in Tucuman and P. maculata collected in Setubal Lake had clade C and clade M sequences, respectively; these therefore represented pure lines of P. canaliculata
and of P. maculata. On the other hand, P. canaliculata collected in
La Plata had both clades or the opposite (clade M) sequences
and P. maculata collected in Santa Fe possessed the opposite
(clade C) sequence, suggesting that these snails were hybrids on
the basis of their gene structures (Matsukura et al., 2013). The
pure line of P. canaliculata (Tucuman) revealed the highest tolerance of cold and desiccation stresses and the pure P. maculata
(Setubal Lake) were the weakest. Interestingly, two hybrid
populations showed intermediate tolerance, except that the cold
tolerance of the hybrid P. maculata from Santa Fe was high,
similar to pure P. canaliculata.
The much lower desiccation tolerance of P. maculata collected
from Setubal Lake may be related to the habitat in which the
snails lived. A large water body like Setubal Lake has a much
smaller risk of desiccation than the small man-made ponds
from which the other three snail populations were sampled. It is
probable that snails inhabiting a more stable environment have
not evolved desiccation tolerance. No previous report has
highlighted the difference between the respective habitats of
P. maculata and P. canaliculata. However, P. maculata is often
observed in large rivers (Hylton-Scott, 1958; Bachmann, 1961;
N.T. Cazzaniga, personal observations), whereas P. canaliculata,
Desiccation tolerance
Pomacea canaliculata descendants from snails collected at
Tucuman showed the highest desiccation tolerance among the
four populations (Table 2). Eighteen per cent of the snails were
still alive at the end of the 48-d desiccation experiment. Pomacea
maculata from Santa Fe revealed the second highest tolerance
and they were significantly more tolerant to desiccation than the
other two Pomacea populations on any census day after day
3. Pomacea maculata collected at Setubal Lake showed poor desiccation tolerance, as all snails died after 6 or more days of desiccation. The order of desiccation tolerance among the populations
was similar to that recorded for cold tolerance. The populations
from the Tucuman and Santa Fe strains showed the highest tolerance to cold and desiccation, followed by the population from
the La Plata strain. The population from the Setubal Lake
strain exhibited the lowest tolerance to both stresses.
DISCUSSION
Matsukura et al. (2013) suggested hybridization between the two
Pomacea species in field populations in Asia and in the populations in Argentina used in this study. They investigated nucleotide sequences of nuclear elongation factor 1-alpha and found
64
COLD AND DESICCATION TOLERANCE OF POMACEA SPP.
although having a broader range of habitats, seems to be better
adapted to shallow, calm, water bodies. In fact, P. canaliculata
generally inhabits shallow, quiet, turbid environments in
southern Buenos Aires province (Martin et al., 2001). Also, in
Japan, shallow-water habitats reportedly favour overwintering
success for lake-inhabiting P. canaliculata (Ito, 2002). Therefore,
P. canaliculata may be better adapted than P. maculata to ephemeral environments with a high desiccation risk. It has been
observed that the operculum of P. canaliculata is more flexible
and provides a better seal than that of P. maculata (Bachmann,
1961; Hayes et al., 2012), supporting our hypothesis. We obviously need further studies to confirm this hypothesis, because we
examined only a limited number of populations in the present
study. In particular, research that separates the influence of
habitats on environmental stress from the influence of hybridization will be required.
After treatment for acquisition of cold acclimation, P. canaliculata collected in Argentina showed enhanced cold tolerance. The
same phenomenon has been reported for this species in invaded
areas of Asia (Wada & Matsukura, 2007, 2011). This indicates
that the character of this seasonal adaptation of P. canaliculata in
invaded areas was brought from the area of origin. After cold acclimation, both populations of P. maculata also showed increased
cold tolerance upon exposure to 08C for 2 d. This is the first
report to indicate that P. maculata, like P. canaliculata, possesses
the ability to acquire enhanced tolerance after exposure to cold
stress.
The two P. canaliculata populations (Tucuman and La Plata)
and the population of P. maculata collected in Santa Fe showed a
cold tolerance similar to that reported for P. canaliculata in Japan
(Matsukura & Wada, 2007; Wada & Matsukura, 2007). Since
cold tolerance in P. canaliculata depends on shell size (Syobu
et al., 2001; Wada & Matsukura, 2007), we used juvenile snails
that were almost the same size as those employed in previous
studies. After cold acclimation, most snails in the three populations survived cold treatment at 08C for 5 d, but almost none
survived treatment at 08C for 10 d. These figures are similar to
those reported for Japanese P. canaliculata. On the other hand,
P. maculata collected from Setubal Lake, which is considered to
be the pure line of this species, was apparently less tolerant to
cold (no survivors after exposure to 08C for 5 d). Pomacea maculata
( pure line) collected on an island in southern Japan also showed
lower cold tolerance than Japanese P. canaliculata (K.
Matsukura, unpubl.). Although P. maculata occurs sympatrically
with P. canaliculata in some regions of South America, its distribution extends further towards the north (i.e. into warmer
regions) in comparison with that of P. canaliculata (Hayes et al.,
2009a, 2012). Therefore, the original P. maculata population may,
in general, be less tolerant to cold weather than P. canaliculata,
probably due to the climates of their respective original areas in
South America.
Life history traits of alien species profoundly influence their colonization success in new habitats (Herborg et al., 2007; Hayes
et al., 2009b). The difference in tolerance to low temperature and
desiccation between the two Pomacea species, as suggested in this
study, probably influenced their colonization ability in invaded
regions. In this respect, P. canaliculata may be a better colonizer of
Asian ephemeral agro-ecosystems than P. maculata. The primary
habitat of Pomacea in invaded areas of Asia is paddy fields, where
submerged and dry conditions alternate according to rice cultivation and fallow periods. In this ephemeral environment, P. canaliculata, which has a higher tolerance to desiccation, may be a
better colonizer than P. maculata. In fact P. canaliculata populations
have become established in many more regions and countries in
Asia, including Japan, in comparison with P. maculata (Hayes
et al., 2008; Matsukura et al., 2008a). Of course, many other
factors may also influence the invasive process by both species
(e.g. fecundity, propagule pressure, timing, behaviour,
environmental management, etc.; Lockwood, Cassey &
Blackburn, 2005; Horgan, Stuart & Kudavidanage, 2012).
Nevertheless, our results contribute some evidence on one of the
influential traits for paddy field colonization by apple snails.
ACKNOWLEDGEMENTS
We sincerely thank Yoich Yusa, Mercedes Marchese and Jorge
Liotta for their help to collect snails. Thanks are due to
S. Gyoutoku for maintaining snail populations. N.J. Cazzaniga
is a staff researcher of CIC (Buenos Aires Province, Argentina).
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