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873
Comparing the freezing susceptibility of thirdinstar larvae of Gnorimus variabilis (Cetoniidae:
Trichiinae) from three distant geographical regions
D. Renault, P. Vernon, and G. Vannier
Abstract: We compared the freezing susceptibility of three populations of Gnorimus variabilis (L., 1758) (Coleoptera:
Cetoniidae) sampled from three distant locations in France. To separate the effects of habitat quality from those of genetics, we reared imagos from all field populations in a common garden experiment until the emergence of the thirdinstar larvae. The local climate appeared to determine the cold hardiness of the third-instar larvae, which live exclusively in cold seasons. The geographical location had an effect on the frost resistance (supercooling point) of the thirdinstar larvae of G. variabilis. We found no significant differences between the supercooling points of the populations
originating from separate latitudes but from the same longitude, Sare (–10.9 ± 4.1 °C) and Husson (–11.5 ± 3.8 °C).
Nonetheless, significant differences occurred between the larvae originating from the same northern latitude but from
separate longitudes, Husson and La Robertsau (–17.8 ± 2.9 °C). It is the first time that a highly significant difference
in the supercooling point of one stage within a single species has been observed along geographical gradients. Moreover, 19 of the 24 larvae originating from La Robertsau were alive after they were kept for 22 h at –10 °C compared
with only 7 of the 24 larvae originating from Sare. When the three populations were reared in the laboratory under the
same experimental conditions, the differential cryoresistance was preserved. It is likely that the greater freezing resistance found in the third-instar larvae of G. variabilis from La Robertsau could have a genetic component.
Résumé : Nous avons comparé la résistance à la congélation de trois populations de Gnorimus variabilis (L., 1758)
(Coleoptera : Cetoniidae) récoltées en France dans trois régions distinctes. Afin de séparer les effets liés aux caractéristiques de l’habitat de ceux inhérents aux individus, les insectes des trois populations naturelles ont été élevés à Brunoy
dans un jardin commun jusqu’à l’émergence du troisième stade larvaire. La localisation géographique génère des différences de résistance au froid chez les larves de troisième stade de G. variabilis. Le climat local semble jouer un rôle
déterminant dans la résistance à la congélation du troisième stade larvaire qui vit exclusivement durant les périodes hivernales. La température de cristallisation est similaire chez les souches prélevées à différentes latitudes, mais à une
même longitude, Sare (–10,9 ± 4,1 °C) et Husson (–11,5 ± 3,8 °C). A l’inverse, une différence significative a été obtenue entre les larves originaires de la même latitude nordique, mais de longitudes distinctes, Husson et La Robertsau
(–17,8 ± 2,9 °C).De telles différences significatives de résistance à la congélation en fonction d’un gradient géographique entre des stades de développement identiques d’une même espèce sont démontrées pour la première fois.
D’autre part, 19 des 24 larves originaires de La Roberstau étaient en vie après avoir été maintenues 22 h à –10 °C
contre seulement 7 des 24 larves originaires de Sare. Bien que les trois souches aient été soumises aux mêmes conditions climatiques lors de leur élevage, des différences de résistance à la congélation persistent. Nous pouvons donc
nous demander si la résistance à la congélation du troisième stade larvaire de G. variabilis originaire de La Robertsau,
qui est supérieure à celle des autres souches, possède un support génétique.
Renault et al.
Introduction
Temperature is generally of prime importance in the distribution and abundance of ectothermic species, even if
many other stressing factors may define the limits of the
geographical ranges (Chown and Clarke 2000). Several studies have attempted to couple the geographical distribution
879
and thermal tolerance of ectothermic species, as well as to
determine how temperature could have an effect on the distribution of species along latitudinal and altitudinal gradients
in either plants or insects (Hill et al. 1999; Shafer et al.
2001; Pither 2003). Thus, the northward distribution of fire
ants from the USA was restricted by the low temperatures
that the fire ants may endure during the winter (Bird and
Received 7 October 2003. Accepted 28 April 2004. Published on the NRC Research Press Web site at http://cjz.nrc.ca on 5 August
2004.
D. Renault1 and P. Vernon. Unité mixte de recherche (UMR) 6553 – Centre national de la recherche scientifique (CNRS),
Université de Rennes-I, Station biologique, 35380 Paimpont, France.
G. Vannier. UMR 5176 CNRS, Muséum national d’histoire naturelle, Laboratoire d’écologie générale, 4 avenue du Petit-Château,
91800 Brunoy, France.
1
Corresponding author (e-mail: [email protected]).
Can. J. Zool. 82: 873–879 (2004)
doi: 10.1139/Z04-062
© 2004 NRC Canada
874
Hodkinson 1999), whereas there was no significant differences between the cold hardiness of congeneric species of
the genus Craspedolepta Enderlein, 1921 from distant geographical locations (i.e., UK and Norway) (Francke et al.
1986).
The supercooling point (SCP) temperature of a wide range
of insect species has been studied (Block 1982; Sømme
1982; Zachariassen 1985; Vannier 1986, 1994; Vannier and
Verdier 1988; Sinclair 1999; Salin et al. 2000), and large
range or hemisphere-related differences in insect responses
to cold have been observed (Addo-Bediako et al. 2000;
Sinclair et al. 2003a). Although some studies have concluded that SCP temperatures were not indicative of cold
hardiness (e.g., Turnock et al. 1983; Bennett and Lee 1989;
Kostal et al. 2001), this parameter was used in many studies
(Sinclair 1999; Block and Worland 2001). It is assumed that
freezing was the cause of insect death and that the insects remained alive and were able to recover as long as the SCP
temperature was not reach (Renault et al. 2002). On the
other hand, survival experiments included the cumulative effects of the cold, which may lead to sublethal injury and
prefreezing mortality.
Various acclimation regimes may have great effects on the
SCP temperatures (Salin et al. 2000; Renault et al. 2002)
and may obscure the differences inherent to the populations.
In Delia radicum (L., 1758) (Diptera: Anthomyiidae), a Canadian population was more cold hardy than a British one,
as its SCP temperature was slightly lower (Block et al. 1987;
Turnock et al. 1990). However, the two populations were not
sampled during the same year and the experimental procedure used to measure the SCP temperatures was slightly different (Block et al. 1987, Turnock et al. 1990). Up to now,
no study has investigated the freezing susceptibility of postembryonic stages within a single species across a latitudinal
gradient. In Japan, Shintani and Ishikawa (1999) described
the supercooling ability of eggs of an alien longicorn beetle,
Psacothea hilaris (Pascoe, 1857), across a latitudinal gradient (from 24°N to 40°N) and they found no significant differences among populations. In China, Jing and Kang (2003)
confirmed this geographical stability by measuring the SCP
temperatures of eggs of Locusta migratoria L., 1758 belonging to different populations (from 18°N to 41°N).
We compared the freezing susceptibility of three populations of Gnorimus variabilis (L., 1758) (Cetoniidae) collected from three distinct locations in France: Sare (Basque
Land), Husson (Normandy), and La Robertsau (Alsace).
Imagos were then reared in a common garden experiment to
allow the three populations to experience the same acclimation regime. The Cetoniidae family is divided into main
subfamilies: the Trichiniiae, which is freezing susceptible at
all larval developmental stages, except for Osmoderma eremita (Scopoli, 1763), and the Cetoniinae, which is freezing
tolerant at all larval developmental stages, except for newly
hatched first-instar larvae (Vernon et al. 1996; Vernon and
Vannier 2001). In the genera Gnorimus (Le Pelletier de Saint
Fargeau and Audinet-Serville, 1828) and Trichius Fabr.,
1775, the life cycle lasts 1 year and all the developmental
stages, from eggs to adults, are freezing susceptible (yearround susceptible species as defined in Vernon and Vannier
2002). As in the endangered Gnorimus nobilis (L., 1758)
(Ranius and Jansson 2000), the third-instar larvae of
Can. J. Zool. Vol. 82, 2004
Fig. 1. Localities in France where the imagos of Gnorimus
variabilis were collected in May 1997: (A) Sare (Basque Land),
(B) Husson (Normandy), and (C) La Robertsau (Alsace). Insects
were then reared at (D) Brunoy.
G. variabilis are found exclusively during autumn and
winter, but it is in January that they exhibit the greatest
supercooling capacity when their gut is empty. Better
knowledge of the cold hardiness of this species could be
helpful in determining whether the geographical distribution
of these populations of G. variabilis could be related to their
cryothermal tolerance.
Materials and methods
Collection sites and insects
Three adult pairs of G. variabilis were collected in May
1997 from Sare (Basque Land), Husson (Normandy), and La
Robertsau (Alsace); three adult pairs were collected from
each location. In the Basque Land, imagos were collected in
the domanial forest of Sare from the mould inside old cavities near ground level of old chestnut trunks. In Husson
(Normandy), imagos were collected from hedged farmland
within dead branches scattered on the soil surface or in hollow trunks of old trees (oaks, chestnuts). In Alsace, imagos
were collected from the hollow trunks of old willows in the
communal forest of La Robertsau.
Husson (48°34′N, 0°53′W; altitude 115 m), Sare (43°16′N,
1°36′W; altitude 150–300 m), and La Robertsau (48°37′N,
7°48′E; altitude -130 m) are shown in Fig. 1. Sare and
Husson are 600 km apart; Sare and La Robertsau are
1200 km apart; and Husson and La Robertsau are 800 km
apart. Husson and La Robertsau are on the same latitude and
Husson and Sare are almost on the same longitude (Fig. 1).
The mean temperature in January 1998 under a thermometer screen was +8.1 °C (minimal temperature mean =
+5.1 °C; absolute minimal temperature during the last
© 2004 NRC Canada
Renault et al.
875
Table 1. Fresh mass, dry mass, water mass, water content, and supercooling point temperatures of third-instar
larvae of Gnorimus variabilis originating from three distinct locations (Sare, Husson, and La Robertsau).
Fresh mass (g)
Dry mass (g)
Water mass (g)
Water content (%)
Supercooling point temperature (°C)
Sare (N = 20)
0.9242±0.094a
0.2777±0.048a
0.6465±0.071a
238.8±38.3a
–10.9±4.1a
Husson (N = 30)
0.8346±0.1189b
0.2390±0.0687b
0.5956±0.0678b
267.2±71.6a
–11.5±3.8a
La Robertsau (N = 31)
0.9269±0.0635a
0.2748±0.0286a
0.6521±0.0524a
239.6±31.3a
–17.8±2.9b
Note: Values are means ± SD and those followed by different letters across a row are significantly different (P < 0.05).
10 years = –9.0 °C) in Sare, whereas it was + 4.8 °C (minimal temperature mean = +1.7 °C; absolute minimal temperature during the last 10 years = –19.4 °C) in Husson; these
two locations are under oceanic influence. The temperature
mean recorded in January 1998 was colder in La Robertsau,
which is characterized by a more typical continental climate
with a mean temperature of about +0.7 °C (minimal temperature means = –1.9 °C; absolute minimal temperature during
the last 10 years = –23.4 °C).
Common garden experiment
To separate the effects of habitat quality from those of genetics, we reared imagos from all field populations in a common garden experiment. From May 1997 to March 1998,
insects were reared at Brunoy (48°71′N, 2°49′E; south of
Paris, Ile de France, France) under a shelter that was exposed to the fluctuations of the local temperature regime.
Males and females were kept in plastic cylindrical boxes (diameter 10 cm, height 30 cm) that were filled with mould and
in which a new generation was produced within 1 year.
Third-instar larvae were removed from their breeding containers in January 1998 by sieving the mould. Larvae
exhibited the same morphology and the same chaetotactic
arrangement. They were then treated individually for weighing and testing in the experiments. The mean air temperature
during January 1998 at Brunoy was +5 °C.
Mass parameters
Fresh mass and dry mass of third-instar larvae of
G. variabilis were recorded with a SARTORIUS microelectrobalance (readibility 0.1 µg) and converted to grams.
The total water content was determined by subtracting the
dry mass from the fresh mass of each larva, which was obtained after drying the insects at 60 °C for 3 days and then
in a desiccator with potassium hydroxide as drying agent for
6 days. Water contents were calculated as a percentage of
the dry mass.
Determination of SCP temperatures
Cold hardiness was assessed by measuring the SCP temperature (i.e., the lowest temperature attained prior to the release of the latent heat of fusion). An automated cryoscope
designed by Salin et al. (2003) was used; the cryoscope consisted of two calibrated thermocouples (thermocoax and
chrome-alumel, ø = 0.25 mm) positioned inside a vertical
copper tube with a fixed temperature gradient (from +4 to
−40 °C). The thermocouples were moved along the tube by a
computer-controlled motor so that the larvae were cooled at
a constant rate of 0.5 °C/min instead of the conventional rate
of 1.0 °C/min (Salt 1961). Each larva was carefully wrapped
in aluminium foil and placed in contact with the thermocouple tip to avoid perforation and injury. The decrease in
temperature was continuously recorded graphically and the
SCP temperature with an accuracy of ±0.1 °C was discerned
from the falling temperature curve as an upward peak (rebound), which was caused by the heat of crystallization.
Survival
To investigate the impact of low temperatures on the survival duration of G. variabilis, third-instar larvae obtained
from the imagos collected in Sare (N = 24) and in La
Robertsau (N = 24) were kept at –10 °C for 22 h in January
1998. Three categories of larvae were then observed (Vernon
and Vannier 2001): (1) alive, whereby after 24 h the larvae
buried themselves in the mould; (2) moribund, whereby the
larvae remained motionless on the surface of the mould and
died about a week later; and (3) dead, whereby the cold
killed the larvae and they decomposed rapidly. Survival was
estimated as the number of alive larvae.
Statistical analysis
Values are given as the mean ± SD. ANOVAs were carried
out after testing the data for normality. The statistical analyses were performed using Minitab™ for Windows version
13 (Minitab Inc. 2000).
Results
Mass parameters
Fresh mass, dry mass, water mass, and water content are
presented in Table 1. The ANOVA indicated that the geographical location had a significant impact on the fresh mass
(P < 0.001), dry mass (P < 0.01), and water mass (P <
0.001) of third-instar larvae of G. variabilis, but not on water content (P = 0.15). Larvae originating from Husson exhibited the lowest fresh mass (P < 0.01), dry mass (P <
0.05), and water mass (P < 0.05) (Table 1). No significant
differences were found between the larvae originating from
Sare and La Robertsau for fresh mass, dry mass, water mass,
and water content. No significant differences occurred in
water contents between the three populations (Table 1).
SCP temperatures
The geographical location had a significant effect on the
freezing resistance of third-instar G. variabilis larvae (F[2] =
32.38, P < 0.001). The population originating from La
Robertsau exhibited significantly lower SCP temperatures
(–17.8 ± 2.9 °C) than the other populations (Husson =
–11.5 ± 3.8 °C and Sare = –10.9 ± 4.1 °C; P < 0.001)
© 2004 NRC Canada
876
Can. J. Zool. Vol. 82, 2004
Fig. 2. Supercooling point temperature (SCP) distributions of third-instar larvae of G. variabilis originating from (A) Sare (N = 20),
(B) Husson (N = 30), and (C) La Robertsau (N = 31).
(Table 1). The distribution of SCP temperatures (relative frequencies) according to the geographical location is shown in
Figs. 2A–2C. It showed that no larvae originating from Sare
and Husson had SCP temperatures lower than −18.0 °C,
whereas more than half of the larvae from La Robertsau had
SCP temperatures lower than –18.0 °C.
© 2004 NRC Canada
Renault et al.
Survival
The results indicated that 7 of the 24 larvae originating
from Sare were alive after they were kept for 22 h at –10 °C,
whereas 19 of the 24 larvae originating from La Robertsau
were alive when kept under the same conditions.
Discussion
Body size generally shows large geographical variations
within species. It is generally argued that the body size
of an insect tends to increase according to latitude and (or)
altitude (Ray 1960), even if the converse can be found
(Mousseau 1997). In this study, we found no significant difference in body size between the samples originating from
separate latitudes but from the same longitude (Sare and
Husson). However, a significant variation occurred between
the samples originating from the same latitude but from separate longitudes (Husson and La Robertsau), emphasizing
that several factors might influence the body size of ectotherms. It is already argued that body-size differences across
geographical regions are caused by a combination of abiotic
and biotic factors that may vary along clines (Atkinson
1994; Mousseau 1997).
Geographical locations produced differential effects on
the freezing resistance of third-instar larvae of G. variabilis.
Larvae issued from the imagos collected at La Robertsau
had a greater cryoresistance than larvae originating from
Sare. However, the ability of G. variabilis to survive cold
exposure is reduced when compared with another Trichiinae
species, O. eremita, which is freezing tolerant (Vernon and
Vannier 2001). Third-instar larvae of O. eremita originating
from the Fontainebleau forest (south of Paris, France) and
kept at the same experimental conditions (–10 °C) as thirdinstar larvae of G. variabilis were frozen and were still alive
after 12 days. Of these third-instar larvae of O. eremita,
40% were still alive after being kept 24 days at –10 °C
(G. Vannier, unpublished data). Moreover, it is interesting to
point out that the supercooling limit has some relevance in
G. variabilis because of its trend among the populations that
matches the cold tolerance of this species. Hodkova and
Hodek (1997) and later Kalushkov and Nedved (2000) also
found a good correlation between the values of survival at
low temperatures and the SCP temperatures in the fire bug,
Pyrrhocoris apterus (L., 1758).
Insect thermal tolerance is frequently related to latitude
(Leather et al. 1993). In this study, we found no significant
difference between the SCP temperatures of third-instar larvae of G. variabilis originating from Sare and Husson (i.e.,
adults that were collected at the same longitude but at
separate latitudes). On the other hand, significant differences
occurred between larvae from adults collected at the same
latitude but at separate longitudes (Husson and La Robertsau).
Addo-Bediako et al. (2000) reported that the SCP temperature tends to decrease towards the equator, but these authors
also pointed out an increase in the variation of thermal tolerance with latitude. The extent of the thermal tolerance variation at a given latitude differs considerably (Chown 2001). It
was suggested that it could be related to a latitudinal increase in the climatic variability which promotes or reduces
the risks of cold-temperature injuries encountered by insects
(Bale 1987; Addo-Bediako et al. 2000).
877
The local climate and the microclimate appear to have a
significant effect on the freezing susceptibility of G. variabilis. It is widely recognized that insect thermal tolerance
varies according to the environmental conditions that the
individuals endure (Chown 2001; Sinclair et al. 2003b).
However, such interactions within a single species and contrasting microclimates have rarely been recorded. Variations
in the SCP temperatures are already related to the geographical distribution of the species, but such studies are mostly
performed by comparing the cold hardiness of the insect
across a large geographical gradient between families, between species (Addo-Bediako et al. 2000), and between congeneric species (Bird and Hodkinson 1999). It has even been
reported that most of the variance in SCP temperatures was
partitioned equally among families and among genera, but
least among species (Addo-Bediako et al. 2000; Chown
2001). This study is the first time such highly significant differences in the SCP temperatures have been demonstrated
between identical developmental stages within a single species and on such a relatively short geographical distance.
The stability of the SCP temperatures of eggs was found in
different populations of the longicorn beetle (Shintani and
Ishikawa 1999) and of the migratory locust (Jing and Kang
2003) across much more considerable distances.
Although the larvae were reared from eggs laid by adults
at Brunoy, and therefore, had experienced an identical acclimation regime, there existed a significant difference in the
mean SCP temperatures among the three populations. These
results suggest that the observed difference in the frost resistance of these populations could not be related to a simple
acclimation process and appear to be inherent to the populations. Further studies should aim to determine whether there
are physiological differences between these populations. The
amount of antifreeze agents accumulated, such as polyols,
sugars, and amino acids, should be analyzed. Such compounds are found to significantly influence the supercooling
ability and the survival at low temperatures (Danks 1996;
Kostal et al. 2001; Renault et al. 2002).
As climate was found to have a predominant role in adaptive divergence and speciation (Orr and Smith 1998), could
the greater freezing resistance found in the larvae of G. variabilis that originated from La Robertsau be the result of the
underlying genetic differences among the populations?
It might be possible that there is some form of lowtemperature resistance in the larvae which is triggered by
the conditions experienced by the adults. Repeated crossbreeding between the populations could be helpful in elucidating the cryothermal resistance differences and the genetic
component responsible for the differences. Lee (2002) already reported that the genotype × environment interaction
could promote speciation. Phylogenetic and molecular genetic studies should measure the genetic distance between
these populations, and therefore, contribute to solve this evolutionary ecophysiological question.
Acknowledgements
Thanks are due to J.M. Luce who collected the insects in
the field and reared them at Brunoy, R. Botalla who devised
our automated cryoscope, and N. Vannier who filed our data.
© 2004 NRC Canada
878
We thank two anonymous reviewers for their very helpful
comments on the manuscript.
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