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Biological Control 58 (2011) 83–95

Contents lists available at ScienceDirect

Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Review

Insect parasitoids cold storage: A comprehensive review of factors of variability
and consequences
Hervé Colinet a,⇑, Guy Boivin b
a
b

Earth and Life Institute ELI, Biodiversity Research Centre BDIV, Catholic University of Louvain, Croix du Sud 4-5, B-1348 Louvain-la-Neuve, Belgium
Centre de Recherche et de Développement en Horticulture, Agriculture et Agroalimentaire Canada, 430 Boulevard Gouin, Saint-Jean-sur-Richelieu, Québec, Canada J3B 3E6

a r t i c l e

i n f o

Article history:
Received 2 March 2011
Accepted 21 April 2011
Available online 28 April 2011
Keywords:
Cold storage
Parasitoids
Low temperature
Phenotypic plasticity
Fitness

a b s t r a c t
Storage at low temperature is a valuable method for increasing the shelf-life of natural enemies such as
insect parasitoids. Cold storage is usually performed under sub-optimal temperatures, and therefore it is
generally associated with major fitness costs. Tolerance to cold storage is a very plastic trait influenced by
a wide range of endogenous (biotic) and exogenous (abiotic) factors experienced before, during, or after
cold exposure. In fact, every hierarchical level from inter-species to inter-individuals shows a high plasticity in the response to cold exposure. Mortality represents the ultimate level of a range of sub-lethal
perturbations accumulating during chilling. Even if individuals remain alive after cold storage, a reduction of several fitness-related traits may be observed directly, later in development or even in the next
generation. The present review focuses on cold storage of insect parasitoids. We first consider the
genotypic-based plasticity in cold storage tolerance and the complex network of endogenous and exogenous factors affecting the phenotypic plasticity in cold storage tolerance. We also summarize and examine the wealth of fitness-related traits affected by cold storage in parasitoids. This review provides a
comprehensive list of documented factors that must be taken into account when designing cold storage
protocols.
Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction
Mass production of beneficial insects has long been considered
necessary for biological control programs, especially those based
on augmentative releases (van Lenteren and Tommasini, 2002).
The major obstacle to the successful implementation of inundative
releases is the difficulty and cost of rearing beneficial insects in
large numbers for mass release at the appropriate time. Unlike pesticides, most insects used in biological control programs have a relatively short shelf-life, so they must be produced shortly before
they are used. The development of efficient storage methods can
reduce the cost of biological control by spreading the production
period over several months.
Storage at low temperature has proved to be a valuable method
for increasing the shelf-life of natural enemies and to provide a steady and sufficient supply of insects for biological control programs.
Cold storage also allows synchronized field releases of natural enemies during the critical stages of pest outbreaks (McDonald and
Kok, 1990; Venkatesan et al., 2000). Sterile insect technique
programs (SIT) also require storage of mass-reared insects during
⇑ Corresponding author. Present address: UMR CNRS 6553, Bât 14A, Université de
Rennes1, 263 Avenue du Général, Leclerc CS 74205, 35042 Rennes Cedex, France.
Fax: +33 (0)2 23 23 50 26.
E-mail address: herve.colinet@uclouvain.be (H. Colinet).
1049-9644/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biocontrol.2011.04.014

the processes of collection, distribution and release. The use of cold
storage is not limited to industrial rearing of beneficial insects, it is
also useful for maintaining insect colonies under laboratory conditions for research purpose. Moreover, insect rearing for pet food, fish
bait, forensic indicators and the rescue of endangered species may
all profit from advances in cold storage technology (Leopold, 2007).
Insect parasitoids are used extensively in biological control programs. Because of their scientific and economic interest, studies on
cold storage of parasitoids started over 75 years ago (King, 1934;
Schread and Garman, 1934; Hanna, 1935) and there is a large literature dealing with this topic. Leopold (1998) has provided a comprehensive review of insect cold conservation, however, since then,
a large number of additional species and experimental modalities
have been explored. Insect cryo-preservation techniques will not
be considered here as they have few applications for parasitoid
cold storage (see Leopold, 2007 for review).
Each parasitoid species has a particular evolutionary history
that shapes its current adaptation. Not surprisingly, much variability is found in the capacity of parasitoid species to tolerate low
temperature making any taxonomic generalization unreliable
(Leopold et al., 1998). In addition to this genotypic-based plasticity,
variability in cold tolerance can be partitioned at a number of phenotypical levels (Hawes and Bale, 2007). In fact, every hierarchical
level from inter-species to inter-individuals shows a high plasticity
in the response to cold exposure.

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H. Colinet, G. Boivin / Biological Control 58 (2011) 83–95

To reduce development or metabolic rate for storage purposes,
beneficial insects are usually placed under sub-ambient temperatures generally above 0 °C. Even at non-freezing temperature, the
cold-induced extension of insect shelf life is generally associated
with major fitness costs (van Baaren et al., 2005, 2006; Chown
and Terblanche, 2006; Hance et al., 2007). Mortality represents
the ultimate level of a range of sub-lethal perturbations accumulating during chilling. However, reduction of fitness-related traits
in surviving individuals can be observed immediately after storage,
later in development or even in the next generations. The present
review first considers the complex network of endogenous and
exogenous factors that affect the phenotypic plasticity in cold storage tolerance. The second part summarizes and examines the fitness traits that are affected by cold storage in parasitoids.
2. Genotypic variability
There is a large interspecific variability in cold storage tolerance,
even between closely-related sibling species (Nakama and Foerster,
2001; Rundle et al., 2004; Lopez and Botto, 2005; Foerster and Doetzer, 2006; Colinet and Hance, 2010). Such variation in cold storage
tolerance was found between closely-related species of Trichogramma sp. (Jalali and Singh, 1992; Kumar et al., 2005) and between five
sympatric Aphidiinae species (Colinet and Hance, 2010). Cold storage tolerance may also show significant inter-populations variability as observed between four populations of Trichogramma chilonis
(Ishii) collected in three different districts of India (Khosa and Brar,
2000) and between six Chinese populations of Trichogramma dendrolimi (Matsumura) (Shi et al., 1993). Geographical origin can have
a strong influence on insect cold tolerance. Because of specific climatic adaptations, temperate species/populations are expected to
be better adapted to low temperature than their counterparts from
tropical lowlands (Chen et al., 1990; Gibert et al., 2001). Geographical gradients in climate should translate into biological gradients,
and clines in life history traits related to cold tolerance are observed
as in cold stress resistance of Drosophila melanogaster (Meigen)
(Hoffmann et al., 2002; Ayrinhac et al., 2004). Because there is a high
natural variability among species and populations, individual studies are necessary to determine species-specific parameters for cold
storage.

After approximately 200 generations in laboratory, the originally
cold-hardy stock became cold-susceptible. While initially 66% of
the puparia survived six months of cold storage at 1 °C, only 18%
survived the same treatment after 200 generations in laboratory.
In T. chilonis, wild populations were found to be three times more
cold tolerant than populations that were laboratory-reared for
more than 200 generations (Nagarkatti, 1979). Similarly, Khosa
and Brar (2000) compared cold storage tolerance in field versus laboratory populations and found that laboratory-reared populations
had lower emergence after cold storage.
A recent example involves Binodoxys communis (Gahan), an
aphid parasitoid that was introduced in 2007 from China to North
America to control the soybean aphid, Aphis glycines (Matsumura)
(Desneux et al., 2009; Heimpel et al., 2010). Field samples following its release in Minnesota failed to recover overwintered B. communis and laboratory data suggest that the strain released lost its
ability to diapause probably following a period of quarantine and
laboratory rearing prior to its release (Gariépy and Brodeur, personal communication).

4. Phenotypic plasticity
Within a genotype, phenotypic plasticity occurs when the
expression of a trait changes with the environment in which the
organism is raised (Roff, 1992; Gotthard and Nylin, 1995). Phenotypic plasticity is thus the ability of a genotype to produce distinct
phenotypes by altering its physiology, morphology, or development in response to changes in the environment (Pigliucci, 2005;
Colinet et al., 2007a) and is a major factor affecting cold tolerance
of insects. In D. melanogaster, phenotypic plasticity accounts for
80% of variance in cold stress tolerance whereas genetic variability
accounts for only 4% (Ayrinhac et al., 2004). Changes in an abiotic
factor such as developmental temperature may change a phenotype from cold tolerant to cold sensitive. Cold tolerance is thus a
very plastic trait that may be influenced by a range of endogenous
and exogenous factors experienced before, during, or even after the
cold exposure (see Table 1). It is therefore essential to understand
the effects of these factors on cold tolerance to successfully store
parasitoids at low temperature.

3. Laboratory adaptation

4.1. Exogenous factors

Undesirable genetic selection is a problem that frequently
occurs during continuous laboratory rearing of biological control
agents (Wang and Grewal, 2002). Selective forces, interacting with
inbreeding, bottleneck and drift can reduce field adaptedness and
vigor when insect populations are reared for several generations
under laboratory conditions (Hopper et al., 1993). Inbreeding can
be problematic in groups of Hymenoptera with a single locus multiple-allele sex determination (Hopper et al., 1993). Genetic drift of
laboratory populations was observed in the parasitoid Aphidius ervi
(Haliday), expressed as a rapid decline in allozyme variation after
few generations (Unruh et al., 1983).
Laboratory colonies of parasitoids can undergo changes following both phenotypic plasticity and genetic adaptation. On a short
time basis, populations can adapt to laboratory conditions causing
non-permanent acclimation (phenotypic plasticity) that is essentially non-genetic. Population can also adapt genetically to laboratory conditions (Mackauer, 1976) and this unintentional laboratory
adaptation could result in rapid decline of stress tolerance.
Long-term rearing at constant optimal temperatures could lead
to selection of strains that have limited tolerance for non-optimal
temperatures. House (1967) reported a case of long term laboratory adaptation in the parasitoid Pseudosarcophaga affinis (Fall).

4.1.1. Temperature
In order to extent their shelf-life, parasitoids can be stored at
low temperatures ranging from 0 to 15 °C. Even under these moderately low temperatures, most species show some level of mortality (Leopold et al., 1998; van Lenteren and Tommasini, 2002).
Generally, the lower the storage temperature, the higher the mortality (e.g. Ballal et al., 1989; Jalali and Singh, 1992; Bueno and Van
Cleave, 1997; Lacey et al., 1999; Easwaramoorthy et al., 2000;
Venkatesan et al., 2000; Lysyk, 2004; Rundle et al., 2004; Lopez
and Botto, 2005; Pandey and Johnson, 2005; Luczynski et al.,
2007; Bernardo et al., 2008). In some cases however, temperature
had no effect on survival (Al-Tememi and Ashfaq, 2005; Marwan
and Tawfiq, 2006). Temperature is indubitably one of the main abiotic factors affecting survival during cold storage. The design of
cold storage protocols often involves the use of specific temperatures and the determination of the lower threshold temperature
for development (To) (Leopold, 2007). The temperature chosen
for cold storage should be based upon the relative balance between
the need to reduce metabolic rate and/or development and the risk
of chilling injuries accumulation. If temperature is too high, undesirable emergence might occur during storage (e.g. Hofsvang and
Hagvar, 1977; Shalaby and Rabasse, 1979; Pitcher et al., 2002).

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H. Colinet, G. Boivin / Biological Control 58 (2011) 83–95
Table 1
List of endogenous and exogenous factors that affect the phenotypic plasticity in cold storage tolerance. Fitness-related traits that may be
affected in cold-stored parasitoids.
Factors of variability

Fitness consequences

Exogenous factors

Endogenous factors

Temperature
Duration of exposure
Rate of cooling or heating
Gradual acclimation
Rapid acclimation
Acclimatization
Developmental temperature
Constant or fluctuating cold exposure
Combined cold exposure
Humidity
Photoperiod
Chemicals
Oxygen concentration
Handling

Mass and body reserves
Life-history strategy
Nutrition
Mode of reproduction
Age/stage
Dormancy status
Gender

An important concept with regard to cold storage is the threshold
temperature. For each species, there is a temperature at or above
which permanent detrimental effects will not occur. In certain species, it seems that this threshold temperature is situated around
development threshold To. Pandey and Johnson (2005) analyzed
cold storage of Anagyrus ananatis (Gahan) immatures below and
above To (12.6 °C) and found that these could be stored for over eight
weeks at 14.8 °C without affecting eclosion rate, but storage at
10.1 °C killed most immatures after less than two weeks. In
Telenomus podisi (Ashmead) (To = 13.1 °C; Yeargan, 1980), exposure
at 12 °C is completely lethal but when pupae were stored at 15 °C,
development continued and adults emerged (Foerster et al., 2004).
Similarly, cold storage of Leptomastix dactylopii (Howard)
(To = 13.2 °C; Tingle and Copland, 1988) at 10 °C resulted in no survival even after only five days, whereas mummies could be stored
at 15 °C for 35 days (Krishnamoorthy, 1989). In some cases, when
storage temperature is around the To, even a very small variation
can result in very large variation in cold storage tolerance. Leopold
et al. (2004) reported that in Gonatocerus ashmeadi (Girault)
(To = 3.8 °C; Chen et al., 2006) exposure at 4 °C resulted in a very
low survival, whereas at 4.5 °C larvae tolerated cold storage for
20 days. In some species, lethal chilling injuries seem to take place
due to the fact that no development occurs below To. However, in
other species, cold storage was successfully performed at temperatures below To. For example, Lysiphlebus testaceipes (Cresson)
(To = 7.5 °C; Tang and Yokomi, 1995) can be stored for 90 days at
4.4 °C (Archer et al., 1973), Aphidius matricariae (Haliday)
(To = 4.5 °C; Miller and Gerth, 1994) tolerates up to three weeks at
3.5 °C (Marwan and Tawfiq, 2006), Encarsia formosa (Gahan)
(To = 12.7 °C; Osborne, 1982) survives up to 28 days at 4.5 °C
(Lopez and Botto, 2005), and Muscidifurax raptor (Girault and Sanders) (To = 9.3 °C; Lysyk, 2000) can be stored up to 12 weeks at 5 °C
(Lysyk, 2004). These examples emphasize the large temperaturedependent variability in cold storage tolerance. The diversity of observed responses stresses the need to experimentally determine the
optimal temperature zone for each species.
Parental (or grandparental) thermal history may also have a
significant influence on offspring’s cold tolerance. This crossgenerational effect is another form of phenotypic plasticity that
has been documented in D. melanogaster (e.g. Watson and Hoffmann, 1995; Magiafoglou and Hoffmann, 2003) but poorly studied
in parasitoids. Hance and Boivin (1993) reported a maternal effect
in the parasitoid Anaphes sp. When parental generation was exposed to a combination of low temperature and short photoperiod,
the F1 progeny showed significant increase in cold hardiness.
Though parental effects are presumably less important than the

Development time and patterns
Lifespan
Mortality
Fecundity
Oviposition period
Sex ratio
Sterility
Mating behavior
Mobility and flight capacity
Foraging behavior
Parasitism
Intergenerational effects
F1 progeny biomass
Morphological alterations
Beneficial effects

direct effect of the generation exposed (Crill et al., 1996), a certain
amount of variability may be expected.
4.1.2. Duration of exposure
In addition to temperature, duration of exposure is also an
essential component of cold survival in insects. Kostal et al.
(2004, 2006) defined the ‘dose of cold exposure’ as a combination
of exposure duration and temperature. In other words, when temperature decreases and/or duration of exposure increases, the dose
of cold exposure increases, and when it exceeds a specific threshold
chilling injuries accumulate and become progressively irreversible
and eventually lethal. Therefore, exposure duration x temperature
interaction ultimately determines mortality rate. In most cold
storage studies where different storage durations were tested, survival decreased with storage duration (Krishnamoorthy, 1989; Okine et al., 1996; Langer and Hance, 2000; Khosa and Brar, 2000;
Pitcher et al., 2002; Ozder and Saolam, 2004; Bayram et al., 2005;
Colinet et al., 2006a, 2006b; Foerster and Doetzer, 2006; Ayvaz
et al., 2008; Chen et al., 2008a; Abd El-Gawad et al., 2010; Colinet
and Hance, 2010). Because chilling injuries are cumulative, the
gradual decline in parasitoid survival may follow linear and thus
predictable patterns (Turnock and Bilodeau, 1992; Jalali and Singh,
1992; Okine et al., 1996; Rodrigues et al., 2003; Abd El-Gawad et al.,
2010) or show complex nonlinear patterns (McDonald and Kok,
1990; Lysyk, 2004) with time of exposure.
4.1.3. Rate of cooling or heating
The rate of temperature change can also influence insect survival at low temperatures. Obviously, the use of slower cooling
rates is more ecologically relevant and provides an opportunity
for cold-hardening (Chown and Nicolson, 2004). Likewise, the rate
of heating also influences thermal tolerance (Chown and Nicolson,
2004; Chown et al., 2009). In some cases however, it seems that a
slow rate of temperature change can be unfavorable resulting in
lower thermal tolerance. In D. melanogaster, survival after cold
exposure is higher when larvae are placed directly to room temperature, rather than a slow or stepwise re-warming (Sinclair
and Rajamohan, 2008).
Whether cold storage tolerance is affected by cooling/heating
rates has not been addressed for most parasitoid species. Some
data suggest that a lower mortality after storage can be obtained
when thermal variation between pre-storage and storage temperature is small (Shalaby and Rabasse, 1979; Singh and Srivastava,
1988). In Aphidius rhopalosiphi (De Stefani–Peres), mummies directly transferred from room temperature to storage temperature
had a lower survival rate than those submitted to treatments using

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step temperature reduction (Levie et al., 2005). These examples
suggest that cold storage tolerance might be affected by the rate
of temperature change in parasitoids.
4.1.4. Gradual acclimation
Gradual acclimation is a form of phenotypic plasticity where
cold tolerance can be increased by long-term pre-exposure (days
or weeks) to sub-lethal low temperature (Hoffmann et al., 2003;
Chown and Terblanche, 2006; Anguilletta, 2009). This thermal
acclimation results from various physiological and biochemical
modifications that include changes in proteins, metabolites, membrane structures, and metabolic rate (see Hoffmann et al., 2003;
Bowler, 2005 for review). In the cold-storage literature, the terms
pre-storage, pre-treatment, pre-conditioning, or pre-cooling, all refer to acclimation. In parasitoids, acclimation generally has a positive impact on cold storage tolerance (Polgar, 1986; Singh and
Srivastava, 1988; Bueno and Van Cleave, 1997; Pandey and Johnson,
2005; Marwan and Tawfiq, 2006; Luczynski et al., 2007), but detrimental effects of acclimation have also been reported. For instance,
in Aphidius colemani (Viereck), acclimation of mummies at 10 °C for
two days resulted in very low emergence compared with no acclimation (Hofsvang and Hagvar, 1977). In Thripobius javae (Girault),
pupae acclimation at 10 °C for ten days doubled the time to emergence in comparison to non acclimated pupae (Bernardo et al.,
2008). In both cases, the beneficial effect of acclimation was probably offset by a development effect, individuals reaching a more
cold-susceptible stage/age during acclimation (see below for discussion on age effect). Because all the cited studies performed thermal acclimation on immature parasitoids, the phenotypic changes
might actually result from a combination of reversible and developmental acclimation (as defined by Anguilletta (2009)).
4.1.5. Rapid acclimation
Rapid acclimation (also called rapid cold-hardening) is another
form of phenotypic plasticity that has been observed in numerous
insect groups. It refers to the increase in cold tolerance after a short
pre-exposure (minutes to hours) to sub-lethal low temperature
(Hoffmann et al., 2003; Chown and Nicolson, 2004; Anguilletta,
2009). Gradual and rapid acclimations involve different physiological bases and their combination can result in positive effects (Shintani and Ishikawa, 2007). Rapid acclimation increases survival after
both cold shock (acute stress) and prolonged cold exposure (chronic
stress) (Lee and Denlinger, 2010). The use of rapid acclimation
might be useful for improving cold storage tolerance. In A. rhopalosiphi, it was observed that different short-term acclimation treatments on mummies reduced mortality after storage as compared
to treatment without rapid acclimation (Levie et al., 2005).
4.1.6. Acclimatization
The term acclimatization has been used as a synonym for acclimation. It describes similar processes than gradual acclimation, but
has been mostly used when referring to acclimation under natural
conditions (Hoffmann et al., 2003; Chown and Nicolson, 2004;
Chown and Terblanche, 2006). The use of field acclimation is often
more difficult to realize than laboratory acclimation and it is likely
the reason why there is no report of such a practice for increasing
tolerance to cold storage, except Rigaux et al. (2000) whose results
suggest a positive effect of acclimatization on survival of parasitoids during cold storage. A seasonal acclimatization has also been
reported in parasitoids with cold storage tolerance varying with
collection seasons (Bai et al., 2009).
4.1.7. Developmental temperature
Developmental temperature can induce permanent phenotypic
changes (Chown and Nicolson, 2004; Anguilletta, 2009) that affect
morpho-physiological characteristics such as size or cold tolerance

(Chown and Terblanche, 2006). As a result, susceptibility to cold
storage is likely to vary according to temperature conditions during development. Colinet et al. (2007b) observed a reduction of
mortality in A. colemani mummies developed at low versus high
temperature. In Trissolcus basalis and T. podisi the opposite was
noticed, a high developmental temperature (25 °C) was beneficial
for cold storage (Foerster and Doetzer, 2006).

4.1.8. Constant or fluctuating cold exposure
The use of fluctuating thermal regimes (FTR) instead of constant
low temperature (CLT) has proved to significantly affect cold survival in several insect species (Lee, 2010, Renault et al., 2004). In
parasitoids, a remarkable reduction of cold storage injuries was observed when pupae of Aphidiidae were periodically exposed to
short pulses to optimal temperature (Colinet et al., 2006b). These
periodic transfers reduce the speed and the amount of accumulated injuries and give opportunities for physiological repair (Colinet et al., 2007c, 2007d; Kostal et al., 2007; Lalouette et al., 2007).
Subsequently, the use of FTR instead of CLT has proved to be beneficial in several different Aphidiinae species (Colinet and Hance,
2010), suggesting that the positive impact of FTR may apply to a
wide range of parasitoid species. Exposure to constant sub-optimal
temperature usually negatively affects reproductive potential of
parasitoids (Hance et al., 2007). Apart from beneficial impact on
survival, FTR also allows preservation of reproductive ability and
conservation of mobility performances in A. colemani (Colinet and
Hance, 2009), two important features for successful biological
control.

4.1.9. Combined cold exposure
The use of combined cold treatment on different stages can dramatically affect cold storage tolerance. In the parasitoid T. javae,
longevity decreases radically when parasitoids are submitted to
combined treatments (i.e. storage at pupal stage followed by storage at adult stage) compared with storage as pupal or adult stage
only (Bernardo et al., 2008).

4.1.10. Humidity
De Bach (1943) suggested three reasons for death during cold
storage in insects: low temperature, starvation and desiccation.
When the body water activity is higher than the atmospheric water
activity, water evaporates from insect cuticle (Warthon, 1985).
Therefore maintenance of body water homeostasis may be a great
challenge under prolonged cold storage. In Aphidiinae, reductions
of water mass and content were noted during storage (Colinet
et al. 2006a; Ismail et al., 2010). Only a few studies have investigated the effect of humidity on cold storage performance of parasitoids. Waggoner et al. (1997) observed a reduced longevity of
adult Aphelinus asychis emerging from mummies exposed to low
humidity. Lacey et al. (1999) found a significant reduction of emergence in E. formosa pupae stored at low humidity (30% RH) compared with high humidity (70% RH). Detrimental effect of low
temperature may thus increase if combined with low humidity,
but excess of moisture can also be detrimental increasing the risk
of fungus contamination (e.g. Wilding, 1973; Chen and Leopold,
2007).
Maintaining, and measuring, relative humidity at low temperature is challenging (Paull, 1999). Rather than relying on refrigiration evaporator, calcium sulfate can be used to remove humidity
from the air and then salt solutions or glycerol–water mixtures
can be used to generate a range of relative humidities (Boivin
et al., 2006). To measure humidity at low temperatures near the
condensation point of air, platinum resistance elements are recommended (Paull, 1999).

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4.1.11. Photoperiod
Photoperiod represents a reliable information signal for daily or
seasonal changes. Photoperiod trigger physiological and biochemical changes that can increase cold tolerance, often via diapause,
but not always (Tauber et al., 1986; Hodkova and Hodek, 2004).
Many insect species show a photoperiodic response in cold tolerance (e.g. Kim and Song, 2000). There is a large literature dealing
with photoperiodic induction of diapause in parasitoids (e.g.
Brodeur and McNeil, 1989; Langer and Hance, 2000; Li et al.,
2008). However, the effect of photoperiod on cold tolerance of
non-diapausing parasitoids has received little attention. In A.
asychis, females reared under short-day conditions better tolerate
prolonged exposure to cold (5 °C) compared to long day-reared
females (Tatsumi and Takada, 2005). In Anaphes sp. cold hardiness
increases when parasitized eggs are maintained under low temperature combined with short photoperiod compared to long photoperiods (Hance and Boivin, 1993). Photoperiod may also affect
reproductive capacity, as observed in cold-stored E. formosa
(Ganteaume et al., 1995).
4.1.12. Chemicals
Exposure to chemical stress can significantly affect cold-storage
susceptibility. Unstored Trichogramma cordubensis (Vargas and
Cabello) prepupae exposed to Deltamethrin and Lambda-cyhalothrin at 22 °C show 65% and 51% emergence, respectively (Vieira
et al., 2001). When these prepupae are exposed to the same dose
of insecticide during cold storage at 3 °C, a drastic increase in mortality is observed resulting in less than 25% and 20% emergence for
Deltamethrin and Lambda-cyhalothrin respectively; whereas coldstored parasitoids not exposed to pesticides show about 90% emergence (Garcia et al., 2009). This shows that cold storage alone is not
detrimental but associated with another stress, such as pesticide
exposure, it resulted in a very low survival, maybe as a result of
low detoxification enzyme activities at low temperature.
4.1.13. Oxygen concentration
Parasitoids live within the oxygen-poor tissues of their host,
therefore they might be adapted to hypoxic conditions. However
this question has not been tested (Hoback and Stanley, 2001). Lowering the oxygen partial pressure has proved to be beneficial for
cold storage of non-diapausing Simulium ornatum (Meigen) eggs
(Goll et al., 1989). In contrast, Leopold (2000) found that hypoxic
and hyperoxic environments were ineffective in improving survival of cold-stored house fly eggs. In parasitoids, the oxygen concentration might affect variability in cold storage susceptibility but
this factor has never been tested either.
4.1.14. Handling
In the pupation process, parasitoids often stick their cocoon to
the host or to the host plant. For practical reasons, parasitoids
may have to be detached from their substrate before cold storage.
This handling stress may affect cold storage tolerance. In E. formosa, cold-stored pupae left on plant leaves show higher emergence than their counterpart detached from plant leaves (Liu and
Tian, 1987). The handling of parasitoids may alter cocoon’s integrity, a feature that is important against cold and desiccation (e.g.
Tagawa, 1996; Rivers et al., 2000).
4.2. Endogenous factors
4.2.1. Mass and body reserves
During cold exposure, an individual metabolism relies on body
energy reserves accumulated during development. Energy reserves, particularly from fat body, are consumed during this starvation period at low temperature (e.g. Renault et al., 2003; Colinet
et al., 2006a). Consequences of cold storage may result from a

87

combination of both chill-injuries and exhaustion of energy reserves (Colinet et al., 2006a). In parasitoids mass/size and lipid reserves are positively correlated (e.g. Rivero and West, 2002; Colinet
et al., 2007a, 2007b), so that larger individual are generally fatter.
Positive association between body size/lipid and cold tolerance
have been observed in insects (e.g. Renault et al., 2003; Liu et al.,
2007). In the context of cold storage, it has also been suggested
that larger and fatter A. colemani mummies better tolerate cold
storage than smaller individuals (Colinet et al., 2007b). The mass/
size of parasitoid is a highly variable phenotype that can be manipulated via modulation of host species or age (e.g. Cloutier et al.,
2000) or rearing conditions (Colinet et al., 2007a).
4.2.2. Life-history strategy
Endoparasitoids develop and feed within the protective tissue
of their host, while ectoparasitoids live externally (Godfray,
1994). Koinobionts allow hosts to continue feeding and growing
after parasitism, whereas idiobionts permanently paralyze or kill
their host at parasitism (Godfray, 1994; Brodeur and Boivin,
2004). Koinobionts are thus physiologically constrained by their
host and their development is finely tuned to that of the host. Generally koinobiosis is associated with endoparasitism, and idiobiosis
with ectoparasitism although some groups are exception i.e. egg
parasitoids that are idiobiont endoparasitoids (Brodeur and Boivin,
2004). Cold storage technique and tolerance depends on which
strategy is used by the parasitoid. In koinobionts, the decline in
survival may result from low temperature acting either directly
on the parasitoid or indirectly through the host, while idiobionts
should not be affected by host survival. Ectoparasitoids can develop on a dead host, which allows the possibility of rearing them on
frozen hosts (e.g. Onagbola and Fadamiro, 2009). In contrast, insectaries rearing koinobionts do not have this option and must continuously supply fresh hosts for parasitoid rearing. Additionally,
because ectoparasitoids are more exposed to abiotic factors, they
are expected to be more resistant to environmental stresses (e.g.
desiccation or temperature) (Tagawa, 1996; Rivers et al., 2000). Indeed, the ectoparasitoid Hyssopus pallidus tolerates cold storage
and no detrimental effects of low temperature are observed
(Hackermann et al., 2008). Some ectoparasitoids feed externally
but remain within the host puparium which provides them a significant protection. Rivers et al. (2000) underlined the importance
of this protective role in the ectoparasitoid, Nasonia vitripennis
(Walker). Naked larvae, unprotected by the host’s puparia, had a
reduced cold tolerance compared to larvae enclosed within the
puparium.
4.2.3. Nutrition
Nutritional resource is another factor that affects cold storage
tolerance. This factor has been studied in predators, in which cold
storage tolerance varies according to the diet (Coudron et al.,
2007). Variation in food quality can influence cold tolerance by
modulating low-molecular-weight sugars and polyols levels (e.g.
trehalose and glycogen), body size and lipids levels (Liu et al.,
2007). In parasitoids, the host represents the nutritional resource,
and it has been shown that host biochemical conditions significantly influence cold tolerance. In N. vitripennis, cold survival is
significantly higher in parasitoid reared on diapausing hosts, which
contains more cryoprotectants (glycerol, alanine) than nondiapausing ones (Rivers et al., 2000). It might thus be advantageous
to rear parasitoids on diapausing hosts before cold storage. However, when hosts are themselves cold-stored prior parasitization,
some decrease in quality can occur and affect negatively the parasitoid during exposure to low temperature. In some species, it is
sometimes more appropriate to store adults rather than immatures, as reported in Cotesia marginiventris (Cresson) (Riddick,
2001). In this case feeding of adults with nutrient (sucrose or

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honey) and water, before, during and after storage significantly increases cold storage tolerance (Shalaby and Rabasse, 1979; Ganteaume et al., 1995; Riddick, 2001; Uçkan and Ergin, 2003).
4.2.4. Mode of reproduction
In some parasitoid species both sexual (i.e. arrhenotokous) and
parthenogenetic (i.e. thelytokous) reproduction can be found in different strains living in sympatry. Differences in mode of reproduction are associated with differences in life-history traits such as
longevity, development, and fecundity (Barke et al., 2005; Pelosse
et al., 2007). Variation in thermal tolerance has also been reported
between reproduction modes. For example in Aphytis mytilaspidis
(LeBaron), thelytokous individuals are more adapted to higher temperatures than arrhenotokous individuals (Rössler and DeBach,
1972). In Trichogramma minutum (Riley) thelytokous parasitoids
tolerate better cold storage than arrhenotokous ones (Wang and
Smith, 1996). The physiological basis of these variations is not yet
elucidated.
4.2.5. Age/stage
In insects, ontogeny-related variation in cold tolerance has been
described (see Bowler and Terblanche, 2008 for review) and a
marked variability occurs among life stages. In Diptera for exemple,
eggs are more cold tolerant than adults (Bowler and Terblanche,
2008). It is difficult to generalize this pattern to other genera/species as exceptions often occur. When cold storage of parasitoids is
planned, it is essential to determine which developmental stage is
the most appropriate. Storage during the adult stage might lead
to higher and faster reduction in fitness than with immatures,
and pupal stage is often considered to be more suitable for shortterm storage (van Lenteren and Tommasini, 2002). There are experimental evidences showing that pupae are actually more cold-tolerant then eggs, larvae or adults (Jalali and Singh, 1992; Nakama and
Foerster, 2001), however, in some cases adults have been reported
to be more tolerant to cold storage than immatures (e.g. Krishnamoorthy, 1989; Riddick; 2001; Bayram et al., 2005).
In addition to the differences among life stages, a large variability in cold tolerance results from a within-life-stage effect (Bowler
and Terblanche, 2008). Here again, a general guideline cannot be
provided since this variability depends on the species and the interactions with other factors, such as the temperature or the duration
of cold storage (e.g. Bayram et al., 2005). In T. basalis and T. podisi, a
positive linear relationship was observed between pupal age and
emergence rate (Foerster et al., 2004). It was suggested that parasitoids stored as young pupae spent more energy to complete their
development which affected their emergence and longevity (Foerster et al., 2004). However, this trend is not true for all parasitoid
species. Several studies have reported a higher cold tolerance when
young versus old parasitoid pupae where stored (Hofsvang and
Hagvar, 1977; Polgar, 1986; Whitaker-Deerberg et al., 1994; Levie
et al., 2005). The opposite trend was also noticed, old parasitoid pupae showing a higher tolerance to cold storage than young counterparts (Bueno and Van Cleave, 1997; Venkatesan et al., 2000).
Finally, intermediate pupal stage (middle age) have also reported
be more appropriate than young or old pupae (Chen et al., 2005;
Luczynski et al., 2007). Parasitoids are often stored as pupae because this stage is immobile, and is well protected from handling
and desiccation inside its cocoon. Although pupae are virtually
inactive, this stage is metabolically very active. The larval tissues
undergo histolysis and are reassembled in the adult form. It is
therefore not surprising to observe a large variability in cold storage
tolerance within this specific stage. A demonstration of the age-related variation in cold storage tolerance was provided by Leopold
et al. (1998) using the housefly. In this study, pupae were coldstored at different ages and the middle-aged ones were the most
cold tolerant. Interestingly, these pupae displayed the lower meta-

bolic activity (respiration rate) suggesting an association between a
metabolic/developmental rate and cold tolerance.
4.2.6. Dormancy status
In nature, insects respond to seasonal temperature change
through a range of adaptations such as dormancy which includes
diapause, a programmed obligatory or facultative interruption in
development, and quiescence which refers to an immediate interruption in development in response to adverse conditions (Hodkova and Hodek, 2004; Tauber et al., 1986). Besides its role of
synchronization with the host cycle, diapause might also facilitate
survival over harsh periods by increasing cold-hardiness (Tauber
et al., 1983). For species which exhibit a diapause-related increase
in cold-hardiness, storage of dormant stages may be advantageous
(Boivin, 1994), assuming that diapause induction and termination
can be controlled. The induction of diapause may be under the control of several factors and the understanding of how diapause starts
and ends in parasitoids is still very incomplete (Polgar and Hardie,
2000; Colinet et al., 2010). Only few practical applications of diapause for cold conservation are reported because of the mortality
occurring during the artificially induced diapause (van Lenteren
and Tommasini, 2002).
Depending on the species, cold-hardiness and diapause may or
may not be linked (e.g. Hodkova and Hodek, 2004). However, based
on the few examples found in the literature, it appears that diapausing parasitoids show increased tolerance to low temperature. In
N. vitripennis non-diapausing larvae are more susceptible to freezing temperature than diapausing larvae (Rivers et al., 2000). In A.
rhopalosiphi and A. ervi, diapausing mummies better survived cold
storage (Langer and Hance, 2000). In T. cordubensis, no mortality
was observed in diapausing parasitoids, while an increasing
mortality occurred in quiescent parasitoids during cold storage
(Ventura Garcia et al., 2002). Similarly, in A. asychis (Walker), diapausing individuals had a reduced mortality during cold storage
compared to non-diapausing counterparts (Tatsumi and Takada,
2005). These examples underline the large variability in cold storage tolerance that can be observed between dormant and nondormant phenotypes. Diapause is not always an option for cold
storage since the ability to enter diapause is not present in all species (Rundle et al., 2004) but when diapause is controlled, it can
allow prolonged (several weeks or months) period of storage (e.g.
Turnock and Bilodeau, 1992; Ventura Garcia et al., 2002; Foerster
and Doetzer, 2006; Hun et al., 2005).
4.2.7. Gender
Cold storage may affect the gender differentially. It is it is difficult to draw a conclusion about the most cold-susceptible sex because there is no general rule. In some cases, a shift toward
production of a higher proportion of males has been reported after
storage, suggesting a differential mortality during development
where females would be more susceptible than males (e.g. Ballal
et al., 1989; Okine et al., 1996; Riddick, 2001; Bayram et al., 2005;
Chen et al., 2008a). Storage as adult also results in sex-specific
responses. Several studies reported that adult males were more
susceptible to cold storage than females (Jayanth and Nagarkatti,
1985; Gautam, 1986; Jackson, 1986; Uçkan and Gulel, 2001);
although no evidence of a sexual difference has also been reported
(Leopold and Chen, 2007a). The fact that female parasitoids generally live longer than males may explain why adult females generally
withstand cold storage longer than males.
5. Fitness consequences of cold storage
Both the host and the parasitoid are affected by prolonged
exposure to cold resulting in lethal or sub-lethal consequences

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(Hance et al., 2007). Chilling injuries accumulate at low temperature following various physiological dysfunctions (reviewed by
Chown and Terblanche (2006)). Low temperature also induces
depletion of energy reserves following prolonged starvation
(Renault et al., 2003; Colinet et al., 2006a). Hence, consequences
of cold-storage result from chilling effect, starvation, or a combination of both phenomena. Organisms acquire and transform energy
resources obtained from the environment in order to maintain
their homeostasis and to produce offspring (Brown et al., 1993).
There is therefore, in parasitoids as in other insects, a trade-off between survival and reproduction (Ellers and van Alphen, 1997; Ellers et al., 2000; Jervis et al., 2003; Colinet et al., 2007a) and fat
reserves play a central role in the physiology of resource allocation
for this trade-off (Ellers and van Alphen, 1997), especially so in parasitoids that are unable to synthesize lipids de novo (Visser and Ellers, 2008; Visser et al., 2010). The consumption of energy reserves,
particularly lipids, during cold exposure is thus expected to translate into fitness cost on survival and/or reproduction (e.g. Renault
et al., 2003; Colinet et al., 2006a). The effect of a stress such as cold
storage might not be immediately detectable, but might carry over
to another stage, negatively influencing either development, fecundity, survival, or some combination thereof (Chown and Nicolson,
2004). Even if parasitoids remain alive after cold storage, a reduction of fitness-related traits may be observed directly, later in
development or even in the next generation. We will examine here
the diversity of fitness-related traits that might be affected in coldstored parasitoids (see Table 1).
5.1. Life history traits
5.1.1. Development time and patterns
Cold exposure can affect different aspects of development,
including the time to emergence and the distribution pattern of
emergence. Post-storage development is expected to resume normally, but in some species the time necessary to complete development increases after cold storage (e.g. Ballal et al., 1989; Jalali and
Singh, 1992; Tezze and Botto, 2004; Pandey and Johnson, 2005;
Luczynski et al., 2007; Bernardo et al., 2008). In some studies,
developmental delay increases with cold storage duration (Bueno
and Van Cleave, 1997; Lysyk, 2004; Colinet and Hance, 2010) suggesting that, likewise chilling injuries, delay is a cumulative process. In addition to delaying emergence time, cold storage may
also affect the distribution pattern of emergence. In G. ashmeadi,
cold storage of parasitized host eggs induces multi-peak emergence of adults (Chen and Leopold, 2007; Chen et al., 2008a,
2008b). In Praon volucre (Haliday), cold storage induces diapause
in some individuals and multi-peak emergences were observed
for up to 115 days (Colinet et al., 2010). Developmental responses
to cold storage may thus vary from complete arrestment of development to slower development.
5.1.2. Lifespan
After cold storage as immatures, parasitoids that were able to
complete development and emerge generally suffer from a severe
reduction in lifespan. The inability of parasitoids to accumulate
fat reserves as adults implies that they are constrained in resource
allocation strategies (Visser and Ellers, 2008; Visser et al., 2010),
and the trade-off between survival and reproduction may be
severely affected by any lipid consumption. In A. colemani, the
amount of fat reserves available for emerging adults declined linearly with duration of cold exposure, and a corresponding linear decrease of adult longevity was observed (Colinet et al., 2006a).
Similarly, Ismail et al. (2010) observed an association between consumption of lipids during storage and reduction of longevity in A.
ervi. Constant decline of adult longevity with storage duration
has been observed in many species including L. dactylopii (Krishna-

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moorthy, 1989), Trichogramma carverae (Oatman and Pinto)
(Rundle et al., 2004), Bracon hebetor (Say) (Al-Tememi and Ashfaq,
2005), Trichogramma evanescens (Westwood) (Ayvaz et al., 2008),
T. chilonis (Nadeem et al., 2010), Campoletis chlorideae (Uchida)
(Patel et al., 1988), and Aphidius picipes (Nees) (Amice et al.,
2008). In the extremes, lifespan may be so short that adults die
during the first few hours after emergence (Waggoner et al.,
1997; Levie et al., 2005).
5.1.3. Mortality
Mortality represents the ultimate cost of prolonged cold exposure. In addition to chilling injuries, parasitoids stored as immature
may not have sufficient energy resources to complete their development and/or to emerge. In some solitary species, dissection of
non-emerged parasitoids after cold storage has shown that mortality occurred mainly after metamorphosis in pharate aduts (Okine
et al., 1996; Colinet et al., 2006a). In other studies, the occurrence
of fully formed adults dying during the eclosion process has also
been reported (Levie et al., 2005; Luczynski et al., 2007). The process of emergence is energy-consuming as it requires strong muscle contraction (Yocum et al., 1994) and chilling is known to induce
muscular dysfunctions (Yocum et al., 1994; Kelty et al., 1996). Lack
of energy together with muscular perturbation is likely the reasons
why pharate aduts fail to emerge after cold storage. In other species, death mainly takes place during pupation (Chen and Leopold,
2007). In both solitary and gregarious parasitoids, mortality generally increases with duration of cold storage (e.g. Okine et al., 1996;
Langer and Hance, 2000; Ozder and Saolam, 2004; Bayram et al.,
2005; Colinet et al., 2006b, 2007b; Foerster and Doetzer, 2006;
Colinet and Hance, 2010) but in gregarious species, this temporal
effect can be seen as a decreasing number of emerging parasitoids
per host as duration of storage increases (Lysyk, 2004).
5.2. Reproduction
5.2.1. Fecundity
The reproductive organs are particularly vulnerable to low temperature effects (Flanders, 1938; Denlinger and Lee, 1998). In
Euchalcidia caryobori (Hanna), low temperature may either cause
retardation of egg maturation or, in its extremes, malformation
of reproductive organs in both sexes (Hanna, 1935). Numerous
observations have been made on the reduction of parasitoid fecundity after cold storage. Like most of the traits affected by cold storage, the fecundity cost is generally proportional to both the
temperature and the duration of exposure (Jayanth and Nagarkatti,
1985; Ballal et al., 1989; Jalali and Singh, 1992; Venkatesan et al.,
2000; Prasad and Ansari, 2000; Foerster and Nakama, 2002; Ozder,
2004; Al-Tememi and Ashfaq, 2005). Absence of reproductive cost
(i.e. fecundity) has been reported in only a few rare cases such as in
L. testaceipes (Archer et al., 1973), Aphidius gifuensis (Ashmead)
(Chen et al., 2005) and in A. ervi (Ismail et al., 2010) but these resuts may have been obtained under conditions that cause little
stress to the stored individuals.
5.2.2. Oviposition period
The temporal pattern of oviposition is another important bionomic trait that can be affected by cold storage. In Anaphes ovijentatus (Crosby and Leonard), the length of oviposition period
decreases with storage duration. Moreover, cold stored females
laid the majority of their eggs on day 1 while control females took
about three days to oviposit the majority of their eggs (Jackson,
1986). Hun et al. (2005) also reported a reduction of egg-laying
period after prolonged storage of diapausing Microplitis mediator
prepupae. Like fecundity, reduction of egg-laying period is probably a symptom of the reproductive cost frequently associated with
cold storage.

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5.2.3. Sex ratio
Low temperatures frequently distort insect sex ratios (Denlinger
and Lee, 1998). Hymenopteran parasitoids usually have a haplodiploid sex-determination system, female being able to choose
the sex of their progeny by controlling fertilization (Flanders,
1956). Distortion of sex ratio may come from different origins. First
a modification of the proportion of fertilized eggs oviposited (i.e.
primary sex ratio) may be observed following modification of the
female reproductive strategy or the inability of males to mate (Colinet and Hance, 2009) or to produce viable sperm (Lacoume et al.,
2007) after a cold stress. Some authors reported a shift towards
production of more males in the F1-progeny from cold-exposed
parasitoids (Riddick, 2001; Foerster and Nakama, 2002; Pitcher
et al., 2002; Levie et al., 2005; Leopold and Chen, 2007a; Chen
et al., 2008b; Ismail et al., 2010). Sex ratio distortion can also result
from a differential mortality between sexes when immatures are
exposed to cold (i.e. secondary sex ratio). Depending on the species,
cold storage has been shown to increased developmental mortality
of females (Jayanth and Nagarkatti, 1985; Gautam, 1986; Ballal
et al., 1989; Okine et al., 1996; Riddick, 2001; Bayram et al., 2005;
Chen et al., 2008a), of males (De Bach, 1943) or to be neutral (Jarry
and Tremblay, 1989; Turnock and Bilodeau, 1992; Rodrigues et al.,
2003; Foerster et al., 2004; Levie et al., 2005; Lopez and Botto, 2005;
Ayvaz et al., 2008; Colinet and Hance, 2010). Distortions of sex ratios in favor of females would have minimal impact on the storage
of parasitoids for biological control purposes (Leopold, 1998).
5.2.4. Sterility
Cold exposure is known to interfere with the normal reproductive process and a commonly observed response is a progressive
reduction of fecundity (as described above). Ultimately this reduction can reach the level of sterility where females do not lay any egg
during their lifetime. There are several examples where female sterility has been reported in parasitoids (Archer and Eikenbary, 1973;
Easwaramoorthy et al., 2000; Foerster and Nakama, 2002; Levie
et al., 2005; Foerster et al., 2004). Prolonged cold exposure may
cause either decrease in maturation rate of oocytes or ultimately
malformation of ovarioles resulting in female sterility (Hanna,
1935).
After mating with cold-stored males, female parasitoids may
produce only male progeny (i.e. unfertilized eggs), possibly
because of male sterility (Ballal et al., 1989; Rigaux et al., 2000; Levie et al., 2005; Pandey and Johnson, 2005) as cold stress may
cause alteration of sperm production or reproductive organs. In
E. caryobori, low temperature causes delay in spermatogenesis
and degeneration of premetamorphic cells resulting in male sterility (Hanna, 1935). In Tetrastiehus sp. dissection of cold-stored
immatures revealed either reduction of sperm viability in seminal
vesicles or empty spermatheca in females (Flanders, 1938). More
recently, Lacoume et al. (2007) showed that sperm production of
Dinarmus basalis (Rond) was affected by cold shocks, inducing a delay in sperm production and seminal vesicle replenishment.
5.2.5. Mating behavior
The possibility that the absence of females in the progeny could
arise from a mating behavior disorder (which might be considered
as a form of sterility) rather than a sperm production/transfer
anomaly has been examined in A. colemani. Male reproductive
potential (mating success, pre-mating period, and competitive
mating ability) was significantly affected when mummies were exposed to constant low temperature. Moreover, when cold-stored
males were able to mate, insemination and fertilization were effective showing that the suspected male sterility was related to mating failure rather than any physio-anatomical alteration in sperm
production (Colinet and Hance, 2009). In Sturmiopsis inferens
(Townsend), cold storage of pupae significantly reduced mating

occurrence (more than 50% reduction) (Easwaramoorthy et al.,
2000). In A. picipes, a reduction in the proportion of males successfully mating after cold exposure of mummies has also been
reported (Amice et al., 2008). It is not know whether mating disorder results from a behavioral alteration of the male, the female or
both sexes. Ismail et al. (2010) did not observe detrimental effect of
cold storage on mating success of A. ervi, however the temperatures chosen were voluntary very permissive to avoid any mortality. It was suggested that cold-induced morphological alterations
of antennal structure (e.g. Pintureau and Daumal, 1995; Bourdais
et al., 2006; Amice et al., 2008) could negatively impact partner
acceptance during mating because antennae play an important role
during courtship (Battaglia et al., 2002). Mobility reduction after
cold storage (e.g. Tezze and Botto, 2004; Ayvaz et al., 2008; Colinet
and Hance, 2009) is another factor that may contribute to the
reduction of mating success. Indeed, for parasitoid species, the success of courtship likely relies on proper locomotion as in Drosophila
flies (Partridge et al., 1987).
5.3. Efficacy
5.3.1. Mobility and flight capacity
Low temperature can strongly affect insect locomotion performances by inducing neuro-muscular dysfunctions (Yocum et al.,
1994; Kelty et al., 1996) that result in uncoordinated movements
(Kostal et al., 2006). There are only a few studies that analyzed
mobility of cold-stored parasitoids though it is an important
parameter for efficient biological control. Storage of Trichogramma
sp. pupae decreased mobility (Tezze and Botto, 2004) and walking
speed of adults (Ayvaz et al., 2008). In A. colemani, locomotion
parameters of adults decline under constant cold exposure,
whereas mobility is not affected under fluctuating temperatures
because of periodic opportunities to repair chilling injuries (Colinet
and Hance, 2009). Flight capacity is another important trait that
may be affected by cold storage. Luczynski et al. (2007) reported
a constant decline of flight with storage duration in E. formosa
and Eretmocerus eremicus (Rose & Zolnerowich). Reserve of fat
and glycogen are known to serve as sources of energy for flight
in various insects. Energy consumption together with muscle alteration may explain why mobility (flight and walking) is reduced
after storage.
5.3.2. Foraging behavior
Parasitization of host depends on a series of complex behaviors
expressed by the female parasitoid. Host acceptance and oviposition
depend on whether females are capable of recognizing their hosts
and assess their suitability for their progeny (Godfray, 1994). Within
a host patch, female reproductive success can be affected by associative learning and by discrimination capacities. There are few studies
that dealt with effects of cold storage on foraging behavior. Female
Anaphes victus (Huber) exposed to cold-storage as larvae oviposited
fewer eggs, were less able to learn external marks, superparasitized
more and distributed their time in the patch differently from controls. The intensity of these foraging behavior changes was positively correlated with the duration of cold exposure (van Baaren
et al., 2005). The use of learned, conspecific chemical cues was more
affected by cold exposure than was the use of learned personal cues
(van Baaren et al., 2006). Foraging behavior of A. picipes also showed
modifications after cold storage of mummies, patch residence time
and host handling time were reduced after two to four weeks of cold
storage (Amice et al., 2008). These modifications may be viewed as a
consequence of the reduced longevity of cold-stored wasps, as theory predicts longer patch residence times for females with shorter
expected lifespan (Charnov, 1976), an effect that was measured in
A. victus females (Wajnberg et al., 2006). Females of the wasp
Microplitis demolitor (Wilkinson) emerging from chilled pupae were

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unable to respond to an attractive odor, even after just four days of
chilling (Hérard et al., 1988).
5.3.3. Parasitism
The rate of parasitism is usually quantified by providing parasitoids unlimited numbers of hosts. It represents another means of
testing fecundity. Contrary to fecundity, which refers to an absolute number of eggs or progeny produced, the percent of parasitism refers to the proportion of host parasitized among a colony.
It thus takes into account the steps of foraging behavior such as
host recognition, acceptation, and discrimination. Several studies
have reported reduction of percent parasitism with increasing
length of cold storage (Khosa and Brar, 2000; Pitcher et al., 2002;
Ozder, 2004; Al-Tememi and Ashfaq, 2005; Bayram et al., 2005;
Kumar et al., 2005; Chen and Leopold, 2007; Chen et al., 2008b;
Garcia et al., 2009; Abd El-Gawad et al., 2010; Nadeem et al.,
2010). Similar reduction in the ability of individuals to parasitize
eggs has also been noted in the fields (Rundle et al., 2004).
5.4. F1 effects
5.4.1. Intergenerational effects
In some cases, parental (or even grandparental) exposure to
cold can have substantial influences on the fitness of the
unstressed offspring. These trans-generational effects are phenotypic modifications transmitted either maternally or paternally
(e.g. Crill et al., 1996). The influence of parental or grandparental
environmental history has been examined for insect life history
traits, but the physiological basis remains indefinite (Chown and
Nicolson, 2004). The plastic responses across generations have
been extensively studied in D. melanogaster and it is recognized
that parental cold stress can influence offspring fitness (Magiafoglou and Hoffmann, 2003; Rako and Hoffmann, 2006).
The parasitoids G. ashmeadi has been examined under different
situations: (i) cold storage of host eggs result in detrimental effects
on the generation reared on these cold-stored eggs (F1) though
these effects did not extend to the next generation (F2) (Chen
and Leopold, 2007), (ii) cold storage as immature within a host induced many adverse effects on the parental generation exposed to
cold and also influenced F1 progeny (Leopold and Chen, 2007b),
(iii) finally, cold storage as adult reduces several fitness traits
(e.g. fecundity, longevity, emergence, development) of the parents
and of their F1 progeny, but these effects did not extend to F2 and
F3 generations (Leopold and Chen, 2007a; Chen et al., 2008b).
When T. evanescens adults are cold-stored, a reduction of longevity
is observed on both parental and F1-progeny generations (Yilmaz
et al., 2007). The mechanism for passing maternal (carryover)
effects of cold stress to the next generation is unknown. Cold stress
may induce some damages to immature oocytes or change maternal metabolism affecting the fitness of the next generation (Rako
and Hoffmann, 2006). Trans-generational effect are not always
observed, for instance the F1 progeny of cold-stored E. formosa
and Eretmocerus corni (Haldeman) pupae are not affected by parental cold treatment (Lopez and Botto, 2005).
5.4.2. F1 progeny biomass
Cold-storage can also affect progeny size/weight as in Muscidifurax sp. where the dry weight of F1 progeny increased following
cold-storage of the parents (Legner, 1976). Similarly, in H. pallidus
(Askew), offspring of larger size emerged when the parental generation was cold-stored as pupae (Hackermann et al., 2008). In both
studies, cold storage of the parental generation did not result in
any detrimental effect on the performances and survival of the parent generation. The increase in size of the F1 could be the result of
the extended development time of the parent generation resulting
in emergence of larger individuals with larger eggs. Indeed, body

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size parameters generally increase as temperature decreases and
vice versa, a pattern commonly referred to as the temperature-size
rule (TSR) (Atkinson, 1994; Anguilletta, 2009) and which is verified
in parasitoids (e.g. Bazzocchi et al., 2003; Colinet et al., 2007a; Boivin, 2010). There is a body of evidence that this thermal plasticity
also occurs on offspring size. Temperature experienced by the female often results in increased offspring size trough variation of
egg size (Anguilletta, 2009).
5.5. Morphological alterations
Low temperature can influence tissue differentiation and/or hormonal balance resulting in morphological defects (Sehnal, 1991).
Several types of developmental abnormalities have been reported
in parasitoids submitted to cold storage. In Trichogramma sp. proportion of wing deformity increases with cold storage duration
eventually reaching 100% in some cases (Schread and Garman,
1934; Tezze and Botto, 2004). Morphological alteration of wings
can critically diminish some fitness-related traits such as field flying/dispersal ability (Hewa-Kapuge and Hoffmann, 2001). Malformations of reproductive organs have also been observed in coldstored E. caryobori (Hanna, 1935), a defect that might have a serious
impact on mating behavior. Alteration of antennal structure has also
been described as a result of cold storage. In A. rhopalosiphi, abnormal sensillae were detected after mummy cold storage (Bourdais
et al., 2006) and in A. picipes antennal fluctuating asymmetry increased with duration of cold exposure (Amice et al., 2008). In three
Trichogramma species, exposure to low temperature (with or without diapause) modified the morphology structure of antennae (Pintureau and Daumal, 1995). Morphological alteration of antennae
may negatively impact olfactory/behavioral processes. Storage as
immature may also results in reduction of adult size, as observed
in T. carverae (Rundle et al., 2004). The size of parasitoid is an important trait generally correlated with fitness (Godfray, 1994; Jervis
et al., 2003) and field performances (Bennett and Hoffmann, 1998).
Finally as in other insects (Sehnal, 1991; David et al., 1990), body
pigmentation of parasitoids darkens as a result of low temperature
exposure, as noted in E. caryobori (Hanna, 1935) and A. colemani (Colinet and Hance, 2009). This might affect some behavioral attributes
such as mate recognition, but this needs further testing.
5.6. Beneficial effects
A few studies have reported beneficial effects of cold storage of
immatures on life history traits of adult parasitoids. For instance, in
Mormoniella vitripennis (Walker), the lower the temperature at
which immatures were subjected, the larger the increase of adult
fecundity (De Bach, 1943). In M. raptor and M. zararaptor, cold storage of larvae increased lifespan and fecundity of adults (Legner,
1976), possibly because of a TSR effect (see before). More recently,
two additional studies reported puzzling data where fecundity increased after a prolonged period of storage. In T. basalis, fecundity
significantly decreased with storage duration but, after seven
months of storage as pupae, fecundity unexpectedly increased to
reach control level (Foerster et al., 2004). Similar observation was
made by Bernardo et al. (2008) who noted no reduction in progeny
production when adult T. javae were stored for up to 35 days, on
the contrary adults stored for ten days produced more progeny
than unstored ones. These beneficial effects are rare and reasons
are so far unsolved.
6. Conclusion
Cold storage tolerance is thus a very plastic trait influenced by a
range of endogenous (biotic) and exogenous (abiotic) factors


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