1. Introduction
The natural distribution range of the spurge hawkmoth
Hyles euphorbiae Linnaeus covers large parts of the Palearctic region in Europe, Asia, and North Africa, involving diverse climate and vegetation zones. Although extensive genetic and whole genome studies show that all populations of
H. euphorbiae constitute one genetic entity [
1,
2], previous investigations focused on morphological as well as genetic variability [
3,
4,
5,
6,
7] revealed a correlation between the distribution of larval and adult morphotypes, mitochondrial DNA (mtDNA) lineages, and the geographical occurrence of the populations. Moreover, based on mtDNA and nuclear DNA, Mende et al. [
1] found two main genetic lineages with geographical correlation, suggesting environmental and climatic barriers as main factors influencing their distribution and the location of the contact zone between them. The lineage ‘euphorbiae’ (according to Hundsdoerfer, Lee et al. [
2]) is distributed in the area of Central, Western, and Eastern Europe with seasonal frosty climatic conditions, while ‘tithymali’ is prevalent across regions without pronounced cold seasons in Southern Europe and Northwestern Africa. Diverse environmental adaptations must not necessarily lead to obvious morphological differences during all life cycle stages, even though two main larval patterns have been observed [
6]. A subdivision of the species into geographically delimited entities with ecologically distinct preferences appears to be common in Lepidoptera [
8] (reviewed in Aardema et al. [
9]), and a correlation with the occurrence of mitochondrial lineages [
1,
5] could indicate an incipient stage of speciation [
1].
Recent gene and protein expression studies notably suggest distinct physiologically adapted ‘euphorbiae’ pupal phenotypes [
10,
11]. Especially the pupal stages, although morphologically uniform, are predestined for the study of physiological plasticity, because of their presumed variability in the ability to overcome periods of various unfavorable environmental conditions [
5]. Depending on local seasonal weather patterns, populations can establish one or more generations per year. The final larval stage before pupation (fifth instar) shows a high sensitivity to differences in photoperiod and temperature, which are the main factors determining the kind of the pupa stage [
12]. The pupae of the ‘tithymali’ populations from the most southwestern localities in the subtropical climatic zone (e.g., Macaronesia), where the daylight length varies only slightly and the coldest winter temperatures do not fall below an average of 10 °C [
13], are not confronted with distinctive seasonal climatic fluctuations during the year. Although the appearance of all developmental stages throughout the whole year is documented, the most abundant occurrence of adult individuals comprises more or less the winter months (e.g., predominantly September–April on Canary Islands or February–October on Cape Verde). This timing corresponds to seasonal availability of leaves on larval food plant (Euphorbia). In the summertime, the leaves drop off, the food is scarce, and the majority of larvae do not survive (own observations MG, [
14]).
In ‘euphorbiae’ populations adapted to four seasons (e.g., in temperate Central Europe), the presence of frost-sensitive developmental stages is clearly delimited to the warmer part of the year due to the regular advent of subzero temperatures. During the favorable vegetation period characterized by the ‘long day’ conditions (up to over 16 light hours per day) together with warm outdoor temperatures, metamorphosis proceeds in quick-hatching (subitan) pupae. This pupal stage develops within only about 2–5 weeks, omitting the stage with externally induced developmental delay - the diapause [
14,
15,
16]. During the annual advent of inconvenient environmental conditions, the day length shortens (to slightly less than 8 light hours per day). Simultaneously, the temperature decreases successively, reaching its minimum markedly below 0 °C in winter months [
13]. These two external changes initiate cold hardening and trigger diapause of the pupae, the overwintering stage, which may insist up to 2 years [
11,
17] (own observation). Furthermore, the remarkable temperature and daylight length variation not only influence but also synchronize the annual population cycle [
12].
Diapause in insects is a complex process [
11,
18,
19,
20,
21], which is characterized by modifications in the metabolism. The resulting pupa phenotype is manifested by its increased capability to survive ambient conditions [
10,
11,
21].
Focusing on diapause induced by decreasing external temperatures in freeze avoiding insects, which do not tolerate the formation of internal ice inside of their bodies [
22], the ability to supercool their body fluids is crucial to cope with subzero temperatures [
18,
23]. This means, they are able to depress the temperature, at which freezing is initiated spontaneously, below the melting point of water, without changing the phase due to the lack of nucleation sources [
18]. Cryoprotectants such as polyols (e.g., glycerol), sugars (glucose, trehalose), and proteins (e.g., antifreeze proteins) facilitate supercooling in the organism and prevent freezing damage [
24]. The supercooling point (SCP) is the temperature at which spontaneous crystallization cannot be further suppressed. Freeze-avoidant insects are not able to survive SCP temperatures or below [
25]. The physiological phenotype of the pupae is thus expected to be indirectly detectable by calorimetric measurements of the SCP. This in combination with the direct experimental detection of the survival ability (mortality) of individuals at various ambient temperatures may discern overwintering diapause stages from subitan pupae, as well as differentially cold-hardy phenotypes of distinct
H. euphorbiae populations.
That diapause pupae of ‘euphorbiae’ populations survive subzero temperatures is well known. In contrast, no cold-induced diapause as well as no ability of pupae to survive at lower temperatures (under 7 °C) was observed in eremial North African ‘tithymali’ populations [
14,
26] (and own observations MG). For ‘euphorbiae’, Harbich (pers. comm.) reported that gradual cooling to 4 °C under laboratory conditions allowed overwintering of diapause pupae under subzero temperatures. Precooled pupae were able to reach stronger cold hardiness than un-acclimated ones. Harris and Alex [
15] demonstrated almost 100% survival of the diapause pupae at 2 °C and 80% mortality at −17 °C after gradual cooling. A low number of pupae survived even −20 °C, which was in accordance with their field experiments and previous studies (e.g., [
27,
28]). Thus, we assume that all ‘euphorbiae’ pupae that entered diapause are able to gradually enhance their cold hardiness and cope even with subzero temperatures [
11], while for ‘tithymali’, this ability should be remarkably constricted (own observations MG).
Despite scattered evidence of differences in cold survival and the indications of an underlying genetic regulation [
10,
11], a clear association between temperature regime and phenotypic cold hardiness in different populations has not yet been systematically investigated. This would be an important basis to understand different physiological adaptations between lineages/ecotypes or formerly recognized species and subspecies in an elsewise over large geographic areas genetically uniform species [
2]. The main goal of our study is thus to increase our understanding of how the two incipient subspecies, ecotypes, or lineages (not homologous with mitochondrial lineages) of
H. euphorbiae, which are geographically as well as climatically constrained (e.g., [
5], own observations MG) and genetically distinguished by two main mtDNA haplotype lineages, change their phenotype in biophysical characteristics, while being exposed to various experimentally simulated winter conditions. We use pupae for our tests, as the life stage with two different states, namely subitan (non-overwintering, cold sensitive) and diapause (overwintering, cold resistant).
The Hyles euphorbiae individuals of the ecotype consisting of ‘euphorbiae’ populations are expected to precisely follow the annual temperature and daylight cycle resulting in accentuated phenotypic differences between subitan and diapause pupae. In contrast, those from ‘tithymali’ populations are expected to be less synchronized with the daylight annual cycle and distinctly less cold tolerant, even if these pupae could be able to undergo cold hardening to some extent. To our knowledge, this has not yet been investigated in detail to date.
The null hypothesis H
0 states that the environmental conditions of the pupae are the only factors determining cold hardiness: (i) Differences in light treatment leads to cold-hardy diapause (short-day regime) or cold-sensitive subitan (long-day regime) pupae; and (ii) differences in temperature, i.e., precooled or not precooled pupae, influence the degree of cold-hardiness. Thus, (i) larvae bred under long-day conditions develop to cold-sensitive subitan pupae with no difference between the two ecotypes
H. euphorbiae ‘euphorbiae’ and ‘tithymali’. Larvae bred under short-day conditions would be cold hardy, since they produce diapause pupae for overwintering, irrespective of which population they were from. (ii) Precooled diapause pupae (cold acclimation) would be cold hardier than pupae that were not precooled (irrespective of the population of origin). Hypothesis H
1 states that certain differences in the genetic constitution of each ecotype determine the degree of cold hardiness in reaction to environmental conditions. The exact mechanism of underlying genetic/epigenetic determination (e.g., [
10]) is beyond the scope of this paper. Thus, (i) pupae from ‘euphorbiae’ populations would differ from those of ‘tithymali’ in that they are generally more cold resistant. Conversely, they are able to react more appropriately to environmental signals and cold acclimation so that (ii) light regime and precooling would increase cold hardiness in ‘euphorbiae’ more than in ‘tithymali’. (iii) Long-term winter conditions would lead to a stronger increase in cold hardiness in ‘euphorbiae’.
We measure supercooling ability and mortality of the pupae after different light and cooling regimes in order to test these hypotheses within and between the ecotypes. We expect that a combined effect of light (determines pupa type) and temperature (degree of acclimation) in the cold-adapted ‘euphorbiae’ populations would lead to a cold-resistant diapause pupae phenotype with the highest degree of cold hardiness.
With our experiments, we focus on the following basic questions for biological understanding:
Do we find evidence of phenotypic differences (supercooling, mortality) between ecotypes of H. euphorbiae, suggesting physiological adaptation as a basis for the observed lineage distribution? To which extent is H. euphorbiae (subitan as well as diapause pupae) resistant to decreasing (even subzero) external temperatures? Which conditions or their combination are crucial for triggering the switch between subitan and diapause pupae? How high is diapause mortality, and how long is diapause duration in different populations?
5. Conclusions
Based on the results of our analyses, we propose two regulation mechanisms controlling low-temperature resistance in H. euphorbiae.
The first approach includes short-term hemolymph changes regulated through the sensitivity of the late larva instar and/or early pupa (supposedly the first activated response in
Hee) to daylight duration shifts, also induced by low-temperature stress alone (proposed main responsible pathway for the cold-stress feedback in
Het). This defense is present in all individuals regardless of population origin and may evoke an opportunistic or short-term cold tolerance [
42,
43,
44]. The resulting phenotype is slightly more cold acclimated than the subitan pupae but possibly not able to provide long-term cold resistance. Despite this, the ability for cold acclimation, including subzero temperatures, leading to cold-induced diapause was ascertained also for
Het populations, which was not known before.
Only the additional effect of cold/lowering temperature following short day conditions may trigger the second mechanism, which is necessary to reach an appropriate efficient and long-persisting cold tolerance in terms of a seasonal cold hardening [
21,
22,
47,
48,
49]. This ability, due to a combined effect of light and cooling regime, was detected only for the
Hee ecotype. Thus, our results support the hypothesis H
1, stating that there are differences between the ecotypes of
H. euphorbiae in their responses to cold stress, which can be detected as different extents of cold hardiness in each of the two ecotypes. These phenotypic differences correspond with the influence of diverse climatic living conditions of each population. In the frosty winter season, the appropriate longstanding cold tolerance is crucial. By contrast, in rather moderate climates with less predictable weather changes, but no hard winter, the occasional change to diapause helps to survive stochastic unfavorable conditions.
Even though these distinguished environmental conditions may facilitate an incipient species differentiation process [
1], persisting similar optima of living requirements, climatic fluctuations, and good mobility of adult individuals can act in the opposite way, enabling contact, hybridization, and gene flow between ecotypes. This may explain the apparent genetic unity of all populations of
H. euphorbiae despite adaptation to different climatic conditions [
2]. For the accurate understanding of ongoing processes, further studies are needed.