Temperature-dependent regulation of diapause and hatching success in aphids: implications for climate change

TL;DR

This study shows how temperature affects aphid diapause and hatching, with implications for how climate change might influence their survival and population dynamics.

Contribution

The study reveals temperature-dependent changes in diapause and hatching success in aphids, providing new insights into climate change impacts.

Findings

Higher temperatures accelerated embryonic development and led to earlier hatching.

Hatching success was highest at 0 °C and lower at 10 °C.

Premature hatching at higher temperatures caused developmental abnormalities and egg cracking.

Abstract

Diapause is a crucial adaptive strategy that enables insects, including aphids, to survive unfavorable environmental conditions, particularly during winter. Diapause of aphids takes the form of facultative and embryonic diapause occurring in the eggs. This study investigates the diapause dynamics of aphids, based on Maculolachnus submacula (Walker), at 3 different temperatures to assess the impact of elevated temperatures on embryonic development and hatching success. To determine the diapause strategy and to detect dividing cells, immunostaining was performed. M. submacula exhibited a slow embryonic development strategy during diapause, with mitotic activity and body growth occurring at all tested temperatures. Embryos incubated at higher temperatures achieved greater body length and leg proportions. Higher temperatures accelerated embryonic development, leading to earlier hatching. Hatching success was temperature-dependent, with the highest percentage of hatched individuals at 0 °C and lower success at 10 °C. Premature hatching at higher temperatures resulted in increased rates of developmental abnormalities of the embryo bodies and egg cracking disorder. Survival of nymph rates was low, with mortality reaching 63.5% in natural conditions. These findings highlight the significant influence of temperature on diapause progression, embryonic development, and survival. The conducted studies indicated that an increase in ambient temperature in winter affects aphids with a diapause strategy, characterized by slow development of embryos during winter diapause. Rising global temperatures due to climate change make the understanding of the effects of increasing temperatures on aphid diapause essential for predicting shifts in population dynamics, pest outbreaks, and ecological interactions.

Graphical Abstract

Figures

Oviparous female (A) of Maculolachnus submacula, oviparous females and eggs on host plant (B). Scale bar 1 mm long.

Body parts of Maculolachnus submacula with arrows showing cell divisions at 35 days of embryo development at different temperatures (A) 0 °C, (B) 8 °C, (C) 10 °C. All scale bars are 100 μm long.

Changes in mean body length (A) and third leg (B) of aphid embryo during the diapause process in 3 different temperatures (n = 20 at each temperature, on each measurement day). Statistical differences in the maintenance phase of diapause were marked with different letters at the level of P < 0.05 (Tukey test). All scale bars are 100 μm long.

Development of embryos of Maculolachnus submacula in 3 different temperatures (A) and developmental disorders at temperature 10 °C (B). All scale bars are 100 μm long.

Success of hatched nymphs in 3 different temperatures (A), nymph’s mortality caused by winter heat waves (B), hatching egg (C), arrow shows egg burster.

The impact of climate change on the course of life cycle and diapause in aphids.

Full text

Introduction

According to the latest reports, we are currently facing the largest climatic changes in almost 2,000 yr; the global temperature has risen very sharply over the last 50 yr. Despite the measures aimed at protecting living organisms from global temperature increases, it is very difficult to protect ecosystems from rising temperatures, so it is very important to know how temperature increases will affect living organisms (Lee et al. 2023, Uzelac et al. 2024). Global temperature changes have a strong impact on insects by affecting their life cycles, reproduction, survival, and wintering. Aphids, due to short generation time and high reproductive rate, react and adapt faster to climate change than other, poikilothermic organisms (Harrington et al. 2001, Bale et al. 2002, Menéndez 2007, Hulle et al. 2010). A longer and warmer autumn, as well as increasing temperature in winter (often appearing as long-period heat waves), have an influence on species that enter diapause as a strategy to survive winter.

Diapause is a crucial adaptive strategy that allows insects, including aphids, to survive unfavorable environmental conditions, particularly during winter. This programmed state of developmental arrest and metabolic suppression enables insects to endure seasonal fluctuations in temperature and resource availability (Koštál et al. 2009, Denlinger 2022). The main features of diapause are arrest of development, metabolism inhibition, and an increase in stress resistance (Koštál et al. 2009, Saulich and Musolin 2017). Insects may enter diapause in different developmental stages, such as embryo, pupa, larval, or imago and this is a species-specific feature (Shingleton et al. 2003, Denlinger 2022). In aphids, embryonic diapause occurs in overwintering eggs and is primarily induced by photoperiodic signals, although temperature and food availability also play significant roles in its regulation (Hand et al. 2016, Karp 2021). Rising global temperatures due to climate change make the understanding of the effects of increasing temperatures on aphid diapause essential for predicting shifts in population dynamics, pest outbreaks, and ecological interactions.

Temperature plays a key role in the induction, maintenance, and termination of diapause in insects (Hodek 1996, Denlinger 2002, Koštál 2006). Recent studies suggest that higher temperatures can disrupt diapause by shortening its duration, or even preventing its initiation entirely (Bale and Hayward 2010, Diniz et al. 2017, Barberà et al. 2019, Cuti et al. 2021). Global climate change, with rising winter and summer temperatures and prolonged warm autumns, affects insect development rates and population dynamics (Kroschel et al. 2013). The most significant effects occur in autumn, when extended warmth influences physiological processes such as developmental rates and diapause induction (Marshall and Roe 2021, Roe et al. 2024). Prolonged warm autumns can lead to increased generations per season, delayed migration, postponed or skipped diapause (Gallinat et al. 2015). Entering diapause while temperatures remain high can result in increased energy expenditure, reduced survival rates, and post-diapause fitness (Williams et al. 2012, Roe et al. 2024). Although aphid diapause is primarily triggered by photoperiodic cues received by the maternal generation, temperature modulates this process by influencing the intensity and duration of diapause. This can have profound consequences for aphid species that rely on diapause for overwintering, as premature diapause termination may expose developing embryos to unfavorable early spring conditions (Bale et al. 2002, Bale and Hayward 2010).

Aphids exhibit 2 main types of reproductive cycles: holocyclic and anholocyclic. Holocyclic species alternate between parthenogenetic reproduction during the growing season and sexual reproduction in autumn, which leads to the production of diapausing eggs (Ogawa and Miura 2014). In contrast, anholocyclic species reproduce parthenogenetically year-round in warm climates, skipping the diapause phase entirely. The switch from parthenogenesis to sexual reproduction in holocyclic species is induced by a shorter photoperiod and decrease in temperatures (Simon et al. 2002, Durak et al. 2020, Cuti et al. 2021). Despite similar environmental conditions in spring and autumn, aphids do not produce sexual morphs in spring, due to an internal timing mechanism known as the “interval timer” (Lees 1960, Lushai et al. 1996).

In aphids, unlike most insects, photoperiodic regulation of diapause is transgenerational and is inextricably linked to a switch in reproduction mode, rather than merely a seasonal halt in development (Le Trionnaire et al. 2008, Denlinger 2022). The transition of aphids from parthenogenetic to sexual reproduction is regulated by a photoperiodic circadian clock located in the brain of adult females (Colizzi et al. 2024). Shortening day length is perceived by the visual system and integrated by molecular oscillators based on clock genes such as period (per), timeless (tim), and cryptochrome (cry). Changes in circadian signaling affect neurons of the pars intercerebralis, leading to alterations in the secretion of neuropeptides and a reduction in the activity of the insulin/insulin-like signaling pathway. As a consequence, the levels of juvenile hormone (JH) decrease and ecdysteroid signaling is modified, which determines the developmental fate of the embryos. Hormonal changes do not directly affect the female, but are transmitted to the embryos developing inside her (the phenomenon of “telescopic generations”). As a result, the embryos are developmentally programmed into sexual morphs (females and males), which, after copulation, lay eggs that enter embryonic diapause. Thus, photoperiod functions as the primary seasonal cue initiating sexual reproduction and the production of overwintering eggs. Temperature acts as a modulatory factor, enhancing the effect of short photoperiods under low temperatures and attenuating it under warmer conditions. Emergence from diapause occurs with the rise in temperature in spring, which reactivates metabolism and hormonal pathways, including insulin. In this way, the integration of information about the photoperiod in adults and the temperature acting on the eggs ensures the seasonal synchronization of the aphid life cycle (Barberà et al. 2019, Cuti et al. 2021, Colizzi et al. 2024).

The relationship between aphid diapause and host plant phenology is another key factor influenced by temperature changes. Many aphid species synchronize their life cycle with the seasonal development of host plants to ensure food availability upon hatching (Borowiak-Sobkowiak and Durak 2012, Durak et al. 2023). Rising temperatures can alter host plant budburst timing, or aphid egg hatching time, potentially leading to a mismatch between aphid hatching and resource availability. This mismatch may have significant ecological consequences, affecting aphid survival, reproductive success, and interactions with predators and parasitoids (Denlinger 2022). Moreover, periods of high temperatures (heat waves) in late winter, followed by a return to low winter temperatures, may have a particularly negative effect, causing early hatching and increased mortality of nymphs. Heatwaves are short-term episodes of unusually high temperatures (lasting several consecutive days), including those occurring in winter, that exceed the normal thermal range for a given season and cause physiological stress and increased mortality in overwintering insects (Abarca et al. 2019, Gudowska et al. 2025).

Embryonic development is characterized by 2 distinct phases: the first phase, termed anatrepsis, involves the fully segmented embryo migrating posteriorly and becoming embedded within the yolk; the second phase, known as katatrepsis, during which the embryo rotates to assume its final orientation, with the head directed toward the anterior pole of the egg. Diapause of most species of aphids starts when the embryo is fully segmented, during the anatrepsis phase (Shingleton et al. 2003 Durak et al. 2020).

The most distinctive characteristic of insect diapause is the cessation of development, a phenomenon extensively documented in numerous insect species (Denlinger 2002, Koštál 2006, Diniz et al. 2017). However, the embryonic diapause has been well described only in Bombyx mori (Linnaeus 1758) (Yamashita 1996). Previous studies on various aphid species have shown that diapause can follow different developmental pathways. In aphids, 2 types of diapause have been identified (Durak et al. 2023). Research on Acyrthosiphon pisum (Harris 1776) (Shingleton et al. 2003), Cinara spp. (Curtis 1835) (Durak et al. 2020), and Brachycorynella asparagi (Mordvilko 1929) (Durak et al. 2023) demonstrated that these species continue embryonic development, with mitotic cells visible throughout diapause. However, some species, such as Appendiseta robiniae (Gillette 1907) (Durak et al. 2023), undergo a true arrested stage during diapause. The effects of increasing temperatures on these diapause strategies remain poorly understood. It is hypothesized that species with slow development during diapause, due to increase of temperature in winter, may experience accelerated embryonic growth, leading to mortality, earlier hatching, and desynchronization with host plants.

Maculolachnus submacula (Walker 1848) is a monoecious holocyclic species belonging to the subfamily Lachninae. It is a dark brown aphid feeding on different Rosa species. Colonies occur in the lower parts of the shoots near the ground (Mehrparvar and Lashkari 2021). Sexual generations occur in September to October. Oviparous females lay eggs on shoots. Initially, the eggs are dark yellow (Fig. 1), and over time they turn black. Aphid colonies live symbiotically with ants. During dry spells, aphids migrate to the root collars where ants build covers of soil particles around them (Gottschalk 1989).

Oviparous female (A) of Maculolachnus submacula, oviparous females and eggs on host plant (B). Scale bar 1 mm long.

This study aims to determine the type of diapause of M. submacula, analyze its progression at 3 different temperatures, and assess the impact of elevated temperatures on diapause dynamics. By examining changes in the diapause duration, we seek to understand how climate change may affect aphid population dynamics, pest management strategies of these pests, and related plant diseases. Understanding these mechanisms is crucial for predicting future shifts in aphid behavior and developing adaptive agricultural and ecological management practices.

Materials and Methods

Aphids and Egg Material

This experiment used a modified methodology from the Durak et al. (2020) paper. Aphids were collected from the natural environment in the south-eastern part of Poland and bred in natural conditions on rose variety “the fairy.” In summer, parthenogenetic females were divided into clip cages of 20 individuals and observed until the appearance of a sexual generation. In autumn, when the temperature dropped to 13 °C to 15 °C and the photoperiod shortened to 10:14LD, a sexual generation of males and oviparous females appeared. The timing of egg laying was strictly monitored. From the day the females laid eggs, the eggs were collected daily with a brush and transferred to vials. The samples were labeled with the date of harvest, which later allowed for precise division of eggs of the same age. Until the 21st day of embryogenesis, the eggs were kept in the environmental chambers at a temperature of 10 °C and 10:14LD, which corresponded to the natural conditions prevailing in autumn. On day 21 (when the embryos had complete body segmentation), the eggs were transferred to 3 different temperatures, that is, 0 °C, 8 °C, and 10 °C. These temperatures were selected to represent winter thermal regimes characteristic of continental, oceanic, and Mediterranean regions of Europe, and were set approximately 1 °C above current regional mean winter temperatures to reflect ongoing climate warming. On days 21, 35, 49, 63, 70, and 112, samples were collected, with each sample containing 20 eggs.

Fixing the Embryos, Dissection, and Staining

To remove chorion, the eggs were placed in 50% bleach for 2 min and then transferred to a scintillation vial containing 5 ml 4% formaldehyde in PBS (phosphate buffered saline solution) and 5 ml heptane, and vibrated for 30 min. In the next step, formaldehyde was switched to methanol, and the vials were succussed until the vitelline membrane broke. After rinsing the eggs with methanol a couple of times, the embryos were positioned on double-sided sticky tape under PBS. Membranes were removed using syringe needles and embryos were transferred to PBS to be cleaned, and finally to methanol for storage at −20°C. To detect dividing cells, embryos were subjected to immunostaining using Anti-phospho (Ser10)-acetyl (Lys14)-Histone H3 rabbit polyclonal antibody which detects Histone 3 phosphorylated at serine 10 and acetylated at lysine 14. To perform staining, embryos were washed twice in ethanol in Eppendorf tubes for 3 min on a rotating wheel and 3 times in PBT (0.1% Triton-X 100 in PBS) for 5 min. To block nonspecific protein binding sites, 1% BSA (bovine serum albumin) in PBT was used. After incubation and removal of the BSA, the primary antibody (Millipore) was added to the tubes in a dilution of 1:5000. After incubating the samples at room temperature for 2 h, the primary antibody was removed, and the embryos were washed 3-fold in PBT, each lasting 5 min. A secondary antibody (Alexa Fluor 647; Invitrogen, Carlsbad, CA, USA) was added in a dilution of 1:1000, and the samples were once more incubated for 2 h at 25 °C. After the removal of the secondary antibody, the embryos were washed 3 times in PBT and DAPI (fluorescent DNA stain) was added to the last wash, incubated for 10 min, and then microscope slides were prepared.

Embryo Size Measurement and Morphology

The size of the embryos, which developed at 3 different temperatures (n = 20 at each temperature, on each measurement day), was measured on the basis of photos taken with a confocal microscope (Zeiss LSM710, Carl Zeiss Microscopy GmbH, Jena, Germany) and scanning electron microscope (SEM). We used a commonly used program to measure the size and length of the embryo’s legs using Microscope Software Zeiss Zen 2009 Light Edition (Minocha et al. 2017). For the distorted embryos, the length was estimated as the sum of several linear measurements. Based on these photos, the morphology of the embryos was assessed.

Dependence of Egg Hatching Success on Temperature

In each of the tested temperatures (0 °C, 8 °C and 10 °C), 50 eggs were reared until the 112th day of embryo development as this period allows the observation of all embryonic developmental stages in aphid species exhibiting diapause in the form of slow, continuous development (Durak et al. 2023). The hatching success of the nymphs at each temperature was then counted.

Dependence of Nymph Mortality on Late Winter Heat Waves

The effect of late winter heat waves on the survival of nymphs was analyzed on material collected from the environment (samples collected in Rzeszów, Poland, 8 March 2025). We verified the hypothesis that nymphs hatched as a result of late-winter heat waves exhibit reduced survival when temperatures subsequently drop sharply, including exposure to subzero conditions, leading to high mortality shortly after hatching. The research used freshly hatched nymphs that appeared as a result of a 1-wk heat period at the end of winter. In order to precisely recognize the impact of late winter heat waves on nymph mortality rates, 2 complementary, 5-day experiments were conducted. It is worth noting that nymphs hatching usually at the beginning of April are typically exposed to temperatures around 8 °C.

In natural conditions after 1 wk of temperatures exceeding an average of 13.8 °C during the day (and maximum temperature exceeding 19 °C) and average night temperatures above 6 °C minimum temperatures, a field experiment was carried out. One hundred freshly hatched nymphs were isolated on the plants for the following 5 days. During this time, the air temperature dropped. The daily average temperature was 2.1 °C (with 4.2 °C maximum temperature), and average night temperature was 0.6 °C, but the minimum night temperature reached −8°C.

At the same time, 100 freshly hatched nymphs were placed in a breeding chamber when the temperature at night was −7°C, for 2 h, and 0 °C for the remaining 10 h, for 5 days. During the day, the temperature was 4 °C. In both variants of the experiment, after 5 days of its duration, all living and dead nymphs were counted.

Statistical Analysis

Collected data were presented as means ± SD, n = 20 for each temperature. We used 1-way analysis of variance (ANOVA) and Tukey post hoc tests to show the difference between samples collected in different temperatures, with significance level P ≤ 0.05. For all statistical analyses, Statistica ver.10 was used.

Results

The experiments started on day 21, a week after the embryos were fully segmented. Changes in development were observed at the analyzed temperatures from the beginning of the experiments. Mitotic cells were observed at each temperature from day 21, and in the subsequent days (Fig. 2). Mitotic divisions were observed in the whole body and an increase of the body length, as well as differentiation in body parts, proved that embryos develop continuously. On this basis, we have determined that M. submacula is an aphid species that has the ability to develop continuously and slowly during winter diapause.

Body parts of Maculolachnus submacula with arrows showing cell divisions at 35 days of embryo development at different temperatures (A) 0 °C, (B) 8 °C, (C) 10 °C. All scale bars are 100 μm long.

At 8 °C and 10 °C, a decrease in the number of visible mitotic cells was observed from day 49, which correlates with the appearance of the cuticle (Durak et al. 2023), which in higher temperatures, appears earlier than in lower temperatures.

Both the growth of the embryo body and the third pair of legs were observed at all temperatures (Fig. 3). At 0 °C from day 21 to 112, the embryo’s body length increased 1.4 times; in higher temperatures, growth proportion was similar, 1.75 at 8 °C and 1.79 at 10 °C. On hatching day, in aphid eggs which were bred at 10 °C, the average length of the third leg was 64% of the body length. At the same time, embryos bred in 0 °C, showed an average third leg length of 48% and those bred at 8 °C, 53%.

Changes in mean body length (A) and third leg (B) of aphid embryo during the diapause process in 3 different temperatures (n = 20 at each temperature, on each measurement day). Statistical differences in the maintenance phase of diapause were marked with different letters at the level of P < 0.05 (Tukey test). All scale bars are 100 μm long.

Between day 21 and 70 in all examined temperatures, we did not observe statistically significant differences in body and third leg length (P > 0.05, Tukey test). After katatrepsis, the embryos grew significantly and statistical differences appeared between temperatures. In higher temperatures, embryos were bigger and started hatching earlier. In 0 °C on day 112, embryos were about 0.8 times the body length of the embryos bred at 10 °C. Differences in third leg length were observed on day 70 (P < 0.05, Tukey test) (Figs 3 and 4A). On day 112 of development, embryos reared at higher temperatures differed significantly in size and leg length compared with those reared at 0 °C (P < 0.05, Tukey test).

Development of embryos of Maculolachnus submacula in 3 different temperatures (A) and developmental disorders at temperature 10 °C (B). All scale bars are 100 μm long.

Embryos developing at different temperatures during diapause differed in timing and hatching success. The percentage of hatched individuals was lower in higher temperatures. At 8 °C and 10 °C, hatching occurred earlier, which in natural conditions corresponds to the middle of winter. On day 112 at the highest analyzed temperature (10 °C), 40% of the eggs hatched, and at 8 °C, 60% of the eggs were successfully hatched (Figs 4A and 5A). At 0 °C, embryos continued to develop and did not hatch on the 112th day of development (Fig. 4A). The hatching success rate at a temperature of 0 °C at 112 was 0%. However, all the embryos that were reared at 0 °C hatched at an appropriate time after completing their development (Figs 4A and 5A).

Success of hatched nymphs in 3 different temperatures (A), nymph’s mortality caused by winter heat waves (B), hatching egg (C), arrow shows egg burster.

Additionally, we observed that at temperature of 10 °C on the 112 day, a number of the eggs were cracked, which suggested that the embryos had started hatching, but many of them did not hatch successfully, had morphological disorders such as changes in body proportions, disproportionately long limbs, distortion of egg burster, and developmental malformations (Fig. 4B). We observed hatching disorders, mainly due to nymphs not being able to discard the egg casings (Fig. 5C).

We also tested the survival success of hatched fundatrix nymphs. The results demonstrated that nymphs hatched during late-winter heat waves exhibited significantly reduced survival when followed by abrupt temperature declines, including exposure to subzero conditions. Mortality of fundatrix nymphs following heat-wave events and subsequent low-temperature stress was very high under both natural and laboratory conditions, averaging 63.5% in natural conditions and 87.8% in laboratory experimental conditions (Fig. 5B).

Discussion

In temperate zones, embryos of aphids will remain in their eggs until spring, when the temperature allows them to develop and the nymphs have access to food. Temperature plays a significant role in initiating diapause, for example, high temperature could cause skipping diapause (Pittendrigh and Takamura 1987, Lankinen et al. 2023) or a delay in egg laying dates. Deferring the start date of egg laying by a few weeks would probably result in a reduction of egg quality, shorten the time when sexual forms copulate, and reduce the number of eggs. These changes, combined with those discovered in this research, reduce the hatching rate in higher temperatures, which may lead to a significant reduction in the number of founders in spring. A reduced number of aphids could cause significant changes in the functioning of the environment. Aphids are the food source for ladybugs, flies, and larvae of Syrphidae flies, which means that a reduction in food source equals a reduction in the number of predators (Nelson et al. 2004).

Our studies have shown that M. submacula develops continuously during winter diapause, as evidenced by the presence of cells in a state of mitotic division and the continued slow growth of embryos in the eggs (Figs 2 and 3). Mitotic cells were observed in embryos developing at the 3 temperatures tested throughout the sample period (Fig. 2). This is consistent with previous research on Cinara genus (Durak et al. 2020), B. asparagi (Durak et al. 2023), and A. pisum (Shingleton et al. 2003) that embryos of some species of aphids can develop slowly and continuously during diapause.

We also observed that M. submacula embryos developed at a similar slow rate regardless of rearing temperature through the first 70 days post-oviposition. This suggests a period of temperature insensitivity, during which development rate is fixed (Fig. 3). In contrast, after day 70 significant differences were observed, embryos at higher temperatures began to rapidly accelerate their development. Previous studies on the aphid species, A. robiniae and B. asparagi, showed that during diapause there are 2 periods dependent on temperature: at the beginning of diapause until day 16 and after katatrepsis and temperature-independent period starting when the embryo is fully segmented, lasting until katatrepsis (Durak et al. 2023). These findings provide further support for the existence of analogous diapause periods in other aphid species. Similar shifts from temperature-sensitive to temperature-insensitive phases during diapause are common across insect taxa (Koštál 2006, Denlinger 2022). In our research, it is clearly shown that between day 21 and 70, the body and leg length of the embryos increased steadily, but at a similar rate at all temperatures. After day 70, we saw significant increases in body and leg size, especially in the higher analyzed temperatures (Fig. 3). The bodies of embryos developing at 10 °C were as much as 1.9 times longer than those developing at 0 °C, and the length of the third pair of legs was as much as 3.5 times longer. Our research shows that the rate of diapause course strongly depends on ambient temperature, which is consistent with Shingleton et al.’s (2003) research on pea aphids.

Higher than normal temperatures cause disturbances in biochemical and metabolomic processes that could cause malformation and developmental defects. Shingleton et al. (2003) observed abnormalities in embryos of A. pisum maintained at 16 °C on day 42, such as disturbances in the process of katatrepsis, that is, the change in position of the embryo within the egg. Embryos also had red eyes and egg burster, characteristic of embryos about to hatch. We also observed abnormalities of M. submacula in 10 °C, such as malformation, and changes in body proportions. The embryos were characterized by disproportionately long legs. It is known that the metabolic rate of insects is sensitive to temperature. Entering diapause in autumn when the ambient temperature is still relatively high exposes diapausing insects to increased energy expenditure (Roe et al. 2024). Also, keeping the insect in the diapause stage results in increased energy expenditures. For example, for Pieris napi (Linnaeus 1758) undergoing pupal diapause, high temperature in autumn has a significant impact on increasing the metabolic rate in diapausing individuals, which translates into their significant weight loss and worse condition after diapause (Williams et al. 2012). Therefore, increased energy expenditures may result in the death of embryos after diapause. We observed dead nymphs in 10 °C. It could be caused by malformation, katatrepsis disorder, or egg cracking disorder. We also observed an inability to crack shield eggs properly. Lower hatching success could be related to loss of one of the functions of the cuticle, namely, protection against water loss and excessive evaporation (Wigglesworth 1945, Gibbs 1998, Gullan and Cranston 2007). Higher temperature during development could cause an increase in evaporation, and even cause changes in the chemical composition of the cuticle (Michelutti et al. 2018) which, in consequence, leads to a decrease in hatching success (Figs 4A and 5A).

Earlier research showed that the strategy of aphid diapause is correlated with a host plant phenology (Durak et al. 2023). In the presented study, we observed early hatching embryos maintained in 10 °C and 8 °C. In higher temperatures, they started hatching earlier than embryos reared in 0 °C. On day 112, all embryos reared in 0 °C still remained in the eggs, while at higher temperatures some of the embryos hatched (Fig. 4). Thus, our research shows that early hatching caused by higher temperature during winter probably could lead to asynchrony between hatching of aphids and host plant development. Early hatching could expose the founders to low temperatures, which are too low for development, as well as a lack of food. Mortality of early hatched founder nymphs would be an additional factor indicating disturbance in the development of spring populations (Fig. 5B).

All vital activities of aphids, as in other insects, including growth, development, survival, reproduction, and migration, are strongly dependent on ambient temperature. Consequently, ongoing global warming is expected to directly affect aphid populations (Bale et al. 2002, Hulle et al. 2010). Climate change may influence both the development and survival of particular aphid species, and their defensive responses can be expressed through physiological as well as behavioral mechanisms (Ma and Ma 2012, Dampc et al. 2020, Durak et al. 2020). However, most known defense mechanisms have been studied primarily in adult stages. Numerous studies have demonstrated a significant acceleration of the first spring flights of aphids in recent years, which is mainly attributed to earlier onset of spring and faster attainment of the minimum population threshold required for migration (Wu et al. 2020). In contrast, the effects of changes in ambient temperature on eggs and embryonic development remain poorly understood. In light of our results, it appears that different aphid species may respond differently to changes in ambient temperature (Fig. 6).

The impact of climate change on the course of life cycle and diapause in aphids.

The early appearance of favorable spring temperatures may cause earlier hatching of overwintering eggs, and also advanced first occurrence dates, which have notably been observed in other species of aphids (Durak et al. 2016, Wu et al. 2020). Fully formed embryos can start hatching quickly if the temperature conditions are favorable (Fig. 6). The consequence of early spring hatching of aphid founders will be an increase in the number of generations of the species. Triggering resumption of development of embryos in early spring could be the function of elevated temperature and water absorption, which was observed in Mindarus abietinus (Koch 1857) (Doherty et al. 2018). On the other hand, the extension of the growing season will increase the number of parthenogenetic generations, later emergence of sexual generations, and entering diapause later. Both phenomena—later entering diapause and earlier exiting diapause—have already been observed in many species of insects, for example, butterflies or flies (Roy and Sparks 2000, Van Asch and Visser 2007, Maurer et al. 2018). An increase in temperature was found to increase the number of generations in, for example, European species of butterflies and moths (Altermatt 2010). It seems that an increase in temperature will affect the development of aphid embryos in eggs (Shingleton et al. 2003). Embryos developing throughout the winter diapause are ready for hatching earlier than species that go through the resting stage and develop later. From this, it follows that climate warming and an increase in spring temperatures will benefit especially slow-growing species during diapause, which will be ready to hatch in early spring. On the other hand, these species will be more susceptible to developmental disorders resulting from increased temperatures, that is, body deformities, body part disproportions, cuticle defects, and also hatching problems. These species will also be most exposed to the impact of heat waves and subsequent late-season freezes in winter, which may additionally cause high mortality among prematurely hatched nymphs.

Understanding the sensitivity of seasonal diapause in aphids is important, not only for understanding the mechanisms regulating aphid bionomy, but may also have important implications for limiting aphid numbers, especially those considered to be serious plant pests and vectors of viral diseases. Accurately predicting egg hatching times is crucial for determining the optimal moments to implement pest control strategies. For example, the original model for predicting nymph emergence was used as a decision-support tool to determine the optimal timing for controlling Philaenus spumarius (Linnaeus 1758) (Lago et al. 2023). Predicting nymphs’ emergence should support timely implementation measures to reduce the spread of aphids.

Conclusion

M. submacula’s diapause strategy is characterized by the slow development of the aphid embryos during winter diapause, with mitotic activity and body growth observed at all analyzed temperatures. Embryos incubated at higher temperatures achieve greater body length and limb proportions, with statistically significant differences. At hatching, the proportion of third leg length relative to total body length varies by temperature, being highest at 10 °C (64%). Higher temperatures (8 °C and 10 °C) accelerate embryo development, leading to earlier hatching of about 40% to 60% of embryos depending on the temperature, while at 0 °C, development progresses more slowly, and no hatching occurs by day 112. Premature hatching at higher temperatures, corresponding to mid-winter under natural conditions, leads to increased rates of developmental abnormalities, such as disproportionate legs, malformation, and failure to fully emerge from the eggshell. Survival rates of hatched fundatrix nymphs are low, with mortality reaching 63.5% in natural conditions and 87.8% in laboratory conditions, indicating that premature hatching negatively affects post-hatching survival. These findings highlight the negative impact of rising temperature on diapause dynamics, embryo development, and hatching success. The research provides valuable evidence on how temperature influences diapause and embryonic development, shedding light on the vulnerability of aphids to environmental change. The observed temperature-dependent differences in hatching success and post-hatching survival suggest that global warming may pose a significant threat to aphid populations by increasing developmental instability and mortality. These insights contribute to a broader understanding of the effects of climate change on other insect species with diapause-dependent life cycles, and thus, ultimately, contributing to conservation strategies and ecological forecasting.