# Insights into the triggering factors and determination mechanisms of host alternation in aphids

Aurelie Etier, Rituparna Ghosh, Théo Vericel, Romuald Cloteau, Gaëtan Denis, Maïwenn Le Floch, Yannick Outreman, Gaël Le Trionnaire,

Jean-Christophe Simon

► To cite this version:

Aurelie Etier, Rituparna Ghosh, Théo Vericel, Romuald Cloteau, Gaëtan Denis, et al.. Insights into the triggering factors and determination mechanisms of host alternation in aphids. Entomologia Generalis, 2025, 10.1127/entomologia/2025/3028. [https://hal.science/hal-05076879v1](https://hal.science/hal-05076879v1) . [hal-05076879](https://hal.science/hal-05076879v1)

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Submitted on 21 May 2025

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# Insights into the triggering factors and determination mechanisms of host alternation in aphids

Aurelie Etier<fnref n="1" />,**, Rituparna Ghosh<fnref n="1" />,**, Théo Vericel<fnref n="1" />, Romuald Cloteau<fnref n="1" />, Gaëtan Denis<fnref n="1" />, Maïwenn Le Floch<fnref n="1" />, Yannick Outreman<fnref n="2" />, Gaël Le Trionnaire<fnref n="1" />, Jean-Christophe Simon<fnref n="1" />,*

With 5 figures

**Abstract:** The bird cherry-oat aphid *Rhopalosiphum padi*, is one of the ten percent of aphids that are host alternating. The annual life cycle of *R. padi* entails an obligatory migration between a primary woody host, *Prunus padus* (Rosaceae), where sexual reproduction occurs, and many secondary hosts of the Poaceae family, where successive generations reproduce parthenogenetically. Migration between the two types of host plants is achieved by specialized migrant morphs. Very little is known about the cues that trigger migrant development, host alternation establishment and the underlying mechanisms. In this study, we aimed to elucidate the key steps involved in migrant induction and determination of host alternation, and to shed light on these intricate processes. We found that when first or second instar larvae of *R. padi* were placed on mature leaves of *P. padus* they developed into migrant morphs, while exposure to young leaves resulted in the development of wingless individuals that remain on the primary host. We also demonstrated the gradual establishment of acclimation to the secondary hosts that precedes the migration step. Finally, we showed that the switch to feeding on the alternative hosts is irreversible once emigrants have reached adulthood. These results not only allowed us to define the critical periods of the leaf stage associated with morph induction and determination, but also to pinpoint the emigrants' developmental stages when acclimation to secondary hosts begins. These novel insights of these critical periods will facilitate the investigation of molecular changes associated with host alternation in aphids.

**Keywords:** *Rhopalosiphum padi*; *Prunus padus*; nutritional polyphenism; primary and secondary host; morph induction

## 1 Introduction

Aphids (Hemiptera; Aphididae) represent a diverse group of insects comprising more than 4700 identified species (Blackman & Eastop 2007). The phenotypic plasticity exhibited by aphids enables them to quickly cope with varying seasonal, crowding, or dietary conditions, thereby facilitating their remarkable capacity to colonize and utilize plants (Blackman 2000). A striking illustration of this plasticity is their ability to produce alternative phenotypes in response to fluctuating environmental factors, a phenomenon referred to as polyphenism (Moran 1992). Environmental cues serve as pivotal triggers for polyphenism, initiating phenotypic divergence through neurochemical and hormonal pathways that induce short- to long-term transcriptional reprogramming, often mediated by epigenetic regulatory mechanisms (Srinivasan & Brisson 2012). Polyphenism in aphids can be categorized into four main types: caste (defensive vs reproductive morphs), reproductive (sexual vs parthenogenetic morphs), dispersal (winged vs wingless morphs), and **nutritional** (Braendle et al. 2006; Shibao et al. 2010; Yan et al. 2020). The latter enables them to transition typically from woody host plants (during winter) to herbaceous host plants (during summer), a phenomenon known as host-alternation (Blackman & Eastop 2007; Dixon 1998). It has been proposed that host-alternation represents an adaptation to seasonality by allowing nutritional complementation between hosts whose quality and presence/availability fluctuate across the seasons (Dixon 1998). However, the fact that only ten percent of all aphid species can alternate hosts has led some authors to propose that host alternation is only advantageous under certain conditions and subject to various types of evolutionary constraints (e.g. ecological, physiological) that prevent this trait from becoming widespread (Moran 1992; Blackman 2006; Blackman 2000). In addition to their fascinating life history traits, aphids pose a significant threat

<footnote n="1">¹ UMR 1349 IGEPP, INRAE, Institut Agro, Université Rennes 1, Le Rheu, France</footnote>

<footnote n="2">² UMR 1349 IGEPP, INRAE, Institut Agro, Université Rennes 1, Rennes, France</footnote>

<footnote>* Corresponding author: jean-christophe.simon@inrae.fr</footnote>

<footnote>** Authors contributed equally to this work</footnote>
to crop yields, both directly by consuming plant nutrients and indirectly by transmitting phytopathogenic viruses (Dedryver et al. 2010). Although many economically important aphids are host alternating species (e.g. *Aphis fabae*, *Myzus persicae*, *Rhopalosiphum padi*), the cellular and molecular mechanisms underlying host alternation in aphids are currently unknown. Gaining a deeper comprehension of host alternation is imperative for understanding the biology of these economically important insects.

Among host-alternating species, *R. padi* stands out as a prominent cereal pest in northern Europe, causing a 15% yield loss of cereals (Jarošová et al. 2019; Leather & Dixon 1982). *R. padi* undergoes host alternation, transitioning between a primary woody host within the genus *Prunus* during winter (*Prunus padus*, exclusively in Europe) and numerous secondary hosts in the Poaceae during summer (e.g. wheat, barley, oat, maize, ryegrass and numerous wild grasses) (Dixon 1971b). The life cycle of *R. padi* begins with oviposition on *P. padus* in the early winter (see Supplementary data Fig. S1). Wingless *fundatrix* emerges from overwintering eggs in the early spring. The *fundatrix* produces wingless *fundatrigeniae* by parthenogenesis (asexual reproduction). Both *fundatrix* and *fundatrigeniae* are specialized to the primary host. After two to three parthenogenic generations of *fundatrigeniae*, spring emigrants appear, which move to secondary hosts belonging to the Poaceae (Wiktelius 1984). Emigrants colonise the secondary hosts and produce wingless *virginiparae*, which are specialized to the secondary hosts. During the summer, both wingless and winged *virginiparae* are produced on the secondary hosts. In response to the shortening of day length, *virginiparae* produces *sexuparae* parthenogenetically. *Sexuparae* sequentially produce two distinct winged morphs in their offspring: first the *gyno-parae*, followed shortly by the males, both of which fly to the primary host. On the primary host, sexual reproduction occurs between male and the *oviparae* (wingless sexual female produced by the *gynoparae*), to produce eggs.

Upon the spring migration, environmental cues are perceived by the *fundatrigeniae* that induce emigrant development (see Supplementary data Fig. S1). Aphid development consists of four larval stages followed by an adult stage. The first and second larval stages (L1 and L2) of *fundatrigeniae* and emigrant are morphologically identical. However, L3 and L4 (*fundatrigeniae*) differ morphologically from N3 and N4 (emigrant) stages, with visible wing buds in the latter. In the *fundatrigeniae*, L3 develops into a wingless adult that remains on *P. padus*. In contrast, in the emigrant, N3 develops into a winged adult that feeds on *P. padus* until migrating to the secondary hosts (Dixon 1971b; Glinwood & Pettersson 2000).

The nutritional polyphenism in *R. padi* is influenced by changes in environmental conditions, such as crowding and plant-associated signals (Dixon & Glen 1971; Wiktelius 1984). Dixon & Glenn (1971b) underscored the significance of plant signals in triggering nutritional polyphenism

in *R. padi*, specifically by showing the involvement of plant maturity. However, their study did not provide details regarding the specific age or maturity level of the leaf responsible for instigating this morph switching. A time-course analysis is essential for precisely delineating age or maturity stage responsible for triggering the development of the emigrant. Ultimately, we aim to identify the changes in phloem compounds responsible for inducing host alternation, although this is not addressed in the present study.

We also lack information regarding the temporal window during larval development wherein perception of plant-emitted signals and/or crowding trigger the development of emigrants. It remains unclear whether these signals can be detected and responded to as early as the first generation of *fundatrigeniae*, or if a few generations must pass before the development of emigrants. Emigrant development becomes apparent from the third larval instar stage, indicating two potential sensitive time frames. The first possibility involves a pre-natal sensitive period when aphid embryos are still within the mother's ovarioles, facilitating the transmission of signals from the mother to her offspring (Dixon 1998). The second possibility corresponds to a post-natal sensitive period, where signal perception occurs during the first and/or second larval instar stages and initiates the switch in only a few days.

Our understanding of the mechanism of nutritional polyphenism during emigrant development remains elusive. Previous investigations have primarily focused on this process in emigrant adults, revealing a heightened preference for secondary hosts in 24 to 48 hours old emigrant adults compared to newly emerged individuals within the same generation (Glinwood & Pettersson 2000). In aphids, there is a well-established phenomenon of mutual exclusivity between flight development and settling on the secondary hosts (Dixon 1971a). This suggests a sequential activation pattern in which flight development precedes acclimation to the secondary host. Emigrants can only encounter the new host once they possess the ability to fly and locate it. After selection of the secondary hosts these aphids need to acclimate to the new environment (acclimation being the process of adjusting to new environmental conditions). In the context of nutritional polyphenism, we hypothesize that host acclimation does not necessarily begin only after the aphid reaches the secondary host, as such a delay would compromise its fitness on the new host. Consequently, it can be postulated that the developmental machinery governing host switching and acclimation operates in parallel rather than in series. Further investigation is warranted to elucidate the initiation of nutritional polyphenism during larval development. We hypothesize that a complex dynamic interplay of molecular mechanisms involving flight readiness and host acclimation may exist, which could potentially allow for the reversal of host switching. Host switching in *R. padi* may be essential in natural habitats to cope with the decline in the nutritional quality of *P. padus* trees; however, in the persistent presence
of spring leaves, *fundatrigeniae* may remain longer on the primary host (Dixon & Glen 1971).

Building on this background, we identified two critical stages in the host alternation of *R. padi* during its association with the primary host: (1) the induction of the emigrant morph triggered by specific plant-derived cues, and (2) the development of the capacity to migrate to and feed on the secondary host. This host-shifting capacity may involve acquiring both a pre-migration preference for the secondary host and a pre-migration ability to thrive on it. The existence of such a pre-acclimation phase could ensure the immediate and successful utilization of the secondary host upon arrival.

This study aims to precisely identify each of these stages of aphid development on the primary host. Furthermore, the existing literature remains unclear whether emigrants leave the primary host due to an abrupt loss of the ability to feed on it or because the primary host gradually becomes unsuitable, therefore prompting migration to the secondary host.

To address these knowledge gaps, we formulated four specific research questions: (1) What is the critical period of leaf maturity required to induce emigrant production and facilitate the transition from the primary to the secondary host? (2) At what developmental stage or generation do *R. padi* larvae perceive environmental signals that initiate emigrant morph development? (3) Does pre-acclimation occur during the larval development of *R. padi* on the primary host, facilitating subsequent acclimation to the secondary host? (4) Is host switching in newly emerged winged adults a reversible mechanism if the nutritional quality of the primary host becomes favorable again?

This study is crucial for understanding the developmental and environmental factors that drive host alternation in *R. padi*, providing insights into how aphids acclimate and transition between hosts. By identifying the critical stages and cues involved, it sets the stage for exploring the molecular mechanisms underlying these processes, such as gene expression changes and hormonal regulation.

# 2 Materials and methods

## 2.1 Insect and plant sources

Ten *R. padi* *fundatrices* at the L1/L2 developmental stages (i.e. few days after egg hatching) were collected on *Prunus padus* in the beginning of March 2023 from Le Rheu, France (48.1011°N, 1.7954°W). *Fundatrices* were individually reared immediately upon collection onto young leaves in isolation within clip-cages (1.5 cm diameter) to mitigate potential crowding effects, which could interfere with the perception of plant cues. They were placed onto the back of uninfested leaves. Young *P. padus* plants of 75–100 cm hight were purchased from local nurseries. These saplings were planted singly into pots (3 litres) with soil (60% white peat,

40% sand, 14% coconut fibre medium, 6% clay). Wheat (*Triticum aestivum*, Geny variety) seeds were used as the secondary host and were germinated individually in the same soil inside 15 mL plastic tubes with holes for water penetration. 15-days old seedlings were used for experiments. Plants and insects were grown inside climatic chamber with constant temperature (18°C) and photoperiod [16:8 h (L:D)], to eliminate seasonal effect. The plants were used for growth measurements, insect culture maintenance and assays. No fertilizers or pesticides were added after planting to avoid any change in plant growth.

## 2.2 Defining leaf age

*Rhopalosiphum padi* emigrants are produced in late spring. It was hypothesized that changes in leaf biochemistry is responsible for induction of winged morph. To mitigate environmental impact on this phenomenon, plants were grown at constant temperature and photoperiod. Leaf growth was monitored by precisely measuring their length (mean ± SE; in mm). Only the first flush of leaves was used in this experiment to avoid high variance in data due to (1) nutrient deficiency with time, and (2) influence of already existing leaves on the growth of second or next flushes of leaves. Measurements of the same leaves (n = 8) were noted, three times a week, over a period of 30 days post bud-burst.

## 2.3 Wing induction experiment

From five *fundatrices* reared in clip-cages, the first four individuals produced by each *fundatrix* were collected at the first larval instar (L1). The larvae were introduced to two contrasted categories of leaves based on the growth pattern analysis of *P. padus* leaves: 15 days-old (considering the bud-burst event as day zero) leaves (hereafter referred to as young leaves) and 30 days-old leaves (hereafter referred to as mature leaves) (see Supplementary data Fig. S2 for a graphical representation of the experimental protocol). The larvae were distributed as follows to the two conditions: out of the four larvae collected from one *fundatrix*, two were deposited within clip cages onto young leaves (referred to as condition 1) and two onto mature leaves (referred to as condition 2). Larvae reared exclusively on young leaves until reaching the L3 stage were maintained in clip cages on young leaves until larviposition. From one *fundatrigeniae*, two L1 were then subjected to two conditions: one in Condition 3 placed on mature leaves immediately after larviposition, and one in Condition 4 initially maintained on young leaves and transferred to mature leaves after reaching the L2 stage. In total 20 freshly born larvae were deposited individually for each condition as replicates.

After reaching the third larval instar (4 days after transfer) the ratio of L3 (future *fundatrigeniae*) / N3 (future emigrants) was monitored across all the tested conditions under a binocular microscope (magnification 6×).
## 2.4 Choice and no-choice preference tests

From the *fundatrices* maintained in clip-cages, 100 *fundatrigeniae* were reared on actively growing plant for one generation. The second generation was exposed to mature leaves and were collected at six stages: (i) L1, (ii) L2, (iii) N3, (iv) N4, (v) winged adults before leaving *P. padus* leaves, and (vi) emigrant 24 h after leaving *P. padus* leaves. A settling choice bioassay was used to determine host plant preference at each stage. Whole-plants for *P. padus* and 15 days old wheat seedlings were used. One mature leaf of *P. padus* still attached and two-branch tip of wheat leaves were placed on each side of a Petri dish (Ø 90 mm). The Petri dish was covered with a lid and sealed with parafilm. Additionally, the wingless *fundatrigeniae* specialized to the primary host, and wingless and winged *virginiparae* specialized to the secondary hosts were used as controls to validate our experimental approach (i.e. we expect wingless *fundatrigeniae* to be more attracted by *P. padus* than wheat and the opposite for *virginiparae*). To stimulate movements, aphids were starved for two hours, then a total of 20 individuals per stage were released into the centre of the Petri dish and the experiment was repeated six times. Given the high vulnerability of the L1 stage, we conducted tests both with and without prior starvation. However, despite these efforts, we observed high mortality rates in both conditions, and the results were excluded from our analysis. The plant on which they settled was recorded after 12 hours, a time which is generally enough for choice establishment in aphids (Sochard et al. 2021a; Sochard et al. 2021b).

## 2.5 Survival study of the emigrants on wheat

From the 100 *fundatrigeniae* reared on actively growing plant for one generation, the produced larvae were collected at six stages after experiencing conditions inducing emigrant production (mature leaves): (i) L1, (ii) L2, (iii) N3, (iv) N4, (v) winged adults before leaving *P. padus* leaves, and (vi) emigrant 24 h after leaving *P. padus* leaves. Forty individuals per stage were individually transferred and reared on one single 15-days old wheat seedling. After wheat infestation, survival was monitored daily for seven days.

## 2.6 Testing the reversibility of the host alternation in *R. padi* emigrant adults

One hundred *fundatrigeniae* were reared on actively growing *P. padus* plant for one generation. Their progeny was reared on mature leaves and were collected as winged adults before leaving *P. padus* leaves and individually placed into plastic bags containing one leaf either young or mature or one single 15-days old wheat seedling in a no choice experiment. A total of 40 adults were placed per bag. Survival rates of the *R. padi* adults were monitored daily for a period of seven days to investigate the viability and the reversibility of the host alternation.

## 2.7 Data analysis and statistics

All the statistical analyses were performed using R software R v4.2.2, with value of p < 0.05 considered as significant. The statistical tests and analyses employed in this study are detailed in the supplementary data section. Results of GLM, GLMM and Cox hazard survival model are summarized in supplemental data table S1, S2, and S3.

# 3 Results

## 3.1 Characterization of the leaf growth pattern of *P. padus*

*Prunus padus* leaves followed a growth pattern, which could be divided into four phases based on their length (Fig. 1): (1) initial slow growth phase with a growth rate of 1.9 mm/day up to day four (7.75 ± 0.96 mm), (2) fast growth phase with an average growth rate of 5.7 mm/day from the fifth to 19th day (94.5 ± 4.73 mm), (3) final slow growth phase of 2.1 mm/day from 19th to 23rd day (103.25 ± 2.88 mm), and (4) very slow/no growth phase. With these results, 15-day and 30-day old leaves were selected as representatives of young and mature leaves, respectively.

## 3.2 The role of leaf maturity in inducing emigrant development and the responsiveness of generations and first and second instar larvae

Based on the growth pattern analysis, we hypothesized that young leaves would induce the production of *fundatrigeniae*, while mature leaves would induce emigrants. Freshly born first instar larvae placed onto young leaves (condition 1) all developed into *fundatrigeniae*. Conversely, the majority (90%) of L1 reared on mature leaves (condition 2) developed into emigrants (Fig. 2). The effect of leaf age on winged/wingless proportion was highly significant ($\chi^2$ = 12.8, df = 1, p = 0.007) and the maturity of leaves was sufficient in inducing emigrants’ development. The L1 from the first (condition 2) or second (condition 3) generation following *fundatrix* stage showed no significant difference in their responsiveness to plant maturity cues (binomial GLMM,

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** *Prunus padus* leaf growth kinetics. The line (mean ± SE) represents the leaf length (n = 8 leaves from three plants). Growing and mature leaves were used for further experiments.</figcaption></figure>
$\chi^2 = 0.24$, df = 2, p = 0.63). Additionally, larvae exposed to mature leaves from the first instar (condition 3) or from the second instar (condition 4) did not reveal a significant difference in the morph determination (binomial GLMM, $\chi^2 = 2.13$, df = 2, p = 0.14). These findings indicate that both first and second generations after *fundatrix* are sensitive to leaf maturity exposure. Furthermore, in the second generation, L1 and L2 were both able to perceive the leaf-based signal, leading to the development of emigrants (Fig. 2).

## 3.3 Nutritional polyphenism is gradually established during the larval development in *R. padi*

### 3.3.1 The initiation of the switch in the preference of host-plant

In order to investigate the temporal dynamics of nutritional polyphenism concurrent with emigrant development, the preference for primary and secondary hosts was evaluated. We found in our host plant choice assay that the mean proportion of *fundatrigeniae* opting for wheat was approximately 5%, whereas more than 85% of secondary host-specialized wingless/winged adults exhibited this

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Influence of the plant maturity on wing induction in *R. padi*. Monitoring of wing induction under four experimental conditions. Condition 1 involved placing individual L1 larvae produced by *fundatrix* in clip cages on young leaves, while Condition 2 entailed placing them on mature leaves. Furthermore, L1 larvae from the second generation (after Condition 1) were subjected to two additional conditions: transfer to mature leaves immediately after birth (Condition 3) or after reaching the L2 stage (Condition 4).</figcaption></figure>

preference, consistent with anticipated outcomes and showing the relevance of our experimental design. Due to a high mortality rate observed after the manipulation and starvation of the first larval instar upon their deposition in Petri dishes (98%, data not shown), the analysis of their preferences was not feasible. Furthermore, the different stages or morphs significantly affected the preference for wheat in *R. padi* (binomial GLMM, $\chi^2 = 132.32$, df = 7, p < 0.001 (Fig. 3)), with *fundatrigeniae* again serving as the reference. The GLMM's coefficients (Supplementary data table S1) showed that *fundatrigeniae* and the emigrants at the second and third larval instars had a similarly very low preference for wheat. However, the wheat preference in *fundatrigeniae* was significantly lower than for the fourth larval instar onward, but no significant difference with the third larval instar. These results indicate an initiation of host plant preference switch from the fourth larval stage.

### 3.3.2 The initiation of the pre-acclimation to wheat

The last observations support the hypothesis of pre-acclimation of host switching, potentially occurring from the fourth larval instar onwards. To investigate possible pre-acclimation to wheat feeding, the settling behavior of emigrants was studied by monitoring their survival rate daily for seven days after their transfer to wheat at the six stages described previously. Stage significantly affected the survival of *R. padi* on wheat (Cox proportional hazards model, $\chi^2 = 68.46$, df = 5, p < 0.001) (Fig. 4). Of all the different stages tested, the first and second larval instars exhibited significantly lower survival rate, with approximately half of the individuals dead 24 hours after transfer to wheat (Supplementary data table S2). Stage N3 displayed moderate survival over the seven days, significantly higher than that of the L1 and L2 stages. The survival rate for the N4 stage was significantly higher than that of the N3 stage and was comparable to that of the emigrants before leaving *P. padus* and the emigrant after the flight. These findings indicate a pre-acclimation to the secondary host initiated from the third larval instar and firmly established from the fourth one.

## 3.4 Host switching is irreversible in adult emigrants

The potential reversibility of host alternation following exposure to new favorable conditions of *P. padus* was investigated in this experiment. Within four days after transfer, half of the emigrants installed on either young or mature leaves of *P. padus* were dead. Furthermore, a significant reduction in survival rate was observed in emigrant adults reared on either young (32.5%) or mature leaves (20%) compared to those reared on wheat (80%) (Cox proportional hazards model, $\chi^2 = 27.86$, df = 2, p < 0.001) (Fig. 5). In addition, there was no significant change in the survival rates between individuals reared on young or mature leaves of *P. padus* (Supplementary data table S3). These findings underscore
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Proportion of *R. padi* individuals at various morph or emigrant developmental stages selecting wheat as the host plant when presented with a choice between *P. padus* (primary host) and wheat (secondary host). Significant differences compared to the *fundatrigeniae* are labeled as NS for "non-significant", \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.005.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Estimated survival probability of *R. padi* individuals installed on wheat at six different emigrant developmental stages (L1, L2, N3, N4, BL, and AL). BL (Before Leaving) refers to emigrants before leaving *P. padus*, while AL (After Leaving) refers to emigrants 24 hours after leaving *P. padus*. Each vertical step in the curve indicates a death event.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Estimated survival probability for emigrants of *R. padi* (collected before leaving *P. padus*) reared on either young leaves of *P. padus* (YL, light green), mature leaves of *P. padus* (ML, brown), or wheat (W, dark green). Each vertical step in the curve indicates a death event.</figcaption></figure>
the irreversibility of host switch, even under conditions favorable for the growth of *R. padi* adult emigrants, such as young leaves of *P. padus*.

## 4 Discussion

In this study we first characterized the growth pattern of *P. padus* leaves, providing a proxy for assessing leaf maturity. We subsequently demonstrated that the leaf maturity signal, perceived in the first and second larval instar, is sufficient to trigger the development of emigrants. Furthermore, we showed that pre-acclimation to the secondary host begins at the third larval instar of emigrant development, and becomes irreversible upon reaching adulthood.

### 4.1 Leaf maturity alone triggers migrant development

In our study, two distinct leaf growth stages were identified: 15-day-old young leaves and 30-day-old mature leaves. As leaves mature, they transition from being metabolite sinks to sources, which affects their nutritional quality (Coleman 1986). Insects' preference-performance highly depends on plant chemistry, which undergoes changes upon leaf matura- tion and associated biochemical alterations, influencing the plant-insect relationship (Barber & Marquis 2011; Feeny 1970). This change includes a reduction in essential nutri- ents and possibly an increase in toxic compounds, making the leaves less suitable for insect consumption (Coley 1980; Haukioja et al. 2002). Consequently, aphids might respond to the decline in food quality by developing into winged emigrants to seek better habitats. Our findings showed that this transition from young to mature leaves alone was suffi- cient to trigger this emigrant development. This suggests that aphids likely perceive changes in phloem composition asso- ciated with leaf maturity and respond by developing alterna- tive phenotypes. In *P. padus*, leaf maturation coincides with changes in amino acids and phenolics (Czerniewicz et al. 2011; Sandström & Pettersson 2000), while the alteration of other compounds remains unexplored. Further research is needed to identify the specific phloem compounds respon- sible for inducing this response.

### 4.2 First and second larval instars are the critical stages to induce the migrant

The morph-switching mechanism relies on a sensitive period of larval development, identified here as the first and the sec- ond larval instar. The production of wings is the first notice- able change in the establishment of nutritional polyphenism. Wing development in aphids is considered the default devel- opmental pathway, with wing primordia present in the first larval instar. During post-embryonic development, these wing primordia will either degenerate or continue their develop-

ment, depending on environmental and physiological cues. The degeneration of wing primordia typically occurs during the second instar larvae stage (Ishikawa & Miura 2009). This explains that the window of sensitivity mainly resides at the first or second larval instars when exposed to mature leaves. In addition, our results suggest that emigrant determination occurs post-natally, as the mothers and their offspring were reared in clip-cages to avoid crowding effects. Although a maternal effect may also initiate this process, it is not man- datory in *R. padi* (Dixon & Glen 1971; Müller et al. 2001). Taken together, these findings highlight the rapid induction of nutritional polyphenism enabling the exploitation of two complementary seasonal food resources (Dixon 1971a). This ability allows *R. padi* populations to anticipate deteriorat- ing conditions, thereby ensuring the sustained survival of the colony.

### 4.3 Acclimation to the secondary host is established during the larval development and is irreversible

Earlier studies showed that the shift in host preference and acclimation to the secondary host occurs gradually follow- ing the adult molting stage (Glinwood & Pettersson 2000; Leather & Dixon 1982). Our study demonstrates an earlier preference and pre-acclimation of *R. padi* to the secondary host. Indeed, host choice tests showed changes in prefer- ences throughout emigrants' development from the fourth larval stage. Existing literature suggests that the interplay between repulsion and attraction, guided by chemical cues, influences host plant preference. Volatiles such as methyl salicylate from *P. padus* play a key role in primary host repellence (Glinwood & Pettersson 2000). The duration of flight before settling on secondary hosts might be influ- enced by chemosensory pathway activation or repression for different hosts during emigrant development (Peng et al. 2021). Recent studies showed that aphid host-plant adapta- tion involves molecular mechanisms that modulate plant defenses or detoxify host-specific secondary metabolites. The current study indicates a predetermined capacity for host switching establishing during larval development that is irreversible and obligate after reaching the adulthood. Once again, the pre-acclimation to secondary hosts may indicate optimization of nutritional polyphenism, enabling rapid exploitation of favorable habitats by the population. Altogether these observations suggest that the genetic pro- grams needed to exploit the new host are probably switched on before the contact to the new host and even before take- off. The temporal sequence between wing development (first and second larval instar), pre-acclimation to secondary hosts (third larval instar) and preference switching (fourth larval instar) may indicate a finely regulated cascade of molecu- lar mechanisms underlying nutritional polyphenism, which remains to be elucidated.
## 5 Conclusion

To clarify the timing and nature of the critical periods of the nutritional polyphenism in *R. padi*, we demonstrated that plant maturity is a key factor influencing the development of spring emigrants. We also identified that the window of perception occurs during the first and second larval stages enabling the development of emigrant within the same generation. The current study for the first time in a host-alternating aphid species, documents pre-acclimation to secondary hosts, initiated from the third larval instar and becoming irreversible after adult molting. These findings highlight the capacity of *R. padi* to sense environmental cues, particularly the primary and secondary host-associated cues for migration. This study provides a foundation for future research aimed at achieving the broader goal of understanding the molecular mechanisms underlying the nutritional polyphenism of aphids.

**Acknowledgments:** This research was funded by ERC Alterevo, grant number 30001959. This work is dedicated to Professor A. F. G. Dixon, a pioneer in aphid ecology and biology and a source of inspiration to many aphid researchers.

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Manuscript received: September 20, 2024

Revisions requested: December 13, 2024

Revised version received: January 5, 2025

Manuscript accepted: February 2, 2025

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**Fig. S1, S2; Statistical analysis; Table S1–S3**