# Type V: A sex-linked crossed resistance to three Cydia pomonella granulovirus isolates, with different levels of dominance

Léa Gingueneau, Bertrand Gauffre, Miguel Lopez-Ferber, Sandrine Maugin, Christine Blachère-Lopez, Sofian Renoult, Samantha Besse, Myriam Siegwart

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Léa Gingueneau, Bertrand Gauffre, Miguel Lopez-Ferber, Sandrine Maugin, Christine Blachère-Lopez, et al. Type V: A sex-linked crossed resistance to three Cydia pomonella granulovirus isolates, with different levels of dominance. Biological Control, 2025, 205, pp.105777. 10.1016/j.biocontrol.2025.105777. hal-05051405

HAL Id: hal-05051405

https://imt-mines-ales.hal.science/hal-05051405v1

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# Type V: A sex-linked crossed resistance to three *Cydia pomonella* granulovirus isolates, with different levels of dominance

Léa Gingueneau<sup>a,b,*</sup>, Bertrand Gauffre<sup>a</sup>, Miguel Lopez-Ferber<sup>b</sup>, Sandrine Maugin<sup>a</sup>, Christine Blachère-Lopez<sup>b,c</sup>, Sofian Renoult<sup>a,e,f</sup>, Samantha Besse<sup>d</sup>, Myriam Siegwart<sup>a</sup>

## HIGHLIGHT

* A new crossed resistance to CpGV-M, -R5 and -V15 was detected in *Cydia pomonella*.

* Type V resistance to CpGV-M and -R5 is Z located and with variable level of dominance.

* Absence of selection for 17 generations does not lead to a loss of resistance.

## ARTICLE INFO

Keywords:

Pests

Biological control

CpGV

Codling moth

Resistance

Inheritance

## ABSTRACT

The codling moth (*Cydia pomonella*), is known to be resistant to many (bio)insecticides, including the *Cydia pomonella granulovirus* (CpGV). Of the multiple isolates available on the European market, two are already known to have selected resistance: Type I to CpGV-M and type II or III to group E (CpGV-S or -R5). In 2019, a monitoring program revealed three wild populations of insects with reduced susceptibility to the three main used virus isolates (CpGV-M, -R5 and -V15). In this context, our aim was to characterise this new type of resistance, referred to as Type V, and to determine its inheritance pattern and degree of dominance, and to investigate whether and how it differs from previously described resistance mechanisms.

We collected a population previously identified as multiresistant from which several lines were isolated and selected during 13 generations with either CpGV-M, -R5 or no virus. Bioassays clearly showed high levels of resistance to CpGV-M, -R5 and -V15, as well as cross-resistance between the three isolates. Individual crossing experiments showed different inheritance modes for resistance to CpGV-M and CpGV-R in type V. Resistance to CpGV-M appeared dominant Z-linked as in type I, while it was more likely recessive, but still Z-linked for resistance to CpGV-R. Altogether, Type V is a novel type of resistance that cannot be overcome by CpGV-V15, and marked by a distinct inheritance pattern. Further research is needed to understand the relationship between resistance to each isolate, and adapt the future pest management strategies.

## 1. Introduction

In agriculture, the intensive use of pesticides on large populations of pests can select resistant individuals that possess the inheritable ability to survive amounts of pesticides that would kill susceptible individuals. Field management of resistance at the earliest stages of its appearance is

a prerequisite for the sustainability of insecticides efficacy. First, it requires monitoring the occurrence, frequency, and location of resistance in natural pest populations (R4P, 2021). Once identified, resistant phenotypes can be isolated into laboratory strains to facilitate the study of underlying resistance mechanisms, with genomic methods subsequently employed to elucidate the genetic bases of resistance; and serve

<footnote><sup>a</sup> INRAE, UR PSH, Domaine Saint-Paul, site Agroparc, Avignon 84914, France</footnote>

<footnote><sup>b</sup> HSM, Univ Montpellier, IMT Mines Alès, CNRS, IRD, 6 Avenue de Clavières, Alès F-30319, France</footnote>

<footnote><sup>c</sup> SPE, INRAE Rte des Chappes, Sophia Antipolis F-06560, France</footnote>

<footnote><sup>d</sup> NPP by UPL, 35 Avenue Léon Blum, Pau F-64 000, France</footnote>

<footnote><sup>e</sup> Biology Centre CAS, Institute of Entomology, Ceske Budejovice, Czechia</footnote>

<footnote><sup>f</sup> University of South Bohemia, Faculty of Science, Ceske Budejovice, Czechia</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: lea.gingueneau@hotmail.fr (L. Gingueneau).</footnote>
as references for bioassays. Genomics methods could be used on resis-
tant laboratory strains, to determine the genetic bases of the resistance.
Knowing these genes allow developing molecular markers for the large-
scale detection of resistance in the field, offering a faster alternative to
classical bioassays. Laboratory strains could also be used to detect a
fitness cost associated with the resistance. Fitness cost means that
resistant individuals have disadvantages compared to susceptible ones
in absence of selection pressure, such as reduced survival or fecundity,
or delayed larval development. Altogether, knowledge obtained from
resistant laboratory strains might support the development of effective
pest management strategies, enabling their implementation in a timely
manner and on a finer geographical scale (Kliot and Ghanim, 2012; Van
Leeuwen et al., 2020; R4P Network, 2021).

The codling moth (Cydia pomonella) is the most significant pest affecting apple orchards worldwide. This lepidoptera has developed resistance to a large number of chemical insecticides, as well as to a virus used as a biocontrol agent (Ju et al., 2021). The *Betabaculovirus cypo-monellae* (CpGV) was first identified in 1964 in Mexico (Tanada, 1964). Due to its efficacy and high specificity, the CpGV mexican isolate, CpGV-M, was widely used as a bioinsecticide in Europe from the 1990 s on-wards. Several CpGV isolates have been isolated later. They are phylo-genetically classified in 7 distinct groups (Eberle et al., 2009; Arneodo et al., 2015; Wennmann et al., 2017; Fan et al., 2019). CpGV-M belongs to group A.

In 2005, a strong resistance to CpGV-M was detected in France and Germany (Fritsch et al., 2005; Sauphanor et al., 2006). It was called Type I resistance. In both countries, the concentration required to kill resistant larvae reached levels up to 10,000 times higher than that needed to kill susceptible larvae (Fritsch et al., 2007, Sauphanor et al., 2006). On this basis, two CpGV-M resistant laboratory strains were created to study mechanisms and genetics of resistance: RGV was created from a susceptible reference strain, into which the resistance character, coming from wild resistant individuals from south of France was introgressed by 9 successive backcrosses (Berling et al., 2009); CpR was directly collected in a wild orchard in Germany where CpGV-M treatment failed, and then reared (Eberle and Jehle, 2006). Crossing experiments between resistant and susceptible individuals showed that the major determinant of Type I resistance is dominant and Z linked (Asser-Kaiser et al., 2007, Asser-Kaiser et al., 2010). In addition, as the results were not fully following the Mendelian transmission, it was supposed that some autosomes could also be involved in the resistance, with minor effects (Berling et al., 2013). Recent Genome-Wide Associ-ated Studies on wild populations confirmed the association of mutations in Z chromosomes and some autosomes with the Type I resistance (Olivares et al., 2023). Although no fitness cost was detected in labo-ratory conditions for Type I resistant individuals (Undorf-Spahn et al., 2012), this resistance might result in a small fitness cost, as the absence of selection for more than 30 generations led to a loss of the original level of resistance on CpR (Undorf-Spanh et al., 2012; Fan et al., 2019).

In France, the RGV colony was used to develop CpGV-R5, a
resistance-breaking isolate by a co-evolution process of CpGV on resis-
tant insects (Graillot et al., 2014). At approximately the same time,
another isolate, CpGV-V15 was developed in Germany. Two new bio-
pesticides based on these isolates have been commercialised in Europe
since 2012: CARPOVIRUSINE EVO2®TM (NPP by UPL, France) and
MADEX® PRO/MADEX® TOP® (Andermatt, Switzerland), respectively.
CARPOVIRUSINE EVO2®TM is a combination of CpGV-R5 (group E) and
CpGV-M (group A), while MADEX® PRO/MADEX® TOP® is a mixture
of isolates from groups A, B and E (Gueli Alletti et al., 2017; Fan et al.,
2019 a). In 2017, Sauer et al. and Jehle et al. identified two new cases of
resistance in Germany, both to CpGV-M (group A) and CpGV-S (group
E). The so-called Type II resistance is dominant and autosomal. The Type
III resistance is more complex with a combination of autosomal gene for
CpGV-S and Z- linked gene for CpGV-M, both dominants. Individuals
carrying resistance Types II or III remain susceptible to CpGV-E2 (group
B). These two resistance types were found in multiple locations in

Germany but haven't been found elsewhere (Jehle, 2024).

In 2020, Siegwart et al. tested 32 wild populations of codling moths for their resistance to the three CpGV isolates commercially available in France and Italy (CpGV-M, -R5 and -V15). They identified two additional resistances: one to both CpGV-M and CpGV-V15 (Type IV) and one to the three isolates. Two-thirds of the natural populations tested contained a significant number of individuals resistant to CpGV-M. One-third of the populations tested contained a significant number of individuals resistant to CpGV-V15, and all these populations also showed resistance to CpGV-M. In addition, three populations containing a significant number of individuals resistant to CpGV-R5, also showed resistance to CpGV-M and CpGV-V15. This nested pattern of resistance suggests an accumulation of resistance, without providing insight into whether the genetic bases of Type I, IV, and V resistances are interconnected.

This study aimed to characterize the mode of inheritance and level of
dominance of Type V resistance and whether the genetic bases of type I
and type V are overlapped. To this end, one of the wild populations
displaying Type V multi-resistance to the three isolates was adapted to
laboratory conditions, and split into three colonies, selected with either
CpGV-R5, or CpGV-M or not selected. These colonies were used to
determine whether the resistance could be fixed, could reach a stronger
phenotype and if it resulted in a fitness cost. Subsequently, individual
crossing experiments were conducted to determine the inheritance
pattern of Type V resistance. We tested two alternative hypotheses about
type V resistance: (H1) Type V is a combination of the mechanism
conferring type I resistance and another mechanism, Type I resistance
(dominant, Z-linked) is linked to Type V; or (H2) a completely new
mechanism of resistance confers resistance to the three isolates
including CpGV-M.

## 2. Material and methods

2.1. *Viruses*

Three isolates of CpGV, stored at -20 °C, were used: the CpGV-M is an amplification of the first isolate used in commercial products (Tanada, 1964); CpGV-R5 corresponds to the isolate 2016-r16 (Graillot et al., 2014) and is the active substance of the commercial product CARPOVIRUSINE EVO2<sup>TM</sup>® (NPP by UPL, France). The CpGV-V15 was obtained from Andermatt and is the active substance of the commercial product MADEX® PRO (Andermatt Biocontrol AG, Switzerland).

## 2.2. Insects

The susceptible laboratory strain (Sv) is coming from a population
collected in "Les Vignères" in south of France, and maintained in labo-
ratory without any insecticide exposure for more than 25 years. The
other susceptible strain (CpNPP) has been reared in the laboratory for
more than 20 years, for industrial production of virus based products. It
originally comes from northern France. Both are reared on an artificial
diet (Guenelon et al., 1981).

A population from Nantes area in Western France, referred to as 44-
002 in Siegwart et al. (2020) was identified as harboring a significant
proportion of individuals resistant to the three tested isolates. This
population was resampled in 2020 for the present study and is desig-
nated here as "MRV". The wild overwintering larvae were kept in a dark
room at 4 °C throughout winter. In spring, after being placed in a 23 °C
and 16D:8N photoperiod room, emerging virgin adults were sexed and
crossed with susceptible individuals of a susceptible laboratory strain
(Sv) to facilitate adaptation in the laboratory. F1 adults from these
crosses were then mixed in equal proportions for each kind of cross to
obtain F2 (i.e. 50 % of offspring resulting from crosses between wild
females and laboratory males, as well as 50 % of the reciprocal crosses
between laboratory females and wild males). No selective pressure was
applied at this step to prevent elimination of recessive alleles. To prevent
cannibalism, each larva was placed individually in a tube containing an artificial diet (Guunnelon et al., 1981). From F2, the MRV strain was divided into three sub-lines: MRV-T, not exposed to viruses and used as an unselected control to indirectly test fitness cost associated with resistance, and MRV-M and MRV-R selected with variable doses of CpGV-M or -R5, respectively. To that aim, 15 µl of a viral solution was applied to the surface of the diet and allowed to dry before a neonate larva was deposited with a brush. MRV-M and MRV-R were selected with variable doses of CpGV-M or CpGV-R5, respectively, from the 2nd to the 4th generation in order to eliminate gradually susceptible individuals, while minimizing the probability of eliminating low-effect alleles in our population. From the 5th to the 15th generation, selection was applied using constant doses of 6250 OB/µL for CpGV-M and 250 OB/µL for CpGV-R5. After the 14th generation, MRV-M was no longer selected, and MRV-R was split into two: one that continued to be selected, referred to as MRV-Rs, and another that was no longer subjected to selection (Fig. 1).

## 2.3. Bioassays

Microplate bioassays were conducted to evaluate the susceptibility of the initial wild population (G0) and the three experimental strains (MRV-T, MRV-M and MRV-R) on the 17th generation (G17) for the three viral isolates (CpGV-M, -R5 and -V15). Bioassay consisted of a 96-well plate filled with artificial diet (Stonefly Heliophis, Industries Ward's Sciences, USA). Six µL of solution containing viruses or water for control was added to each well. One neonate larva was added to each well and incubated for 7 days at 23 °C, 16D:8N photoperiod. After 7 days, the number of live larvae was checked. The susceptible laboratory strain CpNPP was used as reference. When the number of larvae available allowed it, 24 larvae per dose were tested with 3 replicas. For each bioassay a range of 8 doses from 0 to 3,906,250 OB/µL was tested.

## 2.4. Crossing experiments

To determine the mode of inheritance of Type V resistance, single-pair crosses were made between individuals from the 17th generation of MRV and the susceptible CpNPP strain (Fig. 2). First, to test if the

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Design of experimental selection of resistance to two CpGV isolates in *C. pomonella* colonies. After crossing the initial wild population (G0) with a susceptible laboratory strain, the MRV line was divided into 3 sub-lines: MRV-M selected with CpGV-M, MRV-R selected with CpGV-R5 and MRV-T unselected with CpGV. From the 15th generation onwards, no selection pressure was applied to MRV-M and MRV-R, but MRV-R was split into two sublines, with MRV-Rs remaining under selection with CpGV-R5.</figcaption></figure>

genetic bases of Type V resistance to CpGV-M is identical to those of Type I, the line MRV-M was used for single pairs crossing. 20 Virgin MRV-M females were individually mated with virgin CpNPP males to produce F1 offspring (designated as F1f), and reciprocal crosses, involving virgin CpNPP females and MRV-M males, were conducted to produce the corresponding F1 offspring (designated as F1m). A minimum of 20 F1 larvae per pair were placed in individual tubes containing 15 µL of CpGV-M at a discriminating dose (killing a majority of susceptible while sparing a maximum of resistants) of 6250 OB/µL, and 10 larvae per pair were placed into untreated tubes for control. After 21 days post infection (d.p.i.), the number of alive larvae was checked by visual inspection of the individual tubes. The surviving larvae were kept to reach adult stage, from 30 d.p.i, and until none emerged anymore, the number and sex of adult F1s was counted daily.

Subsequently, at least two virgin F1 adults (one male and one female) surviving the discriminating dose, from each initial couple, were individually backcrossed with a CpNPP virgin partner. As for the first cross, 20 F2 offspring per pair were placed into individual tubes containing a discriminating dose of CpGV-M, and 10 in untreated tubes. The number of surviving larvae was checked 21 d.p.i., then, the sex-ratio was recorded at adult stage.

The same experimental setup was applied to MRV-Rs to investigate whether the resistance to CpGV-R5 in Type V relies on the same mechanism as the resistance to CpGV-M. The larvae coming from the crossing between MRV-Rs and CpNPP were exposed to a discriminating dose of CpGV-R5 of 250 OB/µL. The discriminant dose is lower for CpGV-R5 than for CpGV-M because the resistance ratio is lower in the former case.

Last, to determine the mortality of homozygous genotypes of each line, we made 7 single pure pairs for MRV-M, MRV-Rs and CpNPP each. 20 F1 larvae of each couple of MRV x MRV were exposed to the discriminating dose of virus, according to the isolate used for the selection of the respective lines. For CpNPP x CpNPP, 20 F1 larvae were exposed to CpGV-M, and 20 to CpGV-R5. For each pure couple, 10 larvae remained untreated as control.

## 2.5. Statistical analyses

### 2.5.1. Bioassays

In order to evaluate the evolution of resistance under selection, the mortality rate at each dose for the initial population and the three experimental lines at G17 was analysed using a four-parameter log-logistic regression model, as implemented in the drc package in R (version 4.2.2). The lethal doses required to kill 50 % of the individuals ($LD_{50}$) was estimated using the LD function (drc package). Comparison between the susceptible (CpNPP) and resistants (MRV-M, MRV-R, MRV-T) lines was done using the EDcomp function (drc package). The resistance ratio (RR) was calculated as follows: $RR = LD_{50}$ resistant/$LD_{50}$ susceptible.

### 2.5.2. Crossing experiments

Pairs that produced less than 10 offspring were excluded from analyses. First, to determine whether type V resistance allele is located on autosomes or sex chromosomes we compared the mean mortality rates and sex-ratios of surviving F1 offspring (F1f and F1m), depending on the sex of the resistant parent using a $Chi^2$ test. Under a complete autosomal mode of inheritance, we expected no difference between F1f and F1m mortality rates and sex-ratios. By contrast, because in *C. pomonella*, the sex chromosome is Zw in females and ZZ in males, a Z-linked mode of inheritance would lead to a difference in the mortality rate and sex-ratio between F1f and F1m. A mortality rate of 50 % for F1f, where only the males survive; and of 0 % for F1m is expected with balanced sex-ratio.

Second, to assess whether the resistances exhibits (h1) complete dominance or (h2) complete recessiveness, we compared expected and observed mortalities of heterozygous F1 and F2 under each hypothesis, while considering that both resistance to CpGV-M and CpGV-R5 was
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Design of single-pair crosses between MRV individuals (purple and plain thorax) and CpNPP susceptible individuals (black and empty thorax). For the MRV-M sub-line crossing, the virus inoculated to F1 and F2 offsprings was CpGV-M at the dose of 6250 OB/µL. For the MRV-Rs sub-colony crossing, CpGV-R5 at the dose of 250 OB/µL was used. The mortality of treated and untreated larvae were checked 21 days post-inoculation. The surviving larvae were kept to reach adult stage, to differentiate their sex. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

inherited in a Z-linked manner (see results 3.2)(Table 1). The corrected mortality rate (Abbott, 1925) of uncrossed CpNPP couples exposed to each virus were used as homozygous reference for a susceptible phenotype, and the mortality rate of uncrossed MRV-M or MRV-Rs as homozygous reference for a resistant phenotype (reference.mortality rate). To estimate the expected mortalities, it was necessary to calculate

it for each sex separately, and then add them up. As the number of males and females exposed to the virus from the neonatal stage was unknown, we applied, for each crossing, the sex ratio of untreated larvae to the total number of neonate larvae tested, to calculate Est.Nb.males and Est. Nb.females. We finally rounded off to the nearest integer to obtain the number of expected dead larvae as follows (1):

<caption>**Table 1**</caption>

Expectations of genotype and phenotypes of progeny from crossing between resistant (MRV) and susceptible (CpNPP) *Cydia pomonella*. Z = Z sex chromosome, W = W sex chromosome. The resistance allele is located on the Z chromosome, and two hypotheses about the dominance level are made. The phenotype is predicted from the arrangement of resistance (R) and susceptibility (S) alleles in the progeny. The F1 are the offsprings of the first crossing between MRV and CpNPP. The generation F2 are the offspring of the backcross with the F1 and CpNPP.

<table><thead><tr><th>Generation</th><th>Crossing</th><th>Expected Parental genotype</th><th>Expected Progeny genotypes</th><th>Hypothetical level of dominance</th><th>Expected phenotype</th></tr></thead><tbody><tr><td rowspan="4">F1</td><td>♀MRV x ♂CpNPP = F1f</td><td>♀Z<sup>R</sup>W<sup>S</sup> x ♂ Z<sup>S</sup>Z<sup>S</sup></td><td>Z<sup>S</sup>W<sup>S</sup> Z<sup>R</sup>Z<sup>S</sup></td><td>dominant</td><td>Females susceptible, males resistant</td></tr><tr><td rowspan="3">♂MRV x ♀CpNPP = F1m</td><td rowspan="3">♂Z<sup>R</sup>Z<sup>R</sup> x ♀ Z<sup>S</sup>W<sup>S</sup></td><td rowspan="3">Z<sup>R</sup>W<sup>S</sup> Z<sup>R</sup>Z<sup>S</sup></td><td>recessive</td><td>Females and males susceptible</td></tr><tr><td>dominant</td><td>Females and males resistant</td></tr><tr><td>recessive</td><td>Females resistant, males susceptible</td></tr><tr><td rowspan="6">F2</td><td>♀F1f x ♂CpNPP</td><td>♀Z<sup>S</sup>W<sup>S</sup> x ♂ Z<sup>S</sup>Z<sup>S</sup></td><td>Z<sup>S</sup>W<sup>S</sup> Z<sup>S</sup>Z<sup>S</sup></td><td>dominant</td><td>Females and males susceptible</td></tr><tr><td rowspan="2">♂F1f x ♀CpNPP</td><td rowspan="2">♂Z<sup>R</sup>Z<sup>S</sup> x ♀Z<sup>S</sup>W<sup>S</sup></td><td rowspan="2">Z<sup>R</sup>Z<sup>S</sup> Z<sup>S</sup>Z<sup>S</sup> Z<sup>R</sup>W<sup>S</sup> Z<sup>S</sup>W<sup>S</sup></td><td>recessive</td><td>Females and males susceptible</td></tr><tr><td>dominant</td><td>Females half resistant, half susceptible, males half resistant, half susceptible</td></tr><tr><td rowspan="2">♀F1m x ♂CpNPP</td><td rowspan="2">♀Z<sup>R</sup>W<sup>S</sup> x ♂ Z<sup>S</sup>Z<sup>S</sup></td><td rowspan="2">Z<sup>R</sup>Z<sup>S</sup> Z<sup>S</sup>W<sup>S</sup></td><td>recessive</td><td>Females half resistant, half susceptible, males susceptible</td></tr><tr><td>dominant</td><td>Females susceptible, males resistant</td></tr><tr><td rowspan="2">♂F1m x ♀CpNPP</td><td rowspan="2">♂Z<sup>R</sup>Z<sup>S</sup> x ♀Z<sup>S</sup>W<sup>S</sup></td><td rowspan="2">Z<sup>S</sup>W<sup>S</sup> Z<sup>R</sup>W<sup>S</sup> Z<sup>R</sup>Z<sup>S</sup> Z<sup>S</sup>Z<sup>S</sup></td><td>recessive</td><td>Females and males susceptible</td></tr><tr><td>dominant</td><td>Females half resistant, half susceptible, males half resistant, half susceptible</td></tr><tr><td></td><td></td><td></td><td>recessive</td><td>Females half resistant, half susceptible, males susceptible</td></tr></tbody></table>
$$Nb.Exp.Dead.Larvae = (Est.Nb.males \times reference.mortality rate) + (Est.Nb.females \times reference.mortality rate) \quad (1)$$

To compute the corrected number of observed larvae we multiplied of larvae tested by the mean corrected mortality for each type of crossing as follow (2):

$$Nb.Obs.Dead.Larvae = Nb.larvae.tested \times corrected.mean.mortality rate(2)$$

The observed and the expected mortalities were compared for each hypothesis using a Chi-squared test.

The difference in the expected sex ratio of the surviving individuals, under the two hypotheses, was also compared with the sex ratio observed in F1 and F2 larvae in the same way. So we could obtain the expected number of surviving males and females. The number of males and females observed for each replicate was pooled for each type of couple. Expected and observed sex ratios were calculated as the percentage of males over the total number of surviving individuals. The observed and expected sex-ratio were compared for each hypothesis using a Chi-squared test.

# 3. Results

## 3.1. Evolution of resistance under selection

Dose-response bioassays on the initial wild population (G0) gave no significant differences when comparing its LD50 with LD50 of the susceptible reference CpNPP (Fig. 3a,b,c). For CpGV-M (Fig. 3a), the difference between the LD50 of 44–002 and the susceptible reference was not significant (LD50 CpNPP = 350 OB/µL; LD50 44–002 = 8109 OB/µL; p-value = 0.7). Similar results were obtained for CpGV-R5 (Fig. 3b) (LD50 44–002 = 285 OB/µL; LD50 CpNPP = 218 OB/µL; p-value = 0.9) and for CpGV-V15 (Fig. 3c) (LD50 44–002 = 13075 OB/µL; LD50 CpNPP = 4351 OB/µL; p-value = 0.5).

At G17 more contrasted results between the selected MRV lines and the susceptible reference CpNPP were obtained (Fig. 3):

MRV-M was significantly resistant to CpGV-M (LD50<sub>CpGV-M</sub> MRV-M = 3.48E + 06 OB/µL; LD50<sub>CpGV-M</sub> CpNPP = 339; p-value < 0.05; RR<sub>CpGV-M</sub> = 10 265; Fig. 3d). The same conclusion could be drawn for CpGV-V15 (Fig. 3e), but because the mortality of this colony was too low at the higher doses, the LD50 cannot be calculated (LD50<sub>CpGV-V15</sub> > 3906250 OB/µL). MRV-M was also significantly more resistant to CpGV-R5 than the susceptible reference strain (LD50<sub>CpGV-R5</sub>MRV-M = 5.33E + 04 OB/µL; LD50<sub>CpGV-R5</sub>CpNPP = 128 OB/µL, p-value < 0.05; RR<sub>CpGV-R5</sub> = 418; Fig. 3f).

MRV-R was resistant to CpGV-M (Fig. 3d) and CpGV-V15 (Fig. 3e), but the mortality rate at the higher doses was too low to calculate the LD50 (LD50 > 3906250 OB/µL; RR<sub>CpGV-M</sub> > 10,000 and RR<sub>CpGV-V15</sub> > 7000). MRV-R was also significantly more resistant to CpGV-R5 compared to the susceptible reference (LD50<sub>CpGV-R5</sub> MRV-R = 2.28E + 05 OB/µL; LD50<sub>CpGV-R5</sub> CpNPP = 128 OB/µL; p-value < 0.05; RR<sub>CpGV-R5</sub> = 1789; Fig. 3f).

The line MRV-T showed no significant differences in the LD50 compared to the susceptible reference for CpGV-M (LD50<sub>CpGV-M</sub> MRV-T = 7.41E + 05, RR<sub>CpGV-M</sub> = 2189, p-value > 0.05), for CpGV-R5 (LD50<sub>CpGV-R5</sub> MRV-T = 2.42E + 03, RR<sub>CpGV-R5</sub> = 19, p-value > 0.05) and for CpGV-V15 (LD50<sub>CpGV-V15</sub> MRV-T = 6.39E + 04, RR<sub>CpGV-V15</sub> = 126, p-value > 0.05).

## 3.2. Sex-linkage of CpGV resistance

In the individual crosses with MRV-M, a total of 19 and 18 pairs produced enough larvae to be analysed, for F1f and F1m respectively. The corrected mortality rates differed significantly according to the sex of the resistant parent (X² = 34.64; df = 1; p < 0,001) with 48.3 % and 10.4 % for F1f and F1m, respectively. Consistently, the mean sex ratio of progeny surviving the virus treatment also significantly differed according to the sex of the resistant parent (X² = 62.51; df = 1; p < 0,001), with 96.7 % and 46.2 % of males for F1m and F1f, respectively (Table 2).

For the resistance to CpGV-R5 in MRV-Rs, a total of 20 and 14 pairs produced enough larvae to be analysed, for F1f and F1m respectively.

<figure><img src="image_5.png" /><figcaption>Fig. 3. Dose-mortality curves of *Cydia pomonella* larvae exposed to CpGV isolates. Fitted four parameter log-logistic model for mortality of neonate larvae after 7 days of exposure to a range of doses of CpGV. Solid green line and circles: susceptible reference colony (CpNPP); dashed grey line and crosses: resistant line unselected (MRV) for generation 0. Dashed grey line and crosses: unselected line MRV-T; long-dashed red line and inverted cross: MRV-M line selected with CpGV-M; dotted blue line and triangles: line MRV-R selected with CpGV-R5. Panels a, b, c: tests on the generation 0. Panels d, e and f: tests on the resistant lines obtained after 14 generations of virus selective pressure. Panels a, d: bioassay with CpGV-M. Panels b, e: bioassay with CpGV-V15. Panels c, f: bioassay with CpGV-R5. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
**Table 2**

Mean mortality rate and sex-ratio of *Cydia pomonella* larvae exposed to CpGV. Offspring of individual pairs between resistant colonies (MRV-M or MRV-Rs) and a susceptible laboratory strain (CpNPP) were exposed to a discriminating dose of CpGV (6250 OB/µL of CpGV-M for line MRV-M and 250 OB/µL of CpGV-R5 for the line MRV-Rs). F1 are named F1f when resistant females are crossed with susceptible males, and F1m when resistant males are crossed with susceptible females. N = number of pairs, (n) = total number of larvae tested. The mortality was checked 21 days post infection, and survivors were sexed at adult stage. The number of MRV-Rs progeny developing into adults were insufficient for calculation of a sex ratio.

<table><thead><tr><th>Tested colony</th><th>Offsprings</th><th>N(n)</th><th>Corrected mortality (%)</th><th>Observed Sex-ratio (%♂)</th></tr></thead><tbody><tr><td rowspan="2">MRV-M</td><td>F1f</td><td>19 (398)</td><td>48.3 ± 14.5</td><td>96.7 ± 6</td></tr><tr><td>F1m</td><td>18 (363)</td><td>10.4 ± 7.6</td><td>46.2 ± 12.4</td></tr><tr><td rowspan="2">MRV-Rs</td><td>F1f</td><td>20 (400)</td><td>71.3 ± 16</td><td>–</td></tr><tr><td>F1m</td><td>14 (280)</td><td>46.3 ± 20.3</td><td>–</td></tr></tbody></table>

The corrected mortality rate differed significantly according to the sex of the resistant parent (X2 = 12.90; df = 1; p-value > 0.005), with 71.3 % for F1f and 46.3 % for F1m. The mean sex-ratio couldn't be calculated because for most pairs a too low number of offspring reached adult stage to compute an accurate sex-ratio, but when pooling offspring from all pairs together, 22 % of F1m were males, whereas they represented 64 % of F1f. This difference was significant (Chi2 = 35.99; df = 1, p-value < 0.005) (Table 2).

**3.3. Dominance of resistance to CpGV-M in MRV-M**

The mean corrected mortality rate of F1 from the crossing CpNPPxCpNPP exposed to CpGV-M was 98 % whereas it was 8 % for the F1 from MRV-M x MRV-M.

Over the 398 F1f and 363 F1m larvae tested, based on corrected mortalities, respectively 192 and 38 died (Table 3). These numbers of dead larvae were significantly validating the hypothesis of complete dominance (196 F1f and 29 F1m expected; X² = 2.71, df = 1, p-value > 0.05). This number of dead larvae was also significantly different from the number expected under the complete dominance hypothesis (390 F1f and 180 F1m expected; X² = 212.7, df = 1, p-value < 0.005). Furthermore, the sex-ratio of F1f (95.3 %♂) and F1m (46.8 %♂) were significantly identical to the sex-ratio expected under the complete dominance hypothesis (98.0 %♂ and 46.1 %♂ expected respectively; X² = 3.74, df

= 3, p-value > 0.05) whereas the recessive hypothesis is rejected (50.0 %♂ and 1.6 %♂ expected respectively, X² = 1345.97, df = 3, p-value < 0.005).

The number of observed dead larvae of the F2 from the backcross between F1f or F1m with CpNPP are not following the hypothesis of dominance (X² = 15.85, df = 3, p-value < 0.005) neither the hypothesis of recessivity (X² = 101.87, df = 3, p-value < 0.005). The sex-ratio of the F2 offsprings is also significantly different from the expected sex-ratio for dominant resistance (X² = 45.49, df = 7, p-value < 0.005) or recessive resistance (X² = 1185.66, df = 7, p-value < 0.005) (Table 3).

**3.4. Dominance of resistance to CpGV-R in MRV-R**

The mean corrected mortality rate of F1 from the crossing CpNPPxCpNPP exposed to CpGV-R5 was 82 % whereas for the F1 from MRV-Rs x MRV-Rs it was 33 %.

Over the 400 F1f and 280 F1m larvae tested, we estimate after mortality correction that respectively 285 and 130 died of viral infection (Table 4). The number of dead larvae were significantly different from the number expected under the complete dominance hypothesis (230 F1f and 93 F1m expected; X² = 27.68, p-value < 0.005). These numbers of dead larvae were closer but still significantly different to the expected number of larvae under complete recessivity hypothesis (328 F1f and 154 F1m expected, X² = 9.44, p-value < 0.05). The sex-ratio of F1f (63.83 %♂) and F1m (22.06 %♂) were significantly different from the sex-ratio expected under the complete dominance hypothesis (79.82 %♂ and 44.92 %♂ expected respectively; X² = 34.59, df = 3, p-value < 0.005) whereas the recessive hypothesis was not rejected (50.0 %♂ and 18.25 %♂ expected respectively, X² = 8.62, df = 3, p-value > 0.05).

The number of observed dead larvae of the F2 from the backcross between F1f or F1m with CpNPP are not following the hypothesis of dominance (X² = 14.64, df = 3, p-value < 0.005) neither the hypothesis of recessivity (X² = 25.91, df = 3, p-value < 0.005). The sex-ratio of the F2 offsprings is also significantly different from the expected sex-ratio for a dominant resistance (X² = 175.46, df = 7, p-value < 0.005) and recessive resistance (X² = 28.75, df = 7, p-value < 0.005) (Table 4).

**4. Discussion**

**4.1. Type V: A new type of resistance to CpGV**

Viral selection across 14 generations was performed using either CpGV-M or CpGV-R5 on a wild *C. pomonella* population with reduced susceptibility to CpGV-M, CpGV-R5 and CpGV-V15. This process led to

**Table 3**

Comparison between expected and observed mortality of *Cydia pomonella* larvae exposed to a discriminating dose of CpGV-M (6250 OB/µL) from a single-pair crossing experiment. Individuals from the resistant line (MRV-M) were crossed with susceptible individuals (CpNPP). Surviving F1 were individually backcrossed with susceptible individuals to obtain F2. The number of dead larvae was checked 21 days post inoculation and survivors were sexed at adult stage. The theoretical mortality and sex-ratio are calculated depending whether resistance alleles were hypothesized to be dominant or recessive. * = determined on very few individuals looking at the high mortality expected.

<table><thead><tr><th>Generation</th><th>Crossing</th><th>Number of larvae tested</th><th>Hypothesis</th><th>Expected corrected number of dead larvae</th><th>Corrected number of dead larvae</th><th>Expected sex-ratio (%♂)</th><th>Observed sex-ratio (%♂)</th></tr></thead><tbody><tr><td rowspan="4">F1</td><td>F1f</td><td rowspan="2">398</td><td>Dominant</td><td><strong>196</strong></td><td rowspan="2"><strong>192</strong></td><td><strong>98.02</strong></td><td rowspan="2"><strong>95.33</strong></td></tr><tr><td>= ♂MRV-M x ♂CpNPP</td><td>Recessive</td><td>389</td><td>50.00</td></tr><tr><td>F1m</td><td rowspan="2">363</td><td>Dominant</td><td><strong>29</strong></td><td rowspan="2"><strong>38</strong></td><td><strong>46.11</strong></td><td rowspan="2"><strong>46.78</strong></td></tr><tr><td>= ♂MRV-M x ♂CpNPP</td><td>Recessive</td><td>180</td><td>1.64</td></tr><tr><td rowspan="8">F2</td><td>♀F1f x ♂CpNPP</td><td rowspan="2">60</td><td>Dominant</td><td>59</td><td rowspan="2">49</td><td>50*</td><td rowspan="2">80.00</td></tr><tr><td>Recessive</td><td>59</td><td>50*</td></tr><tr><td>♂F1f x ♀CpNPP</td><td rowspan="2">340</td><td>Dominant</td><td>179</td><td rowspan="2">170</td><td>56.07</td><td rowspan="2">48.89</td></tr><tr><td>♀F1m x ♂CpNPP</td><td>Recessive</td><td>260</td><td>5.00</td></tr><tr><td></td><td rowspan="2">367</td><td>Dominant</td><td>178</td><td rowspan="2">214</td><td>98.22</td><td rowspan="2">94.74</td></tr><tr><td></td><td>Recessive</td><td>360</td><td>57.14</td></tr><tr><td></td><td rowspan="2">259</td><td>Dominant</td><td>136</td><td rowspan="2">144</td><td>42.62</td><td rowspan="2">45.83</td></tr><tr><td></td><td>Recessive</td><td>187</td><td>2.78</td></tr></tbody></table>
<caption>**Table 4**</caption>

Comparison between expected and observed mortality of *Cydia pomonella* larvae exposed to a discriminating dose of CpGV-R (250 OB/µL) from a single-pair crossing experiment. Individuals from the resistant line (MRV-Rs) were crossed with susceptible individuals (CpNPP). Surviving F1 were individually backcrossed with susceptible individuals to obtain F2 larvae. The number of dead larvae is checked 21 days post inoculation and survivors were sexed at adult stage. The theoretical mortality and sex-ratio are calculated on whether resistance alleles were hypothesized to be dominant or recessive.

<table><thead><tr><th>Generation</th><th>Crossing</th><th>Number of larvae tested</th><th>Hypothesis</th><th>Expected corrected number of dead larvae</th><th>Corrected number of dead larvae</th><th>Expected sex-ratio (%♂)</th><th>Observed sex-ratio (%♂)</th></tr></thead><tbody><tr><td rowspan="4">F1</td><td rowspan="2">F1f<br>= ♀MRV-Rs x<br>♂CpNPP</td><td rowspan="2">400</td><td>Dominant</td><td>230</td><td rowspan="2">285</td><td>78.82</td><td rowspan="2">63.83</td></tr><tr><td>Recessive</td><td>328</td><td>50.00</td></tr><tr><td rowspan="2">F1m<br>= ♂MRV-Rs x<br>♀CpNPP</td><td rowspan="2">280</td><td>Dominant</td><td>93</td><td rowspan="2">130</td><td>44.92</td><td rowspan="2">22.06</td></tr><tr><td>Recessive</td><td>154</td><td>18.25</td></tr><tr><td rowspan="8">F2</td><td rowspan="2">♀F1f x ♂CpNPP</td><td rowspan="2">130</td><td>Dominant</td><td>105</td><td rowspan="2">81</td><td>56.52</td><td rowspan="2">70.83</td></tr><tr><td>Recessive</td><td>104</td><td>56.52</td></tr><tr><td rowspan="2">♂F1f x ♀CpNPP</td><td rowspan="2">135</td><td>Dominant</td><td>77</td><td rowspan="2">73</td><td>56.14</td><td rowspan="2">29.03</td></tr><tr><td>Recessive</td><td>131</td><td>35.90</td></tr><tr><td rowspan="2">♀F1m x ♂CpNPP</td><td rowspan="2">196</td><td>Dominant</td><td>107</td><td rowspan="2">134</td><td>81.82</td><td rowspan="2">37.50</td></tr><tr><td>Recessive</td><td>161</td><td>54.29</td></tr><tr><td rowspan="2">♂F1m x ♀CpNPP</td><td rowspan="2">116</td><td>Dominant</td><td>67</td><td rowspan="2">56</td><td>63.27</td><td rowspan="2">55.00</td></tr><tr><td>Recessive</td><td>109</td><td>41.94</td></tr></tbody></table>

the establishment of two similar and highly resistant laboratory strains. Both exhibited a high resistance ratio against these three isolates: from 7000 to 10,000 fold. Based on a discriminating dose (equivalent to the LD90 on a susceptible strain), the original wild population exhibited significantly higher resistance to all three isolates compared to the susceptible reference population in Siegwart et al., 2020. However, in our study, there were therefore no significant differences between the LD50 of the original wild population and the susceptible laboratory strain, despite a resistance ratio of more than 23 fold. Wild populations are genetically more diverse than laboratory strains. This explains the large variance in mortality observed among individuals from the resistant population in generation 0 (Robertson et al., 2017).

The resistance selected in this study differs from all resistance types described so far. Indeed, type I resistance affects replication of CpGV-M only, and resistance types II and III are effective against CpGV-M and CpGV-S only. It has been shown that the isolate CpGV-E2 (genotypic group B) has the ability to overcome types I, II and III resistances (Gebhardt et al., 2014; Sauer et al., 2017; Jehle et al., 2017). However, for type V resistance, the CpGV-V15 isolate, which contains approximately 42 % of CpGV-E2 (genotype group B) (Gueli Alletti et al., 2017; Fan et al., 2021), is unable to overcome the resistance. Our experimental evolution study demonstrates that type V resistance confers cross-resistance among CpGV-M, CpGV-R5 and CpGV-V15. The selective pressure exerted by either CpGV-M or CpGV-R5 increased resistance to all three isolates, suggesting that resistance to CpGV-M, CpGV-R5 and CpGV-V15 in type V resistant individuals is conferred by the same or several linked genetic mechanisms.

The unselected line, MRV-T, exhibited a lower, but not significant, level of resistance compared to the selected ones, thereby demonstrating the efficiency of selection. Surprisingly, it appeared more resistant than the original wild population despite the initial crossing with a susceptible strain and the absence of selection for 17 generations. Based on LD50, we did not find a significant difference in resistance compared to the susceptible reference likely due to large standard deviations deriving from the heterogeneity of its genetic composition (mixing individuals carrying or not the resistance). MRV-T pattern of resistance could also be discussed with regard to fitness cost associated with type V resistance. No fitness cost was detected in individuals carrying type I resistance (Undorf Spahn, 2012). However, after more than 60 generations without selection, a decline in resistance was observed in CpR (Fan, 2019), suggesting a small fitness cost associated with resistance. Here, we cannot definitively reject the hypothesis of a fitness cost, but over 17 generations and in the conditions of our experiment we could not find evidence of fitness cost for Type V resistance.

## 4.2. Type V and Type I resistance mechanisms are linked

The crossing experiments supported our first hypothesis stating that the mechanism of type V resistance is linked to those of Type I resistance. Indeed, results of the crossing experiment are consistent with previous studies that characterized Type I as a resistance to CpGV-M, with a major dominant Z-linked gene (Berling et al., 2013; Asser-Kaiser, 2007). But we found a contrasted mode of inheritance pattern in type V resistance to CpGV-R5, where the level of resistance is different between CpGV-M and CpGV-R5. This pattern contrasts with type III resistance where the resistance to the CpGV-M was Z located, and resistance to CpGV-S autosomal. Indeed, the mortality rate of F1 offspring varied according to the sex of the resistant parent when crossed with a susceptible individual. Specifically, when crossing a resistant MRV-M female (Z<sup>R</sup>S) with a susceptible male CpNPP (Z<sup>S</sup>Z<sup>S</sup>), it was predicted that the F1f female offspring would not carry the resistance allele (Z<sup>S</sup>w), in contrast to the males (Z<sup>R</sup>Z<sup>S</sup>). Half of the F1 larvae died, and almost all of the surviving individuals were males. Assuming that an equivalent number of F1 males and females were tested, it can be deduced that all heterozygous individuals (Z<sup>R</sup>Z<sup>S</sup>) were resistant, indicating a dominant resistance. In addition, the number of dead larvae and sex-ratio of F1 were consistent with the hypothesis of dominance. This suggests that mechanisms of resistance to CpGV-M in Type I and in Type V resistance are likely the same. The ♀F1f (Z<sup>S</sup>w) were expected to be susceptible and not giving offsprings, but few of them gave F2, with a majority of males surviving. Following the dominance hypothesis, the F2 from ♀F1m should survive around equally to ♂F1m, but with a sex ratio imbalance towards males because they are theoretically composed of 50 % Z<sup>R</sup>Z<sup>S</sup> resistant males and 50 % of Z<sup>S</sup>w susceptible females. Whereas F2 from ♂F1m should be composed of 25 % Z<sup>R</sup>Z<sup>S</sup> resistant and 25 % Z<sup>S</sup>Z<sup>S</sup> susceptible males, 25 % Z<sup>R</sup>w of resistant and 25 % Z<sup>S</sup>w of susceptible females. In our test, we observed indeed similar mortality rates of 60 % for F2 from ♂F1m, compared with 59 % for F2 from ♀F1m, with a proportion of males of 95 %. However, despite the close link between the hypothesis and observed results, the number of dead larvae and sex-ratio in F2 were not significantly fitting the dominance hypothesis. As demonstrated by Berling et al. in 2013, in type I resistance to CpGV-M, the resistance is conferred by a major dominant gene Z located, but some other genes with minor effects were also implicated, leading to a little variation between theoretical and experimental observations in level of resistance.

It is well established that Type I resistance is sufficient to confer resistance to CpGV-M. However, the selection of Type I resistance in MRV-M also increased its resistance to both CpGV-V15 and CpGV-R5. This strongly suggests that the mechanism conferring resistance to
these three isolates is physically linked to those responsible for Type I
resistance. It could be either a new mutation in the same gene or may
involve other genes physically linked to the major gene conferring
resistance to CpGV-M. Consistently, individual crosses to determine the
mode of transmission of resistance to CpGV-R5 provided evidence that
the underlying mechanism is also sex-linked. It is common to observe
accumulations of mutations in the same gene, or genetic events in the
same genomic zone, for other resistance cases. For example, some in-
dividuals of *Musca domestica* first developed resistance against pyre-
throids with *kdr* mutation in the sodium channel gene *para*, then another
mutation, *super-kdr*, appeared in the same gene, conferring higher
resistance level (*Soderlund and Knipple, 2003*). Another similar pattern
of accumulation of mutations has been described for the resistance to
nicotine and neonicotinoids in *Myzus persicae*. Initially, a mutation in the
expression of the gene CYP6CY3 led to resistance to nicotine, accom-
panied by a modest resistance to neonicotinoids. Subsequent mutations
resulting in elevated neonicotinoid resistance were identified, observed
exclusively in individuals possessing the CYP6CY3 mutation. This sug-
gests a strong probability of a genetic linkage between resistance to
neonicotinoids and resistance to nicotine (Troczka et al., 2021).

4.3. *Inheritance of the new mechanism conferring resistance to CpGV-R5*

Despite its Z linkage, the resistance to CpGV-R5 showed a different pattern of inheritance when compared to CpGV-M resistance. In the crossing experiments on CpGV-R5, the mortality rate of F1f (F1 offspring of resistant mother and susceptible father) was high: approximately 71 %. The number of F1f surviving was too low to yield a representative mean sex ratio for each couple, but when pooling the number of surviving larvae in all F1f crossing, a majority of surviving males was counted (63.8 %). In the reverse crossing, it was expected that all F1m (F1 offspring of resistant mother and susceptible father) will carry a copy of the resistance allele (Z<sup>RZS</sup> for males and Z<sup>Rw</sup> for females). We observed approximately 50 % mortality with a majority of surviving females (78 %), indicating that heterozygous males (Z<sup>RZS</sup>) were less resistant compared to females (Z<sup>Rw</sup>). This supports the recessivity hypothesis for this allele, as it is expressed in females in the absence of a competing allele, whereas this is not the case in heterozygous males. Alternatively, the expression of an allele required for viral infection may be completely absent in females, but still expressed in heterozygous males.

The number of dead larvae observed did not align with the expected numbers for either complete dominance or complete recessivity in F1 and F2 generations. However, if CpGV-R5 resistance is recessive, F1m females (Z<sup>R</sup>w) should survive more than males (Z<sup>RZS</sup>), as no allelic competition occurs in females, who carry only a single copy. This would result in lower mortality in F1m than in F1f, along with an excess of females in F1m. Our results are consistent with this prediction: we observed 46.3 % mortality in F1m compared to 71.3 % in F1f, with a female proportion of 78 % in F1m. These findings support that resistance to CpGV-R5 appears to be partly recessive and linked to the Z sex chromosome.

As in type V, resistance ratio was lower for CpGV-R5, we used a discriminating dose smaller than for CpGV-M. This might have led some susceptible individuals to survive, confirmed by the 18 % of survival in the reference susceptible homozygotes crossing on CpGV-R5. Consequently, results obtained on the F2 are more difficult to interpret because the F1f survivors probably are false positives (composed of a mix of susceptible and resistant individuals) who do not necessarily carry the CpGV-R5 resistance allele. In this condition it is consistent that neither the hypothesis of dominance nor recessivity fit for the test on F2 from F1f.

However, if the CpGV-R5 resistance allele is recessive, the F2 offspring from F1m crosses should show differences in mortality between sexes. Actually, the F2 from ♂F1m should survive better than F2 from ♀F1m, with a sex ratio imbalance towards females because they are

theoretically composed of 25 % Z<sup>RZS</sup> and 25 % Z<sup>SZS</sup> susceptible males,
25 % Z<sup>Rw</sup> of resistant females and 25 % Z<sup>Rw</sup> of susceptible females.
Whereas F2 from ♂F1m should die at the same rate as the susceptible
homozygotes because they are theoretically composed of 50 % Z<sup>RZS</sup>
susceptible males and 50 % of Z<sup>Sw</sup> susceptible females. In our test,
indeed, we observe a higher mortality rate of 63 % for F2 from ♂F1m,
compared to 53 % for F2 from ♂F1m, but with a proportion of females of
45 %.

Previously identified Types II and III resistance to CpGV isolates belonging to the genotypic group E (like CpGV-R5 and CpGV-S), show different patterns of inheritance when compared to Type V. The type II resistance is autosomal and dominant for both CpGV-M and CpGV-S, and these two resistances were crossed, as the selection with each isolate separately lead to the selection of resistance to both of them. Type III resistance is autosomal and dominant for CpGV-S, and both autosomal and Z-linked for CpGV-M, however, it is not known if this resistance is a combination of Type I and Type II, or if they are completely independent. Our type V resistance appears to be Z-linked for both CpGV-M and CpGV-R5, with different levels of dominance. The presence of the dominant Z located resistance to CpGV-M support the hypothesis of a combination with Type I resistance and a new one conferring resistance to CpGV-R5 and CpGV-V15.

Hypotheses on the inheritance and dominance patterns were based on a simple monogenic resistance. However, the observed results did not fully align with the hypotheses, suggesting the possibility of a more intricate underlying mechanism. This could implicate multiple genes or the interaction between different alleles of one gene, thereby modulating the level of resistance. The level of dominance of an allele is not a fixed characteristic, it has been demonstrated that it can vary from dominant to recessive depending on the dose of the substance used to test the mortality (Bourguet et al., 2000). For instance, it was observed that type I resistance to CpGV-M was dominant at lower doses but tended to be recessive when the dose increased (Asser-Kaiser et al., 2007). The level of dominance is also influenced by environmental parameters. In mosquitoes of the *Culex pipiens* species, a single gene (insensitive acetylcholinesterase) confers resistance to propoxur and chlorpyrifos, and the level of dominance exhibited by this gene varies from dominant to recessive depending on the insecticide and the type of food, water and containers used for the bioassay (Bourguet et al., 1996). The precise genetic mechanism underlying this variation in gene dominance remains to be elucidated in our case.

Type I resistance to CpGV-M is characterised by systemic replication inhibition within the insect body (Asser-Kaiser et al., 2011). A range of strategies against viral infections have been observed in the silkworm, *B. mori*, employed as a model to study Lepidoptera. These include the development of antiviral proteins and the formation of morphological barriers (Hu et al., 2023). The precise mechanism of resistance to all isolates except CpGV-M remains to be elucidated, although it may extend the original blocking of replication or introduce a new mechanism. However, the identification of Z-linked inheritance of resistance represents a preliminary step towards more in-depth studies on the genetic bases of type V resistance, which can be used to develop molecular tools for monitoring the resistances. Furthermore, the analysis of inheritance patterns facilitates the modelling of the dynamics of resistance propagation, thus enabling the adaptation of management strategies. Indeed, the actual physical link between CpGV-M and CpGV-R5 resistances implicated a crossed resistance and necessitated a precise utilisation of viruses to disrupt potential selection. To the end, the ability of codling moths to evolve through the development of multiple resistances offers a new challenge for the development of CpGV based products, with a long term efficacy.

CRediT authorship contribution statement

**Léa Gingueneau:** Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization.
**Bertrand Gauffre:** Writing –
review & editing, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization. **Miguel Lopez-Ferber:** Writing - review & editing, Validation, Supervision, Project administration, Funding acquisition, Conceptualization. **Sandrine Maugin:** Validation, Methodology, Data curation. **Christine Blachère-Lopez:** Writing - review & editing, Validation. **Sofian Renoult:** Validation, Data curation, Conceptualization. **Samatha Besse:** Writing - review & editing, Validation, Funding acquisition. **Myriam Siegwart:** Writing - review & editing, Validation, Supervision, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Lea Gingueneau reports financial support was provided by NPP by UPL. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper].

## Acknowledgment

We thank Elodie Lecerf (INRAE PSH, Avignon, France) and Kenia Quinzoni (INRAE PSH, Avignon, France) for their technical assistance in insect rearing; and the farmer who provided the sample of wild resistant population of *Cydia pomonella*. L. Gingueneau received PhD grant from NPP by UPL (Pau, France) to carry on the study at INRAE, UR PSH, France.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biocontrol.2025.105777.

## References

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