# Heterosis effect for larval performance of fall armyworm interstrain hybrids

Laijiao Lan, Nicolas Nègre

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## To cite this version:

Laijiao Lan, Nicolas Nègre. Heterosis effect for larval performance of fall armyworm interstrain hybrids. Insect Science, 2023, 31 (4), pp.1296-1312. 10.1111/1744-7917.13295. hal-04345584

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*Insect Science* (2024) 31, 1296–1312, DOI 10.1111/1744-7917.13295

ORIGINAL ARTICLE

# Heterosis effect for larval performance of fall armyworm interstrain hybrids

Laijiao Lan ID and Nicolas Nègre ID

DGIMI, University of Montpellier, INRAE, Montpellier, France

**Abstract** *Spodoptera frugiperda*, also known as fall armyworm (FAW), is an invasive crop pest that can feed on a variety of host plants, posing a serious threat to food security. There are two sympatric strains of FAW that are morphologically identical but described with different food preferences: the “rice strain” (SfR) and the “corn strain” (SfC). A few genetic loci exist to identify these two strains. Mitochondrial and Z-chromosome-linked haplotypes are the most used, but the biggest part of the genome displays little polymorphism between strains that could explain their adaptation to different plants. We have previously observed consistent transcription differences between the strains in both laboratory and natural populations. Therefore, we wonder if there are effects from host-strain-associated loci, maternally or paternally inherited, on FAW performance that could explain the divergence between the two FAW strains. To test this hypothesis, we first produced two F₁ hybrid generations (SfR ♀ × SfC ♂, SfC ♀ × SfR ♂). These reciprocal hybrids should be heterozygous for all chromosomes except for the maternally inherited mitochondrial and sexual W chromosomes. To evaluate whether plant preference is determined by these genetic loci, we cultivated the two hybrids and the two parental strains in triplicate on an artificial diet and recorded several phenotypic traits such as weight over time, survival rate, emerging rate, developmental time, and sex ratio. Then, the same performance experiment was carried out on corn plants. Surprisingly, on the artificial diet, the two hybrid genotypes were both more performant than the two parental strains in terms of survival rate, pupal emerging rate, and developmental time, whereas they were intermediate to the inbred parental strains in pupal weight. On the corn plant diet, both hybrid genotypes outperformed the two parental strains in larval weight. Although these asymmetrical results revealed that mitochondrial or sex-linked haplotypes alone cannot explain the performance differences, they suggested a heterosis effect in FAW. A reduction of the female number for the CR genotype and the decreased F₁ offspring reproduction in both hybrids suggested the possibility of Haldane's rule, which might be explained by the dominance model.

**Key words** fall armyworm; Haldane's rule; heterosis; hybrid performance; mitochondria

Introduction

The fall armyworm (FAW), *Spodoptera frugiperda* (J.E.
Smith) (Lepidoptera: Noctuidae), is a polyphagous

Correspondence: Nicolas Nègre, DGIMI, University of
Montpellier, INRAE, Montpellier 34095, France. Email: nico-
las.negre@umontpellier.fr

agricultural pest that is native to tropical and subtrop-
ical America. As a generalist, the pest larvae feed on
the leaves, stems, and reproductive parts of more than
350 plant species (Montezano et al., 2018), includ-
ing economically important crops such as corn, rice,
sorghum, sugarcane, and cotton, to which they inflict
huge production and economic losses (Martinelli et al.,
2006). FAW has a strong capacity for long-distance
migration and reproduction (Johnson, 1987; Mitchell et al., 1991; Westbrook et al., 2016), which results in its rapid invasion and poses a serious economic threat to food production in many countries of Africa, Asia, and Australia (Goergen et al., 2016; Ganiger et al., 2018; Li et al., 2019; most recent report maps at https://www.cabi.org/isc/fallarmyworm). FAW has become a major global threat in just a few years and is now a worldwide concern.

Two FAW strains with different food preferences have been described, the “rice strain” (SfR) which predominantly feeds on rice and grasses, and the “corn strain” (SfC), which is more associated with corn, cotton, and sorghum (Pashley et al., 1985; Pashley, 1988). Though morphologically indistinguishable, the two sympatric strains differ in host range (Pashley, 1986), development performance on plants (Groot et al., 2010; Silva-Brandão et al., 2017; Orsucci et al., 2022), mating behavior (Groot et al., 2010), and pheromone compositions (Groot et al., 2008). These traits have been associated with different genetic haplotypes at a few loci, among which the most used molecular markers for strain discrimination are the mitochondrial gene *COI* (Cytochrome c Oxidase subunit I) (Pashley, 1989; Lu & Adang, 1996; Levy et al., 2002; Nagoshi et al., 2006), and the Z-linked locus *Tpi* (Triose-phosphate isomerase) (Nagoshi, 2010). Phylogenies based on mitochondrial markers recurrently showed that FAW is structured in two populations corresponding to SfC and SfR, with $F_{ST}$ superior to 0.9 (Kergoat et al., 2012; Gouin et al., 2017). Moreover, around 150 genes have been detected to be consistently differentially expressed between SfC and SfR from both laboratory populations and natural populations, and *COI* was a main candidate gene (Orsucci et al., 2022). It has been suggested that mitochondrial genes (including *COI*) might potentially be the original target of host plant selection for FAW (Orsucci et al., 2022).

However, despite all the differences exhibited by the two FAW strains, which were often regarded as a case of ecological speciation (Nosil, 2012) or incipient speciation (Durand et al., 2022), mating success has been reported between SfR and SfC in the laboratory (Dumas et al., 2015; Gouin et al., 2017), and a high prevalence of hybrids has been found in field populations (Nagoshi & Meagher, 2003; Prowell et al., 2004; Nagoshi et al., 2006; Machado et al., 2008), with 16% of FAW showing inconsistencies between multiple strain-specific genetic markers (Prowell et al., 2004). A substantial portion of the invasive FAW populations is dominated by a lineage of interstrain hybrids in Africa (Nagoshi et al., 2022) as well as in China (Zhang et al., 2019; Zhang

et al., 2020). It has been postulated that increasing heterozygosity through balancing selection could help facilitate the widespread success of FAW invasions (Yainna et al., 2020). Furthermore, it appears that the hybrids differ from the parental strains in terms of mating behavior and reproductive compatibility (Pashley et al., 1992; Groot et al., 2010; Dumas et al., 2015; Kost et al., 2016).

Phenotypes expressed by hybrids between populations can help reveal the genetic divergence of populations or species across their geographic ranges (Burton et al., 2013). While some studies have investigated genetic incompatibilities between strains to explain FAW reproductive isolation, few have studied whether, at the caterpillar stages, hybrid genotypes have their performance affected. Thus, there is a crucial need to study the impact of host-strain-associated loci on the performance of FAW and compare the differences between interstrain hybrids and parental strains. In this study, we first performed crosses between FAW laboratory strains to produce two interstrain hybrid populations (SfR ♂ × SfC ♂, SfC ♂ × SfR ♂), then we measured and compared the Life History Traits (LHT) to see if there was an impact of mitochondrial types or sex chromosome types on performance between the two reciprocal hybrid genotypes and the two parental strains. We hereafter conducted an additional breeding experiment to compare the reproductive success of both FAW strains and hybrid $F_1$, $F_2$ generations, since gene action changes between the first and second generation of hybridization (Edmands, 1999). Results obtained under artificial rearing where larvae were given *ad libitum* access to food to ensure high survival cannot represent the real situation in the wild, so we have applied the same experimental design to a subsequent performance experiment by replacing the artificial diet with detached leaves from corn, the most infested plant by FAW. On both diets, we observed striking performance differences between the parental strains as well as between the hybrids and the parental strains. We first observed that in the laboratory, SfC performed better than SfR for most phenotypic traits, and hybrids obtained high survival rates and were more fertile than parental strains. Besides, we have found that hybrid genotypes outperformed the two parental strains in terms of survival rate, pupal emerging rate, and developmental time, and similar results were noticed in larval weight when FAW were fed with corn plants. These performance differences between the hybrids and the parental genotypes may help explain the fact that most invasive populations appear to be interstrain hybrids in the wild.
# Materials and methods

## Biological materials

Insects from two laboratory FAW host strains were used: SfC and SfR. The SfC laboratory population was seeded with individuals collected from Guadeloupe in 2001. The SfR population was seeded in 2012 from 50 pupae collected in Florida (Hardee County). Both strains have been reared under laboratory conditions, at a temperature of 24 °C (T) with a 16 h : 8 h light: dark photoperiod (L : D) and 70% relative humidity (RH) on an artificial diet that is named Poitout (from Poitout *et al.*, 1972, principal components: 77% H₂O, 2% agar-agar, 13% corn flour, 6% other nutrients, 1% vitamins; 1% antibiotics) for many generations. The genomes of these two populations have been sequenced (Gouin *et al.*, 2017) and are available in a dedicated database (https://bipaa.genouest.org/sp/spodoptera_frugiperda_pub/). FAW is not present in metropolitan France and is considered a quarantine pest, thus, all experiments below were conducted in a confined environment on an insect quarantine platform (PIQ, University of Montpellier, DGIMI laboratory).

The corn plants (Zea mays ssp. mays L., corn line B73) were produced from organic seeds in the IRD laboratory (INRAE, l'Institut de Recherche pour le Développement, France) in 5.5 L plastic pots filled with organic Jiffy substrates. Corn cultivations were carried out in a greenhouse at 25 °C, 60% RH during the light time, 20 °C, 50% RH during the dark time, and 14 h : 10 h (L : D) under organic conditions. Biological control was applied to the plants after germination with auxiliaries of the Swirskii-Breeding-System and the Californicus-Breeding-System (Biobest Group NV, Belgium).

## Experimentation

**Performance experiment with Poitout** Following biological containment procedures, we first performed reciprocal crosses between the two stains to produce hybrid populations, SfR ♀ × SfC ♂ (RC), SfC ♀ × SfR ♂ (CR). We produced pure strain populations from the two parental genotypes, SfR ♀ × SfR ♂ (RR) and SfC ♀ × SfC ♂ (CC) at the same time. The four genotypes (Fig. S1) were produced in triplicate for a total of 12 populations. Each population was reared in a 2 L oblong plastic box in triplicate (mass rearing, around 30 larvae per box) on an artificial diet. All the lids were punctured with about 50 small holes for ventilation, and the boxes were covered with an additional insect-proof net (150 µm

sieve) and a piece of paper sheet to limit the possible contamination between each genotype and to prevent the tiny neonate larvae from escaping. To avoid the floor and edge effects, the three boxes for each genotype were placed randomly on three floors inside a controlled incubator in PIQ (T = 25 °C, HR = 40% ± 5%, L : D = 16 : 8). Once the larvae reached the end of L6 and stopped eating and moving, we gently transferred them using a soft tweezer to another clean 2 L plastic box filled with 1/3 sterilized vermiculite. This medium can help facilitate the burrowing and chamber construction processes of the pupae, avoid contamination, maintain humidity, and promote gaseous exchange (Montezano *et al.*, 2019). During the rearing, several Life History Traits (LHT) of the four populations were recorded: larval weight over time, pupal weight, survival rate, emerging rate, developmental time, and sex ratio.

Larval weight was measured individually (10 random larvae from each box, 30 in total for each genotype) for the four genotypes separately. The measurements were conducted every other day from the 3rd larval instar to pupal metamorphosis (d6, d8, d10, d12, and d14). Larval instar was determined by measuring the width of the head capsule (Orsucci *et al.*, 2022).

Sexing of the pupae was carried out based on anatomical features (Butt & Cantu, 1962), and pupal weight was measured individually for both females and males on the second day after metamorphosis to avoid the pupal weight effect on reproductive characteristics (Tisdale & Sappington, 2001).

Larval survival rate was measured from the 1st larval instar to metamorphosis for the four genotypes. It was the number of surviving larvae at the end of the larval stage (before metamorphosis) that was counted over the initial number of larvae.

Pupal emerging rate was measured by dividing the number of emerged female or male adults by the initial number of female or male pupae.

Developmental time was the number of days from egg laying until adult emergence.

Sex ratio was the ratio of adult males to adult females for each population.

**Reproduction comparison experiment** We maintained the two parental genotypes from the performance experiment with Poitout (RR ♀ × RR ♂, CC ♀ × CC ♂) and performed crosses to establish two F₁ (RR ♀ × CC ♂, CC ♀ × RR ♂) and two F₂ hybrid populations (RC ♀ × RC ♂, CR ♀ × CR ♂), so six set-ups in triplicate in total. For each setup, we put 1 ♀ × 2 ♂ pupae to increase the mating chance in a plastic tube (d = 5 cm) with a cap (a small hole was dug in the middle for ventilation) and
attached a piece of wet cotton with 10% honey solution in a small plastic container for nutrition and a long filter paper strip around the tube to stimulate oviposition. All the set-ups were kept on the same shelf of the same incubator (T = 25 °C, HR = 40% ± 5%, L : D = 16 : 8) randomly. Couples were formed with pupae that pupated on the same date to reduce the age effects of the adults on their capacity to copulate (Rogers & Marti, 1994; Rogers & Marti, 1997). The progeny of each setup was calculated to analyze the differences.

**Performance experiment with corn** We subsequently repeated the same experimental design and measurements for these four FAW genotypes in triplicate on the corn plant diet. Corn foliage was excised from the whorl leaves of the plants at V6 (Ritchie *et al.*, 1992), thoroughly rinsed with distilled water, and dried with paper towels. The foliage was cut into sections and then put inside the plastic box. A piece of folded damp filter paper strip was lined around the foliage to retain some humidity so that the excised leaf sections stayed fresher for longer. Using a fine hairbrush, about 30 neonate larvae (<24 h hatching) were placed on the excised leaves per box under the same environmental conditions that were described previously for the insect-rearing experiment with Poitout. Corn foliage was replaced twice a day until publication. The same LHT traits were recorded with the same methods.

## DNA extraction

AllPrep DNA/RNA Mini Kit (50) (Qiagen Cat. 80 204) was used in accordance with the manufacturer's protocol. First, a whole larva at the 4th stage was placed in a 2 mL Eppendorf collection tube with Buffer RLT Plus, and 8 larvae from each genotype (CC, RR, CR, RC, 4 × 8 = 32 samples) were tested separately. Then, each larva was disrupted and homogenized with a TissueLyser II from Qiagen (Cat No./ID: 85 300) by adding a bead (size 5 mm) inside the tube, and the homogenized lysate was transferred to an AllPrep DNA spin column placed in a 2 mL collection tube and centrifuged. Finally, the solution in the AllPrep DNA spin column was used for later DNA purification. The concentration of each sample was measured on a NanoDrop spectrophotometer (Wilmington, DE, USA).

## Genotyping

Genotyping experiments were carried out by Restriction Fragment Length Polymorphism (RFLP) to verify

the genotypes of individuals from the four produced populations (CC, RR, CR, RC). Mitochondrial cytochrome c oxidase subunit I (COI) genotype was used, and a Polymerase Chain Reaction (PCR) on genomic DNA (gDNA) from the 4th instar individual was performed with primer sequences (JM-76: GAG CTG AAT TAG GGA CTC CAG G and JM-77: ATC ACC TCC ACC TGC AGG ATC) to amplify a DNA fragment of 550 bp corresponding to COI, with an initial denaturation of 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 47 °C for 45 s, extension at 72 °C for 45 s, and final extension at 72 °C for 20 min. The MspI enzyme was used for the PCR product digestion to reveal a 500 bp and a 50 bp fragment for CC and CR with gel electrophoresis experiments (1.5% agarose gel). Triose-phosphate isomerase (Tpi) genotyping was also used for PCR amplification with the following primers: Tpi-56F (5'-CAAAATGGGTCGCAAATTCG-3') and Tpi-850R (5'-AATTTTATTACCTGCTGTGG-3') to amplify a DNA fragment of 800 bp that corresponds to Tpi, with the same PCR running program as COI. Digestion of the PCR product was done with AvaII enzyme to produce a 500 bp and a 300 bp fragment in RR and showed the offspring possibilities for RC with a gel electrophoresis experiment (1.5% agarose gel). The purified PCR products, which were amplified with Tpi were sent for Sanger sequencing to detect target fragments by using universal primers. The obtained sequence on the chromatogram was compared with authenticated sequences. According to the cut patterns of both restriction enzymes (Figs. S5, S6) and the Sanger sequencing results, the identification of both parental strains, RR and CC, was confirmed, as was the presence of the hybrid genotypes, RC and CR.

## Statistical analysis

Statistical tests were carried out with R statistical software (version 4.2.2, R Development Core Team, 2022), followed by residual analyses to verify the suitability of the distributions of the datasets. For the experiment with Poitout, the larval weight, larval survival rate, pupal weight (female and male), pupal emerging rate (female and male), sex ratio, and developmental time were analyzed using one-way analysis of variance (ANOVA), with FAW genotype (4) as the factor. The means were separated and compared with Tukey's HSD test. The reproduction difference of female progeny between (RC × RC) and (CR × CR) was determined by Student's t-test. For the experiment on the corn diet, we only obtained partial data for the larval weight due to the
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Larval performance differences among four FAW genotypes with artificial diet. (A–E) Larval weight measurements were done every other day at 3rd instar larvae until pupal metamorphosis (d6, d8, d10, d12, and d14). The number of larvae recorded from the three replicates for each genotype was *n* = 30. Different letters above box plots indicate significant differences in larval weight among these four genotypes (Tukey's HSD test, *P* < 0.05). (F) Mean larval survival rate ± SD was shown for four FAW genotypes. The initial number of larvae observed from the three replicates was *n* = 90. Different letters above bars indicate significant differences in survival rates among these genotypes (Tukey's HSD test, *P* < 0.05).</figcaption></figure>

delayed development, and the statistical analysis was
done with a one-way ANOVA with FAW genotype (4) as
the factor. The means were also separated and compared
with Tukey's HSD test. Codes are available as Supporting
Information.

Results

Larval growth and survival differences

To evaluate performance in hybrid populations, we
measured the larval weight of four FAW genotypes (CC,
RR, CR, and RC) starting at 3rd instar larvae until pu-
pal metamorphosis in triplicate every other day (d6, d8,

d10, d12, and d14, Fig. 1A–1E, respectively, or Fig. S2
and Table 1). As shown in Fig. 1, one of the hybrid pop-
ulations, CR, was systematically the heaviest among all
the genotypes for all the measurements, while one of the
parental genotypes, RR was always the lightest, and CC
as well as RC were intermediate between them. From d6
to d10, there were significant differences between each
genotype (*P* < 0.001), and from d12, the larval weight
of RC caught up with CC and showed no significant dif-
ference from that point. For the last measurement (d14),
there was also no significant difference between CR and
CC (F<sub>3, 116</sub> = 37.99, *P* = 0.16 > 0.05).

During the rearing, the larval survival rate was
around 90% for all set-ups. Interestingly, the two hybrid
<table><caption>**Table 1** Results of one-way ANOVA for the larval weight differences of four FAW genotypes with artificial diet during the larval stage (mean ± SE).</caption><thead><tr><th rowspan="2">Days</th><th colspan="4">Larval weight (mg)</th><th rowspan="2">F</th><th rowspan="2">P</th></tr><tr><th>CC</th><th>CR</th><th>RC</th><th>RR</th></tr></thead><tbody><tr><td>6</td><td>9.77 ± 0.39 b</td><td>11.56 ± 0.22 a</td><td>6.66 ± 0.25 c</td><td>5.13 ± 0.19 d</td><td>F<sub>3, 116</sub> = 113.40</td><td>P < 0.001</td></tr><tr><td>8</td><td>20.32 ± 0.88 b</td><td>23.17 ± 0.78 a</td><td>12.68 ± 0.57 c</td><td>9.54 ± 0.40 d</td><td>F<sub>3, 116</sub> = 87.19</td><td>P < 0.001</td></tr><tr><td>10</td><td>84.81 ± 4.66 b</td><td>112.43 ± 3.63 a</td><td>62.51 ± 2.81 c</td><td>44.62 ± 2.09 d</td><td>F<sub>3, 116</sub> = 72.68</td><td>P < 0.001</td></tr><tr><td>12</td><td>170.64 ± 6.71 b</td><td>239.12 ± 10.44 a</td><td>152.11 ± 6.47 b</td><td>106.44 ± 5.73 c</td><td>F<sub>3, 116</sub> = 53.07</td><td>P < 0.001</td></tr><tr><td>14</td><td>457.86 ± 21.04 ab</td><td>508.15 ± 9.83 a</td><td>416.98 ± 17.27 b</td><td>267.35 ± 17.20 c</td><td>F<sub>3, 116</sub> = 37.99</td><td>P < 0.001</td></tr></tbody></table>

*Note*: Different letters indicate significant differences in larval weight in each day (Tukey's HSD test, *P* < 0.05).

genotypes, CR and RC, had slightly higher survival rates (93.44% and 89.39%, respectively) than the two parental genotypes, CC and RR (88.33% and 89.05%, respectively). However, these differences were not significant ($F_{3,8} = 0.611, P = 0.627 > 0.05$; Fig. 1F).

## Pupal weight differences

The pupal weight of four FAW genotypes was measured separately for females and males 2 d after pupal metamorphosis. Indeed, we observed that there were no significant differences between females and males ($F_{1,279} = 0.096, P = 0.757 > 0.05$), except for CR which showed that males were heavier than females (*P* < 0.05). For both female and male weight, there were significant differences between each genotype ($F_{3,279} = 81.648, P < 0.001$), among which CC was systemically the heaviest, RR the lightest, and the hybrid genotypes, CR as well as RC were intermediate (Fig. 2A).

Sex ratio was the number of emerged live adult males divided by the number of emerged live adult females (Fig. 2B). We noticed that for RR, the ratio tended to be 1 : 1, and both CC and RC were female-biased, whereas CR was male-biased. There were no significant differences ($F_{3,8} = 3.191, P = 0.084 > 0.05$) between each genotype.

Emerging rate was calculated by dividing the number of emerged live female or male adults by the initial number of female or male pupae (Fig. 2C). For all genotypes, there were no significant differences between female and male emerging rate ($F_{1,16} = 2.326, P = 0.147 > 0.05$). However, the two hybrid genotypes, CR and RC had higher emerging rates for both sexes than the two parental genotypes, CC and RR, within which there were significant differences in male emerging rates between CR and the parental genotypes ($F_{3,16} = 10.230, P < 0.001$).

## Developmental time differences among four FAW genotypes

Developmental time was the number of days measured from egg laying to adult emergence (Fig. 3). There were no significant differences between either females and males ($F_{1,16} = 2.161, P = 0.161 > 0.05$), or between each genotype ($F_{3,16} = 2.780, P = 0.075 > 0.05$). All genotypes took about a month to complete the whole life cycle. However, there were still some slight differences. CC took about 1 d less for both sexes (27 d and 28 d) to finish the life cycle than RR (28 d and 29 d), and the hybrid genotypes took about 1 d less (26 d and 27 d) for both sexes than the parental genotypes. Additionally, we have noticed that the longest larval developmental time was recorded on RR, which took two more days (15 d) in the duration of the larval stage than the other three genotypes (13 d).

Furthermore, we have calculated the progenies from different cross set-ups to compare the reproduction differences (Fig. S4), and we noticed that the $F_1$ hybrids (RR × CC and CC × RR) were fertile and could produce viable offspring.

## Larval performance differences among different FAW genotypes with corn diet

Since it is more difficult to maintain the experiment with plants compared to the rearing experiment with an artificial diet, we only obtained consolidated results for the larval weight differences for the four FAW genotypes. Similar to the artificial diet, we observed that CR was still systematically the heaviest with significant differences among the four genotypes for all the measurements over time, and RR was the lightest (Fig. 4, or Fig. S3 and Table 2). From d6 to d12, RC was slightly heavier than CC and there were no significant differences between
<figure><img src="image_5.png" /><figcaption>**Fig. 2** Pupal performance differences among four FAW genotypes. (A) Pupal weight was measured for both females and males, respectively. The number of pupae was shown above the boxes. (B) Sex ratio was measured by dividing the number of adult males by the number of adult females that managed to emerge. The total number of males and total number of females of the three replicates were shown above the boxes. Different letters above box plots indicate significant differences among these four genotypes (Tukey's HSD test, *P* < 0.05). (C) Mean emerging rate ± SD was shown for both females and males. The total number of emerged adults and total number of initial pupae of the three replicates were shown inside the bars. Different letters above bars indicate significant differences in emerging rate among these four genotypes (Tukey's HSD test, *P* < 0.05).</figcaption></figure>

them (*P* > 0.05); however, for the last measurement, both
hybrid genotypes outperformed the two parental geno-
types (*P* < 0.001).

**Discussion**

In this study, we have evaluated several Life History
Traits (weight, survival rate, emerging rate, sex ratio, and
developmental time) on four FAW genotypes (CC, RR,
CR, and RC) to study the effects of host-strain-associated
loci on FAW performance, especially on the hybrid per-
formance at larval stages. We have observed signifi-
cant differences between these four genotypes, which we
could interpret as follows.

Larval stage

Surprisingly, under the artificial rearing condition, one
of the hybrid genotypes, CR was systematically the heav-
iest among the four genotypes from d6 to d10 and there
were significant differences between all four FAW geno-
types for the larval weight (Fig. 1 and Table 1), whereas
RC was always intermediate between the parental geno-
types and not significantly different to CC at the end of
the larval stage. Besides, the two hybrid genotypes also
obtained slightly higher larval survival rates than the two
parental genotypes, and CR turned out to survive the best.
Overall, CR outperformed the parental genotypes and RC
was intermediate when the larvae were fed on the arti-
ficial diet. Furthermore, we observed even better hybrid
performance when FAW were fed with corn plants. Both
<figure><img src="image_6.png" /><figcaption>**Fig. 3** Developmental time differences among four FAW genotypes. Mean developmental time ± SD was shown. The initial number of larvae observed from the three replicates was (n = 90). Different letters above bars indicate significant differences in developmental time among these genotypes (Tukey's HSD test, *P* < 0.05).</figcaption></figure>

<table><caption>**Table 2** Results of one-way ANOVA for the larval weight differences of four FAW genotypes with corn diet during the larval stage (mean ± SE).</caption><thead><tr><th rowspan="2">Days</th><th colspan="4">Larval weight (mg)</th><th rowspan="2">F</th><th rowspan="2">P</th></tr><tr><th>CC</th><th>CR</th><th>RC</th><th>RR</th></tr></thead><tbody><tr><td>6</td><td>4.81 ± 0.21 b</td><td>10.66 ± 0.74 a</td><td>5.30 ± 0.33 b</td><td>2.10 ± 0.20 c</td><td>F<sub>3, 116</sub> = 69.56</td><td>P < 0.001</td></tr><tr><td>8</td><td>16.65 ± 0.96 b</td><td>31.77 ± 2.12 a</td><td>18.04 ± 1.55 b</td><td>6.71 ± 0.64 c</td><td>F<sub>3, 116</sub> = 51.45</td><td>P < 0.001</td></tr><tr><td>10</td><td>50.60 ± 3.68 b</td><td>96.73 ± 6.95 a</td><td>52.02 ± 4.72 b</td><td>23.74 ± 2.20 c</td><td>F<sub>3, 116</sub> = 41.17</td><td>P < 0.001</td></tr><tr><td>12</td><td>98.84 ± 10.53 b</td><td>203.47 ± 16.45 a</td><td>110.91 ± 10.53 b</td><td>50.16 ± 6.01 c</td><td>F<sub>3, 116</sub> = 32.62</td><td>P < 0.001</td></tr><tr><td>14</td><td>166.45 ± 10.79 b</td><td>322.64 ± 19.53 a</td><td>265.88 ± 24.81 a</td><td>103.15 ± 9.41 c</td><td>F<sub>3, 116</sub> = 34.50</td><td>P < 0.001</td></tr></tbody></table>

*Note*: Different letters indicate significant differences in larval weight in each day (Tukey's HSD test, *P* < 0.05).

CR and RC outperformed the parental genotypes in terms of higher larval weight (Fig. 4 and Table 2). CR genotype still performed the best compared to the other genotypes and there were significant differences between the hybrid genotypes and the parental genotypes for the last measurement (*P* < 0.001).

## Pupal stage

For both female and male pupal weight, CR and RC were intermediate between CC and RR, and there were significant differences between the two hybrid genotypes and the two parental genotypes, which suggested
<figure><img src="image_7.png" /><figcaption>**Fig. 4** Larval performance differences among four FAW genotypes with corn diet. (A–E) Larval weight measurements were done every other day from the 3rd instar larvae until pupal metamorphosis (d6, d8, d8, d10, d12, and d14). The number of larvae recorded from the three replicates for each genotype was shown above the boxes. Different letters above box plots indicate significant differences in larval weight among these four genotypes (Tukey's HSD test, *P* < 0.05).</figcaption></figure>

maternal effects (Turelli & Moyle, 2007) between both FAW strains. A surprising discovery was that males were significantly heavier than females for CR, while for the other genotypes, there was no significant difference between females and males (Fig. 2A). This result about CR genotype contradicted the biological results of many studies on FAW (Combs & Valério, 1980; Parra & Carvalho, 1984; Machado *et al.*, 1985; Pantoja *et al.*, 1987; Giolo *et al.*, 2002) and some other Spodoptera species such as *Spodoptera latifascia* Walker (Habib *et al.*, 1983), *Spodoptera litura* (Xue *et al.*, 2010), *Spodoptera eridania* (Montezano *et al.*, 2014), and *Spodoptera cosmioides* (Specht & Roque-Specht, 2016), which found female pupae to be significantly heavier than male ones under most conditions with different temperatures and diets. It is likely because greater female pupal weight helps egg production (Allen *et al.*, 2011; Specht *et al.*, 2016).

Such differences may be attributed to the different employed methods, the rearing conditions, the feeding diets, and the population variations of FAW from different locations since our results are consistent with previous observations (Orsucci *et al.*, 2022) on the same laboratory strains. Besides, more pupae managed to emerge for the two hybrid genotypes than the two parental strains for both sexes, and the only significant difference was observed in males between CR and the two parental genotypes (Fig. 2C). As for the sex ratio, RR was approximately 1 : 1, which might be explained by Fisher's principle (Fisher, 1930). Both CC and RC were female-biased, whereas CR was male-biased (Fig. 2B), which follows Haldane's rule, postulating that in hybrids between species, the heterogametic sex (females on Lepidoptera) is absent, rare, or sterile (Haldane, 1922). That could indicate that the host strains of FAW are indeed at early
stages of speciation, as has been postulated before (Fiteni *et al.*, 2022).

## *Developmental time*

Despite no significant differences, we still noticed that the two hybrid genotypes completed the whole life cycle about 1 d earlier than the two parental genotypes for both females and males (Fig. 3), which suggested a better fitness of the hybrids because the extension of the insect life cycle might cause physiological disorders and expose the insects to their natural enemies for a longer time (Beland & Hatchett, 1976).

## *Reproductive success*

The focus of our study was not to test for reproductive isolation between strains, since it has been extensively studied before (Dumas *et al.*, 2015; Kost *et al.*, 2016). However, we had to verify the outcome of performance differences between genotypes by testing their reproductive ability. We observed the mating absence for some set-ups (Fig. S4), which might be due to inadequate mating pairs as previously reported (Dumas *et al.*, 2015). For the F₁ hybrids (RR × CC and CC × RR), the low mating success might also be explained by the hybrid incompatibility (Lane, 2011; Kaeppler, 2012). Nevertheless, it turned out that interstrain mating occurred and F₁ hybrids were fertile (Fig. S4), in conformity with earlier findings (Whitford *et al.*, 1988; Nagoshi & Meagher, 2003), which can help explain the fact that many interstrain hybrids exist in the wild. The F₂ generations (RC × RC and CR × CR) produced the highest number of larvae, which was consistent with other studies (Velásquez-Vélez *et al.*, 2011).

In summary, when FAW were fed with artificial food, the two hybrid genotypes performed better than the two parental genotypes in terms of larval survival rate, pupal emerging rate, and developmental time, and one of the hybrid genotypes, CR, even outperformed the two parental genotypes in larval weight (whereas both CR and RC larvae were heavier than the two parental genotypes when it comes to corn plant diet) and male emerging rate significantly. Besides, these two hybrid genotypes were intermediate to the inbred parental strains in pupal weight (also larval weight for RC). Taken together, these observations suggested that the host strain-associated loci (particularly mitochondria) did have an impact on FAW performance, and the asymmetric results revealed a surprising finding, namely, a heterosis effect between the two FAW strains.

## *Heterosis effects*

Heterosis, also called hybrid vigor, is a complex biogenetic phenomenon that occurs in progenies of diverse individual mating from different species, isolated populations, or selected strains within species or populations and is manifested in the hybrid offspring performance (Kristensen & Sorensen, 2005; Kaeppler, 2012). The exploitation of heterosis has been commonly applied in domesticated and semidomesticated insects for enhanced genetic and phenotypic quality through selection and hybridization, such as the honeybee and the silkworm (Hoy, 1976). Heterosis is quantified on an individual or population basis as the superiority of a hybrid performance that exceeds its midparent value or its better parent value (Mather & Jinks, 1971; Virmani, 1994), such as most phenotypic traits of the CR genotype in this study. A study on malaria mosquito *Anopheles coluzzii* has found that heterosis improves male fitness and the overall sexual performance of hybrid males (Ekechukwu *et al.*, 2015), which might also explain the results observed in FAW CR genotype that male pupal weight was heavier than female ones and that male emerging rate was significantly higher than the two parental strains.

Heterosis can result from different factors, and several hypotheses explain its genetic mechanisms. The most classical ones are dominance (Davenport, 1908; Bruce, 1910; Jones, 1917), overdominance (Shull, 1908; East, 1936), and epistasis (Sheridan, 1981). The first two hypotheses are based on allelic interactions while epistasis is based on nonallelic genetic interactions (Yu *et al.*, 2021). It is well known that there are three different categories of individuals among the segregating progeny of hybrids derived from individual crosses, namely, AA (homozygote dominants), Aa (heterozygotes), and aa (recessive genotypes), with a ratio of 1 : 2 : 1. According to Fisher (1928), heterozygotes are naturally intermediate between the dominant and the recessive genotypes, which was also confirmed by part of our results about hybrid pupal weight (also larval weight for RC), and the superior phenotypic expression of other traits (CR and RC larval survival rate, pupal emerging rate and developmental time, and CR larval weight) may be explained by the accumulation and maintenance of modifiers in an additive manner. There are many genome-wide studies of the transcriptome showing a great number of genes that are expressed in an additive manner in hybrid genotypes versus inbred genotypes (Stupar & Springer, 2006; Swanson-Wagner *et al.*, 2006; Swanson-Wagner *et al.*, 2009). However, how additive effects act on heterosis is still unknown. As we mentioned before, the F₁ generation of both hybrid genotypes showed reduced
reproductive rates and the CR genotype exhibited a male-
biased sex ratio, and these results suggested Haldane's
rule, which plays a role in the evolution of postzygotic
isolation in Lepidoptera. There was good evidence that
dominance theory is a possible explanation for Hal-
dane's rule (Presgraves, 2002). Besides, the existence of
postzygotic reproductive isolation between the two FAW
strains has been observed in laboratory (Velásquez-Vélez
et al., 2011; Dumas et al., 2015). Thus, the superior
performance of some traits of the hybrid genotypes can
probably be explained by the dominance theory, which
presumes that most hybrid disruption genes are recessive,
so they tend to be expressed in the females of the F₁
generation, as females are the heterogametic sex in FAW.
Moreover, some studies suggested nongenetic factors that
contribute to heterosis such as epigenetics, among which
the most studied mechanisms are DNA methylation (He
et al., 2010; Greaves et al., 2012; Shen et al., 2012),
histone modification (Ni et al., 2009), and small RNA
pathways (Groszmann et al., 2011; Shen et al., 2012).

It is important to highlight that heterosis across hybrids
is not universal but trait-specific (Kaeppler, 2011). In our
results for example, interspecific FAW hybrids CR and
RC displayed higher larval survival rate and pupal emerg-
ing rate as well as faster developmental time, but are more
sterile and therefore have reduced reproductive rates than
the inbred parental genotypes. Similar asymmetric ob-
servations also occurred in other studies (Flint-Garcia
et al., 2009). According to Darwin's Corollary to Hal-
dane's Rule (Turelli & Moyle, 2007), hybrid crosses cause
asymmetric incompatibility frequently, which might be
driven by interactions involving the X/Z chromosome and
other uniparentally inherited factors, such as mitochon-
dria and plastids (Turelli & Moyle, 2007; Burton & Bar-
reto, 2012). It has been suggested (Prowell, 1998) that
evolution in FAW seemed to be explained by maternal
traits and sex-linked genes, which spurs research into mi-
tochondria and sex chromosomes for genome-wide and
locus-independent molecular mechanism studies (Baran-
wal et al., 2012; Kaeppler, 2012).

Role of mitochondria

Heterosis in growth was reported to be relevant
to heterosis of mitochondrial function (McDaniel &
Sarkissian, 1966; Grunder, 1974). The concept of mito-
chondrial heterosis has been proposed when McDaniel
and Sarkissian (1966) found the superior oxidative and
phosphorylative activity of mitochondria isolated from
maize hybrids compared with mitochondria from par-
ents. Mitochondrial heterosis in plants and some animals

has been well documented (Mukherjee *et al.*, 1970;
McDaniel & Grimwood, 1971; Srivastava & Sarkissian,
1972; Barratt & Flavell, 1977; Christie *et al.*, 2011; Liao
*et al.*, 2020). It has been reported that mitochondrial
DNAs (mtDNAs) show a significant effect on the fitness
of *Drosophila subobscura*, and there was hybrid vigor
for one line that indicated nuclear × mtDNA epistatic in-
teraction effects (Christie *et al.*, 2011). Meiklejohn *et al.*
(2013) revealed that interaction between mitochondrial
tRNA and nuclear-encoded tRNA synthetase (aaRS)
leads to fitness loss in hybrids between *Drosophila melanogaster* and *Drosophila simulans*. Similar results
were also observed in the interspecific mtDNA strains
of *Drosophila simulans*, which showed a wide range of
variations (Rand *et al.*, 2006). Therefore, there is a good
reason to assume that mitochondria may also play a role
in affecting larval FAW fitness. However, performance
variations in different phenotypic traits and reduced
reproductive rates were observed in our study, which
suggested that mitochondria alone cannot explain the
consistent difference between FAW strains.

Role of sex chromosome

Research on interspecific hybridization helps examine
the development of reproductive barriers between diverg-
ing populations and therefore contributes to a better un-
derstanding of the speciation process (Harrison, 1993;
Howard & Berlocher, 1998; Howard et al., 1998; Richie
& Phillips, 1998; Jiggins & Mallet, 2000), as specia-
tion is fundamentally driven by the evolution of repro-
ductive isolation barriers which reduce or prevent gene
flow (Coyne & Orr, 2004). Barriers to reproduction can
be divided into prezygotic versus postzygotic isolation,
and both were exhibited by FAW (Groot et al., 2010).
However, contradictory results were obtained from dif-
ferent reports when testing reproductive success, as we
mentioned before. In Lepidoptera, speciation might be
explained by the evolution of sex-linked genes, as plenty
of traits that determine strains and species seem to be lo-
calized on the X/Z chromosome (Grula & Taylor, 1980;
Rockey et al., 1987; Sperling, 1994; Prowell, 1998). FAW
has been used as a model species to study the speciation
process (Groot et al., 2010). FST calculated between FAW
strains showed obvious outliers on the Z chromosomes,
suggesting that genetic differentiation between strains is
promoted by strain-specific divergent selection targeting
Z-linked genes (Durand et al., 2022), consistent with the
Large-Z effects (Coyne & Orr, 1989; Coyne & Orr, 2004;
Turelli & Moyle, 2007). Wing traits might be sex-linked.
It has been found that females of some butterflies tended
to mate with males, which have the same wing colors, and this reproductive isolation based on wing colors was stronger between sympatric rather than allopatric populations (Jiggins *et al.*, 2001). Some researchers have found that there were significant differences in wing shape and size between FAW strains in South American populations (Cañas-Hoyos *et al.*, 2014; Cañas-Hoyos *et al.*, 2016). We have also noticed pigmentation differences in our lab FAW strains for both larval body and adult wing patterns (Figs. S7, S8), though the differences need to be quantified in the wild. Besides, esterases and female calling time were found to be probably sex-linked for FAW (Prowell, 1998). Indeed, a genetic incompatibility involving a Z-chromosome-linked sterility locus has been found to cause the sexual abstinence of RC hybrid females, which were behaviorally sterile although the reproductive rate of RC was not null (Kost *et al.*, 2016), and this incompatibility was unidirectional considering the high viability of RC females in this study. Our results showed that both RC and CR hybrid females were fertile and laid hundreds of eggs (Fig. S4), which was against the RC hybrid female sterility, a phenomenon that has been observed in many other reports (Pashley & Martin, 1987; Whitford *et al.*, 1988; Groot *et al.*, 2010). This inconsistency may be due to the unknown complexity and variability in the nature of the reproductive barriers between strains (Nagoshi, 2019). More research on FAW genetics is required to explain this discrepancy, especially for the traits related to reproductive isolation.

## Conclusion

In conclusion, to test if there are effects from host-strain-associated loci on FAW performance that could help explain the divergence between the two FAW strains, we have investigated the Life History Traits of FAW hybrids and parental populations reared on artificial diet and corn plant leaves. First, we observed that hybrids have high survival rates and higher reproductive rates than parental strains in the laboratory, which is compatible with SfR and SfC strains being at a very early stage of incipient speciation. In addition, we have demonstrated the performance differences between the lab FAW strains and the established hybrid genotypes, with hybrid genotypes performing better than the parental genotypes for most phenotypic traits. These differences in larval development between the hybrids and parental genotypes are also observed when FAW were fed with corn plants, revealing a heterosis effect between the corn strain and the rice strain of FAW. There are many factors that account for heterosis and here we suggested the role played by mitochon-

dria and sex chromosomes, and we assumed the most sought-after dominance theory, namely, Haldane's rule, as a possible explanation for the results of the reduced visibility of both hybrid genotypes and the male-biased sex ratio of the CR genotype. However, the molecular genetics and mechanism of heterosis formation in FAW remain unknown, and identification of certain heterosis-related gene loci is needed in the future studies. Besides, it is important to note that our experiments in laboratory are not designed to recapitulate natural populations' history, but rather control the genotypic background of FAW larvae to investigate its effect on traits associated with performance. All experiments in this study were conducted under a controlled, seemingly benign laboratory environment, whereas conditions are likely to be more stressful and might alter the consequences of hybridization in many ways in the wild. Nonetheless, the results of this study can help shed light on the development of management strategies against this pest.

## Acknowledgments

This work was supported by funding from SPE department of INRAE for NN and by the scholarship from China Scholarship Council (CSC) under the collaborative program of CSC & Agreenium (CSC NO. 202008440426; https://en.agreenium.fr/page/agreenium) for LL. We thank the quarantine insect platform (PIQ) for providing the infrastructure needed for insect pest experiments. We are also grateful to Gaëtan Clabots, Raphaël Bousquet, and Dylan Valenza for maintaining the insect collections of the DGIMI laboratory in Montpellier, France. We also want to thank Antony Champion for providing the corn plants and Laëtitia Leclerc and Anne-Sophie Petitot for helping take care of the plants in IRD. We appreciate the help from Sylvia Abdelmalek in setting up the experiment with corn plants. We acknowledge Emmanuelle d'Alençon and Kiwoong Nam for their reviews and comments on an earlier version of the manuscript.

## Disclosure

NN designed the project. LL and NN conducted the performance experiments. LL performed the statistical analysis of LHT. LL performed the DNA extraction of the samples and the genotyping experiments. LL wrote the manuscript and produced the figures. NN edited the current manuscript. All authors read and approved the present manuscript submission. The authors declare that they have no financial conflict of interest with the content of this article.
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Manuscript received April 13, 2023

Final version received August 30, 2023

Accepted October 16, 2023

**Supporting Information**

Additional supporting information may be found online in the Supporting Information section at the end of the article.

**Fig. S1** Genotypes of FAW lab strains and hybrids. “mt” refers to the mitochondrial genome.

**Fig. S2** Larval weight differences among 4 FAW genotypes with artificial diet.

**Fig. S3** Larval weight differences among 4 FAW genotypes with corn diet.

**Fig. S4** Reproduction differences among different FAW genotypes (Student's *t*-test, *P* < 0.05).

**Fig. S5** Gel images of genotyping results with *COI*.

**Fig. S6** Gel images of genotyping results with *Tpi*.

**Fig. S7** Pigmentation differences among 4 FAW genotypes at larval stage.

**Fig. S8** Pigmentation differences among 4 FAW genotypes at pupal stage.