Clément Gilbert

Florian Maumus

Sidestepping Darwin: horizontal gene transfer from plants to insects

published or not. The documents may come

Introduction

Darwin proposed that evolution occurs through natural selection of intraspecific variations transmitted across generations [START_REF] Darwin | On the Origin of Species[END_REF]. But he was unable to provide a satisfactory explanation for the origin of these variations and the mechanism underlying their transmission. Darwin and Mendel were contemporaries, but they never met and Mendel's work was ignored by Darwin [START_REF] Berry | Mendel and Darwin[END_REF]. About 50 years after Darwin and Mendel died, Darwin's natural selection and Mendelian inheritance, which posits that individuals inherit a combination of alleles from their parents, became two pillars of the modern synthesis. Remarkably, observations made in bacteria as early as 1928 -i.e., before the modern synthesis was initiated -suggested that genetic information could also be transmitted between individuals through horizontal transfer (HT), i.e., through means other than vertical, parent-tooffspring, inheritance [START_REF] Griffith | The Significance of Pneumococcal Types[END_REF]. In the second half of the 20 i eth century it became apparent that HT of genes was rampant in prokaryotes and that it was a major source of bacterial innovation [START_REF] Ochman | Lateral gene transfer and the nature of bacterial innovation[END_REF]. The first hint that HT of genetic material was not restricted to prokaryotes but could also occur in eukaryotes may correspond to the discovery of endogenous retroviruses in vertebrate genomes during the late 1960s and early 1970s [START_REF] Weiss | The discovery of endogenous retroviruses[END_REF]. It was then suggested that the endosymbiotic origin of eukaryotes was accompanied by the relocation (through HT) of many organellar genes to the nuclear genome [START_REF] Timmis | Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes[END_REF], and that transposable elements (TEs) [START_REF] Bingham | The molecular basis of P-M hybrid dysgenesis: the role of the P element, a P-strain-specific transposon family[END_REF][START_REF] Engels | The P family of transposable elements in Drosophila[END_REF], as well as non-organellar genes [START_REF] Martin | Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba)[END_REF][START_REF] Syvanen | Cross-species gene transfer; implications for a new theory of evolution[END_REF] could be acquired horizontally in these taxa. These pioneering works were followed by the observation that the genome of phagotrophic single-cell eukaryotes contained many genes captured from their prokaryote and microbial eukaryote preys [START_REF] Doolittle | You are what you eat: a gene transfer ratchet could account for bacterial genes in eukaryotic nuclear genomes[END_REF][START_REF] Andersson | Lateral gene transfer in eukaryotes[END_REF][START_REF] Keeling | Horizontal gene transfer in eukaryotic evolution[END_REF]. Today, the importance of HT of genes in recent eukaryote evolution remains a matter of debate [START_REF] Martin | Too Much Eukaryote LGT[END_REF][START_REF] Martin | Eukaryote lateral gene transfer is Lamarckian[END_REF][START_REF] Boto | Are There Really Too Many Eukaryote LGTs? A Reply To William Martin[END_REF][START_REF] Leger | Demystifying Eukaryote Lateral Gene Transfer (Response to Martin 2017[END_REF][START_REF] Roger | Reply to "Eukaryote lateral gene transfer is Lamarckian[END_REF]. But as a matter of fact, we are witnessing an increasing number of HT reports in both uni-and multi-cellular eukaryotes, largely fueled by the many high-quality new whole genome sequences that are deposited on a daily basis in public databases [START_REF] Soucy | Horizontal gene transfer: building the web of life[END_REF][START_REF] Aubin | Horizontal Gene Transfers in Plants[END_REF][START_REF] Sibbald | Lateral Gene Transfer Mechanisms and Pan-genomes in Eukaryotes[END_REF][START_REF] Van Etten | Horizontal Gene Transfer in Eukaryotes: Not if, but How Much?[END_REF][START_REF] Irwin | Systematic evaluation of horizontal gene transfer between eukaryotes and viruses[END_REF][START_REF] Shen | Tempo and Mode of Genome Evolution in the Budding Yeast Subphylum[END_REF]. Importantly, many of these transfers likely facilitated the adaptation of the receiving species to a new ecological niche [START_REF] Husnik | Functional horizontal gene transfer from bacteria to eukaryotes[END_REF][START_REF] Keeling | Functional and ecological impacts of horizontal gene transfer in eukaryotes[END_REF][START_REF] Boto | Horizontal gene transfer in the acquisition of novel traits by metazoans[END_REF]. In multicellular eukaryotes, most HT of genes reported so far involve genes of bacterial, fungi, or viral origin [START_REF] Irwin | Systematic evaluation of horizontal gene transfer between eukaryotes and viruses[END_REF][START_REF] Husnik | Functional horizontal gene transfer from bacteria to eukaryotes[END_REF][START_REF] Simion | Chromosome-level genome assembly reveals homologous chromosomes and recombination in asexual rotifer Adineta vaga[END_REF][START_REF] Paganini | Contribution of lateral gene transfers to the genome composition and parasitic ability of root-knot nematodes[END_REF][START_REF] Hotopp | Horizontal gene transfer between bacteria and animals[END_REF][START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF]. Aside from HT of TEs which are not rare [START_REF] Gilbert | Horizontal acquisition of transposable elements and viral sequences: patterns and consequences[END_REF], relatively few HT of genes between multicellular eukaryotes have so far been uncovered. This trend is seemingly changing in plants, in which numerous recent cases of plant-to-plant HT have been characterized [START_REF] Hibdige | Widespread lateral gene transfer among grasses[END_REF][START_REF] Ma | Major episodes of horizontal gene transfer drove the evolution of land plants[END_REF][START_REF] Bartha | Intact ribosomal DNA arrays of Potentilla origin detected in Erythronium nucleus suggest recent eudicot-to-monocot horizontal transfer[END_REF][START_REF] Cai | Deeply Altered Genome Architecture in the Endoparasitic Flowering Plant Sapria himalayana Griff. (Rafflesiaceae)[END_REF]. In vertebrates, a single such event is known so far, which involves transfer of a gene coding an antifreeze protein between the Atlantic herring and the rainbow smelt [START_REF] Graham | Horizontal Gene Transfer in Vertebrates: A Fishy Tale[END_REF]. Here, we provide a comprehensive account of another type of eukaryote-to-eukaryote HT, namely plant-to-insect HT, highlight methodological challenges inherent to the detection of such transfers, and discuss the possible mechanisms underlying these HT as well as their impact on insect genome evolution and plant insect interactions.

How many genes of plant origin in insect genomes?

The first claim of plant-to-insect HT was published in a 2015 article in which hundreds of transcripts from the mosquito Anopheles culicifacies were reported to encode proteins 100% identical to proteins found in plants [START_REF] Sharma | Deep sequencing revealed molecular signature of horizontal gene transfer of plant like transcripts in the mosquito Anopheles culicifacies: an evolutionary puzzle[END_REF]. However, using plant proteins listed in supplementary table 1 of [START_REF] Sharma | Deep sequencing revealed molecular signature of horizontal gene transfer of plant like transcripts in the mosquito Anopheles culicifacies: an evolutionary puzzle[END_REF] [START_REF] Sharma | Deep sequencing revealed molecular signature of horizontal gene transfer of plant like transcripts in the mosquito Anopheles culicifacies: an evolutionary puzzle[END_REF], we were unable to recover plant-derived genes in the genome of A. culicifacies, which is now available in Genbank (accession number: AXCM00000000.1). Two cases of TE transfer between plants and arthropods have been described but in both studies, HT was deemed more likely to have occurred from arthropod to plants rather than in the opposite direction [START_REF] Lin | An Ancient Trans-Kingdom Horizontal Transfer of Penelope -like Retroelements from Arthropods to Conifers[END_REF][START_REF] Gao | Horizontal Transfer of Non-LTR Retrotransposons from Arthropods to Flowering Plants[END_REF]. The first robust case of plant-to-insect HT is thus the one reported in [START_REF] Lapadula | Whitefly genomes contain ribotoxin coding genes acquired from plants[END_REF] [START_REF] Lapadula | Whitefly genomes contain ribotoxin coding genes acquired from plants[END_REF], in which a gene of plant origin encoding a ribosome inactivating protein (RIP) has been acquired at least 80 million years ago by an ancestor of two whitefly species (Bemisia tabaci and Trialeurodes vaporariorum). Whiteflies (family Aleyrodidae; Hemiptera) are important agricultural pests as they are able to feed upon a large spectrum of host plants and they can transmit many viruses to plants [START_REF] Chen | The draft genome of whitefly Bemisia tabaci MEAM1, a global crop pest, provides novel insights into virus transmission, host adaptation, and insecticide resistance[END_REF]. After transfer from an unknown plant donor, the RIP gene was duplicated once and twice in the lineage leading to B. tabaci and T. vaporariorum, respectively. Quickly following this report, two other plant-derived genes encoding BAHD acyltransferases were characterized, again in the whitefly B. tabaci [START_REF] Xia | Whitefly hijacks a plant detoxification gene that neutralizes plant toxins[END_REF].

These studies prompted us to perform a systematic search for plant-to-B. tabaci HT, which yielded no less than 49 plant-derived genes in the genome of the Middle East Asia Minor 1 (MEAM1) whitefly [START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF]. We found that these genes could be clustered in 24 orthogroups, 20 of which have members in another whitefly cryptic species (Sub-Saharan Africa -East and Central Africa [SSA-ECA]), which diverged at least five MYA from the MEAM1 whitefly [START_REF] Chen | Genome of the African cassava whitefly Bemisia tabaci and distribution and genetic diversity of cassava-colonizing whiteflies in Africa[END_REF]. We uncovered another two plant-derived genes in T. vaporariorum, which diverged several dozens of MYA from B. tabaci [START_REF] Misof | Phylogenomics resolves the timing and pattern of insect evolution[END_REF], but we were unable to firmly conclude on whether these genes were acquired from plants independently in this species or whether they were transferred in an ancestor of B. tabaci and T. vaporariorum.

In parallel to our study, a large-scale, systematic survey of HT performed by [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] in 218 insect species uncovered no less than 1410 foreign genes. While the vast majority of these genes have a bacterial source, many were transferred from fungi and viruses and a total of 43 were assigned a plant origin. Just by itself, B. tabaci accounts for 24 of these 43 genes, most of them being also reported in our study (see next section). The reason why B. tabaci has a much higher number of plantderived genes than other insects is not immediately evident but we note that this species also has a higher number of genes transferred from bacteria and the higher overall number of horizontally acquired genes (170 versus 68 in Contarinia nasturtii, the species with the second higher number of foreign genes). Whether horizontal gene flux is higher in B. tabaci or whether it is similar to other insects but foreign genes have a higher chance to be retained in B. tabaci remains to be assessed. Interestingly, [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] also uncovered plant-derived genes in three other species of Hemiptera, one Coleoptera, three Diptera, one Hymenoptera, one Orthoptera and two Thysanoptera.

In total, and accounting for the redundancy between our results [START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF] and those of [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] for B. tabaci, a total of 156 genes of plant origin have been so far uncovered in the genome of 14 insect species belonging to six orders (Figure 1). It is noteworthy that nine plant-derived genes are annotated as plant transposons in Aedes mosquitoes [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF], begging the question of whether these genes correspond to recently acquired TEs that may still be able to transpose or whether they arise from molecular domestication of ancient plant TEs horizontally acquired by an ancestor of Aedes mosquitoes.

Figure 1. Number of plant-derived genes identified in insect genomes.

The number of plant-derived genes in Bemisia tabaci MEAM1 results from our merging of results from [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] and [START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF]. Details regarding which genes were found in which study and on the predicted function of these genes are given in Supplementary Table 1. The tree and divergence times were taken from REF. 46 and 79.

Numbers at nodes in Aleyrodidae correspond to putative numbers of horizontal transfer events that took place in the ancestors of Aleyrodidae, as inferred in [START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF] based on the delineation of orthogroups.

Nine of the plant-derived genes from Aedes mosquitoes are annotated as encoding plant transposon proteins in supplementary table 2 of [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF].

Methodological challenges to detect horizontal gene transfer

Inferences of HGT are plagued by methodological issues and several hypotheses alternative to HGT must be considered before HGT is favored [START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF][START_REF] Salzberg | Horizontal gene transfer is not a hallmark of the human genome[END_REF][START_REF] Koutsovoulos | No evidence for extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini[END_REF]. It is for example essential to check that what looks like a horizontally acquired gene is not instead a contaminant. This may be done using fluorescent in situ hybridization, as in Dunning [START_REF] Hotopp | Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes[END_REF] [START_REF] Hotopp | Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes[END_REF] or PCR as in [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF]. Another way to discard the possibility of contamination is to find orthologs of horizontally acquired genes in a group of species, the genome assembly of which were produced independently in different labs. Other recommendations on HGT validation have been discussed in previous papers [START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF][START_REF] Salzberg | Horizontal gene transfer is not a hallmark of the human genome[END_REF][START_REF] Koutsovoulos | No evidence for extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini[END_REF][START_REF] Hotopp | Grafting or pruning in the animal tree: lateral gene transfer and gene loss?[END_REF]]. Yet, even when greatest care is taken, large-scale studies of HGT may yield different results due to differences in the bioinformatics approach being implemented. This is particularly well-illustrated by the two recent reports of HGT from plants in B. tabaci (MEAM1) [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF][START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF]. Both studies used overall similar workflows, being based on sequence similarity search between B. tabaci query proteins and a target database followed by the analysis of the taxonomic distribution of hits to identify candidate HT proteins. Homologs of each candidate were then retrieved and used to build phylogenetic trees that were manually examined for HGT validation. However, the two workflows present key differences (Figure 2). First, they rely on different target databases. Li for each HGT candidate, measures the extent to which the best hit against targets from the query taxonomic group (ingroups) differs from that obtained against targets from other groups (outgroups).

To limit the number of false positives, Li et al. (2022) [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] also calculated the percentage of outgroup species among the top hits. This percentage will be low if the homolog is not conserved in outgroups or if the gene family is comparably distant to homologs across several taxa. By contrast, Gilbert & Maumus (2022) [START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF] have used an original approach in which the UniRef90 target database is depleted of ingroup proteins (here Aleyrodidae) to infer the last common ancestor (LCA) of each query protein.

Using this taxonomic exclusion, the placement of a protein LCA in Viridiplantae (plants) occurs when a significant proportion of its best homologs are plant proteins, suggesting a plant origin. The study applied three different modes of LCA inference implemented in the taxonomy module of the MMseqs2 program [START_REF] Mirdita | Fast and sensitive taxonomic assignment to metagenomic contigs[END_REF], in which the taxonomical assignment of the LCA can be inferred using two methods derived from the 2bLCA protocol [START_REF] Hingamp | Exploring nucleo-cytoplasmic large DNA viruses in Tara Oceans microbial metagenomes[END_REF] or using lowest common ancestor of equal-scoring top hits (this last mode being theoretically similar to AI-based measures). To focus on most robust HGT candidates, only the proteins with LCA in Viridiplantae below the genus level were considered as a filter attesting for their conservation in plants. Using deep protein clustering (35% identity), Gilbert & Maumus (2022) [START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF] found that the 49 plantderived proteins from B. tabaci MEAM1 correspond to 24 clusters. Here, we applied the same clustering to the 24 plant-derived proteins from Li et al. (2022) [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF], which resulted in 18 clusters. We found that only 13 clusters comprise proteins from the two studies while 11 and 5 are unique to REF.

Functional impact of plant genes in insects

Only one experimental study characterized the function of a plant-derived gene in insect [START_REF] Xia | Whitefly hijacks a plant detoxification gene that neutralizes plant toxins[END_REF]. Together with the predicted function of all plant-derived genes in B. tabaci, it suggests that much like bacteriato-insect HGT [START_REF] Wybouw | Horizontal Gene Transfer Contributes to the Evolution of Arthropod Herbivory[END_REF], plant-to-insect HGT was a source of important new functions to insects, which may have facilitated adaptation to their environment. REF. [START_REF] Xia | Whitefly hijacks a plant detoxification gene that neutralizes plant toxins[END_REF] showed that the protein encoded by the BtPMaT1 plant-derived gene in B. tabaci has malonyl-transferase activity, that is, it catalyzes the transfer of a malonyl group onto several phenolic glucosides, which are secondary compounds produced by many plants to defend themselves against herbivorous insects. Malonylation detoxifies phenolic glucosides and renders them harmless to whiteflies. In agreement with this, whiteflies feeding on transgenic tomato plants that express small interfering RNAs targeting BtPMaT1 are unable to survive [START_REF] Xia | Whitefly hijacks a plant detoxification gene that neutralizes plant toxins[END_REF]. Thus, whiteflies harnessed a plant gene which allows them to thwart plant defenses and was perhaps essential in the evolution of these hemipterans towards becoming generalist herbivores [START_REF] Xia | Whitefly hijacks a plant detoxification gene that neutralizes plant toxins[END_REF][START_REF] Whiteman | Whiteflies weaponize a plant defense via horizontal gene transfer[END_REF].

In addition to this functional study, indirect evidence including evolution under purifying selection and expression suggests that most if not all other plant-derived genes in B. tabaci encode important functions [START_REF] Lapadula | Whitefly genomes contain ribotoxin coding genes acquired from plants[END_REF][START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF]. To help explore these putative functions, we provide a table compiling predicted functions of all B. tabaci plant-derived proteins reported in Li et al. (2022) [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] and Gilbert & Maumus [START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF] (Supplementary Table 1). Several of these proteins have predicted functions previously shown to play a role in plant-parasite interactions and response to pathogens, not necessarily insects but rather fungi and oomycetes. It remains unclear however if and how these genes enabled whiteflies to adapt to different host plants. The protein functions inferred are based on the annotation of homologs and only speculative. Only three plant-derived gene clusters have a potentially evident role in helping the insect bypassing host defenses. These include BtPMat1 [START_REF] Xia | Whitefly hijacks a plant detoxification gene that neutralizes plant toxins[END_REF] as well as a predicted plant cell wall degrading enzyme (PCWDE) and a predicted alpha-(1,4)-fucosyltransferase which could be involved in cell wall patterning [START_REF] Zhang | The plant cell wall: Biosynthesis, construction, and functions[END_REF]. Perhaps more surprisingly, many plant-derived genes have putative functions linked to the production and activation of plant defenses. For instance, plant-derived genes annotated as BAHD acyltransferase and phenylalanine ammonialyase have putative functions in the production of phenolic secondary metabolites [START_REF] Auria | Acyltransferases in plants: a good time to be BAHD[END_REF], many being defense compounds. That said, the B. tabaci plantderived BAHD acyltransferase has best BLAST hits against Dcr-like (Defective in Cuticular Ridges) homologs. Dcr is not involved in production of phenolic compounds but is instead required for the assembly of cutin polymers, which are the major components of the outermost layer of plant defense, the cuticle [START_REF] Panikashvili | The Arabidopsis DCR Encoding a Soluble BAHD Acyltransferase Is Required for Cutin Polyester Formation and Seed Hydration Properties[END_REF][START_REF] Li | Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers[END_REF]. The whitefly plant-derived genes also comprise homologs of pathogen-related proteins (which are produced by plants upon biotic attack) and proteins that have known roles in plants in pathogen recognition and/or defense signaling pathways including Thaumatin [START_REF] Woloshuk | Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans[END_REF], subtilisin-like protease [START_REF] Figueiredo | Subtilisin-like proteases in plantâ€"pathogen recognition and immune priming: a perspective[END_REF] or glucan endo-1,3-beta-glucosidase [START_REF] Veronese | In Defense against Pathogens. Both Plant Sentinels and Foot Soldiers Need to Know the Enemy[END_REF]. An advantage to herbivory could generally make sense if the encoded proteins are inactive and simply tether plant enzymes or occupy receptors to limit the defense response. It is also possible that some plant-derived genes may be recycled by insects to protect themselves against pathogens, parasites or predators. In agreement with this scenario, the RIP genes reported in REF. 41 are toxic N-glycosidases playing a role in plant defense against pathogens and insect predators [START_REF] Zhu | The Plant Ribosome-Inactivating Proteins Play Important Roles in Defense against Pathogens and Insect Pest Attacks[END_REF]. As pointed out by Lapadula et al., the whitefly RIPs might play a role similar to those produced by Spiroplasma endosymbionts in Drosophila, known to protect their host against nematodes [START_REF] Hamilton | A ribosome-inactivating protein in a Drosophila defensive symbiont[END_REF] and parasitic wasps [START_REF] Ballinger | Generality of toxins in defensive symbiosis: Ribosomeinactivating proteins and defense against parasitic wasps in Drosophila[END_REF].

Other plant-derived genes in B. tabaci may have predicted functions in nutrient and micronutrient assimilation, glycoprotein secondary modifications, and fatty-acid desaturation including a large family of delta [START_REF] Andersson | Lateral gene transfer in eukaryotes[END_REF] fatty acid desaturases (Supplementary Table 1). It will be interesting to develop experimental approaches to characterize these genes and how they may have been beneficial to whiteflies compared to genes acquired from bacteria [START_REF] Wybouw | Horizontal Gene Transfer Contributes to the Evolution of Arthropod Herbivory[END_REF]. In this context, testing the function of the B. tabaci plant-derived pectinesterase should be relatively straightforward as it has a clear predictable function in degrading plant cell walls. It would also be worth addressing whether the catalytic sites in predicted plant-derived enzymes are conserved to assess their catalytic potential and which plantderived genes are specifically expressed in the insect saliva or gut to suggest a direct link with herbivory.

How can plant DNA be transferred to insects?

The mechanisms underlying HT in eukaryotes remain poorly understood. That most foreign genes so far reported in eukaryotes are from prokaryotes (this is especially true for insects [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF][START_REF] Nakabachi | Horizontal gene transfers in insects[END_REF]) may in part be due to the capacity of several bacteria to transfer their DNA to eukaryotic cells through conjugation [START_REF] Lacroix | Transfer of DNA from Bacteria to Eukaryotes[END_REF]. Furthermore, many arthropod and other metazoan species harbor obligate or facultative intracellular symbionts [START_REF] Mouton | Analyses of symbiotic bacterial communities in the plant pest Bemisia tabaci reveal high prevalence of Candidatus Hemipteriphilus asiaticus on the African continent[END_REF], some of which are in close contact with the host germline genome, facilitating HT [START_REF] Hotopp | Horizontal gene transfer between bacteria and animals[END_REF]. The higher proportion of foreign prokaryotic versus eukaryotic genes so far uncovered in eukaryote genomes may also have methodological underpinnings. Transfers are easier to infer between distantly related species (e.g. between prokaryotes and eukaryotes) than between more closely related ones (e.g. between two eukaryotes). Systematic studies focusing on detecting eukaryote-to-eukaryote HT remain scarce. In any case, this later type of transfer is more difficult to explain than prokaryote-to-eukaryote transfers because no mechanism dedicated to HT is known in eukaryotes (but see [START_REF] Muller | Genome-Wide Patterns of Bracovirus Chromosomal Integration into Multiple Host Tissues during Parasitism[END_REF]). Plant-to-plant HTs have been proposed to occur accidentally through whole genome transfer in heterospecific grafts [START_REF] Fuentes | Horizontal genome transfer as an asexual path to the formation of new species[END_REF], illegitimate pollination, haustoria in parasitic plants [START_REF] Wickell | On the evolutionary significance of horizontal gene transfers in plants[END_REF],

or root-to-rhizome contact [START_REF] Hibdige | Widespread lateral gene transfer among grasses[END_REF]. Regarding plant-to-insect transfers, one might argue that the close ecological interactions existing between plants and most insects facilitate HT. Indeed, all insect species in which plant-derived genes have been found so far (Figure 1) are herbivorous or nectarivores.

Whether DNA can be transferred from plants to insects through feeding, as proposed in the case of bacteria-to-single cell eukaryotes HT [START_REF] Doolittle | You are what you eat: a gene transfer ratchet could account for bacterial genes in eukaryotic nuclear genomes[END_REF] remains to be tested. In this context, it is noteworthy that insects can internalize dsRNA produced by plants through feeding [START_REF] Xia | Whitefly hijacks a plant detoxification gene that neutralizes plant toxins[END_REF][START_REF] Kunte | Prospects, challenges and current status of RNAi through insect feeding[END_REF], so one could speculate that DNA from their food may also occasionally penetrate into insect cells. However, the « you are what you eat » hypothesis may not be fully sufficient to explain plant-to-insect HT as the majority of herbivorous insects included in REF. [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] are devoid of plant-derived genes. Another, non-mutually exclusive hypothesis is that vectors such as exosomes or viruses are involved in plant-to-insect HT [START_REF] Ono | Exosome-mediated horizontal gene transfer occurs in double-strand break repair during genome editing[END_REF][START_REF] Gilbert | Viruses as vectors of horizontal transfer of genetic material in eukaryotes[END_REF]. If true, the known exposure of B. tabaci to a very large diversity of plant viruses, several of which are known to replicate in whitefly cells [START_REF] Hogenhout | Insect Vector Interactions with Persistently Transmitted Viruses[END_REF], may in part explain the higher number of foreign genes found in this, compared to other insect species [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF][START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF]. In this context, it is noteworthy that whiteflies are the main insect vectors of dozens of Begomovirus species, which are highly successful plant viruses. Some of these circular single-stranded DNA viruses are able to replicate in insect cells, persist during the entire lifetime in the insect and can undergo transovarial transmission [START_REF] Fiallo-Olivé | Begomoviruses: what is the secret(s) of their success?[END_REF].

Acquisition of foreign genes by B. tabaci under the form of DNA, rather than RNA, through the intermediate of DNA viruses is consistent with the fact that most plant-like genes uncovered in whiteflies contain at least one intron [START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF]. However, it is also possible that these genes were transferred through an RNA-mediated mechanism and later acquired introns once in the B. tabaci genome.

Conclusions and outstanding questions

The first plant-to-insect HGT was reported recently [START_REF] Lapadula | Whitefly genomes contain ribotoxin coding genes acquired from plants[END_REF] and since then 156 plant-derived genes have been found in 14 insect species from six orders. The reasons why the whitefly B. tabaci concentrates most of these genes, as well as most non-plant foreign genes among insects, are unknown. Based on a comparison between two recent studies [START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF][START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF], we contend that the number of horizontally acquired genes in insects, including those acquired from plants, is likely strongly underestimated. In addition to improve current HGT pipelines that rely on the use of annotated protein-coding sequences, it would be interesting to perform more comprehensive HGT searches using whole genomes. This would allow assessing whether the currently observed higher number of horizontally acquired plant genes versus plant TEs in insect genomes has biological underpinnings or is merely due to methodological limitations. Should the current trend hold, it would be in striking contrast with the trend observed for insect-to-insect HTs, as most if not all currently known such events involve TEs and not genes [START_REF] Gilbert | Transposable Elements and the Evolution of Insects[END_REF]. Future large-scale examination of plant-to-insect HGT may also shed light on the factors possibly influencing such transfers and on whether some plant and/or insect life history traits correlate with HT numbers. Of particular relevance will be to identify recent plant-to-insect HGT events, as this may help identifying donor plants species and the mechanisms underlying transfer. A deeper understanding of the impact of plant-to-insect HGT will only be reachable through multiple functional studies of insect plant-derived genes, such as the one performed in REF. [START_REF] Xia | Whitefly hijacks a plant detoxification gene that neutralizes plant toxins[END_REF]. In addition to help decipher the intricacy of plant-insect interactions, such studies have the potential to unveil new targets for controlling insect pests, which may in some contexts be less problematic than pesticide-based strategies [START_REF] Wang | Silencing horizontally transferred genes for the control of the whitefly Bemisia tabaci[END_REF]. *Together with ref 43, this study provides a proof of principle that horizontally acquired genes may be good targets to develop new strategies to control insect pests. The impact of such strategies on the environment however remains to be assessed compared to pesticide-based approaches.

Highlighted references

Figure 2 .

 2 Figure 2. Graphical comparison of the HT detection workflows used in Li et al. (2022) [31] and Gilbert & Maumus (2022) [44]. The main steps of the workflows are separated in grey boxes and the methods used at each step by REF. 31 and REF. 44 are indicated in salmon and blue rectangles, respectively.

44 .

 44 Gilbert C, Maumus F: Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci. Genome Biology and Evolution 2022, 14:evac141. *A systematic characterization of plant-derived genes in the genome of four whiteflies, uncovering 24 plant-to-whitefly HGT events.54. Wybouw N, Pauchet Y, Heckel DG, Van Leeuwen T: Horizontal Gene Transfe r Contributes to the Evolution of Arthropod Herbivory. Genome Biol Evol 2016, 8:1785-801. *A comprehensive review of arthropod genes acquired horizontally from bacteria, showing that most of them are involved in overcoming plant defenses, assimilation of intracellular plant metabolites or digestion of plant cell wall components. 51. Koutsovoulos GD, Granjeon Noriot S, Bailly-Bechet M, Danchin EGJ, Rancurel C: AvP: A software package for automatic phylogenetic detection of candidate horizontal gene transfers. PLoS Comput Biol 2022, 18:e1010686. *A useful tool to rapidly identify candidate HGT in any genome of interest.

78 .

 78 Wang T-Y, Luan J-B: Silencing horizontally transferred genes for the control of the whitefly Bemisia tabaci. J Pest Sci 2022, doi:10.1007/s10340-022-01492-6.

  [START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF] or REF.[START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF], respectively (Figure2and Supplementary Table1). One cluster unique to REF.[START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] comprises two genes (XP_018917389.1 and XP_018909952.1) for which we could not confirm plant origin. Two other clusters unique to REF.[START_REF] Li | HGT is widespread in insects and contributes to male courtship in lepidopterans[END_REF] have no homolog in the MEAM1 proteome used in REF.[START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF]. This comparison shows that the HT detection workflow we used in REF.[START_REF] Gilbert | Multiple Horizontal Acquisitions of Plant Genes in the Whitefly Bemisia tabaci[END_REF] is more sensitive, but it is not entirely comprehensive. Understanding the causes of reciprocal false negatives will help improving the sensitivity of HT detection. This comparison also shows that the number of insect HGT reported in REF. 31 may be largely underestimated and that HGT, including from plants, may have had an even stronger impact on insects than what is apparent from this study.

HGT is widespread in insects and contributes to male courtship in lepidopterans.

  31. Li Y, Liu Z, Liu C, Shi Z, Pang L, Chen C, Chen Y, Pan R, Zhou W, Chen X, et al.: Cell 2022, doi:10.1016/j.cell.2022.06.014. *A large-scale characterization of foreign genes in 218 insect genomes, showing that HGT is widespread in insects, and that one of the horizontally acquired gene plays a role in male courtship in lepidopterans. The first report of a plant-to-insect horizontal gene transfer event, involving a plant ribosome inactivating protein encoding gene acquired by the whitefly Bemisia tabaci.43. Xia J, Guo Z, Yang Z, Han H, Wang S, Xu H, Yang X, Yang F, Wu Q, Xie W, et al.: *An in-depth functional characterization of BtPMat1, a plant-derived gene encoding a malonyltransferase in whiteflies, showing that this gene detoxifies plant phenolic glucosides, allowing whiteflies to feed on many plants at no cost.

	41. Lapadula WJ, Mascotti ML, Juri Ayub M: Whitefly genomes contain ribotoxin coding
	genes acquired from plants. Sci Rep 2020, 10:15503.
	Whitefly

**hijacks a plant detoxification gene that neutralizes plant toxins. Cell 2021, 184:1693-1705.e17.

*

Acknowledgements

CG is supported by a grant from Agence Nationale de la Recherche (ANR-18-CE02-0021-01 TranspHorizon).

Legends of additional elements

Supplementary Table 1. Annotation of MEAM1 plant-derived proteins found in REF. 31 and REF. 44.

The alternating shades of grey distinguish the clusters established at 35% amino-acid sequence identity using MMseqs2. The false positive cluster from Li et al. ( 2022) is not indicated. The Genbank protein identifiers begin with "XP" while the WhiteflyDB identifiers begin with "Bta". The annotations correspond to the transfer of annotations of the best hits obtained by BLAST against Genbank. The functional category and putative function are only speculative.