A Horizontally Transferred Plant Fatty Acid Desaturase Gene Steers Whitefly Reproduction

Abstract Polyunsaturated fatty acids (PUFAs) are essential nutrients for all living organisms. PUFA synthesis is mediated by Δ12 desaturases in plants and microorganisms, whereas animals usually obtain PUFAs through their diet. The whitefly Bemisia tabaci is an extremely polyphagous agricultural pest that feeds on phloem sap of many plants that do not always provide them with sufficient PUFAs. Here, a plant‐derived Δ12 desaturase gene family BtFAD2 is characterized in B. tabaci and it shows that the BtFAD2‐9 gene enables the pest to synthesize PUFAs, thereby significantly enhancing its fecundity. The role of BtFAD2‐9 in reproduction is further confirmed by transferring the gene to Drosophila melanogaster, which also increases the fruit fly's reproduction. These findings reveal an extraordinary evolutionary scenario whereby a phytophagous insect acquired a family of plant genes that enables it to synthesize essential nutrients, thereby lessening its nutritional dependency and allowing it to feed and reproduce on many host plants.


Introduction
Polyunsaturated fatty acids (PUFAs), which contain multiple double bonds between carbon atoms, are essential nutrients increasingly recognized as important to the survival of all organisms. [1]PUFAs not only represent building blocks of biological membranes but also provide reservoirs of metabolic energy and serve as precursors for highly bioactive molecules such as prostaglandin E 2 (PGE 2 ). [2]In mammals, PUFAs are associated with various physiological processes, including those in cardiovascular and central nervous systems, [3] whereas in insects PU-FAs are mainly involved in pheromone biosynthesis, cuticle formation, and immunity processes. [4]PUFAs also contribute to the synthesis of reproductive tissues and can alter reproductive function and fertility. [5]4b,6] Therefore, the acquisition of PUFAs by organisms is of key importance for their metabolic activities, especially those related to reproduction.
The complex biosynthesis of PUFAs requires the participation of multiple catalytic enzymes, [7] among which Δ12 desaturase is the key enzyme that catalyzes the transformation of monounsaturated fatty acids (MUFAs) into PUFAs.However, the absence of enzymes like Δ12 desaturase in most organisms results in widely divergent PUFA acquisition capacities among species. [8]The current dogma is that photosynthetic plants, heterotrophic protists, and bacteria account for most of the natural PUFA production because they have the enzymatic components necessary for de novo synthesis, unlike higher trophic organisms. [9]Most animals acquire PUFAs through their diet in order to satisfy their requirements for essential fatty acids.However, exceptions have been reported for arthropods, including acarid mites (Acari), copepods (Crustacea), springtails (Collembola), the house cricket Acheta domesticus (Orthoptera), termites (Blattodea), the red flour beetle Tribolium castaneum (Coleoptera), the soldier beetle Chauliognathus lugubris (Coleoptera), a parasitic wasp Nasonia vitripennis (Hymenoptera), and the whitefly Bemisia tabaci (Hemiptera), all being able to biosynthesize PUFAs. [10]Recent research has also revealed genes that encode key enzymes for PUFA synthesis in 80 invertebrate species, including several terrestrial arthropods such as Locusta migratoria (Orthoptera), Sminthurus viridis (Collembola) and B. tabaci (Hemiptera). [11]he whitefly, B. tabaci (Gennadius), is a species complex of at least 30 cryptic species, some of which (e.g., Mediterranean [MED] and Middle East-Asia Minor 1 [MEAM1]) are among the most devastating crop pests worldwide. [12]Whiteflies damage plants by sucking plant phloem sap and transmitting plant viruses. [13]B. tabaci is extremely polyphagous and is known to attack more than 600 plant species and shows exceptional host adaptability. [14]Achieving a sufficient nutrient supply from such a broad range of host plants must confront the whitefly with an exceptional nutritional challenge. [15]The way that the whitefly obtains essential nutrients has been studied to some extent, [16] but their ability to synthesize PUFA's remains poorly understood.
Using a combination of chemical and molecular research tools, as well as insect performance assays, we characterized the horizontally transferred plant BtFAD2 gene family, which encodes Δ12 desaturase-like enzymes in B. tabaci.Of this gene family, the BtFAD2-9 gene is shown to be specifically expressed in the gonads and to play an important role in whitefly reproduction.Our discovery reveals a key molecular mechanism that makes B. tabaci far less dependent on the nutritional quality of their numerous host plants.These findings can be the basis for the development of new strategies to control this exceedingly important pest.

Identification and Characterization of BtFAD2 Gene Family in Whitefly
To investigate the mechanism of nutrient synthesis in whiteflies, we constructed a schematic diagram of a general de novo PUFA biosynthetic pathway based on fatty acid classification and available data in KEGG (Kyoto Encyclopedia of Genes and Genomes) (Figure S1, Supporting Information).Based on a preliminary survey of B. tabaci MED genes related to constructed pathways and on two previous studies, [11,17] we identified the Bt-FAD2 gene family in the B. tabaci MED genome.This gene family consists of 13 genes cloned from B. tabaci by specific PCR primers (Figure S2B and Table S1, Supporting Information).These genes are distributed across four scaffolds of the B. tabaci MED genome and share synteny between the MED and MEAM1 genomes, and some of them form gene clusters and have largely undergone tandem gene duplication during genome evolution (Figure 1A).The observed synteny was also found in the B. tabaci MED chromosome and B. tabaci SSA genome (Figure S2A, Supporting Information). [17,18]Although FAD2 genes in plants usually have only one exon, the BtFAD2 genes exhibit 2-4 exons (Figure 1B).All BtFAD2 proteins carry three conserved histidine box motifs (HXCGH motif, HXXHH motif, and HXXHH motif), which are conserved domains of plant Δ12 desaturase proteins (Figure S2C,D, Supporting Information). [19]BtFAD2 proteins share 20-70% sequence similarity, but uniformly exhibit > 30% similarity compared to the only FAD2 gene in the model plant Arabidopsis thaliana, particularly 68% for BtFAD2-9 (Figure S3A, Supporting Information).

Expression Profiling and Phylogenetic Analysis of BtFAD2 Genes
The expression pattern of BtFAD2 genes was monitored with real-time quantitative PCR (qPCR) for all developmental stages (eggs, 1st-2nd, 3rd, 4th instar nymphs, female and male adults) of B. tabaci MED.The expression was particularly high in adults, especially males, suggesting that these genes play important roles at this stage.Most of the genes were differentially expressed in male and female adults, which implies they have crucial sex-biased functions in B. tabaci (Figure 1B).Importantly, our initial transcriptome data from male and female adults indicated that the expression of BtFAD2-9 was significantly higher than all the other BtFAD2 genes (Figure S3B, Supporting Information).
Our Bayesian phylogenetic analysis showed that all BtFAD2 proteins, as well as FAD2 proteins from other Aleyrodinae insects, Trialeurodes vaporariorum, Aleyrodes proletella, Dialeurodes citri, Aleurocanthus spiniferus and Aleuroclava psidii, clustered together with plant FAD2 proteins, while other functional insect Δ12 desaturases form a separate clade (Figure 1C; Figure S4, Supporting Information).Among all BtFAD2 proteins, BtFAD2-9 clustered with BtFAD2-10 had an ortholog in each Aleyrodinae species included in our analysis and was most closely related to plant FAD2s.The BtFAD2-9 protein was highly similar to other B. tabaci cryptic species (MED_009496 in MED and Bta09295 in MEAM1) but also shared high protein similarity (61%−85%, except for the DcFAD2 partial protein) with other Aleyrodinae FAD2s (Figure S5 and S3A, Supporting Information).These results prompted us to further focus on the role of the BtFAD2-9 gene in B. tabaci.

Horizontal Transfer of BtFAD2-9 From Plants to Whiteflies
A BLAST search against the GenBank database revealed that except for homologs of B. tabaci MEAM1 (XP_018898615.1 in B. tabaci MEAM1), BtFAD2-9 closest homologs were all plant proteins.Genomic analyses were performed to verify whether BtFAD2-9 was inserted into the genome of B. tabaci MED.Results showed that the BtFAD2-9 genomic region located at scaffold 11 was accurately assembled and highly consistent among different B. tabaci cryptic species (Figure 2A).Genomic regions of the BtFAD2-9 gene and their surrounding genes of B. tabaci MED share highly conserved synteny with B. tabaci MEAM1 (Figure 2B).Furthermore, overlapping PCR amplicons of those genomic regions confirmed the assembling accuracy and ensured that BtFAD2-9 is indeed integrated into the B. tabaci MED genome (Figure 2C).Like plant FAD2 proteins, BtFAD2-9 has six transmembrane domains (Figure 2D) and, similar to other Aleyrodinae FAD2 proteins, exhibits three typical histidine clusters (Figures S2C,D, Figures S6A,B, Supporting Information), which is distinct from the other functional insect Δ12 desaturases.Similar to a previous report, [17] our phylogenetic analysis showed that FAD2 genes were present before the split of the Aleyrodinae and, most likely, were not acquired independently by whitefly-species (Figure 2E).Further, BtFAD2 gene duplication events seem to have occurred before the divergence of the B. tabaci cryptic species (≈35.3MYA).Overall, our analyses show  B) Genes are organized according to their phylogenetic analysis constructed by the Bayesian-based phylogenetic analysis based on the optimized WAG + G model at 700 aligned amino acid positions.Gene architectures are shown by a green rectangle (exon) and a black line (intron).The constitutive transcription profiles of BtFAD2 genes in eggs (EG), 1st-and 2nd-instar nymphs (N1-2), 3rd-instar nymphs (N3), 4th-instar nymphs (N4), adults (AD), female adults (FA) and male adults (MA) as determined by qPCR.For each gene, the expression fold changes are color-coded according to the gradient, magenta rectangles indicate significant up-regulation (ratio > 1.5-fold), while yellow rectangles indicate no significant transcription variations.Data are presented as means, n = 3 biologically independent samples.C) Bayesian-based phylogenetic analysis of BtFAD2 with JTT + I + G model at 859 aligned amino acid positions.After midpoint rooting, evolutionary branches were formed by 13 FAD2 proteins, within a group of Aleyrodinae FAD2 proteins.Only the Bayesian posterior probabilities (× 100) at phylogenetically important nodes are shown.BtFAD2-9 is indicated by a red star.
that the BtFAD2-9 gene is not a plant gene contaminant, but that Aleyrodinae ancestors must have horizontally acquired it from a host plant.

Spatio-temporal Expression Profiling of the BtFAD2-9 Gene
To further study the functional role of the BtFAD2-9 gene, its spatio-temporal expression patterns were monitored by qPCR and immunofluorescence.qPCR analysis showed that BtFAD2-9 was expressed in various parts of B. tabaci adults (head, thorax, and abdomen).It is most highly expressed in the abdomen, which suggests that BtFAD2-9 mainly active its functions in this insect body part (Figure 4A).Immunofluorescence showed that the BtFAD2-9 protein is specifically located in the gonads and not in the midgut and salivary glands (Figure 4B).Because of the special structure of the insect's ovariole (Figure 4C), we examined the BtFAD2-9 localization in different developmental stages of oogenesis in B. tabaci. [20]The results showed that BtFAD2-9 is expressed in all different stages of oogenesis, especially in phase I and phase II (Figure 4D).Moreover, the specific localization of BtFAD2-9 in follicular cells suggests that BtFAD2-9 plays an important role in follicular cells during oogenesis (Figure 4D).Together, these results indicate that BtFAD2-9 is functional in the whitefly's gonads and highly expressed in its testes and follicular cells.

Functional Analysis of the BtFAD2-9 Gene Using RNAi and VIGS Assays
To further verify that BtFAD2-9 participates in PUFA biosynthesis (Figure 5A), a series of in vivo RNA interference (RNAi) experiments were performed.Using tailored feeding capsules (Figure S7B, Supporting Information), B. tabaci MED adults were fed on gene-specific dsRNA targeting BtFAD2-9.qPCR analysis confirmed that the transcript and protein levels of BtFAD2-9 were both significantly decreased upon silencing for 96 h, while expression of other 12 BtFAD2 genes did not change sig-nificantly (Figure 5B,C; Figure S7A, Supporting Information).Silencing BtFAD2-9 dramatically reduced the PGE 2 content of whitefly males and females (Figure 5D).As PUFAs are ultimately converted to PGE 2 , these results substantiate the notion that BtFAD2-9 participates in PGE 2 biosynthesis.Subsequent mating assays revealed that silencing of BtFAD2-9 not only remarkably decreased the fecundity of female adults but also affected the mating success of male adults (Figure 5E; Figure S7C, Supporting Information).Also, although silencing of BtFAD2-9 had no effect on egg hatchability, it resulted in a highly skewed sex ratio of the progeny (Figures S7D,E, Supporting Information).Importantly, after exogenous PGE 2 supplementation in the artificial diet, the fecundity and offspring sex ratio of dsBtFAD2-9-fed whiteflies was restored (Figure 5E; Figure S7E, Supporting Information).Furthermore, to examine the effect of other BtFAD2 genes on the fecundity of the whitefly, RNAi was also performed on .Metabolic analyses of BtFAD2-9 enzyme activity.A) BtFAD2-9 was ligated to the pYES2 vector and transformed into yeast for heterologous expression.Yeast metabolites were extracted and analyzed using a GC-MS system.B) Chromatograms of fatty acid methyl esters (FAME) standards (FAME of linolenic acid, 18:3 Δ9, 12, 15 ; linoleic acid, 18:2 Δ9, 12 ; oleic acid, 18:1 Δ9 ; stearic acid, 18:0).C-F) Secondary mass spectrometry chromatograms of FAME standards related to (B).G) Chromatograms of empty vector transgenic yeast metabolites (FAME of oleic acid, 18:1 Δ9 ; stearic acid, 18:0).H-I) Secondary mass spectrometry chromatograms of yeast metabolites related to (G).J) Chromatograms of BtFAD2-9 transgenic yeast metabolites (FAME of linoleic acid, 18:2 Δ9, 12 ; oleic acid, 18:1 Δ9 ; stearic acid, 18:0).K-M) Secondary mass spectrometry chromatograms of yeast metabolites related to (J).N) Fatty acid catalytic processes in BtFAD2-9 transgenic yeast.
another higher-expressed gene, BtFAD2-2 (Figure S7F, Supporting Information).The results showed that silencing BtFAD2-2 has no significant effect on the whitefly's fecundity (Figure S7G, Supporting Information).Subsequently, we tested the function of BtFAD2-9 in an ecologically relevant experiment using a virus-induced gene silencing (VIGS) technique.We constructed VIGS vectors and infiltrated these into tobacco plants (Figure 5F,G).After two weeks, both silencing fragments of the BtFAD2-9 and EGFP genes were detected in the tobacco seedlings by PCR (Figure 5H).Next, BtFAD2-9 expression in B. tabaci adults that had been feeding on the tobacco seedlings for 7 days was assessed by qPCR.Relative to expression in adults feeding on pTRV2-EGFP tobacco plants, BtFAD2-9 expression in adults feeding on pTRV2-BtFAD2-9 tobacco plants was reduced by 62.3% (Figure 5I).Assessment of protein levels confirmed that BtFAD2-9 was reduced in whitefly samples after feeding on specific VIGS tobacco plants (Figure 5J).Continuous silencing of BtFAD2-9 by VIGS for 7 days significantly decreased PGE 2 levels and reduced fecundity of B. tabaci male and female adults (Figure 5K-M).The VIGS experiment also confirmed that BtFAD2-9 gene affects the sex ratio but not egg hatchability (Figures S7H,I, Supporting Information).

Transgenic Expression of BtFAD2-9 in Drosophila
To further confirm the metabolic function of BtFAD2-9, we also ectopically expressed BtFAD2-9 into Drosophila melanogaster, which is a model insect that lacks the capacity for Δ12 desaturation, through the UAS-GAL4 system (Figure 6A).We confirmed the BtFAD2-9 gene was expressed in transgenic D. melanogaster by PCR and Western blot (Figure 6B).Second, to test the effect of the BtFAD2-9 gene on the fecundity of D. melanogaster, we recorded the egg production of transgenic and control flies.The results show that the transfer of the BtFAD2-9 gene enhanced egg production in D. melanogaster (Figure 6C).Moreover, higher levels of PGE 2 were detected in the D. melanogaster expressing BtFAD2-9 line (UAS-GAL4-BtFAD2-9) compared to the control line (W 1118 ) (Figure 6D).Taken together, the transformation with the BtFAD2-9 gene enhanced the ability to synthesize PUFAs thereby allowing D. melanogaster to acquire more PGE 2 and produce more eggs (Figure 6E).

Fertility of Whiteflies
To investigate whether BtFAD2-9 contributes to the exceptional host adaptability of B. tabaci, the gene-specific dsRNA expressed vector (pCAMBIA-RNAi-dsBtFAD2-9) expressing hairpin RNA of BtFAD2-9 was constructed (Figure S8A, Supporting Information) and transferred into tobacco (Figure S8B, Supporting Information).Positive transgenic lines were identified by PCR amplification (Figure 6F).Northern blot analyses confirmed that the positive transgenic lines generated target small interfering RNAs (siRNAs) (Figure 6F).The transcript level of the BtFAD2-9 gene was significantly reduced after whiteflies had fed on such transgenic-BtFAD2-9 tobacco plants (Figure 6G), and assessments of protein levels confirmed that their BtFAD2-9 content was markedly reduced (Figure 6H).Most importantly, feeding on transgenic plants significantly reduced the whiteflies' capacity to reproduce, either parthenogenetically or sexually (Figure 6I,J).In a long-term experiment, transgenic plants significantly altered the whitefly's sex ratio but not egg hatchability (Figures S8C,D, Supporting Information).In addition, immunofluorescence assays revealed a significant reduction in BtFAD2-9 protein levels within the gonads of male and female whitefly adults feeding on the transgenic plants (Figure 6K,L).They also showed a significant reduction in BtFAD2-9 during oogenesis, especially in the first phase (Figure 6M,N; Figure S9A-C, Supporting Information).These results show that BtFAD2-9 is of great importance for whitefly reproduction.

Discussion
PUFAs are indispensable nutrients for all living organisms.It has long been thought that most animals are incapable of de novo biosynthesizing PUFAs and that they can only acquire them through their diet.Unlike plants and certain microorganisms, animals mostly lack Δ12 desaturase, a key enzyme responsible for PUFA biosynthesis by desaturating MUFAs. [8]10a,b,g] The whitefly B. tabaci is also able to produce PUFAs [10c] and has acquired PUFA synthesis genes from plants via an HGT event, [11,17] which was further confirmed in our analysis (Figure S10, Supporting Information).It is increasingly evident that this uncommon evolutionary route has occurred quite frequently in whiteflies. [14,21]Our phylogenetic analysis revealed that the BtFAD2 gene family and other Aleyrodinae FAD2 genes are distantly related to other functional insect Δ12 desaturase genes (Figure 1C).The results further show that all the examined Aleyrodinae species have acquired plant-derived FAD2 genes, strongly indicating that they might be present in all Aleyrodinae species (Figure 1C) and that the transfer of FAD2 most likely occurred in the whitefly ancestor before the divergence of B. tabaci and T. vaporariorum (> 86 MYA) (Figure 2E).FAD2 is a significant gene family in numerous plants and is expressed at all developmental stages.It participates in processes such as stress resistance and seed germination. [19]The evolution and diversification of the FAD2 gene family in plants is species-specific, and the vast expansion is the result of gene duplication events. [22]However, functionally redundant genes generated by duplication cannot be stably retained in the genome unless they undergo functional evolution, such as pseudogenization, neo-, sub-functionalization, or both. [23]Indeed, a number of plant FAD2 genes are known to exhibit neofunctionalization complementary to the conserved function of Δ12 desaturases, for example, hydroxylation, conjugation, and acetylation. [24]In our study, the BtFAD2 gene family includes 13 transcribed genes that might have been generated via several tandem-repeated gene duplications during whitefly genome evolution (Figure 1A; Figure S2A, Supporting Information), and thereby possibly underwent neofunctionalization accompanied by subfunctionalization, which is similar to the case of plant FAD2.Additional phylogenetic analysis revealed that BtFAD2-9, and its Aleyrodinae orthologs, were most closely related to plant FAD2s.This suggests that the Aleyrodinae FAD2-9 gene might be the ancestral FAD2 gene from plants and, based on the presence of BtFAD2-2 orthologs in several Aleyrodinae species, was subsequently duplicated in the whitefly ancestor.However, further analyses are needed to fully elucidate the complex evolutionary history of BtFAD2-9 and BtFAD2-2.Nevertheless, the available data does suggest that multiple gene duplication events occurred before the divergence of the B. tabaci cryptic species (≈ 35.3 MYA). [17,25]Intriguingly, a previous study indicated that the decrease in gene expression after duplication can be beneficial by rebalancing gene dosage. [23]We therefore speculate that a similar transcriptional regulatory mechanism might modulate the differential expression of these tandem-repeated BtFAD2 genes to optimize their functions, which warrants further study.
10c] Our results further show that si-lencing the BtFAD2-9 gene reduced the level of PGE 2 , the major prostaglandin involved in various undesirable metabolic anomalies. [26]PGE 2 is synthesized from PUFAs as substrate and is known to be involved in mammalian fertility, regulating oviduct ciliogenesis, contractility, and other reproductive processes. [27]We found that silencing of the BtFAD2-9 gene in the whitefly reduced fecundity (Figure 5), while the transfer of BtFAD2-9 into D. melanogaster also enhanced its egg production (Figure 6C).In both cases, fecundity was positively correlated with the insects' PGE 2 levels .Indeed, BtFAD2-9 silencing in whiteflies could be rescued by supplementing their diet with PGE 2 (Figure 5E), and therefore, akin to their role in mammals, the BtFAD2-9 gene most likely affects whitefly fecundity by contributing to the production of prostaglandins.Indeed, levels of the BtFAD2-9 protein are particularly high in the follicular cells of the female ovariole, which is an important composition for oogenesis. [28]Follicular cells provide protection to the oocyte, are involved in material transportation during oogenesis, and serve as precursor tissue for eggshell development in insects. [29]The BtFAD2-9 protein was also found to be highly concentrated in the testis of whitefly males, where sperm development takes place, [30] implying that BtFAD2-9 is also involved in the reproductive system of whitefly males.In insects, males deliver prostaglandins to females through mating, which facilitates egg fertilization and promotes egg-laying behavior. [31]However, our experiments showed that silencing of the BtFAD2-9 gene also reduces the fecundity of whitefly females that reproduce asexually through parthenogenesis.This implies that asexually reproducing whitefly females, also produce prostaglandins themselves with the use of FAD2, without having to rely on their diet or males.Altogether, it is clear from our study that BtFAD2-9 is involved in the synthesis of PGE 2 in gonad cells to promote the sexual reproductive process of whiteflies, but further studies are needed to unravel its precise role in parthenogenesis.
Host plant nutrient quality is a key determinant of the fecundity of herbivorous insects. [32]Our findings suggest that B. tabaci can reduce this host plant dependency thanks to the horizontally transferred FAD2 gene (Figure 7).This echoes the Chinese proverb, "give a man a fish and you feed him for a day; teach a man to fish and you feed him for a lifetime".In this context, it is increasingly evident that HGT is an impetus for biological evolution and genetic innovation, and has provided recipient organisms with highly efficient control over biological processes. [33]For example, several horizontally transferred essential amino acid biosynthesis-related genes have been identified in B. tabaci, [34] whereas the whitefly uses the horizontally transferred genes BioA, BioB, and BioD to compensate for the lack of biotin synthesis, again reducing their dependency on endosymbionts. [35]Whiteflies, as piercing-sucking pests, feed mainly on the phloem sap of plants, which usually contains only trace amounts of fatty acids. [36]Hence, the horizontally transferred FAD2 gene compensates for B. tabaci's limited access to fatty acid nutrients and might have a similar function in all Aleyrodinae species.
Silencing of the BtFAD2-9 gene results in a significant decrease in the fecundity of B. tabaci (Figure 5E), which greatly reduces insect performance and could be exploited for crop protection.Hence, this study not only provides insight into a co-evolutionary process that facilitates nutrient acquisition in insects but also reveals that interfering with laterally transferred genes could be a highly effective way to combat pests. [14,37]Targeting the reproductive process of insects is an important aspect of pest control and currently is applied in various pest management strategies. [38]One of the most popular methods of inhibiting pest fecundity is via Wolbachia, an endosymbiotic bacterium that occurs in a broad range of invertebrates.Wolbachia reduces the fecundity of pests by inhibiting mating success. [39]However, this method is less effective against pests like whiteflies that can reproduce through parthenogenesis. [40]We show that silencing BtFAD2-9 significantly reduces the parthenogenetic fertility of the whitefly, thus providing a mating-independent strategy for pest control.A thorough screening for other such fertility-related genes might yield excellent targets for the promising RNAi-based insect pest control strategy.
To summarize, this study illustrates that important physiological traits do not necessarily originate from the evolution of endogenous pre-existing genes, but can be acquired by exogenous HGT events, highlighting an alternative pathway of evolution.The ability of de novo PUFA biosynthesis has important ecological consequences because it implies that whiteflies are released from a dependency on dietary PUFAs.PUFAs serve important functions in all organisms, such as energy storage, mobilization, and transport, as well as structural components in membranes.They also have a number of functions that are apparently more-or-less unique to insects and have the potential to become emerging areas of interest in insect biochemistry and physiology.Indeed, there is growing evidence that integrative studies on insect PUFAs can reveal important principles of animal metabolism, including mechanisms of PUFA biosynthesis, and causes of metabolic diseases like obesity and cancer. [41]

Experimental Section
Insect Strain: A cotton strain of B. tabaci MED was created from individuals initially collected from poinsettia (Euphorbia pulcherrima Wild.ex Klotz.)plants in Beijing, China in 2009, that were then transferred to cotton (Gossypium herbaceum L. cv.DP99B) plants.A tobacco strain of B. tabaci MED was created in 2017 from the above parental cotton strain, by rearing it continuously on tobacco (Nicotiana tabacum K326). [42]The purity of the B. tabaci MED strain was monitored by sequencing a fragment of the mitochondrial cytochrome oxidase I (mtCOI) gene every three to five generations. [43]All the experiments in this study were conducted using the tobacco strain, which was maintained in a glasshouse at 27 ± 1 °C, 60%-80% relative humidity (RH), and a photoperiod of 14 h light/10 h darkness.
RNA Isolation and cDNA Synthesis: Total RNAs were extracted from various whitefly samples using the TRIzol reagent (TaKaRa) according to the manufacturer's recommendations.Agarose gel electrophoresis was used to determine the integrity of the RNA, and NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific) detection was used to quantify the RNA.cDNAs were synthesized using the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa) and the PrimeScript RT Kit (containing gDNA Eraser, Perfect Real Time) (TaKaRa) for BtFAD2 gene cloning and qPCR analysis, respectively.The synthesized cDNAs were immediately stored at −20 °C until used.
Gene Identification and Cloning: The BtFAD2 genes were originally found in our previously sequenced B. tabaci MED genome (https://www.gigadb.org/dataset/100286)and transcriptome libraries, [44] and they were re-evaluated by BLASTp against the GenBank database (https://www.ncbi.nlm.nih.gov/).The putative coding sequences (CDSs) of these BtFAD2 genes were manually corrected using the previously completed transcriptome data of B. tabaci MED. [45]Specific primers used for gene cloning (Table S1, Supporting Information) were designed using Primer Premier 5.0 (https://www.premierbiosoft.com/primerdesign/).The PCR reactions were conducted using LA Taq polymerase with high GC buffer (TaKaRa).The detailed programs of the PCR analyses are listed below: denaturing at 94 °C for 10 min; cycling 35 times with the following parameters: denaturing at 94 °C for 60 s, annealing at 60 °C for 60 s, and extension at 72 °C for 2 min; final extension at 72 °C for 10 min.The obtained amplicons of BtFAD2 were purified, cloned into the pEASY-T1 vector (TransGen), and sequenced, and the finally obtained full-length cDNA sequences of all the MED BtFAD2 genes have been deposited in the GenBank database (accession nos.OQ291260-OQ291272).
Phylogenetic Analysis: For phylogenetic tree construction, the protein sequences of BtFAD2 genes were used as queries in a BLASTp (with "Expect threshold" set at 1E-15) search against the NCBI-non-redundant protein database to identify homologs.For the top 30 hits in each BLASTp result, we downloaded the complete protein sequence of the representative hits.De novo transcriptomes were assembled for three Aleyrodinae species -Aleyrodes proletella, Dialeurodes citri, and Aleurocanthus spiniferus while a de novo genome was generated for the Aleyrodinae whitefly (Table S1, Supporting Information).The 25 μL PCR reactions included 0.5 μL of 50 × ROX Reference Dye (TIANGEN), 0.75 μL of each specific primer, 1 μL of cDNA template, 9.5 μL of ddH 2 O, and 12.5 μL of 2 × Su-perReal PreMix Plus (SYBR Green) (TIANGEN).The qPCR reactions were performed in an ABI 7500 system (Applied Biosystems) with the following protocol: initial denaturation of 94 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s.The amplification efficiencies were determined by dissociation curve analysis using five two-fold serial dilutions of B. tabaci cDNA template.Only primers with 90%-110% amplification efficiencies were used for the subsequent studies.
Relative quantification was calculated according to the 2 −ΔΔCt method, [47] to accurately analyze the expression of the target genes, the expression data were normalized to the internal gene elongation factor 1 alpha (EF1-a) (GenBank accession number EE600682).Three independent biological replicates and four technical replicates were performed for each whitefly sample.
Yeast Transformation and Enzyme Activity Assays: The function of BtFAD2-9 was in vitro characterized in the yeast Saccharomyces cerevisiae system.Briefly, the predicted open reading frame (ORF) of BtFAD2-9 gene was amplified by PCR using primers containing restriction enzyme sites (HindIII and EcoRI) for further cloning into the yeast expression vector pYES2 (Invitrogen) (Table S1, Supporting Information).The pYES2 construct containing BtFAD2-9 ORF was sequenced prior to being used to transform the InvSc1 yeast line (Invitrogen).Yeast transformed with the pYES2 vector was cultured overnight in 2% raffinose, 1% Nonidet P-40, and SC-U medium (uracil dropout medium) at 30 °C with shaking.The cultures were then grown to an OD 600 of 1 and 2% galactose (wt/vol) was added to induce transgene expression.Transgenic yeast expressing BtFAD2-9 were grown in the presence of exogenously added oleic acid substrates (25 mmol L −1 ).Control treatments consisted of yeast transformed with the empty pYES2 and run under the exact same conditions as above.After galactose induction for 48 h, equal amounts of yeast cultures were collected by centrifugation and dried under a stream of oxygen-free nitrogen.To prepare yeast fatty acid methyl ester (FAME), the dried yeast cells were incubated with 2 mL 0.4 mol L −1 potassium hydroxide/methanol solution for 30 min at 37 °C with vortex shaking.Subsequently, 1 mL of 0.9% NaCl and 1 mL of hexane were added for 10 min at 37 °C with vortex shaking.Ultimately, the top phase was collected after phase separation for subsequent assays.Metabolic functions of the BtFAD2-9 were established by comparing the fatty acid profiles of BtFAD2-9 transformed yeast with those of the controls.
GC-MS Analysis: The fatty acid composition was analyzed by the coupled gas chromatography-mass spectrometry (GC-MS).The samples were dried and then dissolved in n-hexane: toluene (1:1) prior to analysis on an Agilent GC-MS instrument (7890B-5977A, Agilent) with an HP-5 MS columns (30 m × 0.25 mm inner diameter, film thickness, 0.25 μm, Agilent) and helium as the carrier gas.GC was performed with temperatureprogrammed automatic injection at 60 °C, holding for 5 min at 60 °C, temperature increase to 230 °C at a rate of 2 °C min −1 , and holding for 40 min at 230 °C.The identity of the desaturation products was determined by comparing their retention times with FAME contained in commercial standards (Sigma-Aldrich, 18919-1AMP).
Western Blots: The antibody of BtFAD2-9 protein used for Western blots was generated from synthetic peptides (Pujian Biotech) derived from respective specific amino acid sequences 316 HHLFPTMPHYHAVEAC 330 , and other specific antibodies targeting -actin and -tubulin were commercially purchased (Abcam, ab115777 and ab18207).The protein level of target proteins was determined with Western blots using -actin or -tubulin as internal controls.The protein samples (ca 30 μg protein extracted from whitefly and Drosophila mixed adult simple, respectively) were isolated using 10% SDS-PAGE and transferred onto PVDF membranes (Merck Millipore).The PVDF membranes were then blocked with blocking buffer containing BSA (CWBIO) at 25 °C for 1 h and incubated with the appropriate primary antibody (1:5000) at 4 °C overnight, followed by incubation with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5000, CWBIO).The protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific), and the images were captured by the Tanon-5200 To detect the effect of heterologous expression of the BtFAD2-9 gene on D. melanogaster fecundity, a 5-day-old male and a virgin female were placed in a test tube for mating.The mated females were kept in food vials (1.06% agar, 3.22% yeast extract, 3.16% brown sugar, 6.32% glucose 7.74% cornmeal, and 1% nipagin), whereby fresh vials were provided every 3 days.The total number of adult progeny was counted in each vial.Fifteen independent assays were performed for each D. melanogaster sample.Finally, the concentrations of PGE 2 in different D. melanogaster lines were measured as above.
Transgenic Tobacco Plants: Transgenic tobacco lines were developed by introducing the hairpin RNA expression vector (pCAMBIA-RNAi-BtFAD2-9) into tobacco (N.tabacum K326).The construction of a hairpin RNA expression vector (Figure S8A, Supporting Information) has been described previously. [48]A 537-bp target fragment of BtFAD2-9 was cloned from B. tabaci MED using sense-BtFAD2-9 primers (Table S1, Supporting Information), and the PCR product was then cloned into XhoI-BglII-cut pCAMBIA-RNAi (pRNAi-Sense-BtFAD2-9).The anti-sense fragment of BtFAD2-9 was cloned from B. tabaci MED using anti-sense-BtFAD2-9 primers (Table S1, Supporting Information), the purified product was then cloned into BamHI-SalI-cut pCAMBIA-RNAi-Sense-BtFAD2-9 (pRNAi-BtBtFAD2-9).A. tumefaciens LBA4404 based transformation was used to transfer the recombinant pCAMBIA-RNAi-BtFAD2-9 plasmid into tobacco in a similar way as described previously for tomato. [14]To verify the success of the transformation, gDNAs of putative transgenic tobacco leaves were extracted using the Plant Genomic DNA Kit (TIANGEN), and the extracted DNAs were subjected to PCR using detection primers (Table S1, Supporting Information).qPCR analyses were carried out to assess the RNAi efficacy on B. tabaci adults feeding on dsBtFAD2-9 transgenic tobacco lines, B. tabaci adults feeding on the dsEGFP transgenic tobacco lines were used as a control.RNAi efficacy was determined every two days for seven days.
For determining the effects of BtFAD2-9 on B. tabaci reproduction, 5 pairs of newly emerged adults or five newly emerged female adults of B. tabaci MED were collected into one clip cage and fixed on the dsEGFP transgenic tobacco or dsBtFAD2-9 transgenic tobacco plants.The newly laid whitefly eggs were recorded after 7 days.The offspring of these mating groups were reared to 35 days, to determine egg hatchability and adult sex ratio.Trans-EGFP tobacco plants were used as controls.
Northern blot: Northern blot analyses were performed to confirm the presence of the generated siRNAs in transgenic tobacco lines.The total RNAs of transgenic tobacco leaves were isolated and purified by TRIzol reagent (TaKaRa).Small RNAs were selectively recovered with 5% PEG8000 and 0.5 m NaCl from the purified total RNAs.The obtained small RNAs were then separated on denaturing 15% polyacrylamide gels and transferred onto Hybond-N + membranes (Amersham), and the membranes were further cross-linked by exposure to UV light and hybridized to specific biotin-labeled DNA probes that were generated by the PCR products labeled with Biotin-dUTP (Beyotime).The results were visualized using the Chemiluminescent Biotin-labeled Detection Kit (Beyotime), and the images were captured by the Tanon-5200 Chemiluminescent Imaging System (Tanon).

Figure 1 .
Figure 1.Genome-wide characterization of the BtFAD2 gene family in B. tabaci.A) Synteny analysis of 13 FAD2 genes among B. tabaci MED and MEAM1.B) Genes are organized according to their phylogenetic analysis constructed by the Bayesian-based phylogenetic analysis based on the optimized WAG + G model at 700 aligned amino acid positions.Gene architectures are shown by a green rectangle (exon) and a black line (intron).The constitutive transcription profiles of BtFAD2 genes in eggs (EG), 1st-and 2nd-instar nymphs (N1-2), 3rd-instar nymphs (N3), 4th-instar nymphs (N4), adults (AD), female adults (FA) and male adults (MA) as determined by qPCR.For each gene, the expression fold changes are color-coded according to the gradient, magenta rectangles indicate significant up-regulation (ratio > 1.5-fold), while yellow rectangles indicate no significant transcription variations.Data are presented as means, n = 3 biologically independent samples.C) Bayesian-based phylogenetic analysis of BtFAD2 with JTT + I + G model at 859 aligned amino acid positions.After midpoint rooting, evolutionary branches were formed by 13 FAD2 proteins, within a group of Aleyrodinae FAD2 proteins.Only the Bayesian posterior probabilities (× 100) at phylogenetically important nodes are shown.BtFAD2-9 is indicated by a red star.

Figure 2 .
Figure 2. Horizontal transfer of BtFAD2-9 into B. tabaci.A) Genomic location of BtFAD2-9 gene in B. tabaci MED.Illumina DNA-read coverage plots resulting from genomic sequencing of different B. tabaci cryptic species and Illumina RNA-seq read coverage plots from diverse B. tabaci cryptic species adults are displayed.The sequence depths are denoted by the numbers on the right of the coverage plots.B) Genome synteny of the BtFAD2-9 gene and their respective two neighboring insect genes in B. tabaci MED (MED_009495 and MED_009497) and MEAM1 (Bta09294 and Bta09296).The black diagonal line indicates more than 95% similarity of two genomic regions.For BtFAD2-9, red rectangles mean exons, red lines mean introns, and green rectangle means untranslated region.For two neighboring insect genes, orange and blue rectangles mean exons, orange and blue lines mean introns.C) Genome fragments cloned by overlapping PCR from B. tabaci MED.Genome fragment of BtFAD2-9 (MED_009496) with its upstream gene (MED_009495) and downstream gene (MED_009497).D) The structure of BtFAD2-9 protein with five transmembrane domains generated by Protter.The N-glycosylation site and the three histidine clusters are labeled in grey, blue, yellow, and green respectively.E) Diagram of the evolutionary history of FAD2 in Aleyrodinae insects.The event (86 MYA) when Bemisia divided from Trialeurodes and the event (35.3 MYA) when B. tabaci divided into different cryptic species are indicated.Abbreviation: Asia II 3: B. tabaci Asia II 3; New World: B. tabaci New World; MED: B. tabaci MED; MEAM1: B. tabaci MEAM1.

FCFigure 4 .
Figure 4. Localization of BtFAD2-9 in different tissues of whitefly.A) Relative expression levels of BtFAD2-9 gene in the head, thorax, and abdomen of adult whitefly.The model below shows the structure and main organs of the whitefly.B) Immunofluorescence (IF) localization of the BtFAD2-9 protein in different tissues of the whitefly using the rabbit polyclonal anti-BtFAD2-9 antibody.Nuclei are shown in blue, red is the positive signal for anti-BtFAD2-9.C) Structure of the ovariole of the whitefly.D) Localization of BtFAD2-9 protein in follicular cells and oocytes of ovarioles at different developmental phases.Nuclei are stained with DAPI (blue), red is the positive signal for anti-BtFAD2-9.Abbreviations are as follows: T, testis; SV, seminal vesicle; AG, accessory gland; TF, terminal filament; NC, nurse cell; IO, immature oocyte; FC, follicle cell; MO, mature oocyte; BC, bacteriocyte.Data are presented as means ± SEM (A), n = 3 (A) biologically independent samples, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey's test was used in (A) for comparison.

Figure 5 .
Figure5.Effect of BtFAD2-9 silencing on B. tabaci performance.A) A proposed pathway from oleic acid to PGE 2 and a role for BtFAD2-9 gene.Shown are proposed chemical structures based on GC-MS data and click chemistry.B) The transcript levels of BtFAD2-9 at 48 and 96 h post-RNAi as determined by qPCR.C) The relative expression levels of BtFAD2-9 proteins at 96 h post-RNAi.Both the detection of BtFAD2-9 protein levels by Western blots (upper row) and quantitative estimation of band intensity by densitometry (graph) are presented.D) PGE2 concentration in B. tabaci adult male and female at 96 h post-RNAi.E) Fecundity of B. tabaci in different mating groups.Whitefly adults were used in four treatments of mating: dsBtFAD2-9 ♂ × control ♀; dsBtFAD2-9 ♂ ×dsBtFAD2-9 ♀; control ♂ ×dsBtFAD2-9 ♀; dsBtFAD2-9 ♀ for parthenogenesis.dsBtFAD2-9 and dsBtFAD2-9 plus PGE 2 were set for each mating group.F) The constructed TRV-based VIGS vectors.G) Procedure for persistent gene silencing of B. tabaci using TRV-based vectors.H) PCR products amplified using cDNA from pTRV2-EGFP (up) and pTRV2-BtFAD2-9 (down) tobacco leaves.M, marker (from top to bottom: 1,200 bp, 900 bp, 700 bp, 500 bp, 300 bp, 100 bp); lanes 1-10, PCR products (Up, 435 bp dsEGFP fragment; Down, 537 bp dsBtFAD2-9 fragment).I) The transcript levels of BtFAD2-9 in B. tabaci adults feeding on VBtFAD2-9 tobacco for 3, 5, and 7 days as determined by qPCR.J) The relative expression levels of BtFAD2-9 proteins from B. tabaci adults feeding on VBtFAD2-9 tobacco for 7 days.Both the detection of BtFAD2-9 protein levels by Western blots (upper row) and quantitative estimation of band intensity by densitometry (graph) are presented.K) PGE2 concentration in B. tabaci adult male and female feeding on VBtFAD2-9 tobacco for 7 days.L,M) Mating (L) and parthenogenesis (M) fecundity of B. tabaci feeding on VBtFAD2-9 tobacco and VEGFP tobacco for 7 days.Values are means ± SEM, n = 3 (B, C, I and J), n = 6 (D and K), and n = 10 (E, L, M) biologically independent samples, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey's test was used for comparison.

Figure 7 .
Figure 7. Schematic overview of how the acquisition of the plant gene BtFAD2-9 empowers the whitefly B. tabaci to enhance its fecundity.In B. tabaci, BtFAD2-9 gene is highly expressed in the gonads.BtFAD2-9 can use oleic acid as a substrate to catalyze the synthesis of linoleic acid, which is eventually used to synthesize prostaglandins E 2 for reproduction.With this ability, B. tabaci adults can synthesize PUFA by themselves, enhancing their reproductive output, which may have contributed to their adaptability to a large range of host plants.