Structural and functional insights into the Diabrotica virgifera virgifera ATP-binding cassette transporter gene family

doi:10.21203/rs.2.9611/v2

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Structural and functional insights into the Diabrotica virgifera virgifera ATP-binding cassette transporter gene family

https://doi.org/10.21203/rs.2.9611/v2

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published 27 Nov, 2019

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Background

The western corn rootworm, Diabrotica virgifera virgifera , is a pervasive pest of cultivated maize in North America and Europe, which has adapted to survive exposure to multiple insecticidal agents. Due to their role in insecticide transport, we sought to identify members of the ATP-binding cassette (ABC) transporter family in D. v. virgifera using a transcriptomics approach.Results

In this study, 65 putative D. v. virgifera ABC ( Dvv ABC) transporters were identified within a combined transcriptome assembly generated from embryonic, larval, adult male, and adult female RNA-sequence libraries. Phylogenetic analysis placed the deduced amino-acid sequences of the Dvv ABC transporters into eight subfamilies (A to H). Of these, eight shared structural and functional conservation with Tribolium castaneum ABC transporter orthologs known to exhibit overt RNA interference (RNAi) knockdown phenotypes. Interestingly, depletion of DvvABCB_19147 and DvvABCG_3712 transcripts in adult females produced detrimental reproductive and developmental phenotypes (egg-laying or -hatching defects), demonstrating the potential of these genes as targets for RNAi-mediated insect control tactics.Conclusions

By combining sequence data from four libraries covering three distinct life stages, we have produced a relatively comprehensive de novo transcriptome assembly for D. v. virgifera . Moreover, we have identified 65 members of the ABC transporter family, and provided the first insights into the developmental and physiological roles of ABC transporters in this pest species.

Epigenetics & Genomics

ATP-binding cassette (ABC) transporter

phylogenetic

transcriptome

RNA interference (RNAi)

corn rootworm

The western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is a major pest of maize in Europe and North America [1-3], where costs of management, as well as crop losses attributed to damage by this pest, are estimated at over 1 billion U.S. dollars annually in North America alone (reviewed in [4]). The notorious difficulty facing efforts to control D. v. virgifera feeding on maize has arisen via intra-species adaptations that overcome various pest management methods [5]. For example, changes in oviposition preference within “soybean variant” populations of D. v. virgifera in the Midwest United States circumvent the cultural-control practice of corn-soybean rotation [3, 5-7]. Additionally, adapted phenotypes within North American D. v. virgifera populations can survive high exposures to organochlorine [8], pyrethroid [9], and carbamate and organophosphate insecticides [10]. In some instances resistant phenotypes have persisted for decades despite the removal of selection pressures [11]. More recently, field populations of D. v. virgifera have developed high levels of resistance to transgenic maize hybrids that express Bacillus thuringiensis (Bt) crystal toxins Cry3Bb1 [12], mCry3A [13], Cry3.1Ab [14], and Cry34/35Ab1 [14-17]. However, RNA interference (RNAi) shows great potential as a novel insect pest control technology [18], especially in instances where target species are sensitive to oral RNAi [19]. D. v. virgifera is highly sensitive to oral RNAi [20-22], suggesting that it could suppress feeding damage caused by this pest [23].

ATP-binding cassette (ABC) transporters comprise one of the largest gene families, are ubiquitous across prokaryotic and eukaryotic organisms [24], and carry out a myriad of transmembrane transport functions in arthropods [25]. ABC transporter proteins have a two-domain structure: a highly conserved nucleotide-binding domain (NBD) and a variable transmembrane domain (TMD) [26]. The NBD binds and hydrolyzes ATP to provide the energy required for translocating a substrate across cell membranes, while the TMD forms a channel through which the substrate is transported [27]. Each NBD possesses several highly conserved, characteristic motifs, including Walker A, Walker B, Q-loop, D-loop, H-loop, and ABC signature motifs, while each TMD is made up of five to six transmembrane α-helices that dictate substrate specificities [27]. ABC transporter proteins require two NBDs and two TMDs for functionality. Some ABC transporters are full-transporters (FT) in that two TMDs and two NBDs are encoded in a single protein, whereas most are half-transporters (HT; one TMD and one NBD) and form functional units following homodimerization or heterodimerization [24, 27, 28]. Due to the relatively conserved sequence of the NBD, this motif has been used for the phylogenetic classification of the ABC transporter superfamily into eight subfamilies designated A to H (ABCA to ABCH) [29].

Among insect species, ABC transporters are implicated in diverse functions, including transportation of eye pigments [30-34], and resistance to chemical insecticides [35, 36]. Interestingly, based on transcriptomic data, Bretschneider et al. [37] suggested that ABC transporters are involved in the degradation of plant secondary metabolites in the cotton bollworm, Helicoverpa armigera. Within the model species for Coleoptera, the red flour beetle, Tribolium castaneum, Broehan et al. [30] reported that RNAi-mediated knockdown of some ABC transporters resulted in mortality, or phenotypes characterized by arrested growth, abnormal cuticle formation, defective eye pigmentation, or abnormal egg-laying or -hatching. Changes in the expression level or structure of some ABCA, ABCC and ABCG subfamily members have been associated with Bt toxin resistance in species of Lepidoptera [38], while paralogs of an ABCB transporter were linked to Cry3Aa resistance in the coleopteran species, Chrysomela tremula [39], and were found to be in proximity to a quantitative trait locus (QTL) for Cry3Bb1 resistance in D. v. virgifera [40].

Similar investigations of ABC transporters in D. v. virgifera are arguably limited due to the dearth of genomic resources available for this species, which are currently comprised of Sanger and Roche 454 read-based transcriptome assemblies [41-44]. Complicating the development of genomic tools is the 2.58 GB size and complex repetitive structure of the D. v. virgifera genome [4, 45]. Regardless, RNA sequencing (RNA-seq) has become an expeditious and cost-effective method for obtaining a wealth of transcriptome sequence data in non-model insects [46]. In the following, a de novo transcriptome assembly approach was used for the first prediction, annotation, and functional analysis of the ABC transporter gene family in D. v. virigifera. Specifically, eight ABC transporters were identified as putative orthologs to those previously reported to have a defining RNAi phenotype in the model coleopteran species, T. castaneum [30] (DvvABCA_18330, DvvABCB_19147, DvvABCE_2830, DvvABCF_2701, DvvABCG_3712, DvvABCG_49457, DvvABCG_13829 and DvvABCH_18290). Subsequent RNAi-mediated knockdown demonstrated conservation of function with T. castaneum, as well as established potential new insecticidal targets for the control of this devastating agricultural pest.

Transcriptome sequencing, assembly, and annotation

Over 22 million raw Illumina (MiSeq) sequencing reads were generated across four libraries (Table 1; NCBI SRA database accession SRP161473: experiments SRX4669438 to SRX4669441). DNASTAR assembled 13,070,671 reads into a combined transcriptome containing 25,296 contigs with an N50 of 1,604 bp (Additional File 1: Table S1). Analogously, assemblies from Trinity and SOAPdenovo-trans respectively produced 162,897 and 133,180 contigs, each with an N50 ≤ 439 bp (Additional File 1: Table S1). Clustering by CD-HIT-EST reduced complexity 3.5 to 34.9% across assemblies, and the number of predicted open reading frames (ORFs) within clustered transcripts ranged from 18,305 to 40,087 (Additional File 1: Table S1). BLASTx query of transcripts by Blast2GO against the arthropod-specific section of NCBI’s non-redundant (nr) protein database generated annotations for 18,343 DNASTAR contigs (E-value cutoff of 10^-6), with a subset of these receiving gene ontology (GO) mapping and additional annotation terms (Additional File 2: Figure S1). Sequences lacking identity to known arthropod proteins above E-value thresholds were attributed to poor sequence conservation and/or novel sequences, as well as non-coding RNAs. The distribution of top BLASTx hits by species showed that T. castaneum was the most frequent, representing 65% of the matches (Additional File 3: Figure S2). Among ontologies assigned via mapping at GO level 2, a majority of the associated terms were assigned to cell structural component, metabolic process, and catalytic activity respectively for GO Cellular Component (C), Biological Process (P) and Molecular Function (M) (Additional File 4: Figure S3). The SeqMan NGen assembly showed a high degree of completeness based on a BUSCO score of 928, or 89.6%, of the 1,066 genes in the arthropod reference set (v. 9.0) being represented, with analogous levels of representation in both SOAPdenovo-trans and Trinity assemblies (Additional File 1: Table S1).

Bioinformatic analysis of the D. v. virgifera ABC transporter family

Results of BLASTx queries identified 65 putative D. v. virgifera ABC transcripts that shared ≥ 37% amino-acid identity with putative T. castaneum orthologs from ABC transporter subfamilies A through H (Table 2; Additional File 5: Table S2). Predictions of protein structural domains identified both FTs and HTs. Four DvvABCA and 32 DvvABCC subfamily members were predicted for D. v. virgifera, all of which are FTs. The DvvABCB subfamily contained seven members, which included both full- and half-transporters. The number of assembled D. v. virgifera paralogs within subfamilies ABCD, ABCE and ABCF contained a smaller number compared to DvvABCB, but each had predicted orthologous relationships to T. castaneum ABC transporters. Specifically, the DvvABCD subfamily contained two predicted ABC transporter proteins which were both HTs. One DvvABCE and three DvvABCF members were identified, and each of these had two predicted NBD motifs with no TMDs, suggesting that, like their counterparts in other species, they probably do not function as transmembrane transporters. The DvvABCG subfamily contained the second largest number of predicted members with 12, all of which were HTs with only a single NBD and a reverse domain organization. The DvvABCH subfamily contained four members, which were similar to those of the ABCG subfamily in being HTs with a reverse domain organization.

The phylogenetic relationships predicted among NBD regions of deduced D. v. virgifera ABC transporter protein sequences formed distinct clades corresponding to the eight known ABC transporter subfamilies (A to H) (node support ≥ 960 of 1,000 bootstrap pseudoreplications; Fig. 1). In addition, a rooted tree constructed from the DvvABC member sequences used in RNAi knockdown experiments confirmed that their corresponding T. castaneum orthologs (Fig. 2).

Gene expression across developmental stages

Since prior research in T. castaneum revealed that only 10 ABC transporters had obvious phenotypic consequences following RNAi-mediated knockdown [30], our study focused on functional analysis of their predicted D. v. virgifera orthologs. From this list, our initial predictions from the D. v. virgifera transcriptome (DNASTAR assembly) identified eight orthologs (Table 3). Differences resided in that T. castaneum has two closely related ABCA genes (TcABCA-9A and TcABCA-9B), for which only one ortholog, DvvABCA_18330, was found in the D. v. virgifera transcriptome. Analogously, while the ABCG genes TcABCG-9A and TcABCG-9B represent two separate, but related eye-color genes in T. castaneum (scarlet and white, respectively), results of BLASTx searches of the DNASTAR assembly resulted only in the identification of an ortholog of white. Semi-quantitative PCR of these eight D. v. virgifera ABC transcripts showed that all are expressed across all of the developmental stages examined (Fig. 3A).

RNAi knockdown phenotypes

Different growth stages of D. v. virgifera were microinjected with dsRNAs (Table 3), after which the level of each corresponding transcript was below or nearly below semi-quantitative PCR detection limits. Specifically, the level of each targeted D. v. virgifera transcript was reduced at 5-days post-injection as compared to buffer-injected controls (Fig. 3B). Moreover, injection of each of the eight dsRNAs resulted in defined phenotypes among dsRNA treated cohorts (Table 3; Fig. 4). The knockdown of DvvABCA_18330 led to approximately 60% mortality among treated pre-pupae, compared to 5% for the buffer-treated control group. In addition, the adults that survived pre-pupal injection and successfully eclosed had defects in their wings and elytra (Fig. 4A), while no phenotypic effects were observed among buffer-injected controls. Injection of DvvABCB_19147 dsRNA into larvae resulted in 100% mortality, and injections into pre-pupae led to defects in their development, which caused individuals to be unable to complete the pupal-adult molt and ultimately resulted in 100% mortality (Fig. 4B). Knockdown of DvvABCB_19147 in newly-eclosed adult females D. v. virgifera resulted in significant reduction in egg laying compared to untreated females (Fig. 5A). Upon further investigation, we discovered that injection of this dsRNA also affected ovary development, causing underdeveloped ovaries, hence the failure to produce eggs (Table 3; Fig. 4J).

RNAi-mediated knockdown of DvvABCE_2830 and DvvABCF_2701 in larvae resulted in 100% mortality. Prior to death, it was noted that the body mass of treated individuals was less than that of similarly-aged larvae treated with buffer alone (Fig. 4G, 4H). Analogously, injection of DvvABCE_2830 and DvvABCF_2701 dsRNA separately into pre-pupae both caused 100% mortality with no adult eclosion (results not shown). Injection of dsRNA specific for DvvABCH_18290 into early-instar D. v. virgifera larvae and pre-pupae caused development to arrest as individuals prepared to molt, thus resulting in 100% mortality (Fig. 4F). Affected individuals appeared to desiccate prior to death (personal observation).

Injection of dsRNA targeting DvvABCG_3712, DvvABCG_49457, and DvvABCG_13829 resulted in phenotypes similar to those seen with RNAi knockdown of the corresponding T. castaneum orthologs [30]. Specifically, injection of dsRNA targeting DvvABCG_13829, which is most closely related to eye-color transporters, gave a white-eye phenotype (Fig. 4E); indeed, DvvABCG_13829 corresponds to the ortholog of white we had identified previously [47]. Injection of DvvABCG_3712 dsRNA into pre-pupae caused developmental defects that resulted in 80% mortality (Table 3; Fig. 4C). Interestingly, adult females treated with DvvABCG_3712 dsRNA produced fewer eggs compared to females injected with buffer alone (Fig. 5B), and the eggs that were laid lacked obvious signs of embryonic development (Fig. 4I) and ultimately failed to hatch (Additional File 5: Figure S4). Injection of DvvABCG_49457 dsRNA into larvae and pre-pupae resulted in molting defects; about 80% of these died during their next molt (Table 3), while the 20% that survived through subsequent larval molts died following pupation (Fig. 4D).

In recent years, ABC transporters have become a major focus for research in arthropods. This is in part due to their overall role in xenobiotic transport and insecticide resistance [25, 48-51], but more specifically, due to their suspected role in susceptibility to Bt toxins [39, 52, 53]. For example, Gahan et al. [54] reported genetic linkage of Heliothis virescens HvABCC2 with resistance to Cry1Ac, while changes in the structure, splicing, or expression level of ABCC2 orthologs were later associated with Cry1Ac resistance in Helicoverpa armigera [55], Bombyx mori [56], and Spodoptera exigua [57]. An ABCC2 ortholog is also linked to Cry1F resistance in Ostrinia nubilalis [58] and S. frugiperda [59]. Additionally, structural mutations in a member of subfamily A, HaABCA2, were implicated in Cry2Ab resistance in H. armigera [60], and reduced expression of ABCG members have been associated with Cry1Ac resistance in P. xylostella [61], as well as Cry1Ac and Cry1Ab resistance in O. furnacalis [62]. More recent studies in species of Coleoptera have implicated ABCB subfamily members in Cry3Aa resistance in C. tremula [39] and in Cry3Ab1 resistance in D. v. virgifera [40].

The study of ABC transporters in several arthropod species have relied on genomic data, including T. castaneum [30], Aethina tumida [63], Bombyx mori [33], D. melanogaster [28], Bemisia tabaci [51], Daphnia pulex [64], and Tetranychus urticae [65]. Due to the status of D. v. virgifera as a major pest of cultivated maize (see Introduction) and current fragmented state of the unpublished draft genome assembly of this species (GenBank accession PXMJ00000000.2), the Illumina-based transcriptome assemblies reported here represent a particularly valuable genetic tool for gene discovery, characterization, and genome annotation. In particular, the 65 ABC transporter genes we identified are expected to be useful in downstream studies on insecticide resistance traits in D. v. virgifera.

Broehan et al. [30] previously identified 73 ABC transporters in T. castaneum, and a 74^th ABC transporter was more recently reported by Grubbs et al. [32]. There are several possible reasons for why the 65 DvvABC transporters we identified are comparatively fewer than in T. castaneum. Firstly, our transcriptome was derived from lower-throughput sequencing data (Illumina MiSeq), therefore genes expressed at very low levels may not have been represented within our raw Illumina data. Secondly, our RNA-seq libraries were not comprehensive of all possible life/growth stages or conditions, such that transcripts not expressed during growth states or under conditions used in this study would have been missed. Regardless, BLASTx analyses of the 65 putative D. v. virgifera ABC transporters identified in this study demonstrate their greatest sequence similarity to T. castaneum and A. glabripennis orthologs. This is probably a consequence of the extensive publicly available genomic data for both T. castaneum and A. glabripennis, as well as their close phylogenetic relationships to D. v. virgifera. Furthermore, the putative one-to-one relationship among orthologs from D. v. virgifera and T. castaneum may suggest the retention of copy number without extensive gene loss or gain across evolutionary time.

Despite the relatively large amount of genomic and transcriptomic data available for model and some non-model coleopteran species, there is a comparative overall dearth of functional data available to support automated computational annotations. To partially address this shortfall, we generated functional information based on RNAi knockdown of eight D. v. virgifera ABC transporters, each of which demonstrated fairly conserved roles relative to their T. castaneum orthologs [30]. While some D. v. virgifera RNAi-mediated loss-of-function phenotypes include visible developmental defects, such as loss of eye pigmentation, others cause growth arrest, and/or death. For example, knockdown of DvvABCA_18330 led to death during the pupal-to-adult molt and also caused deformation of wings and elytra in surviving adult beetles, which was the same as previously seen in T. castaneum [30] (Table 3; Fig. 4A). Since subfamily A transporter members are implicated in mammals with lipid transport, which can impact cell physiology [66], it is conceivable that the effects of the knockdown of DvvABCA_18330, and of its direct orthologs, TcABCA-9A/9B, in T. castaneum [30], could be the result of disrupting critical lipid transport. DvvABCB_19147 RNAi also recapitulated the lethal effects of its T. castaneum ortholog, as well as the effects on female fecundity, which could make this gene a particularly interesting target for RNAi-based pest control. It is worth noting that D. v. virgifera is predicted to have one more ABCB subfamily member compared to other insects [25]. ABCB genes have been implicated in chemical insecticide resistance among insects [48], while in humans, subfamily B genes are known to be mitochondrial transporters with roles in iron metabolism and transportation of Fe/S protein precursors [67, 68]. While these possibilities were outside the scope of our research, future investigations into the function of DvvABCBs could be beneficial for deciphering mechanisms of chemical insecticide resistance in D. v. virgifera.

RNAi knockdown of DvvABCE_2830 and DvvABCF_2701 resulted in 100% larval mortality. ABCE and ABCF subfamilies are highly conserved across all phyla, and due to their lack of TMDs are considered non-transporters. Instead, they appear to play roles in regulating translation [69, 70], indicating that ABCE and ABCF proteins are essential. Thus, given that these genes are highly conserved across taxa in sequence, function, and RNAi phenotype [30], it may not be surprising that lethal RNAi knockdown phenotypes were obtained in D. v. virgifera.

The phenotypes observed following independent RNAi knockdown of DvvABCG_49457 and DvvABCH_18290 involved molting defects that resulted in near complete mortality. While these results are consistent with functional analysis of their T. castaneum orthologs, RNAi knockdown of the DvvABCG_49457 ortholog TcABCG-8A in T. castaneum produced an additional phenotype of premature development of compound eyes [30]. In contrast, we did not observe any analogous eye phenotypes in D. v. virgifera following RNAi knockdown. It is likely that since the injected D. v. virgifera larvae died prior to reaching the next stage of development, there was no opportunity for compound eyes to form. In other species, orthologs of DvvABCH_18290 are known to transport cuticular lipids that are deposited in the outer epicuticle layer to form a waterproof barrier [30, 61]. Therefore, it could be that cuticular lipid deposition may be reduced following RNAi knockdown of this ABCH transporter, which could promote desiccation and subsequent mortality of affected individuals.

The ABCG proteins are HTs, and with 12 predicted members form the second largest subfamily of ABC transporters identified in D. v. virgifera (Table 2). Among insects, some of the first ABCGs to be characterized were the pigment transporters (white, scarlet and brown) in D. melanogaster [34, 71]. Mutants of white are characterized by white eyes (i.e. complete loss of eye pigmentation), scarlet mutants by bright red eyes (i.e. loss of brown pigments), and brown mutants by dark brown eyes (i.e. loss of red pigments) [34]. Studies have revealed that some ABCG proteins perform other crucial physiological roles in the transport of lipids, sterols, and drugs [72]. In the current study, RNAi-mediated knockdown of DvvABCG_13829 resulted in a white-eyed phenotype consistent with prior observations in T. castaneum [30], and with our own previous findings in D. v. virgifera [47]. Our findings support a prediction that DvvABCG_13829 is part of the ommochrome pathway, where it is likely acting within a heterodimeric complex to import ommochrome pigments into the pigment granules of the compound eye. As mentioned above, loss of white function in D. melanogaster, results in white-eyed flies, while mutations in scarlet lead to red-eyed flies. However, RNAi-mediated knockdown of the corresponding gene, ABCG-9A (scarlet), in T. castaneum produces white-eyed beetles [30, 32]. This finding was not surprising, since a previous report of RNAi targeting vermilion, a pivotal gene in the ommochrome pathway, also generates a white-eyed phenotype in T. castaneum [73], leading the authors to conclude that the T. castaneum eye is pigmented by ommochromes alone, and that the ommochrome biosynthetic pathway in T. castaneum produces red pigments as end products, rather than brown pigments as in Drosophila. Unfortunately, our initial survey of the D. v. virgifera transcriptome failed to identify a scarlet ortholog, thus its function was not assessed. It will be interesting to investigate in future studies if pigmentation of the D. v. virgifera eye is more similar to that of T. castaneum or D. melanogaster. Specifically, in D. melanogaster a third ABC-G transporter, brown, is required for wild-type pigmentation of the eye. In flies, Brown heterodimerizes with White and transports pteridine-based pigments into the eye. Although an ortholog of brown has been identified in the T. castaneum genome, no function has been identified [32].

This study provides a relatively large transcriptomic resource comprising genes expressed across several life stages of the arthropod pest species, D. v. virgifera. Due to potential omission of orthologs from our assembly, undoubtedly additional research will need to be performed in order to identify the full compliment of ABC transporters encoded by D. v. virgifera and further functional assays will be needed to validate putative biochemical roles. Regardless, our work represents the initial description of the ABC transporter gene family in D. v. virgifera. Furthermore, the knockdown of ABC transporters DvvABCB_19147 and DvvABCG_3712, each of which reduce egg production and/or prevented embryonic development, could provide novel targets for D. v. virgifera population suppression and use as an insecticidal control agent. This research is a contribution to a growing set of genomic resources for arthropods, and provides information that may facilitate the development of methods to enhance the control of a devastating agricultural pest species.

Insect rearing

All D. v. virgifera used in this study are nondiapausing, from a colony previously established at North Carolina State University using beetles obtained from both Dr. Wade French (USDA-ARS-NGIRL, Brookings, SD) and Crop Characteristics, Inc. (Farmington, MN, USA) (see [74]). Eggs deposited in an oviposition chamber (agar plate with cheese cloth) were collected weekly, pipetted into soil-filled containers, and held at 26°C for 1 week. Larvae were reared on roots of germinated corn seed in 16-oz containers, while adults were maintained in a 30cm³ BugDorm (MegaView Science, Taiwan) at 26°C, 70% relative humidity with an L14:D10 photoperiod and fed an artificial diet (Western Corn Rootworm w/o Pollen Substitute, Frontier Insect Diet, Newark, DE, USA). Injected individuals were reared in small containers with corn seedlings to allow downstream observation.

Transcriptome sequencing, assembly, and annotation

Total RNA was extracted from mixed-staged D. v. virgifera embryos (n = 500 from an overnight egg lay aged up to 14 days), mixed-stage larvae (first-instar larvae (n = 20); second-instar larvae (n = 10); and third-instar larvae (n = 2), as well as an adult male, and an adult female (n = 1 each) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and treated with DNase I (Qiagen) according to the manufacturer’s instructions. The isolated total RNA was submitted to the Genomic Sciences Laboratory (North Carolina State University, NC, USA) for quality assessment, poly(A) selection, fragmentation, selection of ~650 bp fragment sizes, Illumina TruSeq^® library preparation, and 300 bp paired-end sequencing on an Illumina MiSeq sequencer (Illumina, San Diego, CA, USA).

Raw FASTQ reads for each library were assessed using FastQC [75]. Reads were initially imported into SeqMan NGen^® (DNASTAR, Madison, WI, USA), where onboard scripts were used to quality trim and de novo assemble reads into contigs using default settings. Additionally, raw reads from individual libraries were trimmed of Illumina adapter sequence contamination, bases having Phred quality score < 20 (q < 20), and sequence reads < 35 bp using Trimmomatic 0.32 [76]. Resulting trimmed read pairs from each library were concatenated into single R1- and R2-specific FASTQ files using a custom PERL script, and then assembled into contigs using SOAPdenovo-Trans v 1.0.3 [77] (asm-flags = 0; max_rd_len = 301; map_len = 75; avg_ins= 700; kmer (-K= 127)). Trimmed reads were also assembled with Trinity [78] using default parameters, except for adjustment for library insert length (--group_pairs_distance = 700) and minimum read overlap (--path_reinforcement_distance = 75). The complexity of SOAPdenovo-Trans and Trinity assemblies were reduced by clustering allelic variants using CD-HIT-EST [79] with default parameters, except for change of sequence identity (-c 0.95), word length (-n 10), and length of throw-away sequence (-l 11). The relative completeness of each clustered D. v. virgifera transcriptome assembly was evaluated by comparison with the universal single-copy orthologs from Arthropoda obtained from OrthoDB v 9 [80] using BUSCO v 3 [81] (E-value cutoff 0.001). Full- and partial-length open reading frames and corresponding derived amino-acid sequences were predicted from the resulting SOAPdenovo-Trans clusters with TransDecoder v3.0.0 [82] using a minimum length of 100 amino acids.

The transcript sequences assembled by SeqMan NGen® (DNASTAR, Madison, WI) were imported into Blast2GO v4.0 [83, 84] and annotations acquired via BLASTx [85] comparison to the non-redundant (nr) arthropod-specific protein database at the National Center of Biotechnology Information (NCBI). The combined graphs were created at level 2 for Biological Process (P), Cellular Component (C), and Molecular Function (F) categories from Blast2GO.

Bioinformatic analysis of the D. v. virgifera ABC transporter family

A searchable database was created from the combined DNASTAR D. v. virgifera transcript assembly, and subsequently searched with the set of deduced T. castaneum ABC transporter amino-acid sequences [30, 32] as queries using the tBLASTn algorithm in BlastStation software (TM Software Inc., Arcadia, CA, USA). Homologous sequences were selected based on sequence identity and E-value (< 10^-6). Putative D. v. virgifera ABC transporter sequences were then used as BLASTx queries of the non-redundant NCBI protein database using the web blast interface [86], and only sequences that matched ABC transporters were retained. The number and positions of transmembrane domains were assessed via query of the NCBI Conserved Domain Database [87]. Finally, each D. v. virgifera ABC gene was putatively assigned to a subfamily (A-H) based on greatest similarity assigned to orthologs within BLASTx results. This BLAST search procedure was analogously repeated for SOAPdenovo-Trans and Trinity assemblies. The complexity of each ABC gene set was reduced by clustering allelic variants (sequence) across assemblies, and a comprehensive non-redundant set of putative D. v. virgifera ABC transporter contigs were generated. Assembly of origin is denoted in sequence names as follows: DNASTAR (D), Trinity (T), and SOAPdenovo-Trans (S = “scaffold” and C = “contig”) within the FASTA files (Additional Files 6 and 7).

Phylogenetic relationships among derived D. v. virgifera ABC transporter protein sequences were reconstructed from the conserved NBD. A multiple sequence alignment was performed with ClustalW using MEGA 7 [88] (default parameters) and used within a subsequent phylogenetic analysis. Unrooted phylogenetic trees were constructed from 1000 bootstrap pseudo-replications of the aligned data using the Neighbor-joining method as determined by the Best Model of sequence evolution function of MEGA 7 [88]. ABC transporter subfamilies were assigned to D. v. virgifera sequences and clades within this phylogenetic analysis by comparison to similarities from our BLASTx search results and tree topologies among nearest orthologous gene family members in T. castaneum [30], B. mori [89] and D. melanogaster. Additionally, a rooted phylogenetic tree was constructed for a subset of D. v. virgifera proteins that were targeted for RNAi-mediated knockdown and the full set of deduced amino-acid sequences from T. castaneum ABC transporters, with ABCD2 from T. castaneum and Dm-CG2316 from Drosophila melanogaster used as outgroups. Multiple sequence alignments were generated as described above, wherein the deduced D. v. virgifera amino-acid sequences included full-length sequences when possible, but some were incomplete partial-protein sequences. All phylogenetic reconstruction methods were performed as described above.

Gene expression across developmental stages

Preliminary analysis to estimate the relative expression levels for eight transcripts (DvvABCA_18330, DvvABCB_19147, DvvABCE_2830, DvvABCF_2701, DvvABCG_3712, DvvABCG_49457, DvvABCG_13829 and DvvABCH_18290) across growth stages was made via semi-quantitative PCR in order to ensure dsRNA injections would be performed prior to the time of corresponding peak expression. Total RNA was extracted from each developmental stage [embryo (E), larval (L), pupal (P), and adult male (M) and female (F)], from which cDNA was reverse transcribed using the Superscript™ III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) using an anchored poly(T) primer. These cDNA pools were then used individually as template in eight separate PCR reactions each using D. v. virgifera ABC transporter transcript-specific primer pairs (Additional File 8: Table S3). Primers for the D. v. virgifera ribosomal protein S6, DvvRPS6, were used as an external control. PCR reactions were set up using MyTaq™ DNA polymerase according to manufacturer instructions (Bioline, Memphis, TN, USA), and subsequent amplification reactions were performed in a C1000 Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) with the following cycling conditions: (95°C for 3 min), 25× (95°C for 30s, 58°C for 30s, 72°C for 10s), (4 min incubation at 72°C). Amplification products were then visualized and compared using 1.5% agarose gel electrophoresis.

RNAi knockdown phenotypes

Primers were designed for the generation of dsRNA using Vector NTI Advance (VNTI) software (Invitrogen), for all ABC genes whose orthologs are known to produce obvious RNAi phenotypes in T. castaneum [30]. These primer sets targeted regions that encoded transcript-specific TMD domains; this was done in order to potentially reduce unintended off-target effects by avoiding the more conserved NBD domains. Partial cDNAs were amplified for the 8 genes (DvvABCA_18330, DvvABCB_19147, DvvABCE_2830, DvvABCF_2701, DvvABCG_3712, DvvABCG_49457, DvvABCG_13829 and DvvABCH_18290), as described above for developmental stage expression. Nested PCR was performed with an initial denaturation of 95°C for 3 min, 35 cycles at 95°C for 30s, 58°C for 30s, and 72°C for 10s, and then a 4 min incubation at 72°C on a C1000 Thermal Cycler (Bio-Rad). PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions, ligated into the pGEM-T vector (Promega, Madison, WI, USA), and the resulting plasmids were used to transform TOP10 competent E. coli (Invitrogen). All positive clones were cultured in a selective LB medium containing 50 µg ampicillin L^-1. The recombinant plasmid DNAs were isolated using the QIAprep® Spin Miniprep Kit (Qiagen), and the inserts were Sanger sequenced and confirmed by use as BLASTn queries [86]. Purified plasmids with each cloned ABC transporter were used as template in separate PCR reactions primed with the following primers: T7 as a forward primer (due to location in pGEM), and a pGEM-specific reverse primer that was tailed with T7. This enabled all amplification reactions to be performed using the same set of primers under conditions described above. PCR products were analyzed by 1.5% agarose gel electrophoresis, purified using the QIAquick PCR Purification Kit (Qiagen), and then ~1µg of each was used as template for dsRNA synthesis using the MEGAscript T7 in vitro Transcription Kit (Ambion, Austin, TX, USA). Each of the synthesized dsRNAs were purified using the MEGAclear Kit (Ambion) and concentration determined using a Nanodrop 1000 (Thermo Scientific, Waltham, MA, USA) using the single-stranded RNA setting.

RNAi assays were conducted by injecting dsRNA corresponding to each of the 8 specific D. v. virgifera ABC genes individually into the hemocoel of third-instar larvae, pre-pupae and/or newly-eclosed female adults. Before microinjection, experimental insects were anesthetized on ice for 30 min, then injected with a gene-specific dsRNA at a concentration of 1-2µg/µl. Each treatment was replicated three times, with ≥20 individuals in each replicate. Following injection, larvae and pre-pupae were allowed to recover at room temperature for one hour, and then moved to germinated corn for further monitoring and phenotypic analysis. Phenotypes were observed daily using a stereomicroscope, and transcript levels assessed at 5 days post-injection by semi-quantitative PCR using RNA isolated from pools of injected individuals (one individual per replicate, for a total of three individuals per PCR reaction).

Treated females were kept in an oviposition chamber (agar plate with cheese cloth) and maintained on an artificial diet. At two days post-injection, females were mated to untreated males, and generally started to lay eggs ~10 days later. To determine egg viability, eggs were harvested from the oviposition chamber and placed on moistened filter paper in Petri dishes and held at 26°C, 70% relative humidity with an L14:D10 photoperiod. Females were allowed to lay eggs over a two-week period, and eggs were counted every other day to assess the rate of egg laying. Hatch rate counts were made every other day, beginning ten days after the first egg lay (22-days post-injection) and continuing for four weeks until no further hatching was observed.

ATP-binding cassette (ABC); Bacillus thuringiensis (Bt); gene ontology (GO); National Center of Biotechnology Information (NCBI); nucleotide-binding domain (NBD); open reading frames (ORFs); transmembrane domain (TMD)

Ethics approval and consent to participate

Not applicable.

Consent for Publication

Not applicable.

Availability of Data and Materials

The datasets generated and/or analyzed during the current study are included in this published article [and its supplementary information files] or are available at NCBI under BioProject PRJNA490283, with SRA database accessions SRX4669438 to SRX4669441 for the raw reads, and TSA database accessions GHNH00000000 and GHRK00000000 for the Trinity and DNASTAR assemblies, respectively.

Funding

This work was supported by a grant from the Monsanto Corn Rootworm Knowledge Research Program, grant number AG/1005 (to ML), and start‐up funds to ML from NC State University. Portions of this work by BC was supported by a joint United States Department of Agriculture (USDA), Agricultural Research Service (ARS) (CRIS Project 5030-22000-018-00D), ARS SCINet computational resources, and the Iowa Agriculture and Home Economics Experiment Station, Ames, IA (Project 3543). The funding body had no role in the design of the study, collection, analysis, or interpretation of data, or in writing the manuscript.

Competing Interests

The authors declare no competing interests.

Author Contributions

FA and MDL conceived and designed the experiments; FA performed the experiments; FA, NG, BC, BW, MDL analyzed the data; and FA, NG, BC, MDL wrote the manuscript. All authors have read and approved the manuscript.

Acknowledgements

We thank Pei-Shan Wu, Sofia Pinzi, Teresa O'Leary, Lauren Slayton and Wanose Getachew for their expert assistance in rearing WCR. This article reports the results of research only and any mention of products or services does not constitute an endorsement by USDA-ARS. USDA-ARS is an equal opportunity provider and employer.

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Table 1: Paired-end RNA-sequencing libraries and sequencing

			MiSeq	Raw read data		Trimmed read data
ID	Lib_name	Insert	Lanes	Length	Count	Paired	Unpaired
Af1	DvvAdultF_R1	600 to 700-bp	1	300-bp	3,462,470	2,753,272	559,611
Af2	DvvAdultF_R2	600 to 700-bp		300	3,462,470	2,753,272	72,301
Am1	DvvAdultM_R1	600 to 700-bp	1	300	2,223,027	1,859,087	308,133
Am2	DvvAdultM_R2	600 to 700-bp		300	2,223,027	1,859,087	23,335
E1	DvvEggs_R1	600 to 700-bp	1	300	2,690,038	2,146,192	425,023
E2	DvvEggs_R2	600 to 700-bp		300	2,690,038	2,146,192	62,149
L1	DvvLarvae_R1	600 to 700-bp	1	300	2,652,196	2,071,100	445,600
L2	DvvLarvae_R2	600 to 700-bp		300	2,652,196	2,071,100	62,923
				Totals	22,055,462	17,659,302	1,959,075

Table 2: Classification of D. v. virgifera ATP binding cassette (ABC) transporters

Diabrotica virgifera virgifera transcript				Nearest Tribolium castaneum ortholog
Gene ID	Subfamily	Contig	L (aa)	Accession	E-value	Identity (%)
DvvABCA_18330	ABCA	18330	1,644	XP_008199153.1	0	60
DvvABCA_50718	ABCA	50718	1,708	KXZ75645.1	0	58
DvvABCA_49125	ABCA	49125	1,757	XP_008195104.1	0	42
DvvABCA_266167	ABCA	266167	1,641	XP_008195056.1	0	53
DvvABCB_21313	ABCB	21313	1,247	XP_967244.3	0	60
DvvABCB_17742	ABCB	17742	1,257	XP_008191266.1	0	63
DvvABCB_19147	ABCB	19147	667	XP_015834348.1	0	71
DvvABCB_39715	ABCB	39715	716	XP_001813375.1	0	75
DvvABCB_9796	ABCB	9796	834	XP_008194672.1	0	76
DvvABCB_13664*	ABCB	13664	657	XP_008190820.1	0	60
DvvABCB_17837	ABCB	17837	682	XP_972133.2	0	69
DvvABCC_41801	ABCC	41801	1,268	XP_969849.1	0	37
DvvABCC_44708	ABCC	44708	1,257	XP_015836131.1	0	44
DvvABCC_48952*	ABCC	48952	1,252	XP_971802.2	0	48
DvvABCC_17573	ABCC	17573	1,285	XP_015836083.1	0	44
DvvABCC_51687	ABCC	51687	1,556	XP_008197283.1	0	73
DvvABCC_21020*	ABCC	21020	1,297	EFA03948.2	0	52
DvvABCC_222633*	ABCC	222633	1,234	XP_971802.2	0	46
DvvABCC_18126	ABCC	18126	1,343	EFA04472.2	0	57
DvvABCC_49513	ABCC	49513	1,374	XP_008193834.1	0	44
DvvABCC_14070	ABCC	14070	1,343	XP_008194093.1	0	51
DvvABCC_22628	ABCC	22628	1,350	XP_008193834.1	0	56
DvvABCC_20002	ABCC	20002	1,345	EFA04472.2	0	58
DvvABCC_7536	ABCC	7536	1,364	XP_008193834.1	0	61
DvvABCC_47333	ABCC	47333	1,377	XP_008193834.1	0	59
DvvABCC_49618*	ABCC	49618	753	XP_015835265.1	0	76
DvvABCC_45163	ABCC	45163	1,535	XP_008192060.1	0	61
DvvABCC_43960*	ABCC	43960	1,082	XP_973693.2	0	48
DvvABCC_48940	ABCC	48940	1,224	XP_015836125.1	0	42
DvvABCC_217405*	ABCC	217405	1,165	XP_015835266.1	0	50
DvvABCC_10132*	ABCC	10132	871	XP_008200429.1	0	55
DvvABCC_48300*	ABCC	48300	1,258	XP_971632.1	0	44
DvvABCC_47673	ABCC	47673	1,324	XP_969781.1	0	72
DvvABCC_5345	ABCC	5345	1,258	XP_015836093.1	0	40
DvvABCC_22413	ABCC	22413	1,331	XP_969781.1	0	55
DvvABCC_18709*	ABCC	18709	1,260	XP_008193834.1	0	53
DvvABCC_21941	ABCC	21941	1,329	XP_969849.1	0	48
DvvABCC_15305	ABCC	15305	1,324	XP_969781.1	0	64
DvvABCC_12562	ABCC	12562	1,320	XP_972534.1	0	49
DvvABCC_10642	ABCC	10642	1,307	XP_008195764.1	0	60
Table 2 continued
Diabrotica virgifera virgifera transcript				Nearest Tribolium castaneum ortholog
Gene ID	Subfamily	Contig	L (aa)	Accession	E-value	Identity (%)
DvvABCC_41602	ABCC	41602	1,308	XP_008193119.1	0	50
DvvABCC_12703	ABCC	12703	1,318	KYB27686.1	0	54
DvvABCC_14968	ABCC	14968	1,310	EFA03655.1	0	49
DvvBCD_11014	ABCD	11014	755	XP_971218.1	0	77
DvvABCD_11628	ABCD	11628	658	XP_015838765.1	0	78
DvvABCE_2830	ABCE	2830	609	XP_968009.1	0	92
DvvABCF_2701	ABCF	2701	922	XP_971562.1	0	90
Dvv BCF_802	ABCF	802	624	XP_966990.1	0	89
DvvABCF_9935	ABCF	9935	711	XP_972814.1	0	84
DvvABCG_9811	ABCG	9811	660	XP_008192053.1	0	69
DvvABCG_3712	ABCG	3712	668	XP_001813184.1	0	77
DvvABCG_14042	ABCG	14042	720	XP_973458.1	0	76
DvvABCG_10897	ABCG	10897	652	XP_008192849.1	0	63
DvvABCG_22358	ABCG	22358	641	XP_015834971.1	0	63
DvvABCG_23081	ABCG	23081	604	XP_971735.1	0	54
DvvABCG_13051	ABCG	13051	638	XP_015834339.1	0	60
DvvABCG_38769	ABCG	38769	622	XP_973526.1	0	54
DvvABCG_13829	ABCG	13829	658	NP_001034521.1	0	59
DvvABCG_49457	ABCG	49457	941	XP_968472.1	0	73
DvvABCG_36869	ABCG	36869	643	XP_968555.2	0	71
DvvABCG_79525	ABCG	79525	651	NP_001306193.1	0	61
DvvABCH_20789	ABCH	20789	714	XP_015839275.1	0	43
DvvABCH_5118	ABCH	18290	704	XP_008198313.1	0	84
DvvABCH_18290	ABCH	5118	796	XP_973444.1	0	43
DvvABCH_11818	ABCH	11818	763	XP_967359.1	0	72

(*) Incomplete sequences

Table 3: Results of RNAi knockdown of selected ABC transporters

Transcript	Stage	KD	Phenotype	Figure
DvvABCA_18330	Pre-pupal	60%	Deformed wings & elytra	4A
DvvABCB_19147	Larval	100%	Lethal	NS
	Pre-pupal	100%	Defect in pupal-adult molt	4B
	Eclosed females	0%	Malformed ovaries; low egg lay	4J
DvvABCE_2830	Larval	100%	Lethal	4G
DvvABCF_2701	Larval	100%	Lethal	4H
DvvABCG_3712	Pre-pupal	80%	Lethal; pupal developmental arrest	4C
	Eclosed females	0%	Prevented embryonic development	4I
DvvABCG_13829	Pre-pupal	0%	Pigmentation defect; white eyes	4E
DvvABCG_49457	Larval	100%	Lethal at molting	NS
	Pre-pupal	80%	Lethal pupal developmental arrest	4D
DvvABCH_18290	Larval	100%	Lethal at molting	NS

KD = knockdown; NS = image not shown

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Structural and functional insights into the Diabrotica virgifera virgifera ATP-binding cassette transporter gene family

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