- Research article
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An expanded odorant-binding protein mediates host cue detection in the parasitic wasp Baryscapus dioryctriae basis of the chromosome-level genome assembly analysis
BMC Biology volume 22, Article number: 196 (2024)
Abstract
Background
Baryscapus dioryctriae (Chalcidodea: Eulophidae) is a parasitic wasp that parasitizes the pupae of many Pyralidae members and has been used as a biological control agent against Dioryctria pests of pinecones.
Results
This B. dioryctriae assembly has a genome size of 485.5 Mb with a contig N50 of 2.17 Mb, and scaffolds were assembled onto six chromosomes using Hi-C analysis, significantly increasing the scaffold N50 to 91.17 Mb, with more than 96.13% of the assembled bases located on chromosomes, and an analysis revealed that 94.73% of the BUSCO gene set. A total of 54.82% (279.27 Mb) of the assembly was composed of repetitive sequences and 24,778 protein-coding genes were identified. Comparative genomic analysis demonstrated that the chemosensory perception, genetic material synthesis, and immune response pathways were primarily enriched in the expanded genes. Moreover, the functional characteristics of an odorant-binding protein (BdioOBP45) with ovipositor-biased expression identified from the expanded olfactory gene families were investigated by the fluorescence competitive binding and RNAi assays, revealing that BdioOBP45 primarily binds to the D. abietella-induced volatile compounds, suggesting that this expanded OBP is likely involved in locating female wasp hosts and highlighting a direction for future research.
Conclusions
Taken together, this work not only provides new genomic sequences for the Hymenoptera systematics, but also the high-quality chromosome-level genome of B. dioryctriae offers a valuable foundation for studying the molecular, evolutionary, and parasitic processes of parasitic wasps.
Background
Parasitic wasps are in the spotlight for their potential as biological control agents against pests and for their megadiverse biology, positioning them as the subject of biological research [1]. Various parasitic wasps’ species have been widely used as effective natural enemies against numerous destructive pests in agriculture and forestry. For example, Chouioia cunea is used for controlling Hyphantria cunea and Helicoverpa armigera [2], and Trichogramma brassicae is used for controlling Ostrinia nubilalis [3]. Given the diverse biological characteristics of parasitic wasps, they are gradually becoming an ideal model study system for investigating insect genetic diversity [4, 5]. The number of haploid chromosomes in parasitic wasps ranges from 3 to 23 [6]. With the development of new sequencing technologies, numerous insect genome assemblies have now been improved to the chromosome level [7]. To date, data for 14 genome assemblies of parasitic wasps at the chromosome level have been published, and 10 of these assemblies rely on high-through chromosome conformation capture (Hi-C) technology, which include those of Aphidius gifuensis (Hi-C, n = 6) [8], Aphelinus certus (n = 4), Aphelinus atriplicis (n = 4) [9], Cotesia glomerata (Hi-C, n = 10) [10], Cotesia congregata (Hi-C, n = 10) [11], Chelonus formosanus (Hi-C, n = 7) [12], C. cunea (Hi-C, n = 6) [13], Eretmocerus hayati (Hi-C, n = 4) [14], Microplitis manila (Hi-C, n = 11) [15], Nasonia vitripennis (n = 5) [16], Pteromalus puparum (Hi-C, n = 5) [1], Telenomus remus (Hi-C, n = 10) [17], Theocolax elegans (Hi-C, n = 7) [18], and Venturia canescens (n = 11) [19]. This approach provides additional and essential information that is restricted by fragmented genomes and improves comparative genome analyses of parasitic wasps, which is helpful for further analysis of parasitoid lifestyle, behavior, and living habits [7]. Comparative genomics studies have revealed that the oxyglycosylation of the mucin domain in the hemomucin protein plays a pivotal role in the passive immune evasion mechanism employed by parasitoids [20]. Furthermore, a cluster of genes displaying remarkably accelerated evolutionary rates has been identified in these two miniaturized parasitoids, potentially attributable to their convergent adaptation toward miniaturization [17].
The antennae and ovipositors of parasitic wasps have long been linked to the identification of chemical cues pertaining to host localization, inspection, and oviposition [21]. Parasitic wasps antennae have been extensively studied, including ultrastructural studies of sensilla [22], electrophysiological detection of olfactory-active volatiles [23], identification of olfactory-related genes through transcriptome sequencing [24], and functional characterization of the olfactory genes expressed in the antennae [22,23,24,25,26]. Although the sensilla of some parasitic wasps have been successfully identified in the ovipositor [27], and some olfactory-related genes have been detected in the ovipositor; however, the functions of these genes have yet to be characterized [28]. For example, in the fig wasp Apocrypta westwoodi, their ovipositors not only serve as egg-laying organs but also function as olfactory organs, responding to volatile compounds and CO2 in a gaseous form [29]. The olfactory function of insect ovipositors is supported by greater data in other species, and investigations have shown that the ovipositors of several Lepidoptera species express genes encoding olfactory receptors [30]. In addition, through morphological studies, olfactory sensilla have been shown to be present in the ovipositors of Monopis crocicapitella and Homoeosoma nebulella through morphological studies [31, 32]. Functional characterization of neural responses in the sensors on Manduca sexta ovipositors via single sensillum recordings revealed that these sensilla indeed house functional olfactory sensory neurons (OSNs) [33]. The highly expressed odorant receptors (ORs) in the ovipositors were subsequently confirmed to be involved in the detection of host plant volatiles in Helicoverpa assulta [34]. In Diptera, Bactrocera dorsalis was also found to recognize ovipositional preferences by ovipositors [35].
The insect olfactory system primarily encompasses olfactory proteins, generally odorant-binding proteins (OBPs), chemosensory proteins (CSPs), sensory neuron membrane proteins (SNMPs), Niemann-Pick type C2 proteins (NPC2s), ionotropic receptors (IRs), gustatory receptors (GRs) and odorant receptors (ORs) [24]. OBPs are small soluble proteins that are enriched in sensillum lymph; they act as semio-chemical carriers by binding and transporting these chemicals to ORs, and the process mediated by OBPs is considered the first stage of insect olfactory perception [36, 37]. In parasitic wasps, CcunOBP2 in C. cunea and MmedOBP14 in Microplitis mediator participate in the detection of plant volatiles, which may contribute to locating habitats, supplementing nutrients and searching for plant hosts [36, 38]. In addition to their main chemical senses, insect OBPs are also present in other peripheral appendages and internal organs and participate in a variety of insect life activities, such as temperature and humidity perception, mating, taste, and development [39]. OBPs are also expressed within female insect ovipositors, and histological analysis has revealed the expression of OBPs in mosquito ovaries and eggshells [40,41,42]. Furthermore, the female ovary of the oriental fruit fly B. dorsalis has significant expression of OBP19c [43]. OBPs were also shown to be abundantly expressed in the ovipositor of parasitic wasps [28]. Regarding the function of OBPs in Diptera, the female abdomen-biased expression of OBP56d-1 in B. dorsalis significantly increased after stimulation by the oviposition inducer 1-octen-3-ol [44], and the highly expressed OBP56d and OBP56d-2 in the ovipositor were found to be jointly responsible for the oviposition preference of 3-hexenyl acetate (3-HA) [35]. This finding suggests that OBPs on the ovipositor may serve to bring odorants or pheromones into proximity to the odor receptors present in the female reproductive tract. Therefore, characterizing the specific OBPs expressed in the ovipositor will help establish a connection between OBPs and their potential functions.
Baryscapus dioryctriae (Chalcidoidea: Eulophida; Fig. 1) is a gregarious endoparasitoid wasp of many Pyralidae pest pupae [45]. In view of the two important problems in the application of parasitic wasps, reproductive efficiency and field application efficiency, and our previous research revealed that 2-butyl-2-octenal could significantly improve the reproductive efficiency of B. dioryctriae during studying the olfactory function of NPC2s in this wasp [46]. The control efficiency of parasitizing wasps in field applications largely depends on their host-searching ability, which usually relies on their responses to the semiochemicals related to their hosts, and such responses may be influenced by genetic mechanisms and learning behaviors [26, 47]. Parasitic wasps can be trained through learning behaviors to increase their efficiency in locating hosts in laboratory settings or in the field [48, 49]. Therefore, the identification of active compounds associated with host search will be beneficial for the future development of appropriate parasitoid olfactory conditioning methods for the optimization of biological pest control.
In our previous transcriptomic analysis of B. dioryctriae, six OBP genes were found to be more highly expressed in the ovipositor than in other tissues [28]. To obtain more complete genetic information, in our ongoing project, a high-quality chromosome-level genome assembly of B. dioryctriae was generated using a combination of Illumina, PacBio, and Hi-C technologies. A comparative genomic analysis was conducted, leading to the re-identification for odorant-binding proteins of B. dioryctriae’ odorant-binding proteins (BdioOBPs) based on a high-quality genome assembly. Within the expanded OBP genes, a gene exhibiting partial ovipositor expression was discovered and subsequently characterized for its function. Comparative genomic analysis and identification of BdioOBPs were performed based on high-quality genomic assembly. Highly specific expression of the expanded OBP gene was detected in the ovipositor, and then its function was deeply characterized in vitro and vivo.
Results
Chromosome-level genome assembly of B. dioryctriae
K-mer distribution analysis (K = 19) predicted that B. dioryctriae had an estimated genome size of 486.86 Mb with a low heterozygosity rate of 0.39% and a modest GC of 38.18% (Additional file 1: Fig. S1), which indicated that B. dioryctriae has a simple genome and facilitated genome assembly. Single-molecule real-time PacBio reads (10 Gb) were used (Additional file 2: Table S1). We first generated a contig-level assembly with a genome size of 509.39 Mb, a contig N50 length of 2.17 Mb, and a GC content of 38.99% for the genome of B. dioryctriae (Table 1). Hi-C data were subsequently used to improve the assembly, with more than 96.13% of the assembled sequences anchored to six chromosomes; this outcome was further supported by karyotype analysis (Fig. 2A and B). The chromosomal assembly of B. dioryctriae spans 485.5 Mb with a scaffold N50 length of 91.17 Mb, and the length of the largest chromosome was 109.30 Mb (Table 1, Additional file 1: Fig. S2). More than 98.64% of Illumina reads were successfully mapped to the assembly, indicating its excellent integrity and accuracy. Moreover, genome assessment using benchmarking universal single-copy orthologs (BUSCO) indicated that 94.73% of the insect gene sets were present and complete.
Genome annotation and genomic characteristics
A total of 54.82% (279.27 Mb) of the genome of B. dioryctriae was composed of repeat sequences. Transposable elements (TEs) accounted for 48.24% of the genome of B. dioryctriae, of which retroelements were the most abundant TE group (30.41%, 154.90 Mb), followed by DNA transposons (17.84%, 90.86 Mb) (Additional file 2: Table S2). Annotation revealed 33.5 Mb of tandem repeats, which accounted for 6.58% of the whole genome (Additional file 2: Table S2). Genome annotation revealed that 32.02% (155.45 Mb) of the genome sequences encoded proteins in B. dioryctriae, with a total of 24,778 protein-coding genes identified. Notably, this gene count is comparable to those observed in two species characterized by large genomes: G. flavifemur (23,056) and E. hayati (23,911). The average gene length was determined to be 6273.86 bp, while the average coding sequence (CDS) length was 1447.44 bp. The average exon length and average intron length were 1631.1 bp and 96,160 bp, respectively; these values were consistent with those reported for most species except for A. mellifera and N. vestripennis, which presented high intron ratios (Additional file 2: Table S3 and 4). Additionally, approximately 90.48% of the genes (22,419 genes) were functionally annotated (Additional file 2: Table S5). We also identified 95.1% of the BUSCO Insecta database (Insecta_odb9) genes at the protein level.
The analysis of chromosome synteny was carried out by conducting a pairwise synteny search of CDS using the MCScanX pipeline. The results indicate that numerous genome rearrangements have taken place since the split of B. dioryctriae and C. cunea (according to this study, approximately 53.7 million years ago), as shown in the synteny map (Fig. 2C). It can be observed from the collinearity map that Chromosome1 (Chr1) in N. vitripennis shows good collinearity with Chr3 and Chr6 in the Eulophidae wasp genomes (B. dioryctriae and C. cunea). B. dioryctriae and N. vitripenni exhibit lower levels of homology than C. cunea. In this analysis, 253 syntenic blocks were identified between B. dioryctriae and C. cunea, containing 5–411 genes, with an average of 38.27 genes per syntenic block (Additional file 2: Table S6). This comparison revealed 479 syntenic blocks between B. dioryctriae and N. vitripennis, containing anywhere from 5 to 81, with an average of 16.42 genes per syntenic block (Additional file 2: Table S7).
Orthology and phylogenetic relationships
A total of 22,458 orthogonal groups were identified among B. dioryctriae and 13 other Hymenopteran species. The gene family clusters were divided into five categories: generic single-copy genes, multicopy genes, species-specific multicopy genes, species-specific single-copy genes, and other genes. There were 920 species-specific orthogroups and 3939 species-specific genes identified in the genome of B. dioryctriae (Additional file 2: Table S8).
The phylogenetic relationships between B. dioryctriae and the other Hymenopteran insects used in this study were determined with a genome-wide set of 1265 single-copy genes. As expected, the maximum likelihood (ML) phylogenetic analysis demonstrated the separation of the two Symphytas (O. abietinus and A. rosae) from the 12 Apocritas, consistent with expectations. The eight parasitic wasps (B. dioryctriae, C. cunea, N. vitripennis, P. puparum, E. hayati, T. pretiosum, T. remus, and A.gifuensis) clustered together, with four Aculeatas (A. mellifera, O. biroi, G. flavifemur and Polistes dominula) serving as a sister group. The six chalcidoids (B. dioryctriae, C. cunea, N. vitripennis, P. puparum, T. pretiosum and E. hayati) clustered together, and the Eulophidae wasps (B. dioryctriae and C. cunea) were more closely related to the two Pteromalidaes (P. puparum and N. vitripennis) than were Trichogrammatidae (T. pretiosum) and Aphelinidae (E. hayati) (Fig. 3A). B. dioryctriae and other Chalcidoidea species are estimated to have diverged from Cretaceus with a divergence time of approximately 53.71 million years.
Genes under expansion and contraction
The gene family evolution analysis conducted using CAFE identified 309 expanded gene families and two contracted gene families on the terminal branch of B. dioryctriae was separated from other parasitic wasps (Fig. 3A). Notably, several gene families associated with chemosensory activity, including OR, IR and GR were found to be expanded in the genome of B. dioryctriae (Additional file 2: Table S9 and S10). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the terms in which B. dioryctriae expanded gene families were enriched were odorant binding (GO: 0005549, 170 genes, P = 7.38E − 33), nucleotide binding (GO: 0000166, 152 genes, P = 3.40E − 51), DNA-directed DNA polymerase activity (GO: 0003887, 151 genes, P = 6.32E − 66), olfactory receptor activity (GO: 0004984, P = 7.48E − 32), DNA replication (GO: 0006260, 153 genes, P = 1.39E − 40), sensory perception of smell (GO: 0007608, 115 genes, P = 1.61E − 17), deoxyribonucleotide biosynthetic process (GO: 0009263, 120 genes, P = 1.2E − 15), C-type lectin receptor (CLR) signaling pathway (ko04625, 207 genes, P = 3.7E − 63), NOD-like receptor (NLR) signaling pathway (ko04621, 204 genes, P = 9.26E − 65), necroptosis (204 genes, P = 1.75E − 64), and Salmonella infection (ko05132, 204 genes, P = 4.05E − 63). Only two gene families contracted, GO and KEGG enrichment analyses of only two gene families revealed that the terms in which B. dioryctriae contracted gene family was enriched were serine-type endopeptidase activity (GO: 0004252, one gene, P = 0.02) and neuroactive ligand‒receptor interaction (ko04080, one gene, P = 0.04) (Fig. 3B).
Functional analysis of BdioOBP45
A total of 81 OBPs were identified from the genome of B. dioryctriae (Additional file 2: Table S11). All 81 OBP genes were mapped onto six chromosomes (Fig. 4A). and mainly clustered on Chr4 (64 genes, 74.70%), Chr5 (11 genes, 13.25%) and Chr6 (5 genes, 6.02%) (Fig. 4A). Two OBP gene families (OG0000305 and OG0006966) were found to be expanding (Additional file 2: Table S9). The OBP genes were distributed on Chr4 and Chr6; the expression profiles of these OBP genes were relatively scattered; BdioOBP45 was highly expressed specifically on the ovipositor, with a fragments per kilobase million (FPKM) value of 8804.74 (Fig. 4B); and the ovipositor-biased expression pattern of BdioOBP45 was validated by real-time quantitative polymerase chain reaction (RT-qPCR) (Additional file 1: Fig. S3).
The recombinant protein BdioOBP45 was expressed and purified in a bacterial system (Additional file 1: Fig. S4). A binding assay showed that BdioOBP45 could bind efficiently to 1-N-phenyl-naphtylamine (1-NPN) with a dissociation constant (Kd) of 2.324 μM (Fig. 5A). In addition, saturation and linear Scatchard plots show a single binding site for BdioOBP45 and 1-NPN. To investigate the function of BdioOBP45 in odor recognition, gas chromatography-mass spectrometry (GC–MS) analysis was conducted, revealing the presence of a total of 47 compounds in P. koraiensis cones, pest-damaged P. koraiensis cones, mechanically damaged P. koraiensis oleoresin, pest-damaged P. koraiensis oleoresin, and D. abietella pupae (Additional file 1: Fig. S5, Additional file 2: Table S12). Of these, 55 compounds were successfully acquired for subsequent binding assays. Four of the 55 tested compounds (Additional file 2: Table S13) reduced the relative fluorescence intensity of the BdioOBP45/1-NPN complex by > 50%. Three of them strongly bound to BdioOBP45 (Ki < 10 μM), 7.52 ± 0.55 μM caryophyllene oxide, 9.59 ± 0.27 μM g-terpinene and 9.7 ± 0.53 μM α-pinene (Table 2, Fig. 5B).
RNA interference and behavior assays
To further investigate the biological functions of ligands that may have a certain binding affinity for BdioOBP45, we conducted Y-tube olfactory assays. The results showed that α-pinene and g-terpinene could significantly attract B. dioryctriae (Fig. 6A and B). Subsequently, to test the effect of reducing the transcription level of the BdioOBP45 gene on the behavior of B. dioryctriae, dsRNA was injected into female wasps. The optimal timing for behavioral detection was determined by RT-qPCR, and the attractiveness to α-pinene and g-terpinene was compared. Initially, we compared the transcription levels of BdioOBP45 in the control group (non-injected), dsGFP-injected group, and dsBdioOBP45-injected wasps at 12 h, 24 h, 36 h, and 48 h post-injection. Throughout the study period, there was no difference in the transcription level of BdioOBP45 in the control group (non-injected) and the dsGFP-injected group (Fig. 6C). The transcription level of BdioOBP45 was significantly reduced at all time points (P < 0.05), with the greatest reduction observed 24 h post-injection, which was 98% lower than the baseline level. Then, we compared the behavioral changes in B. dioryctriae between different treatment groups 24 h after dsRNA injection, the Y-tube olfactometer test results revealed that the attractiveness to g-terpinene was significantly lower in the dsBdioOBP45-injected group than in the non-injected and dsGFP-injected groups, whereas no significant difference was found between the two control groups. There was no significant change in the response to α-pinene after injection (Fig. 6D).
Discussion
Based on current sequencing projects, Hymenoptera genomes are modest in size (80% are between 180 and 340 Mb), although there are some exceptions; for example, G. flavifemur (636.5 Mb) and E. hayati (692.1 Mb), both of which are relatively large genomes in Hymenoptera [14, 50], were selected for the comparative genome in this study. The expansion of repetitive sequences is one of the most important factors for increasing genome size, and this phenomenon has been reported in many insects [50]. This also applies to Hymenopteran insects, such as E. hayat, with a repeat sequence percentage of 370.8 Mb, and G. flavifemur, with a repeat sequence percentage of 381.7 Mb. The repeat sequences of B. dioryctriae accounted for 54.82% (279.27 Mb) of the whole genome, and the average length of each gene and intron length were moderate, indicating that the TE sequences were amplified. In particular, the amplification of retroelement sequences was a major contributor to the genome enlargement of B. dioryctriae. TE amplification and insertion can cause various changes in the host genome, such as chromosomal rearrangements, gene disruption, and regulation of gene expression; moreover, TEs are associated with a variety of insect adaptations, including resistance to insecticides [51], temperature adaptation [52], adaptive evolution of natural enemy defense [53], and synchronous evolution of phenotypic changes [54]. These retrotransposons may be related to B. dioryctriae’s adaptability to low temperatures and esticides.
Significant expansion or contraction of gene families is often associated with adaptive differentiation of species [55, 56]. For example, Megastigmus duclouxiana and Megastigmus sabinae, which are found in alpine habitats where solar UV radiation increases with altitude, have expanded gene families that are enriched in eye development [57]. The comparative genomic analysis revealed that the expanded genes in B. dioryctriae were primarily associated with chemosensory perception and genetic material synthesis in regions enriched with gene ontology terms. Insects rely on their chemosensory system to discriminate between many attractive or aversive chemical cues in the environment, which is essential for taking appropriate action in response [58]. Parasitic wasps use their sensitive olfactory system to discriminate host-related phytochemicals to determine host location and oviposition site selection [26]. There is a broad positive correlation between the complexity of the chemical ecology of arthropod species and the number of their chemosensory genes [59]. For example, the size of the wasp OR gene family reflects the complexity of chemical cues in its habitat [26]. The expansion of olfaction-related genes in B. dioryctriae might be related to the responsible forest environment in which it lived. KEGG annotation revealed that the expanded gene families were mainly enriched in the C-type lectin receptor signaling pathway, NOD-like receptor signaling pathway, and other immune-related pathways. Unlike vertebrates, which possess adaptive and innate immune responses, insects rely solely on the innate immune system to defend against microbes [60]. Pattern recognition receptors (PRRs) play an important role in innate immunity. The NLR and CLR are six types of PRRS [61]. NLRS are intracellular molecules that recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [62]. CLRs are transmembrane receptors that recognize carbohydrates and mediate immune responses [63]. The expansion of immune-related genes in B. dioryctriae suggests that B. dioryctriae has potential as part of an integrated pest management strategy (pest control combined with elimination of insect pathogens by its strong autoimmune system). Furthermore, a decreasing trend was observed in Ceratosolen solmsi for genes associated with chemical perception, detoxification metabolism, and various immune-related pathways, which may be related to its obligate parasitism [64]. Therefore, we suggest that the amplification of alleles and immune-related genes in B. dioryctriae may be related to its wide host range.
OBPs are crucial components in the olfactory system that mediate odor recognition, and they facilitate the recognition of odors by transporting semiochemicals to ORs through sensillum lymph, ultimately leading to the transmission of electrical signals to the insect brain. Additionally, OBPs play a role in protecting odorants from odorant-degrading enzymes [65]. Research indicates that the number of genes encoding OBPs varies significantly among parasitoid species, and numerous studies have verified that OBPs are vital for the host location of parasitoids through various methods such as gene silencing, fluorescence competitive binding assays, molecular docking, and so on [38]. The number of OBP genes in various parasitoid species ranges from 2 (Scleroderma guani) [66] to 98 (N. vitripennis) [67]. A total of 81 OBP genes were identified in B. dioryctriae, with the relatively high numbers potentially being associated with its complex living environment. Furthermore, the diverse counts of OBP genes in different species may be attributed to the depth of RNA sequencing [68]. In this study, a more comprehensive B. dioryctriae OBP gene family was identified based on genomics in comparison with the previously identified transcriptome-based results (27) [28]. A total of 81 OBP genes were primarily located on Chr4, Chr5, and Chr6. These genes showed a relatively clustered distribution, indicating that they may have undergone family expansion through tandem repeats. There are two expanded gene families among these genes. Through analysis of the expression levels of these two gene families, an interesting gene, BdioOBP45, was found to be highly expressed specifically in the ovipositor. The results of the fluorescence binding assay demonstrated that the main compounds that bind to BdioOBP45 in vitro are caryophyllene oxide, α-pinene, and g-terpinene. All three compounds, which belong to the terpenoid class, are volatile terpenes that mediate interactions between plants and insects, typically benefiting plants while harming herbivores [69]. According to the GC–MS results, all three compounds are typical pest-induced volatile compounds. Herbivore-induced plant volatiles (HIPVs) are important cues for natural enemies to find their hosts [70]. In subsequent behavioral experiments, both α-pinene and g-terpinene could elicit a significant behavioral attraction in B. dioryctriae. After BdioOBP45 was silenced, the attraction of B. dioryctriae to g-terpinene was abolished, whereas the attraction to α-pinene remained, indicating that there may be other BdioOBPs in B. dioryctriae that can bind to α-pinene [37]. Furthermore, certain volatiles that do not inherently elicit a response in parasitoids can act as background volatiles to enhance the parasitoids’ response to other volatiles [71]. Thus, caryophyllene oxide alone does not attract B. dioryctriae directly and may function synergistically in conjunction with other compounds to facilitate host location by parasitoids. To sum up, BdioOBP45 could be used as a potential target to regulate the behavior of the natural enemy parasitic wasp B. dioryctriae.
Conclusions
In this study, a high-quality chromosome-level genome assembly of B. dioryctriae in parasitic wasps was generated using the Illumina, PacBio, and Hi-C combination strategy. Basic genomic analysis revealed the genomic characteristics, phylogenetic position, and gene family evolution of B. dioryctriae. Comparative genome analysis revealed many expanded genes in B. dioryctriae that were mainly involved in olfactory perception, genetic material synthesis, and immune response pathways. Eighty-one BdioOBP genes were identified and located in the genome. Among the expanded OBP genes, a gene named BdioOBP45 was found to be highly expressed specifically in the ovipositor, and functional verification through in vitro and vivo experiments indicated its potential significance in the host-seeking behavior of female parasitic wasps. This work not only provides new genome sequences for Hymenoptera systematics but also supplies a basis for the further protection and utilization of parasitoid resources in pest control through the utilization of B. dioryctriae genomic resources.
Methods
Insect rearing and sampling for sequencing
B. dioryctriae were obtained from the Research Institute of Forest Protection, Jilin Provincial Academy of Forestry Sciences, and then maintained at the School of Life Sciences, Northeast Normal University, Changchun, China. Individuals of B. dioryctriae were cultivated in a climate room with regulated circumstances (25 ± 1 °C, 50 ± 5% R. H., and 16 h L: 8 h D photoperiod) with pupae of G. mellonella as the usual host. Following eclosion, the wasps were raised under the same conditions as previously described while being fed a 10% (V/V) sucrose solution. Large-scale rearing was conducted in the laboratory, yielding a large population. A total of 1000 wasps that emerged within 12 h were collected for subsequent experiments to ensure the uniformity of the mixed sample.
Sequencing and genome size estimation
A modified cetyltrimethylammonium bromide (CTAB) technique was used to extract DNA from 1000 adult male B. dioryctriae. Using an Illumina VAHTSTM Fg DNA Library Prep Kit, a paired-end (PE) library was created, and 150 bp of PE sequencing was performed on an Illumina NovaSeq 6000 platform (Illumina, CA, USA). Using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences), a SMRTbell library was created, and long-read sequencing was performed on the PacBio Sequel II platform. To improve the main genome assembly to the chromosomal level, Hi-C fragment libraries were generated from 300 to 700 bp inserts using adult male samples, and the samples were sequenced using the Illumina platform [72]. Using adult males, a PE RNA-seq library was created, and the PromethION48 platform was used for the sequencing process. Genome size was estimated by K-mer analysis using short reads generated from the Illumina platform with a short insert size of 350 bp and a K-mer size of 19.
Genome assembly and Hi-C scaffolding
To create the genome sequences, high-accuracy circular consensus sequencing (CCS) data were assembled using hifiasm (v 0.16) [73] (The detailed information of the software and database involved in genomic analysis is all organized in Additional file 2: Table S14). After the Hi-C sequencing data were preliminarily filtered and evaluated using Hi-C-Pro v2.10.0 [74], for anchored contigs, BWA v0.7.10-r789 [75] was used to align the clean reads to the draft genome. Next, the contigs were clustered using the hierarchical clustering method in Lachesis, and subsequently, Lachesis was subsequently applied to order and orient the clustered contigs to perform the Hi-C-associated scaffolding [76]. By matching short reads from the Illumina platform to the genome and utilizing a set of 1367 insect BUSCOs with the BUSCO v5.2.2 software [77], the completeness of the genome assembly was evaluated.
To further confirm the chromosome number of B. dioryctriae, karyotype analysis was conducted. A modified variant of the procedure was used to acquire chromosome preparations from the cerebral ganglia of prepupae [78]. Images were obtained using an Olympus orthotopic fluorescence microscope (Olympus Corp., Tokyo, Japan).
Repeat annotation
Using both de novo and homology-based methods, repetitive sequences and TEs were found across the genome. The first step was to use RepeatModeler v2.0.1 [79] to customize a de novo repeat library of the genome. LTRharvest v1.5.9 [80] and LTRfinder v2.8 [81] were then used to create high-quality intact FL-LTR-RTs and nonredundant LTR libraries. The last TE sequences in the genome of B. dioryctriae were located and categorized using RepeatMasker v4.12 [82] to perform a homology search against the library above with the known Dfam v3.5 database [83]. Tandem Repeats Finder TRF 409 [84] and the MicroSAtellite identification tool MISA v2.1 [85] were used to annotate tandem repeats.
Prediction and functional annotation of protein-coding genes
Three techniques were used to predict gene structures: homology-based (Hisat v2.1.0 [86], and Stringtie v2.1.4 [87]), transcriptome-based (Hisat v2.1.0 [86]), and de novo (Augustus v3.1.0 [88], and SNAP 2006–07-28 [89]). In the homolog-based method, the genome of B. dioryctriae was first mapped using GeMoMa v1.7 [90] to contain protein sequences from nine sequenced Hymenoptera insect species: Apis mellifera, A. gifuensis, C. cunea, E. hayati, Gonatopus flavifemur, N. vitripennis, P. puparum, and Trichogramma pretiosum. PASA v2.4.1 [91] updated the gene models that were merged from these various approaches using EVM software v1.1.1 [92]. Genes related to transfer RNA (tRNA) were found using the tRNAscan-SE v1.3.1 [93] method, whereas genes related to ribosomal RNA (rRNA) were found using the barrnap v0.9 [94] program with default parameters. Furthermore, Infernal v1.1 [95] was used to identify microRNAs (miRNAs) and small nuclear RNAs (snRNAs) using default parameters against the Rfam v14.5 [96].
Gene functional annotation was performed based on homolog searches and the best matches to the databases of the National Center for Biotechnology Information (NCBI) Non-Redundant (NR), Evolutionary genealogy of genes: Non-supervised Orthologous Groups (EggNOG), GO, EuKaryotic Orthologous Groups (KOG), TrEMBL, Pfam, KEGG and Swiss-Prot protein databases using diamond Basic Local Alignment Search Tool Protein (BLASTP) v2.0.4.142 [97] with an E-value threshold of 1E−5.
Chromosomal synteny analysis
The gene sequences of B. dioryctriae, C. cunea, and N. vitripennis were aligned using diamond v0.9.29.130 [98] to detect comparable gene pairings (E value < 1e−5, P < 0.05) in order to discover collinear gene blocks among these three species. A C score greater than 0.5 was employed as a filter criterion (the C score was filtered using JCVI software), and MCScan [99] in JCVI v0.9.13 [100] with default parameters was utilized to show high-quality blocks.
Comparative genomics
Using OrthoFinder v2.4 [101], orthologous and paralogous genes were identified in B. dioryctriae and 13 additional insect species (Additional file 2: Table S3), and were subsequently annotated using the PANTHER v18.0 [102]. The single-copy orthologous genes’ protein sequences were aligned using the MAFFT v7.205 software [103]. The bootstrap phylogenetic tree was constructed by ML using IQTree v1.6.11 [104], and the best model, “JTTDCMut + F + I + G4,” was estimated by ModelFinder. Divergence times were computed using the MCMCTREE package included in PAML v4.9i [105]. The calibration times were taken from the TimeTree website (http://www.timetree.org/) and were based on the following six cases: A. rosae vs. C. cunea (223.7–304.0 Ma), C. cunea vs. O. abietinus (187.9–272.5 Ma), O. biroi vs. A. gifuensis (162.4–219.3 Ma), A. gifuensis vs. T. pretiosum (139.2–253.9 Ma), E. hayati vs. T. pretiosum (107.4–241.6 Ma), and O. biroi vs. A. mellifera (100.3–163.5 Ma). The graphical representation was generated using MCMCTreeR v1.1 [106].
Gene family expansion and contraction were assessed using CAFE v4.2 [107] based on the identified gene families and the generated phylogenetic tree with the projected divergence periods of those species. Gene families with notable expansion and contraction with Viterbi P < 0.05 were subjected to KEGG pathway enrichment and GO analysis using the R program cluster Profiler.
Identification and localization of BdioOBP genes in the genome
For OBP gene annotation, a set of reference protein sequences (Additional file 2: Table S15) was used to identify candidate BdioOBP sequences through BLASTx (E value < 1e−5) in the genome of B. dioryctriae. Then, the candidate sequences were identified using PfamScan against the Pfam database. Chromosome genes were mapped and visualized using TBtoolsII's “Gene Location Visualization from GTF/GFF” function [108].
Heatmap construction
Using Bowtie, the sequencing reads were aligned with the unigene dataset, and expectation maximization (RSEM) was used to assess the expression levels in conjunction with RNA-seq. The expression abundance of the relevant gene transcripts was represented by the FPKM. Using log10 (FPKM + 1) values [109], heatmaps of differentially expressed BdioOBPs were created using the OmicShare application (https://www.omicshare.com).
RT-qPCR analysis and validation
RT-qPCR was used to confirm the expression patterns of the highly expressed gene BdioOBP45 in different tissues. To standardize the expression data, ribosomal protein L18 (BdioRPL18) was used as a reference gene. Primer 3 (https://bioinfo.ut.ee/primer3-0.4.0/) was used to design specific primers, which are listed in Additional file 2: Table S16. Subsequently, qPCR was carried out in accordance with the manufacturer’s instructions using the LightCycler 480 II Detection System (Roche, Shanghai, China) and TransStar Tip Top Green qPCR Supermix (TransGen Biotech, Beijing, China). Each qPCR reaction was used according to the manufacturer’s instructions in a 20-µL reaction volume (0.8 µL of primer (forward and reverse, each 0.2 µM), 10 µL of 2 × TransStar Top Green qPCR SuperMix, and 9.2 µL of RT product), and the conditions were as follows: 94 ℃ for 30 s, followed by 45 cycles of 94 ℃ for 5 s, 55 ℃ for 15 s, and 72 ℃ for 10 s. Then, 95 ℃ for 5 s, 65 ℃ for 1 min, 97 ℃ for 10 s, and 60 ℃ for 15 s were used to measure the melt curve. The RT-qPCR analysis was conducted using SPSS Statistics 26.0 to process 2−ΔΔCT results. Comparisons of the quantitative expression levels of BdioOBP45 in different tissues were performed using one-way ANOVA, followed by Tukey’s post hoc analysis (P < 0.05). Three technical and three biological replicates were used for each qPCR.
Gas chromatography–mass spectrometry analysis
The volatile compounds of Pinus koraiensis cones, pest-damaged P. koraiensis cones, P. koraiensis oleoresin subjected to mechanical damage, P. koraiensis oleoresin subjected to pest damage, and D. abietella pupae were analyzed by solid-phase microextraction (SPME). The SPME was performed using an autosampler equipped with a 2-cm 50/30 μm SPME fiber (Supelco, Bellefonte, PA, USA). TRACE 1310 gas chromatograph and TSQ 8000 mass spectrometer (Thermo Fisher Scientific, USA) were used for the analysis. Standard autotunes using perfluorotributylamine (PFTBA) were performed daily [110]. The separation conditions for the DB-5MS capillary column (Agilent Technologies, Santa Clara, CA, USA) were as follows: initial column temperature of 40 °C for 2 min, followed by a 6 °C/min climb to 250 °C, and a 4-min maintenance period. The carrier gas was high-purity helium (99.999%) flowing at a rate of 1 mL/min. Compounds detected in the analyzed samples were identified using the NIST 2014 mass spectral library. Each type of sample is measured with three replicate samples, and a blank control group is set up to ensure the quality of the detection.
Heterologous expression and purification of BdioOBP45
The full open reading frame of the BdioOBP45 gene was amplified by PCR using gene-specific primers (Additional file 2: Table S16) designed based on B. dioryctriae genome data. Putative full-length BdioOBP45 was subcloned and inserted into the expression vector pET30a (Novagen, Germany) with the restriction enzymes EcoRI and XhoI (New England BioLabs, Ipswich, England). The pET30a/BdioOBP45 plasmid was transformed into BL21 (DE3) for expression. After induction with 0.8 mM IPTG, the proteins were expressed and collected from the culture supernatant. Protein purity was confirmed by SDS-PAGE, and to avoid any alterations in protein folding, the 6 × His tag was deleted. For additional testing, the purified proteins were dissolved in buffer B (50 mM Tris–HCl, pH 7.4).
Fluorescence competitive binding assays
Using N-phenyl-1-naphthylamine (1-NPN) as a fluorescently labeled ligand, the binding ability of BdioOBP45 to 55 volatiles (Additional file 2: Table S13) was measured with a Fluoromax-4 fluorescence spectrometer. The excitation wavelength was set to 337 nm, and the emission spectrum was recorded between 380 and 520 nm. BdioOBP45 (2 μM dissolved in 50 mM Tris–HCl, pH = 7.4) and 1-NPN (2–20 μM dissolved in chromatographic methanol) were mixed, and then adding aliquots of a 1 mM methanol solution of ligand to final concentrations of 2–20 μM, record the strongest of each time fluorescence value. The dissociation constants of the competitors were calculated according to previous studies [111]and analyzed by GraphPad Prism v8.0. The results of three repetitions represent the mean ± SEM.
Y-tube olfactometer assay
To examine the biological effect of 1-octen-3-ol on B. dioryctriae, a binary Y-tube olfactometer (1.1 cm in diameter, 6 cm in base, 6 cm in arm length, and 60° angle between the arms) was used for this assay. One arm of the odorant source in this assay was 20 × 50 mm filter paper that contained 20 μL of the test chemicals. The other arm of the olfactometer was 20 × 50 mm filter paper that contained 20 μL of the paraffin oil. When a wasp penetrated an arm by at least 2/3 and remained there for 30 s, it was considered a “choice.” The data were classified as “no choice” if an insect did not decide within 5 min. The choice rate was calculated as Sr = the number of individuals with positive tropism/(all the tested wasps − wasps that did not make a choice) × 100% [38]. The tube was cleaned with dehydrated alcohol after each of the five wasp experiments and allowed to air dry, and the locations of the odor sources were switched. The differences in Y-tube decision behavior between the paraffin oil and test chemical groups were analyzed using a chi-square test.
RNA interference
As directed by the manufacturer, PCR products were transcribed in vitro using the T7 RiboMAX Express RNA interference (RNAi) system (Promega, USA) to produce the dsRNAs of BdioOBP45. To accomplish efficient silence, the newly emerging insects were injected on the first day of the adult stage, while their olfactory system was still developing. 460 ng of dsRNA were injected into the thorax using a NanoLiter 2000 injector. For the negative control, the green fluorescent protein (GFP) gene was utilized as an irrelevant gene. Using the same method as previously used to confirm the expression of olfactory genes, RT-qPCR was employed to measure the effectiveness of gene knockdown at various intervals (12 h, 24 h, 36 h, and 48 h) following interference.
Availability of data and materials
All the data generated in this study has been available at the National Center for Biotechnology Information (NCBI), under BioProject number PRJNA1099397 [112]. The genome assembly has been deposited at GenBank under accession JAGTPF000000000 [113]. The B. dioryctriae transcriptome data for genome annotation are available through NCBI BioProject (PRJNA881758) [114].
Abbreviations
- Hi-C:
-
High-through chromosome conformation capture
- OSN:
-
Olfactory sensory neuron
- OBP:
-
Odorant-binding protein
- CSP:
-
Chemosensory protein
- SNMP:
-
Sensory neuron membrane protein
- NPC2:
-
Niemann-Pick type C2 protein
- OR:
-
Odorant receptor
- 3-HA:
-
3-Hexenyl acetate
- BUSCO:
-
Benchmarking universal single-copy orthologs
- TE:
-
Transposable element
- CDS:
-
Coding sequence
- Chr1-Chr6:
-
Chromosome1-chromosome6
- ML:
-
Maximum likelihood
- IR:
-
Ionotropic receptor
- GR:
-
Gustatory receptor
- GO:
-
Gene ontology
- KEGG:
-
Kyoto encyclopedia of genes and genomes
- CLR:
-
C-type lectin receptor
- NLR:
-
NOD-like receptor
- FPKM:
-
Fragments per kilobase million
- RT-qPCR:
-
Real-time quantitative polymerase chain reaction
- 1-NPN:
-
1-N-phenyl-naphtylamine
- Kd:
-
Dissociation constant
- GC-MS:
-
Gas chromatography-mass spectrometry
- PRR:
-
Pattern recognition receptor
- PAMP:
-
Pathogen-associated molecular pattern
- DAMP:
-
Damage-associated molecular pattern
- HIPV:
-
Herbivore-induced plant volatile
- CTAB:
-
Cetyltrimethylammonium bromide
- PE:
-
Paired-end
- CCS:
-
Circular consensus sequencing
- tRNA:
-
Transfer RNA
- rRNA:
-
Ribosomal RNA
- miRNA:
-
MicroRNA
- snRNA:
-
Small nuclear RNA
- NCBI:
-
National center for biotechnology information
- NR:
-
Non-redundant
- EggNOG:
-
Evolutionary genealogy of genes: Non-supervised Orthologous Groups
- KOG:
-
EuKaryotic Orthologous Groups
- BLAST:
-
Basic local alignment search tool
- RSEM:
-
RNA-seq by expectation maximization
- SPME:
-
Solid-phase microextraction
- PFTBA:
-
Perfluorotributylamine
- RNAi:
-
RNA interference
- GFP:
-
Green fluorescent protein
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Acknowledgements
The authors would like to thank all the reviewers who participated in the review, as well as AJE (https://www.aje.cn/) for providing English editing services during the preparation of this manuscript.
Funding
This research was supported by the National Natural Science Foundation (32302432), the Science and Technology Development Program of Jilin Province (20230202096NC), the Fundamental Research Funds for the Central Universities (2412021QD010, 135111010), and the Young Science and Technology Talent Support Project of Jilin Province (QT202121).
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QC and BZR conceived and designed the experiments. XYZ, QYL, JL, YHZ, HBJ, and LWS collected all samples. XYZ and YY performed the investigation. XYZ, YY, JL, and LD analyzed the data. XYZ and QC wrote the manuscript. All authors have read and approved the final manuscript.
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12915_2024_1998_MOESM1_ESM.docx
Additional file 1: Figure S1. Genome size of B. dioryctriae estimated by k-mer analysis. The k-mer (k = 19) frequency profile was generated by GenomeScope v1.0.0, showing an estimated genome size of 486.86 Mb. Figure S2. Genome-wide Hi-C interaction maps of six pseudochromosomal linkage groups in B. dioryctriae. Figure S3. Expression levels of BdioOBP45 in different tissues. (FA and MA: female and male antennae; FH and MH: female and male heads without antennae; Fab: female abdomens without ovipositors and digestive tracts; Fov: female ovipositors; Mge: male genitalia; Mab: male abdomens without genitalia and digestive tracts; T: male and female thoraxes; L: male and female legs). Columns labeled with different letters are significantly different (p < 0.05). Figure S4. Expression and purification of BdioOBP45. M, protein marker; 1, before induction with IPTG; 2, after induction with IPTG; 3, after purification of the original protein; 4, after the His-tag was removed from the final protein. Figure S5. Chromatographic separation was carried out on a DB-5MS column. (A): TIC of P. koraiensis cones; (B): TIC of Pest-damaged P. koraiensis cones; (C): TIC of P. koraiensis oleoresin after mechanical damage; (D): TIC of P. koraiensis oleoresin after pest damage; (E): TIC of D. abietella pupae.
12915_2024_1998_MOESM2_ESM.xlsx
Additional file 2: Table S1. Genome sequencing of B.dioryctriaeusing PacBio. Table S2. Characteristics of repeat sequences in B.dioryctriae. Table S3. Published genomes of 13 Hymenoptera species used for analysis. Table S4. Comparsion of the assembled genome size and gene structure prediction among 9 hymenopteran insects. Table S5. Functional annotation of B.dioryctriae genome. Table S6. Chromosome synteny based on CDS pairwise alignment between B. dioryctriae and C. cunea. Table S7. Chromosome synteny based on CDS pairwise alignment between B. dioryctriae and N. vitripennis. Table S8. Gene family clusters of fourteen species genes. Table S9. Expanded gene family of B.dioryctriae. Table S10. Contracted gene family of B.dioryctriae. Table S11. Sequence information of identified OBPs in B.dioryctriae. Table S12. Identification of volatile compounds from P. koraiensis cones; Pest-damaged P. koraiensis cones; P. koraiensis oleoresin by mechanical damage; P. koraiensis oleoresin by pest damage and D. abietella pupae. Table S13. Detailed Information of the Compounds for Testing against BdioOBP45. Table S14. Software modes and database sources. Table S15. The protein names and sequences of OBPs that were used in identification candidate BdioOBPs. Table S16. Specific primers of BdioOBP45.
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Zhu, X., Yang, Y., Li, Q. et al. An expanded odorant-binding protein mediates host cue detection in the parasitic wasp Baryscapus dioryctriae basis of the chromosome-level genome assembly analysis. BMC Biol 22, 196 (2024). https://doi.org/10.1186/s12915-024-01998-8
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DOI: https://doi.org/10.1186/s12915-024-01998-8