Research Article
Print
Research Article
Identification and expression of odorant binding proteins in the egg-parasitoid Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae, Telenominae)
expand article infoKendall King, Megan E. Meuti, Norman F. Johnson
‡ Ohio State University, Columbus, United States of America
Open Access

Abstract

Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae) is an egg-parasitoid of the southern green stink bug, Nezara viridula (Linneaus) (Hemiptera, Pentatomidae). Many behaviors associated with female T. basalis host-finding and acceptance are mediated by chemosensory pathways, for which olfactory, gustatory and ionotropic receptors have been previously identified. Odorant binding proteins (OBPs) are small, globular proteins, one of the functions of which is the transport of odorant ligands through the aqueous lymph of chemosensory sensilla to these receptors. We identified 18 classical OBP sequences in the T. basalis genome and transcriptomes sharing an average 26.8% pairwise identity. Gene tree analyses suggest very limited lineage-specific expansion and identify potential orthologs among other scelionids and Hymenoptera. Transcriptome mapping and qPCR comparison of expression levels in antennae and bodies of both sexes determine that at least five TbOBPs are preferentially expressed in the female antennae. These are, therefore, prime candidates for further study to determine their role in detecting host-produced semiochemicals.

Keywords

chemical ecology, chemosenses, expression profiles, OBP, olfaction, Platygastroidea, semiochemicals

Introduction

The subfamily Telenominae (Hymenoptera, Scelionidae) encompasses nearly a thousand species of egg-parasitoids, many of which have proven to be valuable as biological control agents of their host insects (Laumann et al. 2008; Talamas et al. 2015; Sacaccini et al. 2020). Numerous studies have demonstrated that these minute wasps (generally 0.5–2.5 mm in total length) locate eggs to attack by orienting to semiochemicals produced by their adult hosts. These include sex pheromones, alarm pheromones, aggregation pheromones, and cuticular hydrocarbons (Isidoro et al. 2001; Yang et al. 2016). The host specificity of the wasps presumably is mediated by their ability to detect and respond appropriately to these chemicals. In gregarious species (the vast majority), telenomines mate on the host eggs from which the wasps emerge, suggesting that evolution of the chemosensory ability of the female wasp to find eggs may also be a driver of the speciation mechanisms that have resulted in their high species richness (Loch and Walter 2002).

Trissolcus basalis (Wollaston) is a parasitoid of the southern green stink bug, Nezara viridula (Linnaeus) (Hemiptera, Pentatomidae). In captivity, the wasps can successfully parasitize other hosts that are presented to them, but data from specimens collected in the field suggest that they specialize on parasitizing N. viridula (Johnson 1985). Behavioral experiments have demonstrated that females of T. basalis respond to the plant volatiles 3-methylbutanoic acid and p-benzoquinone (Slimani et al. 2016); to the N. viridula kairomones (E)-2-decenal, 4-oxo-(E)-hexenal, and the sesquiterpenes trans- and cis-(Z)-alpha-bisabolene epoxide; the stink bug cuticular hydrocarbon n-nonadecane; and to mucopolysaccharides on the surface of an egg mass (Bin et al. 1993; Mattiacci et al. 1993; Colazza et al. 2007; Laumann et al. 2009).

The chemosensory mechanisms of T. basalis are only beginning to be explored. Previously, we characterized the full repertoire of receptor proteins in this species based on genomic and transcriptomic data: there are 170 olfactory receptors (Ors), 1 copy of Orco, 34 gustatory receptors (Grs), and 23 ionotropic receptors (Irs) (Chen et al. 2021). Several of the Ors are preferentially expressed in the antennae of adult females, suggesting that at least some of these proteins may be tuned to semiochemicals important for host location in the environment. Narrow tuning of the Ors could contribute to the host specificity of this wasp (Andersson et al. 2015).

Before reaching the receptors in the dendritic membrane of the olfactory sensory neurons (OSNs), environmental odorants first interact with another set of proteins, the odorant binding proteins (OBPs). Recent review papers discuss the relevance of OBPs and their role in insect olfaction (Pelosi et al. 2018; Rihani et al. 2021). OBPs may have a generalized function to facilitate the movement of a broad range of hydrophobic odorants to the OSN dendrite through the aqueous sensillar lymph (Vogt et al. 1991; Pelosi et al. 2005; Sun et al. 2016). A number of cases have documented that a subset of OBPs, the pheromone-binding proteins, are themselves narrowly tuned to bind with a small number of compounds (Du and Prestwich 1995; Campanacci et al. 2001; Zhou 2010). OBPs may thus act as an initial filter to limit which odorants can reach and be detected by a specific neuron. Narrow tuning of the response of an OSN may be a function of specificity of the Ors, of the OBPs, or a combination of the two (Andersson et al. 2015).

The objectives of this work were to identify and characterize the OBPs of T. basalis, to compare expression levels between males and females, and to determine which, if any, are more highly expressed in the primary olfactory organs, the antennae. The ultimate goal is to integrate information on OBPs with that of the receptor proteins to better understand the physiological mechanisms by which these specialist parasitoids are able to find their hosts in nature.

Materials and methods

Insects

A Trissolcus basalis colony originating from the southern U.S.A. was reared on eggs of the spined soldier bug, Podisus maculiventris (Say) (Hemiptera, Pentatomidae). The bugs were maintained at 25 °C, 70% relative humidity, and fed a diet of mealworm larvae (Tenebrio molitor Linnaeus, Coleoptera: Tenebrionidae). The Podisus colony was kept in a non-diapause state using a 12–12 hr light-dark schedule. Mated adult female wasps were removed from the colony and introduced to fresh Podisus eggs, and the parasitized eggs were kept separately in vials closed with cotton for roughly 14 days until the adult wasps emerged. Adult wasps were maintained on an alternating diet of simple syrups made from refined white sugar and natural honey. All wasps used for the transcriptome assemblies and gene expression assays were frozen at -80 °C; none of the frozen wasps had been exposed to fresh eggs. Female wasps were frozen the day of their emergence along with any males that may have already emerged.

Protein characterization and gene annotation

OBP amino acid sequences from 14 species across the order Hymenoptera (Table 1) were downloaded from the GenBank non-redundant protein database (https://www.ncbi.nlm.nih.gov/genbank) and used as tblastn queries to search local BLAST databases of the Trissolcus basalis transcriptomes (Genbank accession PRJNA625932) and genome (PRJNA49235) (Altschul et al. 1990; Chen et al. 2021). Putative OBP sequences were annotated in the CLC Main Workbench version 21.0.4 and confirmed as OBPs with searches in Pfam version 34.0 (http://pfam.xfam.org/) and Interproscan version 86.0 (https://www.ebi.ac.uk/interpro/). Signal peptide lengths were predicted with SignalP5.0 (http://www.cbs.dtu.dk/services/SignalP), and the molecular weights of each protein were predicted using BioInformatics calculator (https://www.bioinformatics.org/sms/index.html).

Table 1.

List of taxa of OBPs used to query Trissolcus basalis transcriptome and genome local BLAST databases.

Family Species No. of OBPs from GenBank
Cephidae Cephus cinctus 26
Orussidae Orussus abietinus 5
Bethylidae Sclerodermus sp. 10
Formicidae Solenopsis richteri 8
Apidae Apis cerana 27
Apidae Apis mellifera 34
Braconidae Aphidius gifuensis 11
Braconidae Diachasma alloeum 16
Braconidae Microplitis mediator 7
Aphelinidae Encarsia formosa 39
Encyrtidae Copidosoma floridanum 6
Pteromalidae Nasonia vitripennis 84
Trichogrammatidae Trichogramma japonicum 9
Scelionidae Telenomus podisi 3
Scelionidae Trissolcus japonicus 3

Gene tree analysis

A total of 136 OBP sequences for Trissolcus basalis, Cephus cinctus Norton (Cephidae; Robertson et al. 2018), Apis mellifera Linnaeus (Apidae) (Forêt and Maleszka 2006), Nasonia vitripennis (Walker)(Pteromalidae)(Vieira et al. 2012), Trissolcus japonicus (Ashmead) and Telenomus podisi Ashmead (Farias et al. 2015) were aligned with MUSCLE version 3.8.31 (Edgar 2004) in MegaX version 11.0 (https://www.megasoftware.net/) using the default settings. Accession numbers for each protein included in the analysis may be found in Suppl. material 1 in the HymOBP tab. OBPs of different species are distinguished by prefixing the string OBP## with the first letters of the genus and species name of each taxon: Tb, Cc, Am, Nv, Tj, or Tp. The relationships among the aligned sequences were then analyzed in a maximum likelihood context in MegaX using the amino acid substitution model LG+G (Le and Gascuel 2008, Tamura et al. 2013) based on MegaX’s best-fit analysis, with 1000 bootstrap replicates. CcOBP12 was chosen as the root based on a nearest neighbor analysis of OBPs of Cephus cinctus, the outgroup.

Measurement of OBP expression levels

Preliminary relative expression levels were made with Geneious 20.2.3 (https://www.geneious.com) by mapping RNA-seq reads to the TbOBP sequences for all transcriptomes. Those results were followed up by measuring expression levels using quantitative real-time PCR (qPCR).

Antennae were removed from the bodies from 500 male and 500 female specimens using fine-tipped forceps. These were divided into five biological replicates for each combination of sex and tissue: male antennae (100 antennae), female antennae (100 antennae), male bodies (five bodies), and female bodies (five bodies). RNA was extracted using 500 µL TRIzol Reagent following the manufacturer’s protocol and purified using phenol-chloroform. RNA concentration (0.3–3.0 µL) was measured using Qubit RNA HS Assay Kits, and 20 µL cDNA was synthesized from input of 92 pg of RNA using ThermoScientific Maxima First Strand cDNA Synthesis Kit according to the manufacturer’s instructions and diluted in 80 µL H2O for qPCR.

Two pairs of primers were designed for each OBP using Primer3Plus v2.4.2 (https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Each primer set is 20 bp in length with a melting temperature of 60±0.3 °C (Table 2). Primer specificity and performance were confirmed using melt and standard curves to ensure that each primer set met MIQE guidelines (Bustin et al. 2009). The relatively short sequences for these proteins limited the possibilities for more primer design. Primers for seven TbOBP nucleotide sequences – TbOBP5–9, TbOBP12, TbOBP15 – did not fall within a tested efficiency range between 80–110 %, and no results for those are presented. Features of the primers used in the experiments are presented in Suppl. material 1: qPCR Primers tab.

Table 2.

Odorant binding proteins of Trissolcus basalis.

Gene name GenBank accession Molecular Weight (kD) Signal peptide Scaffold: position Number of exons Length (bp)
TbOBP1 MZ488428 16.15 1–20 scaffold_1144: 9533–10939 5 438
TbOBP2 MZ488429 15.82 N/A scaffold_102: 26609–25696 4 396
TbOBP3 MZ488430 14.56 1–17 scaffold_1037: 13416–15382 5 405
TbOBP4 MZ488431 16.16 1–21 scaffold_159: 35484–34592 6 390
TbOBP5 MZ488432 16.39 1–22 scaffold_159: 41468–40636 6 432
TbOBP6 MZ488433 16.04 1–21 scaffold_4916: 3562–2759 5 420
TbOBP7 MZ488434 18.53 1–20 scaffold_353: 17641–18623 5 498
TbOBP8 MZ488435 16.91 1–16 scaffold_353: 21945–21153 5 446
TbOBP9 MZ488436 15.17 1–19 scaffold_159: 39147–37845 5 411
TbOBP10 MZ488437 15.61 1–20 scaffold_353: 19949–19112 5 417
TbOBP11 MZ488438 14.5 1–20 scaffold_353: 20230–20931 5 390
TbOBP12 MZ488439 15.47 1–22 scaffold_1177: 26917–26077 5 423
TbOBP13 MZ488440 15.2 1–19 scaffold_165: 6291–5476 5 390
TbOBP14 MZ488441 15.76 1–20 scaffold_353: 23655–24502 5 420
TbOBP15 MZ488442 22.71 1–30 scaffold_32: 232386–231315 6 591
TbOBP16 MZ488442 17.17 1–20 scaffold_159: 42798–41880 7 468
TbOBP17 MZ488444 17.14 1–20 scaffold_496: 26802–28032 6 465
TbOBP18 MZ488445 18.96 1–21 scaffold_79: 22955–21677 5 489

Relative expression levels were measured using BioRad CFX qPCR instruments and CFX Maestro software 1.1 (https://www.bio-rad.com). In brief, all reactions were run in triplicate, each in a total volume of 10 µl containing 5 µL of iTaq Universal SYBR Green Supermix (BioRad), 400 nm of primer, and 1 µL of cDNA on a 96-well plate. The relative cycle threshold (CT) of the technical replicates was averaged for each biological replicate. The resulting CT value for each OBP was normalized to the geometric average of the CT values of three reference genes RpL32 (ribosomal protein L32), RpL19 (ribosomal protein L19), and the nuclear ribosomal gene 18S by subtracting the geometric average CT of reference genes for each biological replicate from the CT value for the gene of interest within that biological replicate using the 2–ΔΔCT method (Livak and Schmittgen 2001).

Statistical analysis

All statistical analyses were done in R version 4.0.5 (https://www.R-project.org/). Statistical significance in the mean expression level of each tissue type relative to others was determined with an ANOVA at an alpha value of 0.05 for eleven TbOBPs. Mean separations were assessed with Tukey’s HSD test.

Results

Identification and classification of Trissolcus basalis OBPs>

Eighteen TbOBP sequences were identified, ranging in length from 290–591 bp (96–197 aa), and having an average pairwise amino acid identity of 26.8% (Table 2, Suppl. material 1: 1: TbOBPs tab). Sixteen of these sequences were identified as belonging to the PB_GOBP family in Pfam searches; TbOBP6 and TbOBP7 were not assigned to family in the Pfam query but were characterized as belonging to the PB_GOP superfamily in InterProScan. All TbOBPs exhibit the Classical OBP pattern of C1-X15-39-C2-X3-C3-X21-44-C4-X7-12-C5-X8-C6 (Bohbot and Vogt 2005) with six conserved cysteine residues. The molecular weights range from 14.5–22.7 kD. Although both atypical OBPs and C-plus OBPs (Xu et al. 2003; Spinelli et al. 2012) were used as queries, none of the sequences found in the T. basalis genome or transcriptomes display those motifs. Signal peptides were measured within the first 30 bp of each TbOBP with the exception of TbOBP2, for which no signal peptide or cleavage site was detected. Nine of the TbOBPs were found on just two genome scaffolds (Fig. 1). Scaffold_159 is 79,446 bp in length and contains TbOBP9, TbOBP5, TbOBP16, and TbOBP4 within an extent of 4,555 bp. Scaffold_353 is 86,326 bp in length and contains TbOBP7, TbOBP10, TbOBP11, TbOBP8, and TbOBP14 in a span of 6,861 bp. Only one protein-coding gene was found between any two of these TbOBP sequences within the same scaffold: between TbOBP8 and TbOBP14 on scaffold_353 is gene TRIBAS_03633 of unknown family and function.

Figure 1. 

Position and orientation of syntenic TbOBPs present on genomic scaffolds 159 and 353. Yellow connected ribbon segments represent exon regions of each TbOBP. The black vertical lines labeled with gene names are located at the 5’ end of each sequence.

Phylogenetic analysis of Hymenoptera OBPs

The results of the maximum likelihood analysis of the relationship between OBPs in Trissolcus basalis and those of Cephus cinctus, Nasonia vitripennis, Apis mellifera, and the few OBPs so far characterized for the telenomines Trissolcus japonicus and Telenomus podisi are presented in Fig. 2. The low boostrap values in some internal nodes resulted from low similarities among the analyzed OBP sequences. TbOBP6, TbOBP7, and TbOBP14 form a small lineage-specific expansion in T. basalis (98% support). Similarly, TbOBP16 and TbOBP17 cluster together as a pair (99% support). Possible orthologs with the other telenomines are TbOBP3 and TjOBP1 (100%); TbOBP4 and TjOBP3 (99%); and TbOBP5 and TpOBP3 (99%). TbOBP1 and TjOBP2 (99%) group together along with possible orthologs in Cephus, Nasonia and Apis (86% bootstrap support). Beyond its immediate close relatives in the Telenominae, TbOBP2 groups together with NvOBP64 and CcOBP3 (97%).

Figure 2. 

Maximum likelihood tree illustrating relationships among 136 OBP sequences from Nasonia vitripennis (Nv), Apis mellifera (Am), Cephus cinctus (Cc), Trissolcus japonicus (Tj), Telenomus podisi (Tp), and Trissolcus basalis (Tb). The tree is rooted with CcOBP12. TbOBPs are depicted in bold text and branches highlighted in green, other telenomine species are highlighted in blue. TbOBPs that cluster with single-copy orthologs (SCO), paralog group 2 (P2), and the OBP59a group of McKenzie et al. (2014) are highlighted in yellow, pink, and purple respectively. All bootstrap support values ≥ 85% are indicated at the relevant nodes; lower values are reported to clarify support level at other selected nodes.

Expression of TbOBPs

The number of RNA-seq in each of the four transcriptomes were mapped to the TbOBP sequences as initial measurements of relative expression levels (Table 3). Expression levels are reported as Normalized TPM (transcripts per million, Wagner et al. 2012). Four OBPs are expressed at higher levels in the male antenna than the female antenna: TbOBP1, TbOBP4, TbOBP5, and TbOBP6 (log2 ratio of expression levels > 2). Eight OBPs are expressed at higher levels in the female antenna than the male antenna: TbOBP8, TbOBP10–13, TbOBP15–17. In the rest of the body, male-biased expression levels are suggested in TbOBP4–5, and female-biased expression in TbOBP10–11 and TbOBP16–17. The TbOBPs with sex-biased expression levels in the body also show the same bias in the antennae.

Table 3.

Mapping of transcriptome reads to TbOBP sequences.

Gene Normalized TPM ♂/♀ Ratio (log2)
FA MA FB MB Antenna Body
TbOBP1 37320.61 218172.8 814.31 2380.81 2.55 1.55
TbOBP2 16413.96 19051.54 351804.3 228136.8 0.21 -0.62
TbOBP3 313.56 465.64 13810.42 9442.63 0.57 -0.55
TbOBP4 15474.3 64711.87 7714.45 38576.66 2.06 2.32
TbOBP5 884.06 5663.17 5.62 32.07 2.68 2.51
TbOBP6 9341.61 29195.28 10075 10901.98 1.64 0.11
TbOBP7 27516.63 2332543 417526.7 543738.2 3.08 0.38
TbOBP8 126899.5 1510.92 3215.15 2415.79 -6.39 -0.41
TbOBP9 9551.89 9551.9 84336.73 77414.59 0 -0.12
TbOBP10 17278.82 205.73 738.95 318.47 -6.39 -1.21
TbOBP11 16857.74 165.63 429.27 103.64 -6.67 -2.05
TbOBP12 28.64 20.1 17.2 3344.31 -2.11 7.6
TbOBP13 7260.78 23.43 111.99 29.61 -8.28 -1.92
TbOBP14 84488.98 113486.4 57463.33 101428.3 0.73 0.82
TbOBP15 101067 4035.22 4162.92 4955.34 -4.65 0.25
TbOBP16 429238.4 1467.28 6210.96 323.25 -8.19 -4.26
TbOBP17 524941.4 1504.04 7367.66 511.59 -8.45 -3.85
TbOBP18 0 11.42 5046.14 5970.07 0.24

The transcriptome mapping represents only a single sample of OBP expression levels. Therefore, we used qPCR to confirm that the patterns of expression identified in the transcriptome were consistent in multiple, independent samples.

Expression levels of individual TbOBP genes are presented in Fig. 3. Six of the 11 OBPs show female-biased antennal expression and were significantly more abundant in female antennae than any other tissue: TbOBP1, TbOBP10–11, TbOBP13, and TbOBP16–17. There was no male-biased expression of OBPs in the antennae. Seven OPBs are expressed at higher levels in the male bodies than in the female bodies: TbOBP1, TbOBP4, TbOBP10–11, TbOBP14, TbOBP16–17. None of the qPCR experiments showed female-biased body expression.

Figure 3. 

Expression profiles for 11 TbOBPs across four tissue types as measured with qPCR: female antennae (FA), female bodies (FB), male antennae (MA), and male bodies (MB). Black data points: normalized expression (log2) for each of five biological replicates per tissue; red dots: mean expression level. Significance differences (p < 0.05) are illustrated by differences in letters, calculated using TukeyHSD ANOVA means comparison.

Discussion

The repertoire size of OBP-coding genes varies across characterized Hymenoptera genomes, from 14 genes in Linepithema humile to 90 in Nasonia vitripennis (Vieira et al. 2012; McKenzie et al. 2014). Among insects, the number of OBP-coding genes ranges widely, with over 100 OBPs reported from both of the mosquitoes Culex quinquefasciatus and Aedes aegypti (Manoharan et al. 2013; Rehani et al. 2021). The repertoire of 18 Classical OBPs we found in the genome of Trissolcus basalis, therefore, is on the low end of this spectrum. There are several different motifs among OBPs; beyond the Classical OBPs, non-classical motifs include dimer-OBPs with two sets of 6 conserved cysteines, C-plus with 8 conserved cysteines, C-minus with 4 conserved cysteines, and Atypical with 9–10 cysteines and a long terminus (Zhou 2010). Although it is common to find OBPs that fall within non-classical motifs, it is not unusual to find only sequences with the six conserved cysteine sites consistent with the Classical OBP motif (Gu et al. 2011; Lagarde et al. 2011; Zheng et al. 2016).

The small number of OBPs in Trissolcus basalis limits the possible number and size of lineage-specific expansions in this species. Among the 18 TbOBPs and based on high bootstrap values, we have confidence for the existence of only one pair and one trio of genes. Therefore, in contrast with a species such as Nasonia vitripennis, evolution in the number of OBPs in Trissolcus has been fairly conservative. This is also apparent in our gene tree analysis, which resulted in few lineage-specific expansions among T. basalis OBPs.

McKenzie et al. (2014) recognized four groups of OBPs from ants that they named the single-copy ortholog group, paralog group 1, paralog group 2, and the OBP59a group. They concluded that the two paralog groups were specific to aculeate Hymenoptera. A grouping of Trissolcus OBPs with one member of paralog group 2, AmOBP7, is suggested by the topology of the tree in Fig. 2. The bootstrap values for the nodes linking TbOBP14, TbOBP7, and TbOBP6 to that Apis protein have 0% bootstrap support, indicating that this clustering is spurious. The 4% bootstrap support for the node linking AmOBP7 and TbOBP13 is extremely low and may indicate that this association is also trivial. McKenzie et al. (2014) also reported that the Nasonia genome encodes a gene in the OBP59a group, and on the basis of their tree of hymenopteran OBPs in the supplemental information, we concluded that this was NvOBP64. A key finding in our analysis is that we found that TbOBP2 groups with NvOBP64 (97%), and the two are likely orthologs. Finally, each of the six lineages of OBPs in the single-copy ortholog group identified by McKenzie et al. (2014) were reported to have counterparts in Nasonia as well as in other groups outside of Hymenoptera. Therefore, they concluded that these genes probably date back at least to the origin of Holometabola. In our data, however, three of these lineages –OBP5, OBP10, OBP11 – have no clear ortholog in T. basalis. Of the three remaining, TbOBP1 clusters with AmOBP1 (43%); TbOBP4 with AmOBP6 (12%); and TbOBP12 with AmOBP9 and CcOBP11 (46%). The “single-copy orthologs” may be ancient as suggested by McKenzie et al. (2014), but our data suggest that not all of them have been retained in a recognizable form in the Scelionidae.

Our measures of relative expression levels in tissues and sexes differed in a few cases between transcriptome mapping and qPCR experiments. Only one transcriptome for each combination of sex and tissue was sequenced, and a mapping of transcriptome reads for each such combination is, therefore, only a single point estimate of the true expression level. In contrast, the qPCR experimental levels were calculated on the basis on five biological samples. We have greater confidence in the qPCR results, and some of the values derived from the mappings may be individual extremes. It is also possible that, despite our efforts to maintain constant rearing conditions, the conditions experienced by the wasps prior to collection may have differed in some manner that affected gene expression.

All OBPs found in the genome of T. basalis were recovered in at least one of the adult transcriptomes. This suggests that no OBPs are expressed solely in the immature stages of these parasitoids. Insofar as at least some these proteins are involved in chemosensation, this is not surprising. The larvae of T. basalis develop within the eggs of their hosts, a relatively homogeneous and isolated environment. We would anticipate, though, that in their more generalized function as encapsulins or transporters of hydrophobic ligands, OBPs would still play an important role in nutrition and development in all instars of the wasps (Ishida et al. 2013).

Our motivation in this work is to better understand the chemosensory role of OBPs. Therefore, our attention was focused on those proteins expressed at higher levels in the antennae, the structure that has been demonstrated to be necessary for host-finding and acceptance (Safavi 1968). Transcriptome mapping suggested four male-biased antennal genes: TbOBP1, TbOBP4, TbOBP5, and TbOBP7. However, qPCR measurements resulted in no evidence for higher expression of any OBP in the male antennae. TbOBP1 was, in fact, expressed at significantly higher levels in the female antennae, and TbOBP4 was expressed at relatively high levels (in comparison with the bodies) in the antennae of both sexes. TbOBP5 and TbOBP7 could not be measured by qPCR. The initial transcriptome mapping suggested eight genes preferentially expressed in the female antenna: TbOBP8, TbOBP10–13, and TbOBP15–17. Measurements using qPCR confirmed this expression pattern for TbOBP10–11, TbOBP13, and TbOBP16–17. TbOBP8, TbOBP12, and TbOBP15 could not be measured with qPCR. Genes TbOBP16 and TbOBP17 group together in our gene tree analysis but are found on different scaffolds, suggesting that they did not arise from a simple gene duplication. On scaffold_159, TbOBP16 is flanked on either side by TbOBP4 and TbOBP5, further isolating it from TbOBP17. In contrast, TbOBP8, TbOBP10 and TbOBP11 are syntenic on scaffold_353. TbOBP10 and TbOBP11 also group together on the gene tree and have similar expression profiles, possibly suggesting a common regulatory element.

Up-regulation of a subset of TbOBPs in the female antennae suggests to us that the proteins play a chemosensory role in mediating female-specific behaviors. These may include mate recognition, host habitat recognition, long-range host finding, short-range host acceptance, and recognition of marking pheromones applied after oviposition (Salerno et al. 2017; Slimani et al. 2017; Foti et al. 2019). With the data available, it is still unclear what role these proteins might play in T. basalis. We would predict that TbOBP1 has some general function in olfaction. We base this conjecture on the conservation of this gene across all Hymenoptera. Salerno et al. (2012) identified components of a female-produced sex pheromone in the species Trissolcus brochymenae (Ashmead) that stimulates intense antennation and mounting by males. Notably, we found no indication in the qPCR experiments of any TbOBP that is more highly expressed in the male antenna. We recognize that functionally important olfactory proteins may be expressed in low levels, and that high expression is not a requirement to be important and functional.

McKenzie et al. (2014) concluded that, in ants, the OBPs were “likely involved in conserved aspects of olfaction” and that the OBPs “may not rapidly evolve to recognize species-specific signals” (emphasis added). We need more information on the OBPs in other, related telenomines to be able to assess the birth and death rates of these genes and whether a subset of them have been similarly conserved in the evolution of the subfamily.

Acknowledgments

We thank Gerry Carter (The Ohio State University) for valuable discussions and advice concerning the statistical analysis of the qPCR experiments; and George Keeney and Jeni Ruisch (The Ohio State University) for husbandry of the wasp, stink bug, and mealworm colonies.

References

  • Bohbot J, Vogt RG (2005) Antennal expressed genes of the yellow fever mosquito (Aedes aegypti L.); characterization of odorant-binding protein 10 and takeout. Insect Biochemistry and Molecular Biology 35: 961–979. https://doi.org/10.1016/j.ibmb.2005.03.010
  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J (2009) The MIQE guidelines: minimum information for publication of Quantitative Real-Time PCR experiments. Clinical Chemistry 55: 611–622. https://doi.org/10.1373/clinchem.2008.112797
  • Campanacci V, Krieger J, Bette S, Sturgis JN, Lartigue A, Cambillau C, Breer H, Tegoni M (2001) Revisiting the specificity of Mamestra brassicae and Antheraea polyphemus pheromone-binding proteins with a fluorescence binding assay. Journal of Biological Chemistry 276: 20078–20084. https://doi.org/10.1074/jbc.M100713200
  • Chen H, Lahey Z, Talamas EJ, Johnson NF (2021) Identification and expression of chemosensory receptor genes in the egg parasitoid Trissolcus basalis. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics 37: e100758. https://doi.org/10.1016/j.cbd.2020.100758
  • Colazza S, Aquila G, De Pasquale C, Peri E, Millar JG (2007) The egg parasitoid Trissolcus basalis uses n-nonadecane, a cuticular hydrocarbon from its stink bug host Nezara viridula, to discriminate between female and male hosts. Journal of Chemical Ecology 33: 1405–1420. https://doi.org/10.1007/s10886-007-9300-7
  • Du G, Prestwich GD (1995) Protein structure encodes the ligand binding specificity in pheromone binding proteins. Biochemistry 34: 8726–8732. https://doi.org/10.1021/bi00027a023
  • Farias LR, Schimmelpfeng PH, Togawa RC, Costa MM, Grynberg P, Martins NF, Borges M, Blassioli-Moraes MC, Laumann RA, Bao SN, Paula DP (2015) Transcriptome-based identification of highly similar odorant-binding proteins among neotropical stink bugs and their egg parasitoid. PLoS ONE 10(7): e0132286. https://doi.org/10.1371/journal.pone.0132286
  • Forêt S, Maleszka R (2006) Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera). Genome research 16(11): 1404–1413. https://doi.org/10.1101/gr.5075706
  • Foti MC, Peri E, Wajnberg E, Colazza S, Rostás M (2019) Contrasting olfactory responses of two egg parasitoids to buckwheat floral scent are reflected in field parasitism rates. Journal of Pest Science 92: 747–756. https://doi.org/10.1007/s10340-018-1045-2
  • Gu SH, Wang SP, Zhang XY, Wu KM, Guo YY, Zhou JJ, Zhang YJ (2011) Identification and tissue distribution of odorant binding protein genes in the lucerne plant bug Adelphocoris lineolatus (Goeze). Insect Biochemistry and Molecular Biology 41: 254–263. https://doi.org/10.1016/j.ibmb.2011.01.002
  • Isidoro N, Romani R, Bin F (2001) Antennal multiporous sensilla: their gustatory features for host recognition in female parasitic wasps (Insecta, Hymenoptera: Platygastroidea). Microscopy Research and Technique 55: 350–358. https://doi.org/10.1002/jemt.1183
  • Johnson NF (1985) Systematics of new world Trissolcus (Hymenoptera: Scelionidae): species related to T. basalis. The Canadian Entomologist 117(4): 431–45. https://doi.org/10.4039/Ent117431-4
  • Lagarde A, Spinelli S, Qiao H, Tegoni M, Pelosi P, Cambillau C (2011) Crystal structure of a novel type of odorant-binding protein from Anopheles gambiae, belonging to the C-plus class. Biochemical Journal 437: 423–430. https://doi.org/10.1042/BJ20110522
  • Laumann RA, Moraes MC, Pareja M, Alarcão GC, Botelho AC, Maia AH, Leonardecz E, Borges M (2008) Comparative biology and functional response of Trissolcus spp. (Hymenoptera: Scelionidae) and implications for stink bugs (Hemiptera: Pentatomidae) biological control. Biological Control 44: 32–41. https://doi.org/10.1016/j.biocontrol.2007.10.003
  • Laumann RA, Santos de Aquino MF, Blassioli-Moraes MC, Pareja M, Borges M (2009) Response of the egg parasitoids Trissolcus basalis and Telenomus podisi to compounds from defensive secretions of stink bugs. Journal of Chemical Ecology 35: 8–19. https://doi.org/10.1007/s10886-008-9578-0
  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods 25(4): 402–408. https://doi.org/10.1006/meth.2001.1262
  • Loch AD, Walter GH (2002) Mating behavior of Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae): potential for outbreeding in a predominantly inbreeding species. Journal of Insect Behavior 15(1): 13–23. https://doi.org/10.1023/A:1014423910984
  • Manoharan M, Chong MNF, Vaïtinadapoulé A, Frumence E, Sowdhamini R, Offmann B (2013) Comparative genomics of odorant binding proteins in Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. Genome Biology and Evolution 5: 163–180. https://doi.org/10.1093/gbe/evs131
  • Mattiacci L, Vinson SB, Williams HJ, Aldrich JR, Bin F (1993) A long-range attractant kairomone for egg parasitoid Trissolcus basalis, isolated from defensive secretion of its host, Nezara viridula. Journal of Chemical Ecology 19: 1167–1181. https://doi.org/10.1007/BF00987378
  • McKenzie SK, Oxley PR, Kronauer DJ (2014) Comparative genomics and transcriptomics in ants provide new insights into the evolution and function of odorant binding and chemosensory proteins. BMC Genomics 15: e718. https://doi.org/10.1186/1471-2164-15-718
  • Pelosi P, Iovinella I, Zhu J, Wang G, Dani FR (2018) Beyond chemoreception: diverse tasks of soluble olfactory proteins in insects. Biological Reviews 93(1): 184–200. https://doi.org/10.1111/brv.12339
  • Robertson HM, Waterhouse RM, Walden KK, Ruzzante L, Reijnders MJ, Coates BS, Legeai F, Gress JC, Biyiklioglu S, Weaver DK, Wanner KW (2018) Genome sequence of the wheat stem sawfly, Cephus cinctus, representing an early-branching lineage of the Hymenoptera, illuminates evolution of hymenopteran chemoreceptors. Genome biology and evolution 10(11): 2997–3011. https://doi.org/10.1093/gbe/evy232
  • Safavi M (1968) Étude biologique et écologique des hyménoptères parasites des oeufs des punaises des céréales. Entomophaga 13: 381–495.
  • Salerno G, Frati F, Marino G, Ederli L, Pasqualini S, Loreto F, Colazza S, Centritto M (2017) Effects of water stress on emission of volatile organic compounds by Vicia faba, and consequences for attraction of the egg parasitoid Trissolcus basalis. Journal of Pest Science 90: 635–647. https://doi.org/10.1007/s10340-016-0830-z
  • Slimani T, Chung PK, Foti M, Rostás M, Peri E, Colazza S (2016) Single Sensillum Responses in Trissolcus basalis Females to Companion Plant Volatiles. Congresso Nazionale Italiano di Entomologia, 134 pp.
  • Slimani T, Bagarello V, Colazza S, Peri E, Park DKC (2017) Semiochemical exploitation by two egg parasitoids, Trissolcus basalis and Trissolcus brochymenae (Hymenoptera: Scelionidae). PhD Thesis. Università degli Studi di Palermo.
  • Scaccini D, Falagiarda M, Tortorici F, Martinez-Sañudo I, Tirello P, Reyes-Domínguez Y, Gallmetzer A, Tavella L, Zandigiacomo P, Duso C, Pozzebon A (2020) An insight into the role of Trissolcus mitsukurii as biological control agent of Halyomorpha halys in northeastern Italy. Insects 11: e306. https://doi.org/10.3390/insects11050306
  • Spinelli S, Lagarde A, Iovinella I, Legrand P, Tegoni M, Pelosi P, Cambillau C (2012) Crystal structure of Apis mellifera OBP14, a C-minus odorant-binding protein, and its complexes with odorant molecules. Insect Biochemistry and Molecular Biology 42: 41–50. https://doi.org/10.1016/j.ibmb.2011.10.005
  • Sun L, Wei Y, Zhang DD, Ma XY, Xiao Y, Zhang YN, Yang XM, Xiao Q, Guo YY, Zhang YJ (2016) The mouthparts enriched odorant binding protein 11 of the alfalfa plant bug Adelphocoris lineolatus displays a preferential binding behavior to host plant secondary metabolites. Frontiers in Physiology 7: e201. https://doi.org/10.3389/fphys.2016.00201
  • Talamas EJ, Herlihy MV, Dieckhoff C, Hoelmer KA, Buffington M, Bon MC, Weber DC (2015) Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae) emerges in North America. Journal of Hymenoptera Research 43: e119. https://doi.org/10.3897/JHR.43.4661
  • Vieira FG, Foreˆt S, He X, Rozas J, Field LM, Zhou JJ (2012) Unique features of odorant-binding proteins of the parasitoid wasp Nasonia vitripennis revealed by genome annotation and comparative analyses. PLoS ONE 7(8): e43034. https://doi.org/10.1371/journal.pone.0043034
  • Vogt RG, Prestwich GD, Lerner MR (1991) Odorant‐binding‐protein subfamilies associate with distinct classes of olfactory receptor neurons in insects. Journal of Neurobiology 22: 74–84. https://doi.org/10.1002/neu.480220108
  • Wagner GP, Kin K, Lynch VJ (2012) Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory in Biosciences 131(4): 281–285. https://doi.org/10.1007/s12064-012-0162-3
  • Xu PX, Zwiebel LJ, Smith DP (2003) Identification of a distinct family of genes encoding atypical odorant‐binding proteins in the malaria vector mosquito, Anopheles gambiae. Insect molecular biology 12(6): 549–560. https://doi.org/10.1046/j.1365-2583.2003.00440.x
  • Yang SY, Zhong YZ, Zhang JP, Wang XP, Zhang F (2016) A comparative scanning electron microscopy study on antennal sensilla of Trissolcus japonicus and Trissolcus plautiae, egg parasitoids of stink bugs (Pentatomidae). Annals of the Entomological Society of America 109: 112–120. https://doi.org/10.3389/fphys.2020.01058
  • Zheng ZC, Li DZ, Zhou A, Yi SC, Liu H, Wang MQ (2016) Predicted structure of a Minus-C OBP from Batocera horsfieldi (Hope) suggests an intermediate structure in evolution of OBPs. Scientific Reports 6: 1–11. https://doi.org/10.1038/srep33981

Supplementary material

Supplementary material 1 

TbOBP sequences; OBP sequences for gene tree; qPCR primers

Kendall King, Megan E. Meuti, Norman F. Johnson

Data type: protein sequences, Genbank acccession numbers, primer sequences

Explanation note: TbOBPs tab: amino acid sequences of Trissolcus basalis odorant binding proteins. HymOBPs tab: GenBank accession numbers of hymenopteran odorant binding proteins used in gene tree analysis. qPCR primers: primer sequences used in qPCR measurements of expression levels of Trissolcus basalis olfactory binding proteins.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (19.94 kb)
login to comment