Research Article |
Corresponding author: Norman F. Johnson ( baeus2@yahoo.com ) Academic editor: Elijah Talamas
© 2021 Kendall King, Megan E. Meuti, Norman F. Johnson.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
King K, Meuti ME, Johnson NF (2021) Identification and expression of odorant binding proteins in the egg-parasitoid Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae, Telenominae). In: Lahey Z, Talamas E (Eds) Advances in the Systematics of Platygastroidea III. Journal of Hymenoptera Research 87: 251-266. https://doi.org/10.3897/jhr.87.68954
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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.
chemical ecology, chemosenses, expression profiles, OBP, olfaction, Platygastroidea, semiochemicals
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 (
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 (
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) (
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 (
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.
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.
OBP amino acid sequences from 14 species across the order Hymenoptera (Table
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 |
A total of 136 OBP sequences for Trissolcus basalis, Cephus cinctus Norton (Cephidae;
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
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 (
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.
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
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.
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
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
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.
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.
The repertoire size of OBP-coding genes varies across characterized Hymenoptera genomes, from 14 genes in Linepithema humile to 90 in Nasonia vitripennis (
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.
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 (
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 (
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 (
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.
TbOBP sequences; OBP sequences for gene tree; qPCR primers
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.