Next Article in Journal
Genomic and Transcriptomic Analysis Reveals Cuticular Protein Genes Responding to Different Insecticides in Fall Armyworm Spodoptera frugiperda
Next Article in Special Issue
Stink Bug Communication and Signal Detection in a Plant Environment
Previous Article in Journal
Evaluation of Resistance Development in Bemisia tabaci Genn. (Homoptera: Aleyrodidae) in Cotton against Different Insecticides
Previous Article in Special Issue
Hermetia illucens (L.) (Diptera: Stratiomyidae) Odorant Binding Proteins and Their Interactions with Selected Volatile Organic Compounds: An In Silico Approach
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Insects
Volume 12
Issue 11
10.3390/insects12110998
insects-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Olfactory Sensilla and Olfactory Genes in the Parasitoid Wasp Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae)

by
Basman H. Al-Jalely
1,2 and
Wei Xu
1,*
1
Food Futures Institute, Murdoch University, Murdoch, WA 6150, Australia
2
College of Agricultural Engineering Sciences, University of Baghdad, Baghdad 10071, Iraq
*
Author to whom correspondence should be addressed.
Insects 2021, 12(11), 998; https://doi.org/10.3390/insects12110998
Submission received: 8 October 2021 / Revised: 30 October 2021 / Accepted: 2 November 2021 / Published: 5 November 2021
(This article belongs to the Collection Insect Sensory Biology)
Browse Figures
Review Reports Versions Notes

Abstract

:

Simple Summary

Parasitic wasps are the major natural enemies of many organisms, and therefore they are broadly used in the biological control of numerous agricultural and horticultural pests. For example, Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) is a tiny natural egg parasitoid of various agricultural pest insects, including Plutella xylostella, Helicoverpa armigera, Spodoptera frugiperda and Ectomyelois ceratoniae. However, how T. pretiosum seek and localise host insect eggs is still not clear. The olfactory system is critical in guiding insect behaviours, including mating, feeding and oviposition, which play pivotal roles in the interactions between parasitoid wasps and their hosts. This project aimed to investigate T. pretiosum major olfactory tissue (antennae) and the olfactory genes, including odorant binding proteins (OBPs) and odorant receptors (ORs). T. pretiosum adult antennae were examined under scanning electron microscopy, and four types of olfactory sensilla were observed. Using T. pretiosum genome, 22 OBPs and 105 ORs were identified, which were further compared with olfactory genes of other Hymenoptera insect species. The expression patterns of OBPs between T. pretiosum male and female adults were examined to identify female- or male-specific OBPs. This study enriches our knowledge of T. pretiosum olfactory system and will help better use it in the integrated pest management (IPM) for many insect pest species.

Abstract

Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) is a tiny natural egg parasitoid of several agricultural pest insects, which has been widely used in the biological control for Plutella xylostella, Helicoverpa armigera, Spodoptera frugiperda and Ectomyelois ceratoniae. However, limited studies have been conducted on T. pretiosum olfactory system, which is critical in regulating insect behaviours. In this study, T. pretiosum adult antennae were investigated under ascanning electron microscopy (SEM). Four types of olfactory sensilla were observed, including chaetica sensilla (CS), trichoid sensilla (TS), faleate sensilla (FS) and placoid sensilla (PS). Using T. pretiosum genome, 22 putative odorant binding proteins (OBPs) and 105 odorant receptors (ORs) were identified, which were further compared with olfactory genes of Apis mellifera, Nasonia vitripennis and Diachasma alloeum. The expression patterns of OBPs between T. pretiosum male and female adults were examined by quantitative real time PCR (qRT-PCR) approaches. Three female-specific OBPs (TpreOBP19, TpreOBP15 and TpreOBP3) were identified, which may play crucial roles in T. pretiosum host-seeking and oviposition behaviours. This study enriches our knowledge of T. pretiosum olfactory genes and improves our understanding of its olfactory system.

1. Introduction

Insect behaviours, including mating, foraging, host-finding and oviposition, are guided by their olfactory systems [1]. Hair-like olfactory sensilla distributed over the surface of antennae are utilised by insects to detect chemical signals from the environment [2,3,4]. With the advance of molecular and cellular biology, our understanding of insect olfactory mechanism has progressed. Various gene families have been reported to play pivotal roles in the dynamics, selectivity and sensitivity of insect olfactory systems, including odorant binding proteins (OBPs) [5], odorant receptors (ORs) [6,7], ionotropic receptors (IRs) [8], sensory neuron membrane proteins (SNMPs) [9] and odorant-degrading enzymes (ODEs) [5,10]. Hydrophobic odorants lack solubility, so they have difficulties in passing through the aqueous sensillum lymph and reaching receptors. OBPs, one class of proteins that are highly expressed in insect antennae, can bind, solubilise and deliver semiochemical molecules to ORs [11,12]. ORs are localised on the dendritic membrane in insect olfactory sensilla, detecting odorant compounds and transducing olfactory signals to insect brains to regulate behaviours [13]. ODEs can degrade the odorant compounds after they activate ORs, which clean the olfactory system for the new cycle of detection [5,10,14]. OBPs and ORs are involved in the first step of odorant detection, which are the target proteins in this study.
Parasitic wasps develop in or on various life stages of other arthropod hosts, and chemical signals are critical in guiding their mating, host-seeking and oviposition behaviours. For example, after mating, female wasps utilise host-associated chemical cues, including host pheromones or herbivore-induced plant volatiles to localise hosts [15,16]. Trichogramma pretiosum is a minute (≤0.5 mm long) wasp and female adults lay eggs into a number of lepidopteran eggs, including Plutella xylostella, Helicoverpa armigera, Spodoptera frugiperda and Ectomyelois ceratoniae. After T. pretiosum eggs hatch, the larvae devour the developing caterpillar, pupate and grow into adult wasps inside the host eggs. Adult wasps emerge by chewing holes in the host eggs and are then ready to parasitise other moth eggs. T. pretiosum have been used to control caterpillar pests in a wide range of horticultural and field crops [17].
Generally, egg parasitoid wasps rely on chemical cues originating from the adult host, host products, or the plant that host feeding on to seek host eggs [18], which are a very inconspicuous host stage attacked by parasitic wasps. It was reported that T. pretiosum use volatile host pheromones to locate host eggs [19]. Volatiles of female Helicoverpa zea and synthetic H. zea female pheromone components increased parasitisation rates by T. pretiosum [19]. Later, follow-up studies demonstrated that T. pretiosum responds to calling H. zea females [20]. However, limited attention has been paid to T. pretiosum olfactory system, which plays a critical role in semiochemical detection and guiding T. pretiosum host-seeking behaviours. The completion of the whole genome sequences of T. pretiosum provides an invaluable resource for us to annotate and analyse olfactory genes in this minute parasitoid wasp [21].
In this study, firstly, T. pretiosum antennae and sensilla were investigated by using scanning electron microscopy (SEM). Then T. pretiosum genome database was used to annotate the key olfactory genes, including OBPs and ORs, which were further analysed using bioinformatics, phylogenetics and molecular approaches.

2. Materials and Methods

2.1. Insect Materials

T. pretiosum pupae were sourced from Bugs for Bugs™ (https://bugsforbugs.com.au/, accessed on 27 November 2018) and kept in the lab at 25 ± 1 °C, 70–80% relative humidity (R.H.) and 16:8 h (Light:Dark) photoperiod. Emerged adults were collected immediately using vacuum traps and anaesthetised using carbon dioxide (purity > 99.9%, moisture < 100 ppm) for five minutes, and then sexed under a stereomicroscope (Olympus Corporation, Tokyo, Japan) based on antennae structures. Emerged adults were allocated for RNA extraction and scanning electron microscopy (SEM) (Ted Pella Inc., Redding, CA, USA).

2.2. Total RNA Extraction

After sexing, collected T. pretiosum adults were immediately stored in liquid nitrogen and then homogenised using a disposable homogenising pestle (Sigma-Aldrich, St. Louis, MO, USA). T. pretiosum adult body sizes are tiny (<0.5 mm), so we did not dissect different tissues such as antennae and palps for RNA extraction. Total RNA was extracted using the Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. The purified total RNA was treated by DNase I (New England Biolabs, Ipswich, MA, USA) to remove genomic DNA, quantified, quality checked using NanoDrop™ ND-2000 (Thermo Scientific, Waltham, MA, USA) and stored at −80 °C in the Western Australia State Agricultural and Biotechnology Centre (SABC, Murdoch, Australia).

2.3. Scanning Electron Microscopy (SEM)

T. pretiosum adults were preserved in 3% glutaraldehyde in 0.025 M pH 7.0 phosphate buffer for 24 h and then used for fixation with a Pelco Biowave processor (Ted Pella Inc., Redding, CA, USA). A critical point drying apparatus (Polaron E3000, Quorum Technologies, Lewes, UK) was used in the preparation process at the critical point of CO2 = 31.1 °C and 1071 psi. Dried samples were mounted on SEM stubs under a SZH10 microscope (Olympus Corporation, Tokyo, Japan) and then sputtered with 10 nm gold using a Polaron Sputter coater SC 7640 (Quorum Technologies, Lewes, UK) with argon gas (pressure < 1 × 10−2 mbar, voltage = 1 kV). Samples were examined and photographed under a Zeiss 1555 VP-FESEM SEM instrument (ZEISS Australia, North Ryde, Australia) operated at 10 kV, high current, 10–12 mm working distance, and 30 µm aperture. Sample preparation and examination were conducted at the Centre for Microscopy, Characterization, and Analysis (CMCA) at the University of Western Australia. Five male and five female antennae were observed under SEM (n = 5).

2.4. Gene Identification and Phylogenetic Analysis

Genes encoding for T. pretiosum OBPs, and ORs in the genome (NCBI: PRJNA297592) were identified using BLAST (blastn) searches with reported D. melanogaster and Apis mellifera OBP and OR genes as queries, as previously described [22]. Extensive manual curation was then performed on the T. pretiosum genome according to exon/intron splice site of GT-AG rule. The identified OBP and OR amino acid sequences (Supplementary Materials) were used for validation by NCBI blastp based on the identity and similarity to orthologous genes from other insects. All identified T. pretiosum OBP and OR amino acid sequences are available in an online supporting file (Supplementary Data).
Encoded TpreOBPs (Supplementary Data) were aligned by MEGA-X, a bioinformatics software for sequence data analysis using default settings. Gap Opening Penalty (10.00) and Gap Extension Penalty (0.20) were used for multiple sequence ClustalW alignment with 30% Delay Divergent Cut-off. N-terminal signal peptides were predicted using SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP, accessed on 5 September 2021). Calculated molecular weights (MW) and isoelectric points (pIs) were obtained using the ExPASy proteomics server (http://www.expasy.org/tools/protparam.html, accessed on 5 September 2021) [23]. The amino acid sequences of TpreOBPs were used to search the best blast hit sequences from NCBI using blastp. The Exon/Intron graphics were generated with GSDS (http://gsds.cbi.pku.edu.cn/index.php, accessed on 5 September 2021).
TpreOBP amino acid sequences (Supplementary Data) were used to create an entry file for phylogenetic analysis in MEGA-X with A. mellifera, Nasonia vitripennis and Diachasma alloeum OBP protein sequences [24,25,26,27]. Firstly, the amino acid sequences were aligned using ClustalW alignment with default settings: Gap Opening Penalty (10.00), Gap Extension Penalty (0.20) and 30% Delay Divergent Cut-off. A Maximum Likelihood tree was then constructed using the default settings based on Jones-Taylor -Thornton (JTT) model and Nearest-Neighbor-Interchange (NNI) method. The same phylogenetic analysis approach was also used to investigate T. pretiosum, A. mellifera, N. vitripennis and D. alloeum ORs.

2.5. Quantitative RT-PCR (qRT-PCR)

T. pretiosum cDNA templates were prepared from total RNA samples using the SuperScript™ VILO™ cDNA Synthesis Kit (Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol. Quantitative Real-Time PCR (qRT-PCR) was performed using gene-specific primers (Supplementary Table S1), which were designed using the Primer3web (version 4.1.0) software (http://bioinfo.ut.ee/primer3/, accessed on 10 August 2019). The reference housekeeping gene, glyceraldehyde-phosphate dehydrogenase (GAPDH), was selected here because it has been shown as one of the best candidate reference genes in qPCR analysis [28]. A 2-Step qPCR protocol was performed on Rotor-Gene Q-5 Plex (Qiagen, Valencia, CA, USA) using Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 65 to 95 °C in increments of 1.0 °C for 5 s [29]. For each cDNA sample and primer set, reactions were run in triplicate, and average fluorescence Ct values were obtained. Relative expression levels were determined using the comparative 2−ΔΔCt method for relative quantification [30]. Three biological replicates were performed. Statistical analysis was performed on the expression profiles between male and female adults using the Student’s t-test (SPSS version, IBM, Armonk, NY, USA).

3. Results

3.1. Antennae and Sensilla

T. pretiosum adult antennae are sexually dimorphic (Figure 1), as previously reported for Trichogramma australicum antennae [31]. Both male and female antennae consist of an elongated scape (Sc) with basal radicle (R), pedicel (P) and flagellum (F). The flagellum is differentiated into basal anelli (ring segments), funicle and apical club (C), which are all apparent in the female antenna (Figure 1a,c).
The female club segment is broadest at its midpoint, slightly tapered, curved and is apically blunt. The apex is flattened on the dorsal surface and slightly curved on the ventral surface. The club is covered with numerous types of antennal sensilla on the surfaces, where sensory neurons for the perception of smell, taste, sound, and touch are localised. The male antenna has a distinct thin first anellus. The second anellus and funicular segments are fused with the club to form an elongate tube-like structure (Figure 1b,d). The club is slightly curved with a blunt apex. The surfaces of the male’s scape and pedicel are like that of the female antenna, but the surface of the club is more irregularly corrugated and covered with numerous relatively long sensilla. Based on the shape, three types of sensilla were recognised from male adult antennae, and they are chaetica sensilla (CS), trichoid sensilla (TS) and placoid sensilla (PS) (Figure 1e,f). However, in female adult antennae, four types of sensilla were observed. Besides CS, TS and PS, a new type of sensilla, faleate sensilla (FS), was observed as well (Figure 1e), which was reported as female-specific sensilla in T. australicum [31]. The different antennae structures between male and female adults were used here to distinguish male and female adults.

3.2. Identification of TpreOBPs and TpreORs

A total of 22 OBPs and 105 ORs were identified from the T. pretiosum genome (Table 1), which were used to compare with the olfactory proteins from A. mellifera, N. vitripennis and D. alloeum, three other Hymenoptera insects with available genome sequences. The number of OBPs (22) in T. pretiosum is higher than D. alloeum (15 OBPs), A. mellifera (21 OBPs), but lower than N. vitripennis (90 OBPs). The number of ORs (105 ORs) in T. pretiosum is significantly lower than in A. mellifera (163 ORs), D. alloeum (187 ORs), and N. vitripennis (301 ORs).

3.3. Bioinformatics and Phylogenetic Analysis of TpreOBPs

All 22 TpreOBPs are full-length sequences and exhibit 66–99% identity to known insect OBPs at the amino acid level. For example, TpreOBP2 showed 99% identify to OBP2 (Sequence ID ANG08492.1) from Trichogramma dendrolimi. TpreOBP3 showed 97% identify to OBP3 (Sequence ID ASA40277.1) from Trichogramma japonicum. No signal peptide was predicted from TpreOBP7, while all the other 21 TpreOBPs carry signal peptides. The mature (without signal peptide) TpreOBPs range from 109 to 132 amino acids, and their molecular weights range from 12,279 to 15,033 Da (Table 2). The isoelectric points (pIs) of most TpreOBPs are below 7.0 except TpreOBP1, TpreOBP14, TpreOBP18 and TpreOBP20, whose pIs are higher than 7.0 (Table 2). The alignment of TpreOBP amino acid sequences highlights the six conserved cysteine residues (Figure 2a). Most of T. pretiosum OBPs share the characteristic features of the classic OBP family: small size, presence of an N-terminal signal peptide sequence as well as a highly conserved pattern of six different exon/intron structures were identified from 22 TpreOBP genes, which consist of two exons, four exons, five exons or six exons, respectively (Figure 2b). TpreOBP18 is the only OBP gene consisting of two exons. TpreOBP11 and TpreOBP17 contain four exons in each. TpreOBP2, 4, 6, 7, 8, 13, 14, 16, 20, 21, and 22, contain five exons while TpreOBP1, 3, 5, 9, 10, 12, 15 and 19 contain six exons.
The phylogenetic analysis of OBPs was performed among T. pretiosum and three other Hymenopteran species: A. mellifera, N. vitripennis and D. alloeum (Figure 3). On the phylogenetic tree, various TpreOBPs were clustered closely with N. vitripennis OBPs (NvitOBPs) (Figure 3), suggesting they share high identities at the amino acid level. For example, TpreOBP11 and NvitOBP26 share 68.4% identity. TpreOBP18 and NvitOBP18 share 63.4% identity. These TpreOBPs may play similar roles as clustered NvitOBPs. For example, host-seeking, oviposition or detecting plant compounds for nectar feeding. TpreOBP7 belongs to “plus-C” because it contains seven cysteines in the mature sequence. All other T. pretiosum OBPs belong to the “classic” OBPs, while no “Minus-C” or “Double” OBPs were annotated from T. pretiosum (Figure 3). There are two “Minus-C” OBP groups: one was formed by AmelOBP13-21 while the other was formed by NvitOBP56-62 and NivtOBP27 (Figure 3). One “double” OBP group was formed by NvitOBP38-46, and NvitOBP48 was observed from the tree (Figure 3), as described before.

3.4. Phylogenetic Analysis of T. pretiosum ORs

The phylogenetic analysis of 105 TpreORs, 163 AmelORs, 301 NvitORs and 187 DallORs was performed in a similar way as OBPs. The results revealed a number of species-specific OR subfamilies (Figure 4), which may point to species-specific adaptations during evolution and lifestyles. For example, a T. pretiosum-specific OBP was observed, which did not show similarity to any other ORs from A. mellifera, N. vitripennis and D. alloeum. As one of the most conserved genes in various insect species, TpreORCO shared 72.8% identity with N. vitripennis ORCO (NvitOR1), 60.8% identity with A. mellifera ORCO (AmelOR2) and 51.3% identity with D. alloeum ORCO (DallOR1) at the amino acid level. Various TpreORs were clustered closely with N. vitripennis ORs (NvitORs) (Figure 4), just as observed in TpreOBPs. TpreOR63 and NvitOR105 share 50.6% identity.

3.5. Expression Profiles of T. pretiosum OBPs

OBPs are involved in the first step of odorant detection, so they are our major targets in the expression profile study. Expression profiles between male and female insects can help identify the female- or male-specific OBPs and build the links between OBPs and their potential functions. Here, TpreOBPs were examined between male and female adults by qRT-PCR. To test the designed primers, reverse transcriptase (RT)-PCR was conducted first, and the products were analysed using electrophoresis. All 22 TpreOBPs were successfully amplified, and their band sizes were the same as expected, suggesting the primers were designed appropriately.
To study the expression levels, the qRT-PCR approach was utilised to compare TpreOBP expression levels between males and females with the TpreGADPH gene as a reference gene (Figure 5). The results were presented in three types: Type 1 are female-specific OBPs, which showed the expression levels of OBPs in female adults are over five times higher than in male adults (female/male ratio > 5), including TpreOBP19, TpreOBP15 and TpreOBP3 (Figure 5a). For example, the female/male (F/M) ratio of the expression of TpreOBP19 was 42.6 ± 13.8. The F/M ratio of the expression of TpreOBP15 was 23.7 ± 2.9, while the F/M ratio of the expression level of TpreOBP3 was 23.1 ± 1.4.
Type 2 are male-specific OBPs, including TpreOBP22, TpreOBP5, TpreOBP10 and TpreOBP17, which demonstrated significantly higher expression levels (>5) in male adults than in female adults (Figure 5b). For example, the male/female (M/F) ratio of the expression of TpreOBP22 is 167.7 ± 61.9. The F/M ratio of the expression level of TpreOBP5 was 66.0 ± 3.2. The M/F ratio of the expression level of TpreOBP10 was 18.4 ± 0.8, while the M/F ratio of the expression level of TpreOBP17 was 8.7 ± 0.8. All other OBPs belong to type 3 because either the F/M ratio or M/F ratio of the expression level is lower than 5 (F/M < 5 or M/F < 5) (Figure 5c).

4. Discussion

Parasitoid wasps constitute a large group of hymenopteran superfamilies, which lay eggs on or in the bodies of other arthropods, resulting in the death of their hosts. Trichogrammatids are some of the smallest parasitoid wasps that grow up within-host insect eggs. One of them, T. pretiosum, is frequently used as biological control agent against major lepidopteran pests, including P. xylostella, H. armigera and E. ceratoniae. However, the mechanism of how T. pretiosum localise host eggs is not fully understood yet. Without this knowledge, it is difficult for us to better understand T. pretiosum host-seeking and parasitising behaviours and refine our current application by using it as an efficient and effective biological control agent. This study on T. pretiosum olfactory system provided us with insights into this field.
Firstly, T. pretiosum adult antennae were examined using SEM because they are the major insect olfactory organs. Male and female T. pretiosum showed very different antennae structures. Furthermore, the faleate sensilla (FS) was only observed in female but not male antennae, suggesting significant differences between their olfactory systems. Interestingly, this was not observed in another P. xylostella parasitoid wasp, Diadegma semiclausum, which lays eggs into P. xylostella larvae specifically (Unpublished data).
Completing the T. pretiosum genome project is a significant step towards further understanding its olfactory system and potential applications in pest control. T. pretiosum genome shows rapid genome evolution compared to other hymenopterans (Pereira et al., 2019), reflecting adaptations to miniaturisation and to life as a specialised egg parasitoid. In the T. pretiosum genome, 22 OBPs and 105 ORs were identified, and compared with olfactory proteins of A. mellifera, N. vitripennis and D. alloeum. A. mellifera is one of the model species for Hymenoptera, whose olfaction and social behaviours have been extensively studied. N. vitripennis is the most widely studied of the parasitoid wasps, which is a generalist and parasitises a wide range of dipteran hosts, including blowflies, fleshflies and houseflies. D. alloeum is a specialist parasitoid of the fruit fly, Rhagoletis pomonella. The number of OBPs and ORs exhibited significant differences in these four Hymenopteran species, supporting the differences among their chemosensory systems and biology. For example, A. mellifera has 163 ORs but only ten gustatory receptors (GRs). Presumably a large number of ORs can enhance A. mellifera olfactory ability and facilitate the typical foraging and social behaviours while honeybees have limited need for GRs for plant secondary metabolite discrimination since flowering plants have evolved visual and olfactory cues to attract bees [26]. T. pretiosum has the lowest numbers of ORs in these four species. It may be due to that most of its life stages are inside the host eggs and its tiny body size. A number of OBPs and ORs exhibited high similarities at amino acid level between T. pretiosum and N. vitripennis, suggesting these proteins may play similar roles in both two generalist parasitoid wasps. A recent study compared the OBPs and ORs between T. pretiosum and D. semiclausum because both are important parasitoid wasps and widely used in the biological control for P. xylostella, one of the most destructive insect pests of cruciferous plants [32]. Unlike T. pretiosum, a natural egg parasitoid that lay eggs to various host insect eggs, D. semiclausum lay eggs into P. xylostella larvae specifically and selectively. A number of OBPs and ORs were identified showing high similarities between these two wasps (e.g., TpreOR39 and DsemOR30, TpreOR33 and DsemOR19, TpreOR77 and DsemOR63, TpreOBP2 and DsemOBP8), which may function similarly in these two species. More conserved OBPs or ORs will be identified from D. semiclausum after its genome was available. Further functional characterisation of these OBPs or ORs will provide insights to their roles in T. pretiosum olfactory system and behaviours.
OBPs are involved in the first step of odorant detection, so the characterisation of their sex-specific expression will help build the links between OBPs and their potential functions. For example, female-specific OBPs are more likely to play a significant role in the parasitism behaviours. Male-specific OBPs may be involved in the sex pheromone detection and mating behaviours. Here the whole bodies of male and female T. pretiosum adults were used for RNA extraction rather than antennae because T. pretiosum is too tiny (<0.5 mm), and the antennae collection is extremely time-consuming, which may cause RNA degradation. Therefore, the qRT-PCR results only exhibited the expression profile of TpreOBPs between male and female adult bodies, but not antennae. OBPs have been reported to be expressed in non-olfactory organs rather than antennae, and they may have other functions [12]. For example, it was reported that Aedes aegypti OBP22 is produced in the sperm and transferred to females during mating [33]. Therefore, further localisation of TpreOBPs will be needed by using technologies such as in situ hybridisation or immunohistochemistry.
TpreOBP19, 15 and 3, are female-specific OBPs, which are the candidate OBPs to assist in parasite behaviours. TpreOBP22, TpreOBP5, TpreOBP10 and TpreOBP17 showed male-specific expression, suggesting they may contribute to female seeking and mating behaviours. Other TrepOBPs showed similar expression levels between male and female T. pretiosum, indicating they may play similar roles in both. For example, they are detecting flower odors for nectar-sucking, which are a major sugar resource for insect species. These results help us link the functions of T. pretiosum OBPs for future functional characterisation studies. Further, in vitro (e.g., ligand binding assay) and in vivo (e.g., RNAi or CRISPR) functional characterisation will help demonstrate their roles in T. pretiosum olfactory system. For example, after the knock-down or knock-out of the target OBPs or ORs, the treated insect responses and behaviours will be observed to various chemical compounds [34,35]. On the other hand, the candidate OBPs can be used by a “reverse chemical ecology” approach to identify their ligands and demonstratetheir functions [36].

5. Conclusions

In summary, 22 OBPs and 105 ORs were identified from the genome sequence of T. pretiosum, which were further studied by phylogenetic and bioinformatics methods. The sex-specific expression patterns of T. pretiosum OBPs were analysed using qRT-PCR between male and female adults. This study advances our understanding of the chemosensory system of T. pretiosum at the molecular level and provides a foundation for further research on the olfactory system in T. pretiosum.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/insects12110998/s1, Table S1: TpreOBP Primers designed for qRT-PCR. Supplementary Data S1. The amino acid sequences of TpreOBPs and TpreORs.

Author Contributions

Conceptualisation, B.H.A.-J. and W.X.; methodology, B.H.A.-J. and W.X.; formal analysis, B.H.A.-J. and W.X.; resources, W.X.; writing—original draft preparation, B.H.A.-J. and W.X.; writing—review and editing, W.X.; supervision, W.X.; project administration, B.H.A.-J. and W.X.; funding acquisition, B.H.A.-J. and W.X. All authors have read and agreed to the published version of the manuscript.

Funding

Wei Xu is the recipient of an Australian Research Council Discovery Early Career Researcher Award (DECRA) (DE160100382). Basman Al-Jalely is a recipient of a PhD. Scholarship from the Iraqi Government, Ministry of Higher Education and Scientific research.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets during and/or analysed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

We would like to thank the Western Australian State Agricultural Biotechnology Centre (SABC) and the College of Science, Health, Engineering and Education (SHEE) at Murdoch University for their help supporting this project.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Leal, W.S. Odorant reception in insects: Roles of receptors, binding proteins, and degrading enzymes. Annu. Rev. Entomol. 2013, 58, 373–391. [Google Scholar] [CrossRef]
  2. Keil, T.A. Reconstruction and morphometry of the silkmoth olfactory hairs: A comparative study of sensilla trichodea on the antennae of male Antheraea polyphemus and A. pernyi (Insecta: Lepidoptera). Zoomorphologie 1984, 104, 8. [Google Scholar] [CrossRef]
  3. Keil, T.A. Surface coats of pore tubules and olfactory sensory dendrites of a silkmoth revealed by cationic markers. Tissue Cell 1984, 16, 705–717. [Google Scholar] [CrossRef]
  4. Larsson, M.C.; Hallberg, E.; Kozlov, M.V.; Francke, W.; Hansson, B.S.; Lofstedt, C. Specialized olfactory receptor neurons mediating intra- and interspecific chemical communication in leafminer moths Eriocrania spp. (Lepidoptera: Eriocraniidae). J. Exp. Biol. 2002, 205, 989–998. [Google Scholar] [CrossRef]
  5. Vogt, R.G.; Riddiford, L.M.; Prestwich, G.D. Kinetic properties of a sex pheromone-degrading enzyme: The sensillar esterase of Antheraea polyphemus. Proc. Natl. Acad. Sci. USA 1985, 82, 8827–8831. [Google Scholar] [CrossRef] [Green Version]
  6. Clyne, P.J.; Warr, C.G.; Freeman, M.R.; Lessing, D.; Kim, J.; Carlson, J.R. A novel family of divergent seven-transmembrane proteins: Candidate odorant receptors in Drosophila. Neuron 1999, 22, 327–338. [Google Scholar] [CrossRef] [Green Version]
  7. Vosshall, L.B.; Amrein, H.; Morozov, P.S.; Rzhetsky, A.; Axel, R. A spatial map of olfactory receptor expression in the Drosophila antennae. Cell 1999, 96, 725–736. [Google Scholar] [CrossRef] [Green Version]
  8. Liu, N.-Y.; Xu, W.; Dong, S.-L.; Zhu, J.-Y.; Xu, Y.-X.; Anderson, A. Genome-wide analysis of ionotropic receptor gene repertoire in Lepidoptera with an emphasis on its functions of Helicoverpa armigera. Insect Biochem. Mol. Biol. 2018, 99, 37–53. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Zhang, H.-J.; Xu, W.; Chen, Q.-m.; Sun, L.-N.; Anderson, A.; Xia, Q.-Y.; Papanicolaou, A. A phylogenomics approach to characterising sensory neuron membrane proteins (SNMPs) in Lepidoptera. Insect Biochem. Mol. Biol. 2020, 118, 103313. [Google Scholar] [CrossRef]
  10. Vogt, R.G.; Riddiford, L.M. Pheromone binding and inactivation by moth antennae. Nature 1981, 293, 161–163. [Google Scholar] [CrossRef] [PubMed]
  11. Leal, W.S. Proteins that make sense. In Insect Pheromone Biochemistry and Molecular Biology, the Biosynthesis and Detection of Pheromone and Plant Volatiles; Blomquist, G.J., Vogt, R.G., Eds.; Elsevier Academic Press: Cham, Switzerland, 2003. [Google Scholar]
  12. Pelosi, P.; Iovinella, I.; Zhu, J.; Wang, G.R.; Dani, F.R. Beyond chemoreception: Diverse tasks of soluble olfactory proteins in insects. Biol. Rev. 2018, 93, 184–200. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Fleischer, J.; Pregitzer, P.; Breer, H.; Krieger, J. Access to the odor world: Olfactory receptors and their role for signal transduction in insects. Cell. Mol. Life Sci. 2018, 75, 485–508. [Google Scholar] [CrossRef] [PubMed]
  14. Ishida, Y.; Leal, W.S. Rapid inactivation of a moth pheromone. Proc. Natl. Acad. Sci. USA 2005, 102, 14075–14079. [Google Scholar] [CrossRef] [Green Version]
  15. Vinson, S.B. Host Selection by Insect Parasitoids. Annu. Rev. Entomol. 1976, 21, 109–133. [Google Scholar] [CrossRef]
  16. Turlings, T.C.J.; Erb, M. Tritrophic interactions mediated by herbivore-induced plant volatiles: Mechanisms, ecological relevance, and application potential. Annu. Rev. Entomol. 2018, 63, 433–452. [Google Scholar] [CrossRef]
  17. Pereira, F.; Reigada, C.; Diniz, A.; Parra, J. Potential of Two Trichogrammatidae species for Helicoverpa armigera control. Neotrop. Entomol. 2019, 48, 966–973. [Google Scholar] [CrossRef]
  18. Fatouros, N.E.; Dicke, M.; Mumm, R.; Meiners, T.; Hilker, M. Foraging behavior of egg parasitoids exploiting chemical information. Behav. Ecol. 2008, 19, 677–689. [Google Scholar] [CrossRef]
  19. Lewis, W.J.; Nordlund, D.A.; Gueldner, R.C.; Teal, P.E.A.; Tumlinson, J.H. Kairomones and their use for management of entomophagous insects.13. Kairomonal Activity for Trichogramma Spp Hymenoptera, Trichogrammatidae of Abdominal Tips, Excretion, and a Synthetic Sex-Pheromone Blend of Heliothis-Zea (Boddie) Lepidoptera, Noctuidae Moths. J. Chem. Ecol. 1982, 8, 1323–1331. [Google Scholar]
  20. Noldus, L.P.J.J. Response of the egg parasitoid Trichogramma-pretiosum to the sex-pheromone of its host Heliothis-zea. Entomol. Exp. Appl. 1988, 48, 293–300. [Google Scholar] [CrossRef]
  21. Lindsey, A.R.I.; Kelkar, Y.D.; Wu, X.; Sun, D.; Martinson, E.O.; Yan, Z.; Rugman-Jones, P.F.; Hughes, D.S.T.; Murali, S.C.; Qu, J.; et al. Comparative genomics of the miniature wasp and pest control agent Trichogramma pretiosum. BMC Biol. 2018, 16, 54. [Google Scholar] [CrossRef] [Green Version]
  22. Xu, W.; Papanicolaou, A.; Liu, N.Y.; Dong, S.L.; Anderson, A. Chemosensory receptor genes in the Oriental tobacco budworm Helicoverpa assulta. Insect Mol. Biol. 2015, 24, 253–263. [Google Scholar] [CrossRef] [PubMed]
  23. Cai, L.J.; Zheng, L.S.; Huang, Y.P.; Xu, W.; You, M.S. Identification and characterisation of odorant binding proteins in the diamondback moth, Plutella xylostella. Insect Sci. 2020, 28, 987–1004. [Google Scholar] [CrossRef] [PubMed]
  24. Vieira, F.G.; Foret, S.; He, X.L.; Rozas, J.; Field, L.M.; Zhou, J.J. Unique features of odorant-binding proteins of the parasitoid wasp Nasonia vitripennis revealed by genome annotation and comparative analyses. PLoS ONE 2012, 7. [Google Scholar] [CrossRef] [Green Version]
  25. Robertson, H.M.; Gadau, J.; Wanner, K.W. The insect chemoreceptor superfamily of the parasitoid jewel wasp Nasonia vitripennis. Insect Mol. Biol. 2010, 19, 121–136. [Google Scholar] [CrossRef] [PubMed]
  26. Robertson, H.M.; Wanner, K.W. The chemoreceptor superfamily in the honey bee, Apis mellifera: Expansion of the odorant, but not gustatory, receptor family. Genome Res. 2006, 16, 1395–1403. [Google Scholar] [CrossRef] [Green Version]
  27. Tvedte, E.S.; Walden, K.K.O.; McElroy, K.E.; Werren, J.H.; Forbes, A.A.; Hood, G.R.; Logsdon, J.M.; Feder, J.L.; Robertson, H.M. Genome of the parasitoid wasp Diachasma alloeum, an emerging model for ecological speciation and transitions to asexual reproduction. Genome Biol. Evol. 2019, 11, 2767–2773. [Google Scholar] [CrossRef]
  28. Panina, Y.; Germond, A.; Masui, S.; Watanabe, T.M. Validation of common housekeeping genes as reference for qPCR gene expression analysis during iPS reprogramming process. Sci. Rep. 2018, 8, 8716. [Google Scholar]
  29. Xu, W.; Zhang, H.; Liao, Y.; Papanicolaou, A. Characterization of sensory neuron membrane proteins (SNMPs) in cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae). Insect Sci. 2020, 28, 769–779. [Google Scholar] [CrossRef]
  30. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29, e45. [Google Scholar] [CrossRef]
  31. Amornsak, W.; Cribb, B.; Gordh, G. External morphology of antennal sensilla of Trichogramma australicum Girault (Hymenoptera: Trichogrammatidae). Int. J. Insect Morphol. 1998, 27, 67–82. [Google Scholar] [CrossRef]
  32. Al-Jalely, B.H.; Wang, P.; Liao, Y.; Xu, W. Identification and characterisation of olfactory genes in the parasitoid wasp Diadegma semiclausum (Hellen) (Hymenoptera: Ichneumonidae). Bull. Entomol Res. 2021, 1–10. [Google Scholar] [CrossRef] [PubMed]
  33. Li, S.; Picimbon, J.F.; Ji, S.; Kan, Y.; Chuanling, Q.; Zhou, J.J.; Pelosi, P. Multiple functions of an odorant-binding protein in the mosquito Aedes aegypti. Biochem. Biophys. Res. Commun. 2008, 372, 464–468. [Google Scholar] [CrossRef] [PubMed]
  34. Liu, Q.; Liu, W.; Zeng, B.; Wang, G.; Hao, D.; Huang, Y. Deletion of the Bombyx mori odorant receptor co-receptor (BmOrco) impairs olfactory sensitivity in silkworms. Insect Biochem. Mol. Biol. 2017, 86, 58–67. [Google Scholar] [CrossRef] [PubMed]
  35. Zhang, Y.J. RNAi-induced electrophysiological and behavioral changes reveal two pheromone binding proteins of Helicoverpa armigera involved in the perception of the main sex pheromone component Z11-16:Ald. J. Chem. Ecol. 2017, 43, 207–214. [Google Scholar] [CrossRef]
  36. Leal, W.S.; Barbosa, R.M.; Xu, W.; Ishida, Y.; Syed, Z.; Latte, N.; Chen, A.M.; Morgan, T.I.; Cornel, A.J.; Furtado, A. Reverse and conventional chemical ecology approaches for the development of oviposition attractants for Culex mosquitoes. PLoS ONE 2008, 3, e3045. [Google Scholar] [CrossRef] [Green Version]
Insects 12 00998 g001 550
Figure 1. The scanning electron microscopy (SEM) analysis of T. pretiuosum female and male antennae and sensilla. (a,b) the hand drawings of T. pretiuosum female and male antennae. (c,d), SEM analysis of T. pretiuosum female and male antennae. (e,f), SEM analysis of T. pretiuosum female and male olfactory sensilla on the antennae. Sc, elongated scape; R, basal radicle; P, pedicel; F, flagellum; and C, apical club. Various types of olfactory sensilla were observed, including chaetica sensilla (CS), trichoid sensilla (TS), Placoid sensilla (PS) and faleate sensilla (FS).
Figure 1. The scanning electron microscopy (SEM) analysis of T. pretiuosum female and male antennae and sensilla. (a,b) the hand drawings of T. pretiuosum female and male antennae. (c,d), SEM analysis of T. pretiuosum female and male antennae. (e,f), SEM analysis of T. pretiuosum female and male olfactory sensilla on the antennae. Sc, elongated scape; R, basal radicle; P, pedicel; F, flagellum; and C, apical club. Various types of olfactory sensilla were observed, including chaetica sensilla (CS), trichoid sensilla (TS), Placoid sensilla (PS) and faleate sensilla (FS).
Insects 12 00998 g001
Insects 12 00998 g002a 550Insects 12 00998 g002b 550
Figure 2. The alignment of 22 TpreOBP amino acid sequences (a) and their intron/exon gene structures (b). The six conserved cysteines are highlighted by red colour. Black blocks represent exons, while the lines represent introns.
Figure 2. The alignment of 22 TpreOBP amino acid sequences (a) and their intron/exon gene structures (b). The six conserved cysteines are highlighted by red colour. Black blocks represent exons, while the lines represent introns.
Insects 12 00998 g002aInsects 12 00998 g002b
Insects 12 00998 g003 550
Figure 3. Phylogenetic analysis of OBPs from T. pretiosum (TpreOBPs), A. mellifera (AmelOBPs), N. vitripennis (NvitOBPs) and D. alloeum (DallOBPs). 22 TpreOBPs are marked in red, 21 AmelOBPs are marked in blue, 90 NvitOBPs are marked in black, and 15 DallOBPs are marked in green. Consensus support values are labelled on branches.
Figure 3. Phylogenetic analysis of OBPs from T. pretiosum (TpreOBPs), A. mellifera (AmelOBPs), N. vitripennis (NvitOBPs) and D. alloeum (DallOBPs). 22 TpreOBPs are marked in red, 21 AmelOBPs are marked in blue, 90 NvitOBPs are marked in black, and 15 DallOBPs are marked in green. Consensus support values are labelled on branches.
Insects 12 00998 g003
Insects 12 00998 g004 550
Figure 4. Phylogenetic analysis of ORs from T. pretiosum (TpreORs), A. mellifera (AmelORs), N. vitripennis (NvitORs) and D. alloeum (DallORs). 105 TpreORs are marked in red, 163 AmelORs are marked in blue, 301 NvitORs are marked in black, and 187 DallORs are marked in green. The ORCO (*) subfamily formed by TpreORCO, NvitOR1, DallOR1 and AmelOR2 are highlighted.
Figure 4. Phylogenetic analysis of ORs from T. pretiosum (TpreORs), A. mellifera (AmelORs), N. vitripennis (NvitORs) and D. alloeum (DallORs). 105 TpreORs are marked in red, 163 AmelORs are marked in blue, 301 NvitORs are marked in black, and 187 DallORs are marked in green. The ORCO (*) subfamily formed by TpreORCO, NvitOR1, DallOR1 and AmelOR2 are highlighted.
Insects 12 00998 g004
Insects 12 00998 g005 550
Figure 5. Quantitative real-time PCR (qPCR) analysis of T. pretiosum OBPs (TpreOBPs) from female and male adults. Normalised by TpreGADPH gene. (a), the female/male (F/M) expression ratios of TpreOBP19, TpreOBP5 and TpreOBP3, which are female-specific OBPs with F/M > 5. (b), the male/female expression ratios of TpreOBP22, TpreOBP5, TpreOBP10 and TpreOBP17, which are male-specific OBPs with M/F > 5. (c), other TpreOBPs that are expressed in both male and female adults with F/M or M/F ratios were less than 5.0. Error bars show standard deviation. M, adult males and F, adult females.
Figure 5. Quantitative real-time PCR (qPCR) analysis of T. pretiosum OBPs (TpreOBPs) from female and male adults. Normalised by TpreGADPH gene. (a), the female/male (F/M) expression ratios of TpreOBP19, TpreOBP5 and TpreOBP3, which are female-specific OBPs with F/M > 5. (b), the male/female expression ratios of TpreOBP22, TpreOBP5, TpreOBP10 and TpreOBP17, which are male-specific OBPs with M/F > 5. (c), other TpreOBPs that are expressed in both male and female adults with F/M or M/F ratios were less than 5.0. Error bars show standard deviation. M, adult males and F, adult females.
Insects 12 00998 g005
Table
Table 1. The numbers of OBPs and ORs, in Trichogramma pretiosum, Apis mellifera, Nasonia vitripennis, and Diachasma alloeum.
Table 1. The numbers of OBPs and ORs, in Trichogramma pretiosum, Apis mellifera, Nasonia vitripennis, and Diachasma alloeum.
SpeciesOBPOR
Trichogramma pretiosum22105
Apis mellifera21163
Nasonia vitripennis90301
Diachasma alloeum15187
Table
Table 2. T. pretiosum OBPs.
Table 2. T. pretiosum OBPs.
Gene NameFull LengthSignal PeptideIsoelectric Points PIMolecular WeightMature Amino AcidsExpect ValueIdentSequence ID
TpreOBP1Yes (Y)Y, 1–228.7115,032.731322E-9190%ANG08491.1 odorant-binding protein 1 [T. dendrolimi]
TpreOBP2YY, 1–254.8314,599.411272E-10799%ANG08492.1 odorant-binding protein 2 [T. dendrolimi]
TpreOBP3YY, 1–225.9614,983.561305E-10697%ASA40277.1 OBP3 [T. japonicum]
TpreOBP4YY, 1–195.4512,776.751148E-8490%ANG08494.1 odorant-binding protein 4 [T. dendrolimi]
TpreOBP5YY, 1–195.1413,830.531202E-9293%ANG08495.1 odorant-binding protein 5 [T. dendrolimi]
TpreOBP6YY, 1–234.1513,825.721206E-9996%ANG08496.1 odorant-binding protein 6 [T. dendrolimi]
TpreOBP7YNo5.5614,358.551312E-4463%CCD17854.1, putative odorant binding protein 85 [Nasonia vitripennis]
TpreOBP8YY, 1–226.4613,407.551223E-9897%ANG08498.1, odorant-binding protein 8 [T. dendrolimi]
TpreOBP9YY, 1–275.3613,361.111192E-10399%ANG08499.1, odorant-binding protein 9 [T. dendrolimi]
TpreOBP10YY, 1–185.6313,547.401194E-9093%ANG08500.1, odorant-binding protein 10 [T. dendrolimi]
TpreOBP11YY, 1–177.4812,666.671199E-8898%ANG08501.1, odorant-binding protein 11 [T. dendrolimi]
TpreOBP12YY, 1–185.2713,426.281192E-9698%ANG08502.1, odorant-binding protein 12 [T. dendrolimi]
TpreOBP13YY, 1–177.812,279.111091E-5366%XP_014219837.1, uncharacterized protein LOC106647812 [Copidosoma floridanum]
TpreOBP14YY, 1–199.3213,439.531171E-9297%ANG08504.1, odorant-binding protein 14 [T. dendrolimi]
TpreOBP15YY, 1–195.0613,402.081192E-9797%ANG08505.1, odorant-binding protein 15 [T. dendrolimi]
TpreOBP16YY, 1–194.9612,914.791144E-8292%ANG08506.1, odorant-binding protein 16 [T. dendrolimi]
TpreOBP17YY, 1–204.0413,483.341204E-9193%ANG08507.1, odorant-binding protein 17 [T. dendrolimi]
TpreOBP18YY, 1–178.9512,678.931172E-890.98AZB49386.1, odorant-binding protein 5 [Heortia vitessoides]
TpreOBP19YY, 1–205.513,980.461232E-810.98ANG08509.1, odorant-binding protein 19 [T. dendrolimi]
TpreOBP20YY, 1–167.7513,005.761186E-680.99ANG08510.1, odorant-binding protein 20, partial [T. dendrolimi]
TpreOBP21YY, 1–164.3413,959.621212E-880.96ANG08512.1, odorant-binding protein 22 [T. dendrolimi]
TpreOBP22YY, 1–236.5314,056.711231E-820.82ASA40280.1, OBP6 [T. japonicum]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Al-Jalely, B.H.; Xu, W. Olfactory Sensilla and Olfactory Genes in the Parasitoid Wasp Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae). Insects 2021, 12, 998. https://doi.org/10.3390/insects12110998

AMA Style

Al-Jalely BH, Xu W. Olfactory Sensilla and Olfactory Genes in the Parasitoid Wasp Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae). Insects. 2021; 12(11):998. https://doi.org/10.3390/insects12110998

Chicago/Turabian Style

Al-Jalely, Basman H., and Wei Xu. 2021. "Olfactory Sensilla and Olfactory Genes in the Parasitoid Wasp Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae)" Insects 12, no. 11: 998. https://doi.org/10.3390/insects12110998

APA Style

Al-Jalely, B. H., & Xu, W. (2021). Olfactory Sensilla and Olfactory Genes in the Parasitoid Wasp Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae). Insects, 12(11), 998. https://doi.org/10.3390/insects12110998

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop