Variations in the expression of odorant binding and chemosensory proteins in the developmental stages of whitefly Bemisia tabaci Asia II-1 – Scientific Reports

The globally distributed cotton whitefly, B. tabaci, encompasses 46 cryptic species, with 16 found in Asia²⁸. Despite Asia II-1’s prevalence, knowledge gaps persist regarding its OBPs and CSPs. Our study examined these proteins’ polypeptide sequence motifs and their expression across developmental stages. We assessed highly expressed OBPs and CSPs for interactions with odour compounds via in silico docking, shedding light on their roles in olfaction and host recognition. OBPs and CSPs play crucial roles in insect olfaction by transporting ligands to activate chemosensory transmembrane proteins^1,9,29. They influence insect behaviour and physiological adaptations, including food-seeking, reproduction, and pesticide resistance^1,15,29,30. Prior research on OBP diversity in B. tabaci was confined to the MEAM1 and MED genetic groups. Our study provides insight into stage-specific expression of OBPs and CSPs from B. tabaci Asia II-1 prevalent widely in Asia. Domain analysis revealed that most of the OBPs, excluding OBP10 and OBP13 possess PBP_GOBP superfamily domain suggesting their involvement in odorant binding as per gene ontology. All the CSP proteins have OS-D superfamily domain implying their likely involvement in chemosensory signal transduction³¹.

Phylogenetic analyses offer valuable insights into the evolutionary relationships between the OBPs and CSPs of different Hemipteran insects. Our analysis revealed that the OBP8 of B. tabaci Asia II-1 grouped with the OBP8 of MED and MEAM-1 and remained separated from the OBPs of other Hemipteran insects. It shows that OBP8 may be an ancestral gene in the evolution of all other odorant-binding proteins of B. tabaci. Among the newly reported OBPs (OBP 9 to 14) from B. tabaci AsiaII-1, the OBPs 10 and 13 formed distinct clusters without sharing clades with any other Hemipteran OBPs, implying its exclusivity to the Asia II-1 genetic group of B. tabaci. Our analysis also revealed that the OBP 8 and CSP4, located respectively on chromosomes three and nine of B. tabaci, branched as distinct clades, indicating their uniqueness. The neighbourhood joining analysis of the OBPs of B. tabaci cryptic species also shows that OBP8 remains as a uniclade in the tree without having any shared branches with other OBPs of B. tabaci Asia II-1.

The CSPs of B. tabaci cryptic species grouped together and formed separate clades from those of other hemipteran CSPs. However, the CSP4 of Asia II-1/MED, along with the CSP7 of MEAM1, constituted a unique clade. Branching as a distinct clade and unique motif pattern of CSP4 coupled with the significantly higher expression levels in developmental stages in B. tabaci Asia II-1, MED¹⁸, and MEAM1¹⁷ strongly suggest that CSP4 may play a vital role in the behavioural physiology of B. tabaci.

Chromosomal location and protein motif analysis are valuable tools for investigating gene evolution, including events like duplication, reversal, or skipping, and for assessing functional conservation^32,33. In general, approximately half of the Asia II-1 OBPs and CSPs cluster on chromosomes three, six, and seven, often sharing the same loci on the chromosome. This clustering was likely to have resulted from the high conservation of these gene families in B. tabaci Asia II-1. Notably, the motif patterns display partial conservation among representatives within the same cluster. The evolution of the OBP gene family seems to follow the birth-and-death model, involving pseudogenization or functional divergence of duplicate genes during duplication events^34,35. However, the adjacent genes on the same chromosome may be involved in analogous functions, as observed in other organisms like the fire ant Solenopsis invicta, wherein certain genes on the social chromosome play a critical role in behavioural modulation between monogyne and polygyne colonies³⁶.

Unlike the OBPs, which form multiple clusters with fewer genes within each cluster, the CSPs B. tabaci Asia II-1 primarily formed two clusters, indicating a potential origin through gene duplication. The CSP2 and CSP3 clustered together on chromosome number six (Table S9) of B. tabaci were associated with insect defence¹⁵. However, the OBPs 1 and 8 clustered together on chromosome number three were found to be associated, respectively, with different functions like identification of the oviposition site¹⁶ and host preference³⁷. This supports the birth-and-death model of the evolutionary origin of OBPs. However, further studies may elucidate the functional roles of closely and distantly located OBPs and CSPs on the chromosomes of B. tabaci.

Understanding the expression patterns of OBPs and CSPs at different life stages is crucial for uncovering their roles in various adaptations such as feeding preferences, mate-seeking behaviour, and pesticide resistance^13,38. Interestingly, the BtAsiaII-1 OBPs/CSPs were not detected in the egg stage, suggesting that they may not play a role in embryonic development, unlike in some other insects like Galeruca daurica, where a specific OBP, i.e., GdauOBP28, was highly expressed during the egg stage³⁹. Earlier studies have shown that OBPs like 3 and 8 contribute to the orientation of insects towards their host plants at medium or short distances in the B. tabaci MED and MEAM1 genetic groups^37,40,41. The OBPs like 3,4,5,7,8,9,10,13, and 14 were observed to show good expression in the first instar nymphs, the stage wherein the insect starts feeding by probing their stylets into the phloem tissues of the host plant. We speculate that these OBPs may be primarily involved in host finding and feeding in B. tabaci. It may be probable that whitefly B. tabaci may recruit different OBPs for recognition of chemical cues from host plants, as this pest is recorded as a polyphagous pest with a host range of more than 200 plants. Detailed studies are needed to ascertain the functional roles of these OBP genes. An increased number of OBPs have been identified in non-sensory tissues such as the pheromone glands, wings, legs, fat body, and salivary glands²⁹. They have roles in pheromone delivery, host adaptation, development, reproduction, and insecticide resistance³⁸. OBP 27 in Spodoptera frugiperda^13,38 and OBP 56a in Phormia regina³⁰ have roles in binding and transporting fatty acids. OBP 27 is highly expressed in the reproductive organs of males and is involved in the mating of males in S. frugiperda³⁸. AeOBP22 might transport or sequester a pheromonal component during mating⁴². OBP10 in Helicoverpa armigera and Helicoverpa assulta might act as carriers of oviposition deterrents and mediate the dissemination of eggs in species with larval cannibalism⁴³. Further studies are needed to unravel the non-sensory role of OBPs in the nymphal stages of whitefly B. tabaci.

It is significant to note that OBP 8 displayed significant levels of gene expression in all the developmental stages and particularly a higher expression (5613 log₂ fold change) during the adult stage of B. tabaci Asia II-1 (Fig. 5a). This extremely high level of expression underscores the functional significance of OBP8 in the lifecycle of B. tabaci Asia II-1. Higher levels of expression of OBP8 were earlier recorded in adults of the B. tabaci genetic groups MED³⁷ and MEAM1¹⁷. Consistently higher expression of OBP8 in different genetic groups suggests that the OBP8 may play a crucial role in survival, host recognition, and feeding, and it may as well be involved in other physiological functions of B. tabaci. The role of OBP8 in host recognition in adults of B. tabaci MED had earlier been documented by Wang et al.³⁷. Barring OBP5, all other OBPs in the present study have shown significantly higher expression in adults, and eventually they may be associated with host recognition, feeding, and reproductive physiology in B. tabaci Asia II-1. Contrary to our results, He et al.¹⁴ reported that OBP5showing higher levels of expression in the adult B. tabaci MED was found to be associated with host recognition. Such variations in expression levels of OBPs may be attributed to biotype-specific variations of OBP genes in B. tabaci cryptic species. An earlier study reported that biotype-specific signatures in CSPs 1, 2, and 3 in the MED and MEAM1 genetic groups of B. tabaci, was leading to functional variations¹⁵. The OBPs 1 and 4 expressed higher in adults of B. tabaci were implicated in oviposition site detection¹⁶. The OBPs 8 and 3 of B. tabaci were found to be associated with host findings by Wang et al.³⁷ and Shi et al.⁴¹. Most of the BtAsiaII-1 OBPs showed low levels of expression during the fourth instar/prepupal stage, which is incidentally a non-feeding stage of the insect⁴⁴.

Results of our study show that all the BtAsiaII-1 CSPs except CSP 9 exhibited significantly higher expression in the prepupal and adult stages, suggesting their role in the reproductive physiology of B. tabaci, as reproductive organ development occurs during the prepupal stage⁴⁴, and these may be involved as well in mating and oviposition during adulthood. The lower expression of CSPs in the first instar nymph indicates that they may not be primarily involved in feeding. Expression of CSPs barring CSP 4 and 9 was relatively higher in the second and third nymphal stages compared to the first nymphal stage and adulthood (Figs. 4 and 5) implying that these CSPs may be associated with other physiological functions. Variation in expression of CSPs has earlier been reported in the MEAM1 and MED genetic groups of B. tabaci^15,16,45. The functional role of some of the CSPs including 1, 2, 3, and 11, have earlier been deduced in these B. tabaci genetic groups. CSP1 was found to be associated with insecticide resistance¹⁵, while CSP2 was implicated in olfaction host plant recognition and oviposition preference^15,46. The CSP3 expressed at high levels in nymphs of B and Q biotypes of B. tabaci was found to play a role in odour recognition from host plants¹⁵. The CSP-11 was associated with reproduction⁴⁵. However, there has been no report on the functional characterization of OBPs and CSPs in B. tabaci Asia II-1, the widely distributed in Asia.

Significantly high expression of genes like OBP8 and CSP4 in the developmental stages of B. tabaci Asia II-1 prompted us to explore its possible role in olfaction. We deduced the predicted protein models for OBP8 and CSP4 and the predicted structures conform to the Ramachandran Plot. The secondary structure predictions for OBP8 and CSP4 yielded confidence values of 99.9% and 100%, respectively. It is significant to note that it showed 200-to-5000-fold increased expression compared to other OBPs in the adult stage, implying its significance in eliciting behavioural responses in the whitefly, B. tabaci. Probably, it may be involved in host recognition or play a role in the growth and developmental physiology of the insect. Detailed studies are needed to ascertain its functional role in B. tabaci.

We conducted molecular docking with 10 ligands for both OBP8 and CSP4 in B. tabaci Asia II-1. These ligands included green plant volatiles or HIPVs known to interact with insects in olfaction and host plant recognition. OBP8 showed strong binding with limonene, α-pinene, β-pinene, p-cymene, β-ionone, β-caryophyllene, and 2,4-Phytadiene (> − 5 kcal/mol), while β-ocimene and myrcene exhibited moderate binding, and cis-3-Hexen-1-ol had low binding affinity. Previous studies implicated OBP1 and OBP4 in oviposition site selection due to their strong binding to β-ionone, suggesting its role as a cue for host selection by B. tabaci.

Our findings indicated a significant binding affinity of β-ionone to both OBP8 and CSP4, with binding energies of − 5 kcal/mol and − 5.1 kcal/mol, respectively. Considering the abundant expression of OBP8 in the adult stage of B. tabaci and its robust affinity for β-ionone, we may speculate its possible role in host recognition or reproductive functions. α-pinene and limonene also exhibited high binding energy values exceeding − 5 kcal/mol. Our results are in concordance with the findings of He et al.¹⁴ who reported that these compounds enhance whiteflies’ ability to locate host plants. Furthermore, α-pinene had been implicated in eliciting a positive response from virus-infested plants to B. tabaci facilitating the rapid transmission of cucumber mosaic virus⁴⁷, tomato leaf curl virus⁴⁸ and cotton leaf curl virus⁴⁹. Myrcene bound strongly to both OBP8 and CSP4 (− 4.8 kcal/mol), while β-ocimene showed moderate affinities to OBP8 (− 4.8 kcal/mol) and CSP4 (− 4.3 kcal/mol). Liu et al.⁵⁰ demonstrated the attractancy of these compounds to Encarsia formosa and whiteflies. Other VOCs like β-caryophyllene, p-cymene, and 2,4-phytadiene also showed strong binding affinity to OBP8 (> − 5 kcal/mol), while β-caryophyllene and azulene showed good binding to CSP4 (− 5.3 kcal/mol and − 4.9 kcal/mol, respectively).

As of now, only a few studies have been taken on the functional characterization of OBPs and CSPs in the whitefly, B. tabaci. Our study provides an insight into expression patterns of these OBPs/CSPs across all the developmental stages of B. tabaci Asia II-1, hitherto unexplored. The differential expression patterns of certain BtAsiaII-1 OBPs and CSPs call for unravelling their functional role. Significantly high expression of OBP8 and CSP4 consistently across the developmental stages of B. tabaci Asia II-1 prompted us to explore its possible role in olfaction through in-silico docking analysis with volatile organic compounds having perceived roles in olfaction and host plant recognition. The in-silico analysis particularly identified that compounds such asα-pinene, β-pinene, 2,4-Phytadiene, β-caryophyllene, and β-ionone modulated the orientation behaviour of B. tabaci Asia II-1. However, detailed in vitro ligand binding studies with different OBPs and CSPs may confirm the assertions made in this study. Moreover, functional validation can be done through use of tools such as RNAi or CRSPR-Cas9 evaluation systems. Despite these limitations, our study lays the groundwork for future research into the functional roles of OBPs and CSPs in B. tabaci Asia II-1. Unravelling the functional role of OBP/CSPs may lead to development of novel control strategies for this global invasive pest.

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• Source: https://www.nature.com/articles/s41598-024-65785-9