Two intracellular loops of pheromone receptors of four
Noctuid moth species presumably responsible for ligand
recognition
Merlijn van de Ven (11704934)
Supervised by:P. Roessingh, A. T. Groot
Abstract
In order to mate, moths find each other through species-specific sex pheromones, excreted by adult females, that are perceived by the olfactory receptors (ORs) of the males. The olfactory receptors that perceive pheromones are called pheromone receptors (PRs). Studies have indicated that the ligand recognition of two orthologous PRs from two Noctuid moth species Helicoverpa assulta and Helicoverpa armigera, HassOR14b and HarmOR14b, is determined by two amino acids in the last two intracellular loops of the seven transmembrane protein structure. This study aimed to investigating whether the ligand recognition of other PRs in other Noctuid moths are also determined by those two intracellular loops. The variation in the amino acid sequences of the first two intracellular loops were compared to the variation in the last two intracellular loops. Interestingly, all the orthologous PRs that were tested, showed more conserved sequences in the last two intracellular loops than in the first two intracellular loops. This suggests that the last two loops of multiple PRs of the Noctuid moth species that were tested could possibly be involved of the ligand recognition of other PRs in other
Noctuid moths. These insights broaden the knowledge of the PRs in Noctuid moths and give insight for further studies of olfactory mechanisms of Noctuid moths.
Keywords: Noctuid moths, Helicoverpa assulta, Helicoverpa armigera, Sesamia nonagrioides, Sesamia inferens, Pheromone receptor, ligand recognition
Bachelor Thesis
Institute of Biodiversity and Ecosystem Dynamics University of Amsterdam
Introduction
Olfaction is an extremely important sense for all animals (Ache & Young, 2005) and plays an important role in insect behaviour (Li et al., 2015). Olfactory stimuli serve to detect environmental chemical information that can help the animal locate necessary items such as food, water and nesting sites (Ache & Young, 2005). Nocturnal insects, such as moths, have appeared to be ideal models to study the physiology of olfaction, since the sensory modality is highly developed because of its importance for their survival. Moths have been a model to study the molecular bases of olfaction for a long time. Besides, moths include important and diverse pests of crops, stored products and forests. Because moths are pests, a better
understanding of the molecular mechanisms of their olfactory reception can lead to the identification of targets while developing effective methods for pest control (Legeai et al., 2011).
In moths, mate-finding behaviour is mediated by species-specific chemical cues, usually released by adult females, that can be perceived by olfactory receptors (ORs). The antennae that are located on the head of the insect are able to detect those chemical stimuli. Insect antennae can bear thousands of innervated olfactory structures; the olfactory sensilla, which contain multiple receptor neurons (ORNs). The dendrites of those ORNs extend into the sensillar lumen. These cues are called sex pheromones which are released during ‘calling’ behaviour and can be tracked by the conspecific males over a long distance. Sex pheromones consist of a mixture with precise relative ratios of components and ensure a species-specific recognition. Most of them consist of a blend of one major component together with a few minor components. The pheromones are detected by transmembrane receptors belonging to the OR family, which are called pheromone receptors (PRs). In the OR phylogeny, moth PR-encoding genes form a monophyletic group. This suggests that they all evolved from a single common ancestor (de Fouchier et al., 2015).
Odorant signals are detected by the olfactory receptors (ORs) at the dendritic membranes. The olfactory signal gets transduced into an electrical signal that will be transferred to the brain (Haupt et al., 2010). Those receptors are encoded by genes in the genome - OR genes - and encode seven transmembrane proteins located on the dendrites of ORNs (Haupt et al., 2010). The olfactory capacity of insects thus depend on the amount of expressed OR genes. Now that many insect OR repertoires have been identified (Wanner & Robertson, 2008),unique, lineage-specific expansions of OR clades have been observed in different insect orders (de Fouchier, 2017).
Little is known about the location of the recognition of the receptor to detect the receptor-specific ligand. In the study of Ke Yang et al. in 2017, they found that two single-point mutations are responsible for the ligand selectivity of a pheromone receptor between two closely related moth species, Helicoverpa assulta and Helicoverpa armigera. The PR HassOR14b is found to be tuned to (Z)−9-hexadecenal, which is the major sex-pheromone component in Helicoverpa assulta. The ortholog of HassOR14b is HarmOR14b, from the closely related species Helicoverpa armigera. HarmOR14b is tuned to another chemical (Z)−9-tetradecenal. In their study, they looked into the amino acid residues that determine the ligand selectivity of those orthologous PRs. Two amino acids that are located in the last two intracellular domains of the seven transmembrane receptor together turn out to determine the functional difference between the two orthologs. This showed that the tuning specificity of the PRs in the two Helicoverpa moth species could be achieved with just a few amino acid substitutions in the last two intracellular loops (Yang et al., 2017). It still remains unclear whether the ligand specificity of other ORs (in other Noctuid moths) are also determined by the same intracellular domains.
Therefore, this study will focus on the amino acid sequences of ORs of four Noctuid moth species; H. armigera, H. assulta, Sesamia nonagrioides and Sesamia inferens. H. armigera and H. assulta are important sympatric pests in Asia. Despite of their shared genus, their foraging ranges are different. The usage of a specialist and a generalist is based on the contrast it creates between two closely related species, possibly increasing the overall chance of finding differences in the receptors. H. armigera is a polyphagous species, while H. assulta is an oligophagous species (Zhang et al., 2015). This is also the case with the two other closely related Sesamia nonagrioides and Sesamia inferens. The Mediterranean corn borer S. nonagrioides, an oligophagous species with a fairly wide range of host plants, is a major pest of corn crops around the Mediterranean region (Cruz & Eizaguirre, 2015). Meanwhile, S. inferens, the purple stem borer, is a polyphagous insect pest that has become one of the major rice pests in China since the 1990s (Zhang et al., 2014).
Yang et al. (2017) found that the ligand recognition of HassOR14b and HarmOR14b is determined by the amino acid sequences in the last two intracellular loops (region VI and VIII, see figure 3). The aim of this study was to determine whether the ligand specificity of other Noctuid moths is also localised in these loops in the ORs. I hypothezised that when these loops indeed play a central role in odour binding, they ae expected to show less variation in amino acids than sequences elsewhere in the OR. This expectation is based on the assumption that the sequence in the loops of region VI and VIII have to be relatively conserved to keep function as a binding pocket, while the actual ligand recognition could change by just altering a few amino acids , (Yang et al., 2017). Therefore I predict that in general the intracellular loops of region VI and VIII will be significantly more conserved than the other two intracellular domains I and III.
Materials & methods
Collecting amino acid sequences
The amino acid sequences of the olfactory receptors of S. inferens, S. nonagioides, H. assulta and H. armigera were searched for in the Genbank of NCBI and in the Supplementary Materials in the literature. If the aa sequences were complete, their file was saved and stored to be used for the phylogenetic analyses. 45 completely sequenced ORs were used in this phylogenetic analysis; 17 ORs of H. assulta (Hass), 10 ORs of H. armigera (Harm), 13 ORs of S. nonagrioides (Snon) and 5 ORs of S. inferens (Sinf). Accession numbers can be found in the Supplementary Materials Table 1.
Phylogenetic analyses
Using the program MEGA, the amino acid sequences of the four species were aligned using MUSCLE and a maximum likelihood phylogeny of ORs was created (Figure 1).
Figure 1. Maximum likelihood phylogeny of the ORs of four Noctuid moth species. The amino acid sequences are based on the reported transcriptome data of functionally identified PRs. The tree was rooted on the ORCO lineage. The bar indicates the phylogenetic distance value.
Abbreviations: Hass, Helicoverpa assulta; Harm, H. armigera; Sinf, Sesamia inferens; Snon, S. nonagrioides. The red dots ‘●’ represent the PRs. The GenBank accession numbers of the olfactory receptors used in this analysis are listed in the Supplementary materials Table 1.
Selection of orthologous pairs
The ortholog ORs of closely related species were selected based on this phylogeny (figure 2). In total seven pairs of orthologs, which were chosen based on the phylogenetic tree of the ORs of the four Noctuid species (see Figure 2).
Figure 2. Highlighted ORs in the phylogenetic tree from figure 1. The amino acid
sequences of 2 PRs of S. nonagrioides were paired with 2 orthologous PRs of S. inferens and 5 PRs of H. armigera were paired with 5 orthologous PRs of H. assulta (see Table 1). The ORs were aligned using the program MAFFT.
Statistical analysis
To investigate whether there is a significant difference between the number of different amino acids in region I and III relative to region VI and VIII, single-nucleotide
polymorphisms (SNPs) were counted for region I and III as well as for region VI and VIII (see Figure 3). The intracellular loops of RI and RIII were compared in total and the
comparison of the longer intracellular loops in RVI and RVIII was a total of 41 amino acids, Table 1. Pairs of the 14 ortholog PRs Specialist PR Generalist PR HassOR6 HarmOR6 HassOR3.1 HarmOR3 SnonOR6 SinfOR21 SnonOR15 SinfOR29 HassOR14b HassOR11 HarmOR14b HarmOR11 HassOR13 HarmOR13
20 amino acids before SNP 323 and 355 and 20 amino acids after these SNPs(see Figure 3). To test whether there was a significant result between the variation of the amino acids of loop I and III relative to loop VI and VIII, a sign test was performed. P-value was determined by the binomial distribution table (‘Binomial distibution – probability function’, 2014).
Figure 3. Representation of the seven transmembrane structure of HassOR14b (Yang et al., 2017). The intracellular loops of region VI and VIII indicated by the red squares with the two point mutations responsible for the ligand specificity (F232I and T355I) indicated by the black circles inside the squares. The intracellular loops of region I and RIII indicated by the blue squares.
Results
The available full-length sequenced PRs made it possible to analyze the four Noctuid moth species of which two pairs of closely related species. The amino acid sequences of the intracellular loops of the seven-transmembrane proteins were compared. The differences in amino acids of the intracellular loops of RI&III relative to RVI&VIII of two orthologous PRs of S. nonagrioides and S. inferens and five orthologous PRs of H. assulta and H. armigera species were counted (Table 2). The pairs were chosen based upon the phylogenetic analysis (Figure 2). Seven out of the seven pairs of the orthologous PRs tested positive in terms of the variation of the amino acid sequence in the intracellular loops of region I&III being higher than the variation in region VI&VIII. The sign test (Table 3) showed that this results in a significant difference in variation between the four regions (p=.0078).
Table 2 shows the amino acid sequence comparisons of loop I&III relative to loop VI&VIII of seven pairs of orthologous PRs of two closely related species. The intracellular loops of the four different regions were compared by counting the number is SNPs in the by MAFFT alignt sequences.
Table 2. Amino acid sequence comparisons between orthologous PRs pairs. The
‘Difference’ column representing the amount of different amino acids within the loops of that particular region. The ‘Total difference’ column representing the amount of different amino acids of region I & III and region VI and VIII added up. And the last column represents whether the amount of different amino acids was higher in region I & III than in region VI and VIII, + testing positive, - testing negative. The amino acids marked in yellow represent the SNPs. R OR aa sequence Differences Total difference RI+RIII> RVI+RVIII I HassOR14b -MAGLRDFFFNFEANEAIT 1 + I HarmOR14b -MAGLRDFFFNYEANEAIT +
III HassOR14b CAFTIFGTATYIAIHKKNMTFFELGHLYITLMISCVVVFR 8 9 +
III HarmOR14b CVLTIVGTATYVMIHKKNMTFFELGHLYITLMLSCVVFSR +
VI HassOR14b RGYIFSNILHWFFSYIVSTWFCTLDLFLSVIVFHIWGHFKI 1 +
VI HarmOR14b RGYIFSNILHWFFSYIVSTWICTLDLFLSVIVFHIWGHFKI +
VIII HassOR14b LLECSQMTPEALARYLPLTLTLFQQLIQLSIVFELVGTVSS 1 2 +
VIII HarmOR14b LLECSQMTPEALARYLPLTLILFQQLIQLSIVFELVGTVSS +
R OR aa sequence Differences Total difference RI+RIII> RVI+RVIII I SnonOR15 MLSSLRNFFFNIEPQEGVT 2 + I SinfOR29 MLTSLRNFFFNIEPQEGVN +
III SnonOR15 CLICVFGGVTYVLLHKKSMTFFELGHLYISLLMNAVIFSRI 0 2 +
III SinfOR29 CLICVFGGVTYVLLHKKSMTFFELGHLYISLLMNAVIFSRI +
VI SnonOR15 KWYIAYNFYNWGISYLCATWFCMHDCFLSLMVFHMWGHFKI 0 +
VI SinfOR29 KWYIAYNFYNWGISYLCATWFCMHDCFLSLMVFHMWGHFKI +
VIII SnonOR15 LLECSQMTTAALTRYLPLTVILFQQLIQLSIIFELIGSISD 1 1 +
R OR aa sequence Differences Total difference RI+RIII> RVI+RVIII I HassOR3.1 --MGLRQFLFENEAVEGIN 0 + I HarmOR3 --MGLRQFLFENEAVEGIN +
III HassOR3.1 TFLLSAGFILYVVKHNSELTFLETGHMYIVLLMSFIDVSRV 5 5 +
III HarmOR3 TVSGSAGFILYLVKHNSELTFLETGHMYIVLLMSLIDVSRV +
VI HassOR3.1 RGYSIACILHWIISYLCSTWFCMFDLFLSLMVFHLWGHFKI 1 +
VI HarmOR3 RGYSIACIIHWIISYLCSTWFCMFDLFLSLMVFHLWGHFKI +
VIII HassOR3.1 LLECSQMTAQALIRYVPLTVILTQQLIQLSVIFELVGSESD 1 2 +
VIII HarmOR3 LLECSQMTAQALIRYVPLTIILTQQLIQLSVIFELVGSESD +
R OR aa sequence Differences Total difference RI+RIII> RVI+RVIII I HassOR6 --MSFRKFLFENEAVDGIK 1 + I HarmOR6 --MSFRKFLFENEAVDGIT +
III HassOR6 TIATVVGSILYVVVHVTELTFLEAGLMYLIILISILDAITV 5 6 +
III HarmOR6 TIATVVGSILYVVVHVAELSFLEAGLMYLIILMSFLDALTV +
VI HassOR6 FGYVVACILHWIISYLCSTWFCMFSLFISLMVFNIWGHFKI 3 +
VI HarmOR6 FGYIVACILHWIISYLCSTWFCMFNLFISLMVFNLWGHFKI +
VIII HassOR6 LLECSQMTAQALMRYLPLTIILTQQLIQLSVIFELVGSESE 0 3 +
VIII HarmOR6 LLECSQMTAQALMRYLPLTIILTQQLIQLSVIFELVGSESE +
R OR aa sequence Differences Total difference RI+RIII> RVI+RVIII I SnonOR6 --MILRKFLFENEPVLGIT 2 + I SinfOR21 --MSLRKFLFENEPVIGIT +
III SnonOR6 NIFTLCGGISYVIIHKDDLSFLEVGHMYIIILMNVVIKSRL 14 16 +
III SinfOR21 TIACMFGGVSYVIIHMSELTFLEVGHMYIIILMNTVDMSRV +
VI SnonOR6 KGYIVVCILHWFFSYMCATWFCMFDLFLLLMVFNLWGHFKI 8 +
VI SinfOR21 KGYIVACIIHWSLSYLCSTWFCMFDLFLSLMVFHLWGHFKI +
VIII SnonOR6 LLECSQ-TAQALIRYLPLT--- 2 10 +
VIII SinfOR21 LLECSEMTAQALMRYLPLTIILTQQLIQLSVIFELVGSESE +
R OR aa sequence Differences Total difference RI+RIII> RVI+RVIII I HassOR11 ---MYAGNAVTGIT 0 + I HarmOR11 ---MYAGNAVTGIT +
III HassOR11 SLVYLALGVAYLKKNFHRFDFLELGQLYIVLLMNMLSTSRA 2 2 +
III HarmOR11 SLVYLALGIAYLKKNFHRFDFLELGQLYIVLLMNMLCTSRA +
VI HassOR11 NGYLAATLSGWYGTILCGSSVSMFDLFLCLMIFNLWGHFKI 1 +
VI HarmOR11 NGYLVATLSGWYGTILCGSSVSMFDLFLCLMIFNLWGHFKI +
VIII HassOR11 LLECSQMTAAALTRYLPLTIIMFGELVLLSIIFETIGTMSE 0 1 +
VIII HarmOR11 LLECSQMTAAALTRYLPLTIIMFGELVLLSIIFETIGTMSE +
R OR aa sequence Differences Total difference RI+RIII> RVI+RVIII I HassOR13 ---MKILSDGSDLEGVE 0 + I HarmOR13 ---MKILSDGSDLEGVE +
III HassOR13 NVTTLIGGAIYLRNNTGVLPSFELGHTYITVFMNCITCSRC 4 4 +
III HarmOR13 NVATLVGGAVYLRNNTGVLSSFELGHTYITVFMNCITCSRC +
VI HassOR13 KGYLAMTSFNCFISYTCTSYFCVVDLTVSLVIFHLWGHMRL 1 +
VI HarmOR13 KGYIAMTSFNCFISYTCTSYFCVVDLTVSLVIFHLWGHMRL +
VIII HassOR13 LLECSQLDMKTLVRYGPLTVVIFQQLIQISVIFELLGSSND 0 1 +
Table 3 represents the sign test. As shown in Table 2, for all the seven pairs of the
orthologous PRs, the variation of the intracellular loops of region I&III is higher than the variation in region VI&VIII. In terms of the binomial distribution table (‘Binomial
distribution – probability function’, 2014), this results in a significant difference in variation between the four regions (n=7, k=7, p=.0078).
Table 3 Sign test for 7 pairs of Pheromone receptor proteins. The sign in de
RI+RIII>RVI+RVIII columns represents whether the amount of variation of amino acids was higher (+) or lower (-) in region I & III than in region VI and VIII in that particular
orthologous pair of PRs. Each pair of PRs has shown significantly more variation in the intracellular loops RI and RIII relative to RVI and RVIII. (p = .0078)
Orthologous PRs RI+RIII>RVI+RVIII
HassOR14b vs HarmOR14b +
HassOR13 vs Harm OR13 +
HassOR3.1 vs HarmOR3 +
HassOR6 vs HarmOR6 +
HassOR11 vs HarmOR11 +
SnonOR6 vs SinfOR21 +
Discussion
I have investigated whether there is a generalised ligand specificity in PRs of four Noctuid moths species with the expectation that in order to keep the function of the receptor, the last two defining intracellular loops of the seven transmembrane protein structure will be more conserved than the first two nondefining intracellular loops, because the tuning
specificity could be achieved with just a few amino acid substitutions. In this study I found through comparing seven pairs of orthologous PRs of the closely related H. armigera and H. assulta, and the closely related S. inferens and S nonagrioides, significantly more variation in the first two intracellular loops than the last two intracellular loops. This confirms the
expectation that the amino acids in the loops of RVI and RVIII are more conserved and I can conclude that my results indeed support the hypothesis that RVI and VIII are involved in the ligand recognition of the four Noctuid moth species used in this study.
These results extend the findings of Yang et al. (2017), who discovered that two amino acids located in the last two intracellular domains of the seven transmembrane protein structure determine the functional difference between two orthologous ORs; OR14b of both H. assulta and H. armigera. My results that suggest that the ligand recognition could also be determined by these last two intracellular domains of multiple ORs of S. inferens, S.
nonagioides, H. armigera and H. assulta because of the last two intracellular domains being more conserved than the first two, indicate the generalization of the observation of Yang et al. (2017).
Because I have only been able to compare the amino acid sequences of the loops and look at the conserveness, my results are based on indirect evidence. It cannot be ruled out that the ligand recognition isn’t determined by anything else than the last two intracellular loops. This means my study can only suggest the possibility of the ligand recognition being
determined by the loops of RVI and RVIII. In order to exclude any other ligand
determination points in the protein structure, this study will need an experimental set up in which the OR will be mutated to find out whether the ligand is still able to bind like performed in the study of Yang et al. (2017). The intracellular loops of RVI&RVIII being more conserved than the loops of RI&RIII may not be the result of causal relation between the presence of a binding pocket and a higher level of conservation. Although not very likely, there may be a selection force on the intracellular loops of RI and RIII that promote variation in these loops. Finally, when the specificity of the OR is indeed determined by just a few amino acids, why would the whole loop be conserved? I assumed the whole loop structure would be conserved and needed to create the binding pocket, and apparently this is the case, but direct evidence is definitely needed.
To be able to provide hard evidence for the hypothesis forwarded in this study, a lot of lab work is required including CRISPR-cas (Wang et al., 2013; Zhu et al., 2019), a method in which DNA can be modified with unprecedented precision, or RNAi (Orr et al., 2020), a simple and rapid method of silencing gene expression, to prove the function of the receptor. These techniques go beyond the limitations of a bachelors project.
The availability of full length sequenced ORs on the internet is low. A lot of the amino acid sequenced of ORs are only partial. Also, some olfactory co-receptors are given a number instead of assigning the receptor as a co-receptor. There is not a lot of information on the olfactory receptor co-receptor, but according to Yang et al. (2012), the olfactory receptor co-receptor (Orco) is supposed to form heteromers with its particular OR and functions as a cation channel, which opens when the OR is activated. For example, formerly named OR83b in Drosophila species is actually an olfactory receptor co-receptor, but also OR2 in moths and other insects. I have also noticed that scientists choose their own name for the OR they sequenced, which means there are some possible duplicate ORs that are assigned a different
number. This meant that finding usable information for this study was a struggle and eventually limited to four Noctuid species of which (part of) the ORs sequences were complete and thus usable for this investigation.
Further research could include more Noctuid species or species from other families to maybe generalize the result even more and broaden the knowledge of the ligand recognition in PRs. The ORs of a lot of other moth species that are sympatric pests in different parts of the world are still low in terms of information and investigation. Some studies have been investigating the olfactory mechanisms of moth pest species, like Chang et al. (2017) who analyzed the oriental armyworm Mythimna separata, but more detailed investigation might be of great importance to save the harvest. This study could also be taken into the lab with the availability of sequencing the ORs of the species with more of a free choice rather than being limited by the available information on the internet. The method of mutating the receptors to find out whether the ligand specificity is located used by Yang et al. (2017) could be used on multiple PRs of the Noctuid species used in this study; S. nonagioides, S. inferens, H.
armigera and H. assulta. This may provide more evidence that support these findings. Also, the dN/dS ratio could be analyzed to measure the selection pressure. It is relatively more common for mutations in the DNA sequence to have no effect on the amino acid in a protein that is under selection pressure because the non-silent mutations are deselected. Without selection, the non-silent mutations will stay present (Yang et al., 2000). This could give insights in what ORs are more prone to change more rapid than other ORs, or ORs of other species.
To summarize, this report showed how the last two intracellular loops of multiple pheromone receptors of S. nonagioides, S. inferens, H. armigera and H. assulta could possibly be responsible for the determination of the ligand recognition. Further research should confirm and generalize this suggestion. This information will be an valuable addition to current knowledge of the olfactory mechanism and appears to be a of great potential, for example in terms of pest control.
Acknowledgements
I would like to thank Peter Roessingh for his supervision and his 24/7 availability to help me, during the experimental phase and with the writing of this thesis. I would also like to thank Astrid Groot for the supervision, the open mind and the ambition to make the most out of my project.
Supplementary materials
Document 1. Accession numbers of the olfactory receptors used in the phylogenetic analyses. Supplemantary materials
All raw data can be found in the data repository
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