Functional opsin retrogene in nocturnal moth
- Pengjun Xu†1, 2,
- Roberto Feuda†3,
- Bin Lu4,
- Haijun Xiao1,
- Robert I. Graham5 and
- Kongming Wu1Email author
© The Author(s). 2016
Received: 21 June 2016
Accepted: 21 September 2016
Published: 19 October 2016
Retrotransposed genes are different to other types of genes as they originate from a processed mRNA and are then inserted back into the genome. For a long time, the contribution of this mechanism to the origin of new genes, and hence to the evolutionary process, has been questioned as retrogenes usually lose their regulatory sequences upon insertion and generally decay into pseudogenes. In recent years, there is growing evidence, notably in mammals, that retrotransposition is an important process driving the origin of new genes, but the evidence in insects remains largely restricted to a few model species.
By sequencing the messenger RNA of three developmental stages (first and fifth instar larvae and adults) of the pest Helicoverpa armigera, we identified a second, intronless, long-wavelength sensitive opsin (that we called LWS2). We then amplified the partial CDS of LWS2 retrogenes from another six noctuid moths, and investigate the phylogenetic distribution of LWS2 in 15 complete Lepidoptera and 1 Trichoptera genomes. Our results suggests that LWS2 evolved within the noctuid. Furthermore, we found that all the LWS2 opsins have an intact ORF, and have an ω-value (ω = 0.08202) relatively higher compared to their paralog LWS1 (ω = 0.02536), suggesting that LWS2 opsins were under relaxed purifying selection. Finally, the LWS2 shows temporal compartmentalization of expression. LWS2 in H. armigera in adult is expressed at a significantly lower level compared to all other opsins in adults; while in the in 1st instar stage larvae, it is expressed at a significantly higher level compared to other opsins.
Together the results of our evolutionary sequence analyses and gene expression data suggest that LWS2 is a functional gene, however, the relatively low level of expression in adults suggests that LWS2 is most likely not involved in mediating the visual process.
KeywordsGene duplication Expression Evolution Noctuidae
Gene duplication is a fundamental process in genome evolution generating new biological functions and promoting adaptation to the changing environment [1–6]. The classical model predicts that after gene duplication, one of the two genes usually degenerates in a few million years, or, in rare cases, one of the duplicates might evolve new gene functions . There are four well established mechanisms by which the DNA duplicates. These are: 1) unequal crossing-over, 2) duplicative (DNA) transposition, 3) polyploidization, and 4) retrotransposition . The contribution of these mechanisms, (with the exception of retrotransposition) to the origin of new genes is well-established. Retrotransposition is different to the other three mechanisms as it includes an intermediate RNA step. That is, a mature mRNA is reverse transcribed into a complementary DNA copy without introns and then inserted back into the genome randomly . The location of insertion is presumed to be random, thereby implying that the copy usually lacks the regulatory apparatus responsible of driving the correct gene expression. This is one of the main reasons why, until recently, retrotransposed genes were not considered to be functional [10–12]. However, recent work has challenged this view suggesting that several retrotransposons have been important for the evolution of novel phenotypes . Interestingly, Marques et al. proposed that between 0.5 and two retrogenes are fixed every million years in flies and mammals, respectively [13–15]. The integration of statistical and molecular methods can be employed to assess the retrotransposons functionality. As reviewed by Kaessman et al.  a ratio between non-synonymous and synonymous mutation (ω value) of less than 1 suggests that the gene is under selection and hence still functional. Additionally, other features such as the presence of an open reading frame (ORF), conserved sequence to other species and expression (e.g. qPCR or RNA-seq) are indicative of their functionality.
Opsins are a subfamily of G-protein coupled receptors crucial for the visual process in all the metazoans . In insects, this process is mediated by four paralogs: Long wave sensitive (LWS), Ultraviolet sensitive (UV), Blue sensitive (B) and probably Rh7 [18, 19]. Additionally, it is believed c-opsin might be involved in circadian rhythms . Mechanistically, photoreceptors expressing opsin of different wavelengths signal to the brain, which then perceive colours . The type of wavelength and the separation between maximum peak of absorbance defines the visual capability [18, 19, 22]. Opsin retrotransposons have been identified in various species, including the diurnal moth Callimorpha dominula (superfamily Noctuidea) , the jellyfish Tripedalia cystophora , in cephalopods , and in teleost fish .
In this work, we investigated the evolutionary history of an opsin retrogene in 7 species of nocturnal moth (Noctuidea). Firstly, we sequenced the whole transcriptome for 3 different developmental stages and adults of the pest Helicoverpa armigera, including first instar larvae, fifth instar larvae and adults. We identified that in addition to the traditional opsin repertoire (LWS, B and UV), H. armigera possesses a second intronless LWS gene (that we call LWS2). Subsequently, we investigated the phylogenetic distribution of this gene in six other Noctuidea species, one Crambidae, 15 Lepidoptera and 1 Trichoptera species. Our results suggest that 1) LWS2 evolved within Noctuidea and 2) the ω-value is less than 1, suggesting a relaxed purifying selection acting on LWS2. Finally, expression levels of LWS2 in H. armigera strongly differ between developmental stages, suggesting that most likely this gene is not involved in the visual process.
Material and methods
Transcriptome, annotation and opsin identification
Using RNA-Seq, the transcriptome of the whole bodies of first instar larvae (four groups and n = 30 for each gourp), fifth instar larvae (four groups and n = 10 for each group), and adults (four gourps and n = 10 for each group) of the nocturnal moth H. armigera was sequenced with paired-end and 100-nt read length on the channels of an Illumina HiSeq™ instrument. Assembled contigs were annotated using BLASTx to align with the database of NR, String, Swissprot and KEGG (see Additional file 1 for details). The RNA-Seq data were submitted to the NCBI GEO database (accession number: GSE86914). Primers were designed and PCR undertaken to (Additional file 1: Table S4) amplify the full-length cDNA of LWS2. To understand whether this duplication was specific to H. armigera or species from Noctuidae, we amplified LWS2 in six other species of Noctuidae moth: Agrotis ypsilon, Agrotis segetum, Mamestra brassicae, Mythimna separata, Spodoptera exigua, Spodoptera litura and one species of Crambidae: Ostrinia nubilalis. Additionally, we employed BLASTn algorithm to investigated the presence of LWS2 in other 15 complete Lepidoptera and 1 Trichoptera complete genomes from LepBase , spanning at total of 12 insect families (see Additional file 1: Table S5). Sequences were identified as follow: LWS1 and 2 were used as seed in a BLASTn search. Each sequences with an e-value <10−20 was retained a putative good opsin. To discriminate between opsin and other GPCRs after the translation in protein using TranslatorX  and using InterProScan we identified all the sequences with the retinal binding domain.
Alignment, phylogenetic and evolutionary analysis
The data set including the newly identified LWS2 from Noctuidea, Callimorpha domicula opsins, Ostrinia nubilalis opsins and putative LWS2 from LepBase (81 sequences in total available in Additional file 2) was aligned using the codon model as implemented in PRANK . The resulting alignment was manually curated to remove gap-rich regions. GTR-G was identified as the best-fitting substitution-model accordingly to the AIC as implemented in Modelgenerator . The phylogenetic reconstruction was performed using Maximum likelihood (ML) using Iqtree  and confirmed using Bayesian analysis (BA) under site heterogeneous model CAT-G model as implemented Phylobayes 3.3e . In ML reconstruction the nodal support was evaluated using UltraFast bootstrap (BS) and abayes (aBS)  while in BA using the Bayesian posterior probability (PP). In the BA the convergence among chains was estimated using bpcomp and chains were considered converged when the maxdiff value was < 0.3. The phylogenetic analyses were performed using a rooted tree i.e. the LWS tree was rooted using Rh7, UV and Blue opsin. Finally, in order to account for the possible misleading effect of using distant related outgroup, we repeated the analyses without outgroup.
In order to estimate whether LWS2 is under evolutionary constraint we estimated the ratio between non-synonymous and synonymous substitutions (ω-value) using a maximum likelihood approach  as implemented in CODEML . If the ω < 1, this is indicative of purifying selection However, if the retrogenes were under relaxed purifying selection, we expect an elevated ω-value relative to its paralog LWS1, which suggests that it may be neo-functionality or sub-functionalization. We evaluated five hypotheses: (1) one ω-ratio for all branches (one-ratio model; assuming that all branches have been evolving at the same rate); (2) ω-ratio = 1 for all branches (neutral model; neutral evolution for all branches); (3) moth LWS2 lineage and LWS1 lineage have different ω-ratio (ω2 and ω1; two ratio model; allowing foreground branch to evolve under a different rate); (4) neutral evolution for moth LWS2 lineage (ω2 = 1); and (5) the free-ratio model with free ω-ratio for each branch. In addition, we used branch-site models: moth LWS2 lineage was defined as the foreground, rest lineages were defined as the background branch, and these were then specified in the tree file by using branch labels. Likelihood-ratio test (LRT) was employed to determine if the alternative model, indicating positive selection, was superior to the null model. We also performed CODEML test on H. armigera LWS2 lineage to see if nature selection acted on any of the LWS2 branches.
Quantitative expression of opsin
To test the expression of the opsin in H. armigera, we investigated the relative level of expression in the 3 different developmental stages using fragments per kilobase of exon per million fragments mapped (FPKM) . Differential gene expression between paralogs at different developmental stages was evaluated using STATA v.9.0 and ANOVA. Bonferroni multiple comparisons were used to determine the level of significance between the relative levels of mRNA expression.
Results and discussion
Selective patterns for LWS opsins
Estimates of ω
A: one ratio
ω = 0.06527
B: one ratio ω = 1
ω = 1
B vs. A
C: the LWS2 branch has ω2, the LWS1 branch has ω1
ω2 = 0.08202
A vs. C
ω1 = 0.02536
D: the LWS2 branch has ω2 = 1
ω2 = 1
D vs. C
ω1 = 0.02515
E: each branch has its own ω
Variable ω by branch
A vs. E
G: the LWS2 branch
H: the LWS2 branch has ω = 1
H vs. G
In conclusion we report the existence of LWS2, originating as retrocopies from LWS1, in seven moths from the superfamily Noctuoidea. Furthermore, the intact ORF, the ω < 1, the phylogenetic conservation and expression independently suggests that LWS2 opsins are functional.
a Bayes support
Blue sensitive opsin
Fragments per kilobase of exon per million fragments mapped
Long-wavelength sensitive opsin
Open reading frame
Ultraviolet sensitive opsin
We thanks Dr. Tiantao Zhang, from Institute of Plant Protection, Chinese Academy of Agricultural Sciences, for providing the reference sequence of LWS1 and LWS2 in O. nubilalis.
This research was supported by Science Fund for Creative Research Groups of the National Science Foundation of China (No. 31321004), the National Natural Science Foundation of China (Grant No. 31401752).
Availability of data and material
The RNA-seq datasets generated during the current study are available in the NCBI GEO database (accession number: GSE86914), (https://www.ncbi.nlm.nih.gov/geo/).
KW, PX, RF, BL and HX conceived the study. PX, BL and HX performed the experiments. KW, PX, RF, BL, HX and RIG wrote the manuscript. All of the authors critically reviewed and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The reared or wild-captured moths used in this study were serious pests in China, therefore, no permits were required for the described insect collection and experimentation.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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