Li et al. BMC Genomics 2013, 14:582 http://www.biomedcentral.com/1471-2164/14/582 RESEARCH ARTICLE Open Access Identification of differential expression genes associated with host selection and adaptation between two sibling insect species by transcriptional profile analysis Haichao Li1,2, Hao Zhang1,2, Ruobing Guan1,2 and Xuexia Miao1* Abstract Background: Cotton bollworm (Helicoverpa armigera) and oriental tobacco budworm (Helicoverpa assulta) are noctuid sibling species. Under artificial manipulation, they can mate and produce fertile offspring. As serious agricultural insect pests, cotton bollworms are euryphagous insects, but oriental tobacco budworms are oligophagous insects. To identify the differentially expressed genes that affect host recognition and host adaptation between the two species, we constructed digital gene expression tag profiles for four developmental stages of the two species. High-throughput sequencing results indicated that we have got more than 23 million 17nt clean tags from both species, respectively. The number of unique clean tags was nearly same in both species (approximately 357,000). Results: According to the gene annotation results, we identified 83 and 68 olfaction related transcripts from H. armigera and H. assulta, respectively. At the same time, 1137 and 1138 transcripts of digestion enzymes were identified from the two species. Among the olfaction related transcripts, more odorant binding protein and G protein-coupled receptor were identified in H. armigera than in H. assulta. Among the digestion enzymes, there are more detoxification enzyme, e.g. P450, carboxypeptidase and ATPase in H. assulta than in H. armigera.These differences partially explain that because of the narrow host plant range of H. assulta, more detoxification enzymes would help them increase the food detoxification and utilization efficiency. Conclusions: Thisstudysuppliedsomedifferentiallyexpressedgenesaffectinghostselectionandadaptation between the two sibling species. These genes will be useful information for studying on the evolution of host plant selection. It also provides some important target genes for insect species-specific control by RNAi technology. Keywords: Development, Host plant range, Transcripts, Digital gene expression tag profile (DGE-Tag), Sibling species, Differential expression gene, Helicoverpa armigera, Helicoverpa assulta Background Interestingly, these two species can mate under artifi- Cotton bollworm (Helicoverpa armigera, Hübner) and orien- cial manipulation and produce offspring. However, when tal tobacco budworm (Helicoverpa assulta, Guenée) are two female H. armigera mated with male H. assulta, the first sibling noctuid species of Lepidoptera. They are distributed filial generations are all males [3]. This result further con- in almost same region from 50°S to 50°N and from 45°S to firms that they are two distinct species [4]. Under natural 45°N, respectively. H. armigera are slightly broader than conditions, because of differences in sex pheromone oriental tobacco budworms [1,2]. In the field, similar ex- composition, the two species seldom mate. Their sex pher- ternal morphology makes them easily confused. omones comprise cis-11-hexadecenal (Z11-16: Ald) and cis-9-hexadecenal (Z9-16: Ald), but the compositions * Correspondence: [email protected] are reversed in the two species. The ratio of these two 1 Key Laboratory of Insect Developmental and Evolutionary Biology, Institute components is 97:3 in H. armigera, but it is 7:93 in of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China H. assulta [5-7]. Full list of author information is available at the end of the article © 2013 Li et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Li et al. BMC Genomics 2013, 14:582 Page 2 of 12 http://www.biomedcentral.com/1471-2164/14/582 In addition, the host ranges of these two species are some taxonomic characteristics to distinguish these two significantly different. H. armigera is a euryphagous insect species. Their eggs, larvae and pupae look like nearly same whose host range includes 40 families of over 200 different (Figure 1A-C). Only under the microscope, according to plants. However, H. assulta is an oligophagous insect, they some taxonomic characteristics they can be distinguished are mainly feeding on the plants of the Solanaceae, for (Figure 1E-G). The adult is the easiest stage to be distin- example, tobacco and hot pepper [1,8,9]. Although each guished by entomologist, because there are some special species have their own preferred host plants, both of them speckles and stripes on the wings (Figure 1D, H). love feed on tobacco and hot pepper [4]. These similarity To show the relationship of H. armigera and H. assulta, and difference may be depend on the host plant selection multiple sequence alignments of spanning the 18S rRNA by adult, or depend on the food digestion or detoxification across 26 species from 23 orders (Additional file 1, enzymes from larvae. Host plant selection is a complicated Additional file 2: Figure S1A) and the expansion segment and continuous process. The color, odor and shape of the of the COI gene across 20 species of lepidopteran moths plants will affect the insects choice on host plants, among (Additional file 3, Additional file 2: Figure S1B) were which odors are a critically important element for the constructed and supplied as Additional files. The results lifestyle and reproduction of an insect species [10]. Ac- provide clues about the evolutionary origin of the phyt- cordingly, insects with different feeding habits possess ophagous Noctuidae. The sister group of H. assulta and their own specific odor identification and odor-binding H. armigera is clustered on a clade. Some hypotheses on proteins [11]. At the same time, enzymes for food diges- the sister group relatedness based on morphology are tion and detoxification are also important factors for concordant with our molecular results. insect growth and development. These enzymes probably effect on the survival of insects, consequently affecting on Sequencing of DGE-tag libraries and unique tag annotation the host range of an insect species. Therefore, identifying DGE-tag profile libraries were constructed from total RNA enzymes related to insect development and feeding habits of H. armigera and H. assulta for four development stages will benefit to research on insect host range and on insect (embryo, larva, pupa and adult). The summary sequence pest control. resultsareshowninTable1.Lowfrequencytagswere The two sibling species are non-model insects. Their discounted under the assumption that many could have genome sequences are not available till now. To identify arisen through sequencing errors such as base substitu- differentially expressed genes from H. armigera and tion, deletion or addition at a single position [14]. There- H. assulta, digital gene expression tag (DGE-tag) profile fore, after eliminating low quality tags (containing Ns), libraries were constructed and sequenced using high copy numbers less than two and adaptor sequences, the throughput second-generation sequencing technology remaining reads were called clean tags, of which more [12,13]. A DGE-tag profile is according to the theory and than 50% were singletons (tags with count equal to 1), method of SAGE (serial analysis of gene expression) com- which is typically observed in SAGE experiments [15]. bined with high-throughput sequencing technology [14]. We obtained approximately 23 million 17nt clean tags In this study, eight DGE-tag libraries were constructed from both insect species. Their total unique clean tag and sequenced for four developmental stages (embryo, (Uni-tag) numbers were also similar at approximately larva, pupa and adult) of the two species. Differentially 357,000 (Table 1). Unique tag-to-gene assignments were expressed transcripts or genes between H. armigera and conducted for the four development stages of H. armigera H. assulta were analyzed by bioinformatics. Most growth and H. assulta using SOAPdenovo program just permit- and development related genes have similar expression ting 1 bp mismatch [16]. On average, more than 75% of modes. The differentially expressed genes are mainly focus the uni-tags of H. armigera were mapped on transcripts; on olfactory-related genes and enzymes for food detoxi- however, only 64.5% uni-tags of H. assulta mapped on fication or digestion. Therefore, the two sibling species transcripts. The total numbers of transcripts or genes represent a good model for host plant selection and were 268,145 and 230,591 for H. armigera and H. assulta, adaptability. These results also provide valuable data for respectively, among which the annotated transcripts or insect pest control. genes were 88,857 and 75,157, respectively (Table 1). The Illumina short-reads sequence of H. armigera and Results and discussion H. assulta were submitted to NCBI Sequence Read The main identifying characteristics of H. armigera and Archive under the accession number of SRR628282 and H. assulta SRR620569, respectively. The two insect species in genus Helicoverpa, Cotton Although we obtained similar amounts of total unique bollworm (H. armigera) and oriental tobacco budworm clean tags