Compared to Odorant-binding Proteins in the Reproductive System and Antennae of Athetis Dissimilis using Transcriptome Analysis

Odorant-binding proteins (OBPs) are prevalent in the antennal transcriptomes of different orders of insects. Studies on OBPs have focused on their role in the insect chemosensory system, but knowledge of their functions in the insect testis is limited. We sequenced the transcriptomes of the Athetis dissimilis reproductive organs and analyzed the expressive of OBPs in different tissues. We identied 23 OBPs in the testis and ovaries and 31 OBPs in antennal transcriptomes. The results of real-time quantitative PCR revealed that 23 of the 54 OBP genes were highly expressed in both female and male antennae, including three that exhibited male-biased expression and 15 that exhibited female-biased expression. A total of 24 OBPs were highly expressed in the testis of A. dissimilis, while expression of OBPs in the ovaries was very low. These ndings highlight the functional diversity of OBPs in insects and can facilitate further studies on the OBPs in A. dissimilis and lepidopteran species. execute odorant functions 27,29−33 . In this study, we identied 31 novel OBPs through the analysis of A. dissimilis antennal transcriptomes, expressing ve previously reported AdisOBPs 24 . The number of OBPs in A. dissimilis antennae were similar to the antennal transcriptomes of S. litura (33) 31 and S. littoralis (36) 34 but more abundant than S. exigua (11) 35 , M. sexta (18) 36 and H. armigera (26) 37 . We additionally sequenced the transcriptomes of A. dissimilis ovaries and testis. The alignments against the Nr database showed that 56.87% of the A. dissimilis unigenes were comparable to Helicoverpa armigera sequences. A total of 24 OBPs were identied in the transcriptomes of A. dissimilis reproduction organs. Based on the cluster analysis of the phylogenetic trees, ve AdisOBPs belonged to PBP/GOBP; 35 AdisOBPs belonged to ‘Classic’ OBPs; 7 AdisOBPs belonged to ‘Plus-C’ OBPs; and 9 AdisOBPs belonged to ‘Minus-C’ OBPs. These results were similar to the classications of most insect OBPs 19,31,38 . Interestingly, AdisOBP1, AdisOBP17 and AdisOBP40 did not cluster into these 4 subclass OBPs, but multiple sequence alignments of the A. dissimilis OBPs revealed that 3 of the OBPs contain no conserved cysteines. Their construction requires further to verication. Insect OBPs in the sensory organs 15,19,31,39−41 . Our result showed that 23 AdisOBPs were signicantly expressed in both female and male antennae compared to other tissues. Only the expression of 3 AdisOBPs were signicantly higher in the antennae’s of males compared to females, suggesting that females require more abundant OBPs for spawning. OBPs are also expressed in the non-olfactory organs, such as those required for reproduction . In study, 24 AdisOBPs showed signicant expression in the testis of A. dissimilis compared to other tissues, but the expression


Results
Illumina sequencing and assembly A total of 34,565,866, 32,154,799, and 26,952,526 clean reads containing 10. 35, 9.63, and 8.07 giga base (Gb) pairs of clean nucleotides respectively, were obtained from the three replicates of the A. dissimilis ovaries. A total of 27,752,168, 28,900,040, and 30,838,686 clean reads containing 8.29, 8.65 and 9.23 giga base (Gb) pairs of clean nucleotides respectively, were obtained from the three replicates of A. dissimilis testes. The quality of the transcriptome sequences was high, with Q30 percentages of 94.03%, 94.36%, 94.21%, 94.42%, 94.27% and 94.01% for the three replicates of A. dissimilis ovaries and testes, with a GC content were ~ 50% (Table 1). Then 221,074 transcripts and 82,016 unigenes with N50 length of 1,350 and 1,243 were obtained from assembled using Trinity (Table 2).    Note: Genes beginning with the lowercase letter "c" came from the identi cation of antenna transcriptome, and genes beginning with "Gene" came from testis identi cation.

Expression of the OBPs in the antennae, ovaries and testis of A. dissimilis
To understand the functions of the identi ed OBPs in A. dissimilis, we measured the relative expression levels of OBPs in different tissues of A. dissimilis via uorescence qRT-PCR (Fig. 6).

Discussion
Insects rely on peripheral sensilla on the antennae to distinguish plant odorants and pheromones 25 , a knowledge of the molecular mechanisms of olfaction is essential for better using olfactory-based pest management strategies and the development of novel strategies. OBPs are more accessible targets for research, considering they are small, soluble, stable and easier to manipulate and modify. About exact functions of the OBPs are unclear, but it is widely believed that their function is to capture and transfer outside odorants to ORs located on the membranes of ORNs [26][27][28] . Insect OBPs are present on the antennae where they execute odorant functions 27,29−33 . In this study, we identi ed 31 novel OBPs through the analysis of A. dissimilis antennal transcriptomes, expressing ve previously reported AdisOBPs 24 . The number of OBPs in A. dissimilis antennae were similar to the antennal transcriptomes of S. litura (33) 31 and S. littoralis (36) 34 but more abundant than S. exigua (11) 35 , M. sexta (18) 36 and H. armigera (26) 37 . We additionally sequenced the transcriptomes of A. dissimilis ovaries and testis. The alignments against the Nr database showed that 56.87% of the A. dissimilis unigenes were comparable to Helicoverpa armigera sequences. A total of 24 OBPs were identi ed in the transcriptomes of A. dissimilis reproduction organs.
Based on the cluster analysis of the phylogenetic trees, ve AdisOBPs belonged to PBP/GOBP; 35 AdisOBPs belonged to 'Classic' OBPs; 7 AdisOBPs belonged to 'Plus-C' OBPs; and 9 AdisOBPs belonged to 'Minus-C' OBPs. These results were similar to the classi cations of most insect OBPs 19,31,38 . Interestingly, AdisOBP1, AdisOBP17 and AdisOBP40 did not cluster into these 4 subclass OBPs, but multiple sequence alignments of the A. dissimilis OBPs revealed that 3 of the OBPs contain no conserved cysteines. Their construction requires further to veri cation.
Insect OBPs are expressed in the sensory organs 15,19,31,39−41 . Our result showed that 23 AdisOBPs were signi cantly expressed in both female and male antennae compared to other tissues. Only the expression of 3 AdisOBPs were signi cantly higher in the antennae's of males compared to females, suggesting that females require more abundant OBPs for spawning. OBPs are also expressed in the non-olfactory organs, such as those required for reproduction [42][43][44] . In this study, 24 AdisOBPs showed signi cant expression in the testis of A. dissimilis compared to other tissues, but the expression of AdisOBPs in the ovaries were low. It was previously speculated that OBPs expressed in the testis deliver compounds to the females during mating [18][19] . Hence, it is understandable to presume that such stable proteins could be used in the testis of insect where there is need for transportation of hydrophobic molecules in aqueous media or protection of chemicals from degradation, as well as to assure a gradual release of semiochemicals in the environment. So these proteins have been named for ''encapsulins", to imply the common role of encapsulating small ligands 45 .
Like antennae, insect testes contain a large number of OBP genes. These genes may also be involved in the development of testis or the movement of sperm and so on. The functions of these genes need us to further study. Our results provide a reference for the study of these genes.

Insect rearing and sample preparation
The A. dissimilis strain was collected from Luoyang (province of Henan, China) corn elds (112°26´ E, 34°43´ N) in 2014 and maintained at the Henan Science and Technology University. Colonies were reared on an arti cial diet at 25 ± 1°C, 80 ± 5% relative humidity and a 16-h/8-h light/dark cycle.
Based on preliminary data, we found that the A. dissimilis sperm and eggs began to mature 3 days after emergence. We respectively collected the ovaries and testes of 3-day old virgin females and male adults (n = 40 per treatment) from three biological replications. Dissections were performed in sterile PBS-DEPC and immediately frozen in liquid nitrogen until RNA isolation. cDNA library preparation and sequencing Total RNA from the A. dissimilis ovaries and testis tissues were extracted using RNAiso Plus kit (TaKaRa, Dalian, China) and treated with DNase I (

Assembly and Functional annotation
Raw data (raw reads) in the FASTQ format were rst modi ed into clean data (clean reads) through Perl scripts. This was performed through the removal of reads containing adapter sequences, >10% unknown nucleotides and quality values ≤20. The Q20, Q30, and GC content were then calculated using highquality data.
Transcriptomes were assembled using Trinity (version trinityrnaseq_r20131110) with default settings, except for min_kmer_cov set to 2 46  Sequence and phylogenetic analysis Sequence similarities were assessed using the NCBI-Blast network server (http://blast.ncbi.nlm.nih.gov/). The signal peptides of OBPs were predicted using Expression analysis through quantitative real-time polymerase chain reaction Male antennae, female antennae, ovaries and testes tissue from adults at 3 post-eclosion were excised and frozen in liquid nitrogen. Total RNA was extracted using RNAiso Plus kits (TaKaRa, Dalian, China) and isolated RNA was transcribed to rst-strand cDNA using PrimeScript TM RT reagent with gDNA Eraser (TaKaRa, Dalian, China) following the manufacturer's protocols. Real-time quantitative PCR (RT-qPCR) was performed with SYBR ® Premix Ex Taq TM II (TaKaRa). The A. dissimilis GADPH gene was used as an endogenous control to correct for sample-to-sample variations. A 200 ng/mL cDNA sample was used for per tissue. Primers were designed using Primer Premier 5.0 software and are listed in supportment Table 1. RT-qPCR reactions contained: 10 μL of SYBR Premix Ex Taq II, 20 ng of cDNA template, 0.2 μM of each primer and nuclease-free water. The cycling conditions were 1 cycle of 95°C for 5 min, followed by 40 cycles of 95°C for 5 s and 55°C for 30 s. Melt curve conditions were 95°C for 10 s and 65°C for 30 s. No-template controls (NTC) were included to detect possible contamination. Three biological replicates were analyzed and the relative expression of the OBP genes was measured using the 2 -∆∆CT method 51 . Expression was calculated relative to levels in the female antennae, which were arbitrarily set to 1. Differences in the expression of AdisOBP genes between the different tissues were compared using a one-way nested analysis of variance (ANOVA), followed by a Tukey's honestly signi cance difference (HSD) test using SPSS (SPSS Institute 17.0, SPSS Inc, Chicago, IL, USA).