Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2021, Article ID 1967158, 15 pages https://doi.org/10.1155/2021/1967158

Research Article Sex-Biased Gene Expression of Collected from Gansu Province, China, Reveals Their Different Therapeutic Potentials

Songyu Gao ,1 Feng Wu ,2 Xintong Chen ,1 Ying Yang ,3 Yina Zhu ,1 Liang Xiao ,1 Jing Shang ,4 Xiaowei Bao ,5 Yi Luo ,6 Haihu Chen ,7 and Qing Liu 3

1Faculty of Naval Medicine, Naval Medical University (Second Military Medical University), Shanghai 200433, China 2College of Marine Science, Shanghai Ocean University, Shanghai 201306, China 3College of Science and Veterinary Medicine, Shanxi Agricultural University, Jinzhong, Shanxi 030801, China 4School of Nursing, Naval Medical University (Second Military Medical University), Shanghai 200433, China 5College of Food Science and Pharmacy, Xinjiang Agricultural University, Urumqi 830052, China 6Central Medical District of Chinese PLA General Hospital, Beijing 100120, China 7Department of Intervention, Changhai Hospital, Naval Medical University (Second Military Medical University), Shanghai, China

Correspondence should be addressed to Yi Luo; [email protected], Haihu Chen; [email protected], and Qing Liu; [email protected]

Received 2 June 2021; Accepted 10 August 2021; Published 21 August 2021

Academic Editor: Oluwafemi Adeleke Ojo

Copyright © 2021 Songyu Gao et al. 2is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2e , named Mesobuthus martensii, commonly called Quanxie (全蝎) in Chinese, have been widely used as one of the animal medicines for more than 1,000 years because of the strong toxicity of their venoms. Meanwhile, scorpions are sexually dimorphic in appearance, and many exhibit traits associated with sex-biased gene expression, including maternal care, mating competition, female mating choices, ecology, and even venom composition and lethality. 2is study aims to explore the dif- ferences in composition of the venom of scorpions of different sex using the method of transcriptomics. Whole de novo transcriptomes were performed on the samples of M. martensii captured from Gansu Province to identify their sex-biased gene expression. 2e conserved CO-1 sequences of the captured samples matched that of M. martensii. A total of 8,444 (35.15%), 7,636 (31.78%), 8,510 (35.42%), 7,840 (32.63%), 9,980 (41.54%), and 11,829 (49.23%) unigenes were annotated with GO, KEGG, Pfam, Swissprot, eggNOG, and NR databases. Moreover, a total of 43 metalloproteases, 40 potassium channel toxins, 24 phospholipases, 12 defensins, 10 peroxiredoxins, 9 cysteine proteinase inhibitors, 7 serine protease inhibitors, 6 sodium channel toxins, 2 NDBPs, 1 calcium channel toxin, 1 waprin-like peptide, 1 antibacterial peptide, 1 antimicrobial peptide, and 1 anticoagulant peptide were screened out. With the fold change of 2 and 0.5, p value < 0.01, and q value < 0.05 as thresholds, a total of 41 out of 157 (26.11%) toxin-related unigenes had significant differential expression, and this ratio was much higher than the ratio of differentially expressed unigenes out of all annotated ones (8.84%). Of these differentially expressed toxins, 28 were upregulated and occupied the majority, up to 68.30%. 2e female scorpions showed more upregulated unigenes that annotated with toxins and had the potential to be used as more effective therapeutic drugs. In addition, this method of omics can be further used as a useful way to identify the difference between female and male toxic .

1. Introduction females in both humans and animals in the aspect of sex- specific reproductive tissues and also of size, shape, color, and Variation is very common and important among different behavior [1, 2], which are all sexually dimorphic traits. Sex- individuals of the same species, according to Darwinian biased genes are called sex-specific genes if they are exclu- evolution theory. 2ere are differences between males and sively expressed in one sex, or sex-enriched genes if they are 2 Evidence-Based Complementary and Alternative Medicine expressed at higher levels in one sex compared with the other foam box fed on Tenebrio molitor. 2eir poison glands were [3]. In the process of growth and development, the abundance cut from their telsons and were quickly frozen in liquid of sex-biased genes is constantly changing, which usually nitrogen and kept in −80°C environment. increases and peaks at a certain stage and then decreases in individual cases [4]. In addition, the level of differential 2.2. Chemicals and Reagents. 2e Trizol reagent was pur- expressed genes can be affected by environmental factors, chased from Invitrogen (CA, USA). 2e RNA 1000 Nano such as the diet quality of Drosophila melanogaster and in- LabChip Kit was purchased from Agilent (CA, USA). 2e dividual’s social status of turkeys [5, 6]. mRNASeq sample preparation kit was purchased from Belonging to class Arachnida and phylum Arthropoda, Illumina (San Diego, USA). All other chemicals were of the the ancient terrestrial animal scorpions, we collected, almost highest purity commercially available. have the same morphology characters of the scorpions discovered in the fossils, which began to form 400 million years ago in the Silurian Period [7]. According to the 2.3. Total RNA Extraction. Total RNA was extracted by common treatment strategy called “combating poison with Trizol reagent (Invitrogen, CA, USA). 2e manufacturer’s poison” in traditional Chinese medicine, scorpions were procedures were strictly followed. 2e quantity and purity of used as one of the animal medicines more than 1,000 years total RNA were analyzed by Bioanalyzer 2100 and RNA 1000 ago because of their strong toxicity [8]. Like other animals in Nano LabChip Kit (Agilent, CA, USA) with RIN number nature, scorpions are also sexually dimorphic in appearance >7.0. Poly(A) RNA was purified from total RNA (5ug) using and show many traits associated with sex-biased gene ex- poly-T oligo-attached magnetic beads in two rounds of pression, including maternal care, mating competition, fe- purification. After purification, the mRNA was fragmented male mating choice, venom composition, and lethality into small pieces by divalent cations in the elevated tem- [9, 10]. Male scorpions have smaller bodies with longer tail perature. 2en, the cleaved RNA fragments were reverse- segments, whereas females are larger with shorter and more transcribed to create the final cDNA library in accordance rounded tail segments. 2e males are often faster and more with the protocol for the mRNASeq sample preparation kit flexible in searching for mates, whereas females are more (Illumina, San Diego, USA). 2e average insert size for the sedentary because they are frequently gravid and always paired-end libraries was 300 bp (±50 bp). 2en, we per- carry the young on their backs, making long-distance travels formed the paired-end sequencing on an IlluminaHiseq4000 more difficult. 2e differences in ecological niches between at the (LC Sciences, USA), following the vendor’s recom- males and females have resulted in the fact that females are mended protocol. more apt to sting defensively than males. In the book of Shennong’s Classic of Materia Medica, 2.4. De novo Assembly, Unigene Annotation, and Functional written in the Han Dynasty and regarded as the earliest Classification. First, Cutadapt [14] and Perl scripts in house known work of traditional Chinese medicine, the ancients were used to remove the reads that contained adaptor had found there were many differences in the Chinese contamination, low-quality bases, and undetermined bases. traditional medicines produced in different regions, and this 2en, sequence quality was verified by FastQC (http://www. viewpoint had been proved using modern technologies [11]. bioinformatics.babraham.ac.uk/projects/fastqc/), including 2e concept of genuine medicinal materials formally put the Q20, Q30, and GC content of the clean data. All forward in the book of Collected Essentials of Species of downstream analyses were based on clean data of high Materia Medica completed in the Song Dynasty showed that quality. De novo assembly of the transcriptome was per- the medicinal materials were produced in an area with formed with Trinity 2.4.0 [15]. Trinity groups were tran- specific natural conditions and ecological environment. In scribed into clusters based on shared sequence content. Such the same light, the sex was also regarded as an important a transcript cluster is very loosely referred to as a “gene”. 2e influential factor of many traditional Chinese medicines, longest transcript in the cluster was chosen as the gene such as Tubiechong (Eupolyphaga sinensis Walker), sequence (aka unigene). Hamayou (Oviductus Ranae), Lurong (Cornu cervi panto- All assembled unigenes were aligned against the non- trichum), and the like. [12, 13]. In this article, the technique redundant (Nr) protein database (http://www.ncbi.nlm.nih. of transcriptomics was used to evaluate the differential gov/), Gene ontology (GO) (http://www.geneontology.org), expression levels of the toxins in venoms, which SwissProt (http://www.expasy.ch/sprot/), Kyoto Encyclo- can be used as the main component of medicines, in the pedia of Genes and Genomes (KEGG) (http://www.genome. hope of laying a foundation for the studies on how to choose jp/kegg/), and eggNOG (http://eggnogdb.embl.de/) data- the sex of medicinal animals and improve their therapeutic bases using DIAMOND [16] with a threshold of efficacy. E-value < 0.00001. 2. Materials and Methods 2.5. Differentially Expressed Unigenes Analysis. Salmon [17] 2.1. Sample Collection. A total of 80 scorpions were collected was used to perform the expression level for unigenes by from Gansu Province, China, in the summer of 2020. Half of calculating TPM [18]. 2e differentially expressed unigenes them were female, and the others were male. 2ey were sent were selected with log2 (fold change) > 1 or log2 (fold to the laboratory immediately after collected and kept in a change) <−1 and with statistical significance (p value < 0.01 Evidence-Based Complementary and Alternative Medicine 3 and q value < 0.05) by R package edgeR [19]. Next, GO and 90.43% of all unigenes. 2e three length distribution ranges KEGG enrichment analyses were again performed on the with the largest proportion of unigenes were 200–399 bp, differentially expressed unigenes by Perl scripts in-house. 400–599 bp, and 600–799 bp with a number of 10,771 (44.83%), 3,461 (14.40%), and 1,902 (7.92%), respectively (Figure 1(a)). 2e GC content of all the unigenes was 2.6. Bioinformation Analysis. According to the level-1 do- 33.98%, and 95.79% unigenes had a GC content below 50% mains’ biological process, cellular components, and mo- (Figure 1(b)). 2e TPM, which means the transcripts per lecular functions, the GO (http://geneontology.org/) terms kilobase of exon model per million mapped reads, was used were analyzed. All unigenes were analyzed in contrast with as a normalized parameter to measure and compare the the Kyoto Encyclopedia of Genes and Genomes (KEGG) gene abundance among different samples (Figure 1(c)). database for their signaling pathways. 2e toxins or ven- With a BLASTx alignment e-value threshold of 10−5, we omous transcripts from the scorpion samples were then successfully annotated 8,444 (35.15%), 7,636 (31.78%), searched against a toxin-related subdatabase from SWIS- 8,510 (35.42%), 7,840 (32.63%), 9,980 (41.54%), and 11,829 SPROT (https://www.uniprot.org/program/Toxins). 2e (49.23%) unigenes with the GO, KEGG, Pfam, Swissprot, protein sequences were translated from RNA sequences by eggNOG, and NR databases (Figure 1(f)). From all the the ORFfinder online service (https://www.ncbi.nlm.nih. annotated unigenes, we screened 2,124 of them, which gov/orffinder). From NCBI (https://blast.ncbi.nlm.nih.gov/ showed significant differential between the female and male Blast.cgi), we collected the sequences used for multiple se- scorpions and accounted for a percentage of 8.84%, while 2 quence alignments and performed the alignments with the and 0.5 were chosen as the threshold of the fold change and software Bioedit (version: 7.2.5). Phylogenetic trees were 0.01 for the p value and 0.05 for the q value. 2e majority of constructed in MEGA7 by the neighbor-joining method the differential expression unignens with a number of 1,722 with bootstrap replication set at 1000. 2e 3D modeling was (81.07%) were upregulated, whereas only 402 (18.93%) of carried out in the software Discovery studio 2016. them were downregulated (Figure 1(g)).

3. Results 3.2. Species Identification. 2e molecule mtDNA cyto- 3.1. Quality Control and Overview of Transcriptome. 2e chrome c oxidase subunit I (MT-COI, COI), conventionally samples were collected from Gansu Province of China to considered to be the signature sequence owing to its high explore the differences of the venoms between male and conservation, was screened out from the contrasted tran- female scorpions by the method of transcriptome, which scriptome to make the species identification of the collected was regarded as an effective way to detect the differences in samples in molecular level [20]. With high identity and TPM the gene level of the samples with different characters. 2e in all the groups of samples, sequence DN20344 c1 g2 was samples were sent to our laboratory lively with all body chosen for further research among all the 6 COI sequences parts and with typical morphological characteristics of from annotation result to NR database. By the online tool of scorpions. 2ey were kept in a foam box and fed on BLASTn (nucleotide to nucleotide), the most highly aligned Tenebrio molitor temporarily. 2e collected scorpions were sequence was sequence JF700146.1 from the species Meso- divided into two groups, male and female, according to the buthus martensii with an identity of 97.64%, followed by sex-biased characters, including the hardness of genital sequence DQ340065.1 also from the species M. martensii operculum and the width of sternum. 2en, the body width (Figure 2(b)). Moreover, by the online tool BLASTx (nu- and the metasoma width of the samples were measured and cleotide to protein), the most aligned protein sequence was analyzed. 2e result of body width/metasoma width also from the species M. martensii with an identity of 95.18% showed that female scorpions have higher value than the (Figure 2(c)). By the software BioEdit, the amino acid se- male ones (Figure 1(d)). Conversely, the male scorpions quence YP 001427343.1 was aligned with the CO1 amino have more pectineous teeth in the pecten, which was acid sequences of DN20344 c1 g2 transformed from the consistent with the inherent characteristics of scorpions DNA sequence by MEGA 7 (Figure 2(a)). (Figure 1(e)). Eight groups of samples were tested, in- In conclusion, it is safe to say that all the collected scorpions cluding 4 groups of females and 4 groups of males. A total belonged to the same species M. martensii after identified at the of 47,842,140 (on average) raw reads were obtained with a level of nucleotides and proteins. Compared with the NR GC ratio of more than 40% in all 8 groups of samples, after database, a nonredundant protein sequence database collected mRNA purification, cDNA synthesis, library construction, by NCBI, the 80–95% similarity sequences were the most, and Illumina sequencing. After the adapters and low- accounting for 32.66% of all sequences, followed by the 60–80% quality sequences were removed, whose quality value was similarity sequences with a percentage of 28.91% and the less than 20, a total of 46,756,394 (97.7%, on average) valid 40–60% similarity sequences with a percentage of 18.13% reads were left (Table 1). 2e strategy of mixed de novo (Figure 2(d)). 2e e-value ranges of 0, 0∼1e-100, 1e-100∼1e-60, assembly of all the samples was adopted to get the unigenes. 1e-60∼1e-45, 1e-45∼1e-30, 1e-30∼1e-15, and 1e-15∼1e-5 rep- A total of 24,026 unigenes were obtained with a median resent 9.96%, 32.42%, 12.96%, 7.30%, 10.05%, 14.31%, and length of 458 bp, and the total assembled bases number was 12.99% of all sequences, respectively (Figure 2(e)). 2e top 4 21,528,143 bp using the software Trinity. A total of 21,727 aligned species were Centruroides sculpturatus (69.20%), unigenes had a length from 200 to 2,199 bp and made up Limulus polyphemus (3.07%), Stegodyphus mimosarum 4 Evidence-Based Complementary and Alternative Medicine

12000 100 100 14000 100 1800 10000 2600 12000 80 80 80 1400 10000 8000 1200 60 60 8000 60 6000 1000 6000 40 800 40 40 4000 Accumulative (%) Accumulative Accumulative (%) Accumulative 600 (%) Accumulative Number of Unigene of Number Number of Unigene of Number Number of Unigene of Number 4000 20 400 20 20 2000 2000 200 0 0 0 0 0 0 -- -- 10 20 30 40 50 60 70 80 ≥50 0-1.99 2-3.99 4-5.99 6-7.99 8-9.99 ≥4600 GC Content 200-399 400-599 600-799 800-999 10-11.99 12-13.99 14-15.99 16-17.99 18-19.99 20-21.99 22-23.99 24-25.99 26-27.99 28-29.99 30-31.99 32-33.99 34-35.99 36-37.99 38-39.99 40-41.99 42-43.99 44-45.99 46-47.99 48-49.99 1000-1199 1200-1399 1400-1599 1600-1799 1800-1999 2000-2199 2200-2399 2400-2599 2600-2799 2800-2999 3000-3199 3200-3399 3400-3599 3600-3799 3800-3999 4000-4199 4200-4399 4400-4599 TPM Length (a) (b) (c) 3.5 30 25000 24026 3.0 25 20000 2.5 20 2.0 15000 15 11829 1.5 9980 Number 10000 8444 8510 10 7636 7840 1.0 Pectineous Teeth Pectineous 5000 0.5 5 Body width/Metasoma width Body width/Metasoma 0.0 0 0 Female Male Female Male All NR GO

Gender Gender Pfam KEGG eggNOG Swissprot (d) (e) (f) 40

35

15

10 -log10 (p value) 5

0

-10 -8 -6 -4 -2 0 2 46810 log2 (fold change)

Up Regulation Down Regulation No Difference (g)

Figure 1: Quality control and overview of the transcriptome of male and female scorpions. (a) Length distribution of unigenes. (b) GC content distribution of unignens. (c) TPM distribution of unigenes. 2e horizontal coordinates of A, B, and C are the sequence length, GC content, and TPM of the unigenes, respectively. 2e left vertical coordinates of these histogram represent the number of unigenes, whereas the right vertical coordinates are the cumulative percentage of unigenes. (d) Body width/metasoma width of female and male samples. (e) Pectineous teeth number of female and male samples. (f) Annotation results with the database of GO, KEGG, Pfam, Swissprot, eggNOG, and NR, respectively. (g) Volcano plot of the differential expression unigenes. 2e red dots represent the upregulated unigenes, whereas the dark blue ones represent the downregulated unigenes.

Table 1: Overview of the raw data and valid data. Sample Raw reads Valid reads Valid (%) Q20 (%) Q30 (%) GC (%) F1 51,750,108 50,424,598 97.44 96.28 89.64 42.06 F2 42,467,820 41,604,228 97.97 97.11 91.55 42.93 F3 44,188,454 43,308,140 98.01 97.10 91.49 44.98 F4 45,727,618 44,794,146 97.96 97.03 91.35 43.99 M1 45,499,340 44,613,506 98.05 96.95 91.32 41.38 M2 45,494,584 44,587,118 98.01 96.65 90.41 40.39 M3 41,850,764 40,897,900 97.72 96.40 89.92 41.23 M4 65,758,434 63,821,518 97.05 96.20 89.49 40.07 Evidence-Based Complementary and Alternative Medicine 5

10 20 30 40 50 60 70

DN20344_c1_g2 M. martensii Clustal Consensus

80 90 100 110 120 130 140

DN20344_c1_g2 M. martensii Clustal Consensus

150 160 170 180 190 200 210

DN20344_c1_g2 M. martensii Clustal Consensus

220 230 240 250 260 270 280

DN20344_c1_g2 M. martensii Clustal Consensus

290 300

DN20344_c1_g2 M. martensii Clustal Consensus (a) EU381098.1 C. vittatus 85.66 TRINITY DN20344 c1 g2 93 100 100 EF122643.2 C. vittatus 85.56 YP 001427343.1 I M. martensii 95.18

NC 037222.1 C. vittatus 84.65 AEL96857.1 A. hoggarensis 91.84

MK479167.1 C. borinquensis 86.66 98 YP 001936212.1 B. occitanus 90.94

77 KY982200.1 H. princeps 85.33 AWN56619.1 J. pintoi 88.25

TRINITY DN20344 c1 g2 AWN56586.1 H. bonetti 88.55 100 100 DQ3400653.1:1317-2313 M. martensii 96.49 77 QCS34915.1 C. baergi 88.86 67 JF700146.1 M martensii 97.64 83 AWN56577.1 C. infamatus 89.16 0.02 0.01 (b) (c) 3.07% 1.99% 7.30% 10.05% 32.66% 1.78% 12.96% 14.31% 1.53% 16.16% 0.99% 28.91% 12.99% 21.45% 32.42% 9.96% 18.13% 69.20%

4 .14% <40% 95%~100% 0 1e-45~1e-30 80%~95% 60%~80% 1e-60~1e-45 1e-100~1e-60 C. sculpturatus P. tepidariorum 40%~60% 1e-15~1e-5 1e-30~1e-15 D. melanogaster S. mimosarum 0~1e-100 Others C. secundus L. polyphemus (d) (e) (f)

Figure 2: Species identification. (a) An amino acid sequences of COI from M. martensii aligned with the CO1 amino acids of DN20344 c1 g2 transformed from the DNA sequence by blastx. At the bottom of the columns, asterisks (∗) show conserved positions, and colons () show conserved substitutions and points. (b) Phylogenetic tree of mtDNA sequences of cytochrome c oxidase subunit I (MT-COI, CO1) constructed by the means of MEGA 7 with the Neighbor-Joining method. 2e sequencing DN20344 c1 g2 is marked with red dots and the arthropoda are marked with light blue dots. 2e identity values are indicated on the right of each species name. (c) Phylogenetic tree of the COI amino acid sequences of DN20344 c1 g2 and 7 other species constructed by means of MEGA 7 with the Neighbor-Joining method. 2e sequencing DN20344 c1 g2 is marked with red dots and the arthropoda are marked with light blue dots. 2e identity values are indicated to the right of each species name. (d) Similarity distribution for the annotation of NR database. (e) E-value distribution for the annotation of NR database. (f) Species distribution for the annotation of NR database.

(1.99%), and Drosophila melanogaster (1.78%), and they all domains, namely, biological process (BP), cellular compo- belong to Arthropoda as M. martensii (Figure 2(f)). nent (CC), and molecular function (MF). 2e number of the annotated unigenes in the top three BP domain was 387, 312, 3.3. GO Analysis. GO analyses of unigenes were conducted and 292 accounting for the percentage of 1.78%, 1.44%, and by BLAST2GO software with default parameters, and 8,444 1.34% for the term of biological process, translation, and unigenes (35.15%) were annotated to three primary GO oxidation-reduction process. Cytoplasm, nucleus, and 6 Evidence-Based Complementary and Alternative Medicine integral component of membrane occupied the top three peptide (Figure 5(a), Table S1). Regarded the fold change of terms with 1,509, 1,304, and 958 unigenes, which come up to >2 or <0.5, p value < 0.01 and q value < 0.05 as thresholds, a the percentage of 6.95%, 6.00%, and 4.49% in CC domain. As total of 41 out of 158 (25.95%) had significant differential for the domain of MF, the top three terms were protein expression, and this ratio was much higher than that of binding, molecular function, and metal ion binding with 600 differential expression unigenes in all annotated ones (2.76%), 479(2.20%), and 407 (1.87%) unigenes (8.84%). Of these differential expression toxins, 28 toxins (Figure 3(a)). Subsequently, the differential expression were upregulated and occupied the majority, up to 68.30%. unigenes were enriched in the GO terms, and the top twenty Besides, the ratio of differential expression unigenes varied abundant terms were chosen for further analyses. Proteolysis in different kinds of toxins. As for the toxins we screened, all showed the top differential expression unigenes in the do- the 43 potassium channel toxins showed 24 differential main of BP, reaching to 28, and also showed a rich factor of expression unigenes and 17 of them were upregulated. We 0.16; however, the top rich factor in these terms was RNA- found 6 differential expression genes in 43 metalloproteases dependent DNA biosynthetic process with a rich factor of and only 1 in 24 phospholipases, indicating that the dif- 0.47 and with differential expression unigenes of 7 ferential expression of toxin was mainly on potassium (Figure 3(b)). For the CC domain, there were 55 differential channel toxins. Besides, we found that 4 defensins, 2 serine expression unigenes in the term of cellular component, with protease inhibitors, 1 cysteine proteinase inhibitors, 1 so- a relatively low rich factor of 0.11, whereas the monolayer- dium channel toxins, 1 peroxiredoxin, and 1 anticoagulant surrounded lipid storage body term had a rich factor of 1 but peptide were upregulated, and none of these toxins down- the number of differential expressed unigenes in this term regulated. Besides, the downregulated toxins were only was only 2 (Figure 3(c)). 2e term of zinc ion binding was distributed in potassium channel toxins, metalloproteases, the most one in the MF domain with 31 unigenes and its rich and phospholipases (Figure 5(c)). factor was 0.10, while the maximum rich factor value was As an abundant and diverse family of type-1 trans- 0.54 in the term of RNA-DNA hybrid ribonuclease activity membrane metalloproteinases, a disintegrin and metal- with 7 differential expression unigenes (Figure 3(d)). loproteinase (ADAM) are necessary for the developmental processes of numerous systems, including cardiovascular system, nervous system, immune system, gastrointestinal 3.4. KEGG Analysis. A total of 7,636 unigenes (31.78%) were system, and the like [21–24]. ADAM endopeptidases can successfully annotated by KEGG databases, which was di- hydrolyze adhesion molecules, signaling receptors, cyto- vided into six level-1 parts, namely, cellular process, envi- kines, and growth factors. Besides, the dysfunction of ronmental information processing, genetic information ADAMs are associated with a lot of pathological states, such processing, human diseases, metabolism, and organismal as Alzheimer’s diseases, inflammation, osteoarthritis, kidney systems. 2e top term was translation in genetic information fibrosis, and even cancers [25–28]. In the transcriptome we processing with 644 unigenes, followed by transport and constructed, the sequence Trinity_DN16345_c6_g12 was catabolism and then signaling molecules and interaction aligned with ADAM10 successfully. A model AFB1 (PDB (Figure 4). ID: 6BE6) was used to perform the sequence alignment Cellular processes included transport and catabolism (Figure 6(a)) and 3D modeling (Figure 6(b)) of (586), cell growth and death (120), and cellular community- DN16345_c6_g12. 2e main structural variations contained eukaryotes (81). Environmental information processing a coil in 295KC298, five turns in 143IC146, 176YN178, included signal transduction (530), signaling molecules and 288DGKEC294, 319PT322, and 371NK374, a helix-turn- interaction (108), and membrane transport (34). Genetic coil-helix in 23RSYEPSESSA34, a sheet-coil-turn-coil-sheet information processing included translation (644), folding, in 150YNENVNGRQVQT163, a coil-turn in sorting and degradation (430), and transcription (237). 204VECRPGGS213, a helix-coil-sheet in 252RAV- Human diseases included infectious diseases (127), cancers FNGEGKENCFQ267, a coil-turn-helix in (82), and endocrine and metabolic diseases (78). Metabolism 300RDVKPYKKDNPN313, a sheet in 343MCR347, and a included amino acid metabolism (393), carbohydrate coil-turn-helix-coil in 432KISAMKNISG443, respectively metabolism (313), and lipid metabolism (297). Organismal (Figure 6(a)). Phylogenetic analysis showed that the se- systems included immune system (136), endocrine system quence DN16345_c6_g12 was evolutionally close to the (103), and circulatory system (43) (Figure 4). sequence GBM27081.1 from A. ventricosus (Figure 6(c)).

3.5. Ae Differential Expression of Potential Toxins. By BLAST annotation of the transcriptome with the NR and 3.6. Cathepsin L. As an important interfering factor in SwissProt databases, a total of 158 toxin-related unigenes various biological processes, such as degradation and pro- were screened and sorted according to their classification. cessing of protein, the cathepsins discovered in human body 2e result showed that they included 43 metalloproteases, 40 are sorted into 11 subtypes [29]. Because they can inhibit potassium channel toxins, 24 phospholipases, 12 defensins, enzymatic activity in the process of invasion and metastasis 10 peroxiredoxins, 9 cysteine proteinase inhibitors, 7 serine of tumors, atherosclerosis, renal disease, and viral infection, protease inhibitors, 6 sodium channel toxins, 2 NDBPs, 1 Cathepsin L has become the potential therapeutic target in calcium channel toxin, 1 waprin-like peptides, 1 antibac- modern times. Besides, Cathepsin L is considered as a key terial peptide, 1 antimicrobial peptide, and 1 anticoagulant intracellular lysosomal protease and provides a link between Evidence-Based Complementary and Alternative Medicine 7

1600

1200

800

400 Number of Unignen of Number

0 rR tD rD pR nR pD SCR ICM PHA ICPM cytosol nucleus cytoplasm proteolysis translation membrane ATP binding ATP RNA binding RNA DNA binding DNA nucleoplasma mitochondrion protein binding protein Golgi apparatus zinc ion binding zinc ion hydrolase activity hydrolase metal binding ion extracellular space extracellular biological_process plasma membrane transferase activity transferase nucleotide binding nucleotide signal transduction extracellular region extracellular molecular_function cellular_component calcium ion binding ion calcium nucleic acid binding nucleic extracellular exosome extracellular developmental process developmental endoplasmic reticulum endoplasmic oxidoreductase activity oxidoreductase cytoplasmic translation cytoplasmic protein phosphorylation protein transmembrane transport transmembrane oxidation-reduction process oxidation-reduction Go Term

Biological process

Cellular Component

Molecular Function (a) 0.5 6 1.0 11 12 22 18 0.8 33 0.4 24 44 30 0.6

Number of Unigene of Number 55

0.3 4.920 Unigene of Number 5.398 1.300 5.875 0.4

Rich Factor Rich 2.208 Rich Factor Rich 6.353 3.115 6.830 4.023 0.2 7.308 4.930 7.785 0.2 5.838 8.262 6.745 7.653 8.740 -log10 (p value)

8.560 -log10 (p value) 0.1 0.0 axon I band sarcomere proteolysis pathogenesis cell proliferation DNA integration DNA extracellular space extracellular extracellular region extracellular host cell membrane host cellular_component actin filament bundle actin filament double-strand break ... break double-strand developmental process developmental postsynaptic membrane postsynaptic muscle myosin complex myosin muscle positive regulation of ... of regulation positive ... of regulation positive condensed chromosome condensed negative regulation of ... of regulation negative ... of regulation negative RNA-dependent DNA ... DNA RNA-dependent voltage-gated potassium ... potassium voltage-gated site of double-strand break double-strand of site replication fork processing fork replication respiratory chain complex I complex chain respiratory mitochondrial respiratory ... respiratory mitochondrial histone H3-K4 methylation histone striated muscle thin filament muscle striated respiratory chain complex IV complex chain respiratory histone H3-K36 methylation histone retrotransposon nucleocapsid retrotransposon transposition, DNA-mediated transposition, monolayer-surrounded lipid ... lipid monolayer-surrounded nucleic acid phosphodiester ... acid phosphodiester nucleic histone H3-K36 dimethylation histone postsynaptic density membrane density postsynaptic mitotic DNA integrity checkpoint integrity DNA mitotic

DNA double-strand break processing break double-strand DNA Terms of Cellular Component DNA catabolic process,. endonucleolytic process,. catabolic DNA Terms of Biological Process

(b) (c) 0.6 7 0.5 13 19 0.4 25 31

0.3 Unigene of Number 1.300 2.218 Rich Factor Rich 3.135 0.2 4.053 4.970 5.888 0.1 6.805 7.723 8.640 -log10 (p value) 0.0 serine-type DNA binding DNA H3-K4 specific H3-k36 specific zinc ion binding zinc ion iron ion binding ion iron hydrolase activty hydrolase peptidase activity transposase activity RNA-DNA hybrid ... hybrid RNA-DNA endonuclease activity endonuclease RNA-directed DNA ... DNA RNA-directed single-stranded DNA ... DNA single-stranded NADH dehydrogenase ... dehydrogenase NADH DNA topoisomerase binding topoisomerase DNA protein homodimerization ... homodimerization protein metalloendopeptidase activity metalloendopeptidase stingle-stranded DNA binding DNA stingle-stranded double-stranded DNA binding DNA double-stranded sodium channel inhibitor activity inhibitor sodium channel Terms of Molecular Founction (d)

Figure 3: Gene Ontology (GO) annotation of unigenes. (a) 2e top fifteen terms of the three GO domains. (b) Bubble plots of the differential expression protein annotated in the biological process GO terms between female and male samples. (c) Bubble plots of the differential expression protein annotated in the cellular component GO terms between female and male samples. (d) Bubble plots of the differential expression protein annotated in the molecular function GO terms between female and male samples. 8 Evidence-Based Complementary and Alternative Medicine

Cell motility Cellular community-eukaryotes Cell growth and death Transport and catabolism Membrane transport Signaling molecules and interaction Signal transduction Replication and repair Transcription Folding, sorting and degradation Translation Drug resistance Substance dependence Immune diseases Neurodegenerative diseases Cardiovascular diseases Endocrine and metabolic diseases Cancers Infectious diseases Biosynthesis of other secondary metabolites Metabolism of terpenoids and polyketides Xenobiotics biodegradation and metabolism Metabolism of other amino acids Nucleotide metabolism Metabolism of cofactors and vitamins

KEGG Pathway (Level 2) KEGG Pathway Glycan biosynthesis and metabolism Energy metabolism Lipid metabolism Carbohydrate metabolism Amino acid metabolism Excretory system Sensory system Nervous system Environmental adaptation Development Aging Digestive system Circulatory system Endocrine system Immune system

0 100 200 300 400 500 600 700 Number of Unigenes

Cellular processes Human diseases

Enviromental Information Processing Metabolism

Genetic Information Processing Organismal Systems Figure 4: KEGG annotation of unigenes. 2e level 2 terms of KEEG are cellular process, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems.

Phospholipases, 24 Peroxiredoxin, 9

Metalloprotease, 43 Serine protease (inhibitors), 7 Antibacterial peptide, 1

Antimicrobial Waprin-like peptide, 1 peptides, 1 Others, 22

defensins, 12

NDBPs, 2 Anticoagulant peptide, 1

Ca channel toxin, 1 Cysteine proteinase (inhibitors), 9 Na channel toxin, 6 K channel toxin, 40

(a) Figure 5: Continued. Evidence-Based Complementary and Alternative Medicine 9

14 Anticoagulant peptide 1 12 Cysteine proteinase inhibitor 1 Peroxiredoxin 1 10 Serine protease inhibitors 2 8 Phospholipases 1 6 Metalloprotease 15 Defensins 4 4 Sodium channel toxin 1 -log10 (p value) 2 Potassium channel toxin 17 7 Total 28 0 13 020406080 100 -8 -6 -4 -2 0 2 4 68 Percentage log2(fold change) up Up Regulation down Down Regulation No Difference (b) (c)

Figure 5: Classification of the scorpion venoms. (a) 2e classification of potential toxins screened from captured samples. (b) Volcano plots of differential expressed toxin-related unigenes. Each point in the map represents a differential unigene. 2e red points represent upregulated unigenes and the dark blue ones represent down-regulated unigenes. 2e abscissa represents the fold change, while the ordinate represents the statistical significance. (c) 2e number of up- or downregulated toxin-related unigenes. 2e abscissa represents the per- centage, and the ordinate represents the kinds of the toxins. A Disintegrin and metalloproteinase. lysosomal dysfunction and frontotemporal lobar degener- 4. Discussion ation. Sequence Trinity_DN20786_c0_g1 was aligned with Cathepsin L (CTSL) successfully. A model CTSL (PDB ID: 4.1. Sexual Dimorphism Exists Widely in the Same Species. 6JD8) was used to perform the sequence alignment 2e different selections between sexes lead to sexual di- (Figure 7(a)) and 3D modeling (Figure 7(b)) of DN morphism, and some of them serve for the sex-specific re- DN20786_c0_g1. 2e main structural variations contain 4 productive activities and requirements. Sexual dimorphism turns in 112AG115, 168DWRLL174, 280SG283, and means that the female and the male show differences in 372DN375, 2 coils in 136WTMR141 and 344FG347, 2 he- natural selection, sexual selection, or nonadaptive processes. lixes in 91EH94 and 365NVS269, 2 helix-turns in Like other animal species in nature, sexual dimorphism can 153FHIELY160 and 321GNTND327, a coil-helix in influence the scorpions in the aspect of morphology, defensive 144TTEY149, a helix-coil in 248EDDYG254, respectively behavior, venoms, and the like . 2e most conspicuous dif- (Figure 7(a)). Phylogenetic analysis shows that the sequence ference of their morphology is that the female scorpions have DN20786_c0_g1 was evolutionally close to the sequence bigger bodies with shorter metasomal segments, whereas the BAA86911.1 from P. americana (Figure 7(c)). bodies of male ones were smaller and their metasomal seg- ments were longer and less rounded. 2is feature is directly related to the reproductive function of the females, which 3.7. Peroxiredoxin IV;. Belonging to peroxidases, which can helps identify the sex during courtship [10, 34]. 2e hardness reduce the peroxides, peroxiredoxin is a abundant and of genital operculum, the width of sternum, and the pecti- highly conserved family [30]. Peroxiredoxin IV (PrxIV), one neous teeth in the pecten also differ between sexes. Besides, of this family, is the only known secretory form in this the male scorpions are faster and fed on more kinds of food, family. Besides, peroxiredoxin IV; is considered to have the whereas the female ones are always sedentary because the ability to fight against diabetes mellitus, atherosclerosis, newborn scorpions usually stay on the females’ back and insulin resistance, and nonalcoholic fatty liver diseases by remain there for several days without eating anything [35]. suppressing oxidative damage, inflammatory cytokines, and Dimorphism is also considered deriving from a time- apoptotic activities [31–33]. In the transcriptome we con- honored tradition in biometrics called allometry, which structed, the sequence Trinity_DN19197_c3_g1 was aligned was used to study the relationship between body pro- with peroxiredoxin. A model peroxiredoxin (PDB ID: portions or body shape and overall body size [36, 37]. 3QPM) was used to perform the sequence alignment When the trait size and body size are isometric, with every (Figure 8(a)) and 3D modeling (Figure 8(b)) of unit of the body size increases, there will be a corre- DN19197_c3_g1. Interestingly, there was no difference in sponding unit of the trait size increase. 2erefore, the traits the domain between DN19197_c3_g1 with the model in scale body size. 2is pattern is likely in cases where fitness term of 3D modeling. Phylogenetic analysis shows that the is directly related to trait size and, that is, a larger body size sequence DN19197_c3_g1 is evolutionally close to the se- increases the ability to develop larger traits. In hypo- quence AAY66580.1 from I. scapularis and ABY76309.1 allometry, individuals of all body sizes have more or less from I. ricinus (Figure 8(c)). the same trait size. Hypoallometry may result from 10 Evidence-Based Complementary and Alternative Medicine

10 20 30 40 50 60 70 80

PDB|6BE6 DN16345_c6_g12 Clustal Consensus Helix-Turn-Coil-Helix Turn 90 100 110 120 130 140 150 160

PDB|6BE6 DN16345_c6_g12 Clustal Consensus Sheet-Coil-Turn-Coil-Sheet 170 180 190 200 210 220 230 240

PDB|6BE6 DN16345_c6_g12 Clustal Consensus

Turn Coil-Turn Coil-Turn-Helix 250 260 270 280 290 300 310 320

PDB|6BE6 DN16345_c6_g12 Clustal Consensus Helix-Coil-Sheet Turn Turn Coil 330 340 350 360 370 380 390 400

PDB|6BE6 DN16345_c6_g12 Clustal Consensus

Sheet Turn 410 420 430 440 450 460 470

PDB|6BE6 DN16345_c6_g12 Clustal Consensus

Coil-Turn-Helix-Coil (a) Coil-Turn XP 023243347.1 C. sculpturatus Coil-Turn-Helix-Coil 15 XP 027206139.1 D. pteronyssinus Sheet-Coil-Turn-Coil-Sheet 9 DN16345 c6 g12 40 GBM27081.1 A. ventricosus

7 KFM77108.1 S. mimosarum

24 XP 030529792.1 R. argentea

Turn XP 021000996.1 P. tepidariorum 42 RWS30733.1 L. deliense Turn Turn 50 XP 013772127.1 L. polyphemus Sheet Helix-Coil-Sheet XP 015785243.1 T. urticae 74 XP 017486589.1 R. zephyria

0.2 Helix-Turn-Coil-Helix

Coil-Turn-Helix

Turn Coil Turn

(b) (c)

Figure 6: Sequence alignment, 3D modeling and phylogenetic analysis of a disintegrin and metalloproteinase. (a) A putative sequence Trinity_DN16345_c6_g12 was aligned with a model Adam (PDB ID: 6BE6). At the bottom of columns, asterisk (∗) shows conserved positions, colons (:) shows conserved substitutions, and points (.) shows nonconserved substitutions. Grey lines, green bends, blue-banded arrowheads, and red solenoids represent coils, turns, sheets, and helices respectively. Different fragments are framed by red lines. (b) 3D modeling was simulated by the template ADAM (PDB ID: 6BE6) in SWISS-MODEL and viewed in Discovery Studio 2016. 2e colors grey, green, blue, and red represent coils, turns, sheets, and helices, respectively. Different structures are indicated by red arrows. (c) Phylogenetic tree was constructed with sequence Trinity_DN16345_c6_g12, which is a putative ADAM and 10 other sequences from different species in MEGA 7, by the Neighbor-Joining method. 2e putative ADAM is marked in a red dot, arthropoda are marked in light blue dots, and Angiospermae are marked in a yellow dot. situations where large body size and intermediate trait size individual in good condition devotes more resources to result in the highest fitness. As for hyperallometry, the secondary traits than an individual in poor condition. large individuals express disproportionately large traits compared with smaller individuals. Hyperallometry is 4.2. Ae Differentiation of Venoms between Male and Female predicted when increasing the trait size produces greater Animals. During the process of gestation and childbirth, the relative fitness benefits at large body sizes. 2us, an females always have increases in body mass and a change of Evidence-Based Complementary and Alternative Medicine 11

10 20 30 40 50 60 70 80

PDB|6JD8 DN20786_c0_g1 Clustal Consensus Helix-Turn 90 100 110 120 130 140 150 160

PDB|6JD8 DN20786_c0_g1 Clustal Consensus

Helix Turn Coil Coil-Helix 170 180 190 200 210 220 230 240

PDB|6JD8 DN20786_c0_g1 Clustal Consensus

Turn 250 260 270 280 290 300 310 320

PDB|6JD8 DN20786_c0_g1 Clustal Consensus

Helix-Coil Helix Turn 330 340 350 360 370 380

PDB|6JD8 DN20786_c0_g1 Clustal Consensus

Helix-Turn Coil Turn (a) Helix 35 TRINITY DN20789 c0 g1 BAA86911.1 P. americana Helix-Coil 20 XP 014666952.1 P. caudatus

54 CAB3361155.1 C. dipterum Helix-Turn RWS31480.1 L. deliense

XP 015929473.1 P. tepidariorum 32 Coil-Helix 100 XP 025110869.1 P. canaliculata

95 KFM77975.1 S. mimosarum Coil XP 013412168.1 L. anatina

Turn 23 XP 023712955.1 C.s secundus 69 RWS08299.1 D. tinctorium Turn 0.2

Coil

Helix Turn Helix-Turn Turn

(b) (c)

Figure 7: Sequence alignment, 3D modeling, and phylogenetic analysis of cathepsin L. (a) A putative sequenceTrinity_DN20786_c0_g1 was aligned with a model CTSL (PDB ID: 6JD8). At the bottom of columns, asterisk (∗) shows conserved positions, colons (:) shows conserved substitutions, and points (.) shows nonconserved substitutions. Grey lines, green bends, blue-banded arrowheads, and red solenoids represent coils, turns, sheets and helices respectively. Different fragments are framed by red lines. (b) 3D modeling was simulated by the template CTSL (PDB ID: 6JD8) in SWISS-MODEL and viewed in Discovery Studio 2016. 2e grey, green, blue, and red represent coils, turns, sheets and helices, respectively. Different structures are indicated with red arrows. (c) Phylogenetic tree was constructed with sequence Trinity_DN20786_c0_g1, which is a putative CTSL and 10 other sequences from different species, in MEGA 7, by the Neighbor- Joining method. 2e putative CTSL is marked in a red dot, arthropoda in light blue dots, priapulida in a purple dot, ollusca in a green dot, and brachiopoda in a pink dot. nutrient allocation, which lead to the decrease of locomotor than that of downregulated ones in the toxin of defensins, performance and the decline of predation ability and thus serine protease inhibitors, sodium channel toxins, and other increase their risk of facing natural enemies. Scorpions use toxins. All these indicated that the venom of female scor- their venoms for both feeding and defending, as most pions may have stronger toxicity than that of male ones. venomous animals do. 2ere are also two other usage sit- 2e variation in the component of venoms is common in uations: for hygiene and during courtship [9]. All these other venomous animals. Study on Loxosceles spiders, which differences further suggest that male and female venoms also can cause human injury and more than 3,000 cases of might be optimized for different tasks. 2us, the female may sting are reported every year, revealed that venoms from change the contents of their venoms to compensate for some female spiders are more toxic than venoms from males in defects. In this study, these venoms, such as potassium common [38]. 2e venoms of female Bothrops jararaca channel toxins, had a significant ratio of upregulation. contained four peptide fragments, which did not exist in Besides, the number of upregulated unigenes were more male snakes, and the females always had stronger lethal 12 Evidence-Based Complementary and Alternative Medicine

10 20 30 40 50 60 70 80

PDB|3QPM DN19197_c3_g1 Clustal Consensus

90 100 110 120 130 140 150 160

PDB|3QPM DN19197_c3_g1 Clustal Consensus

170 180 190 200 210 220 230 240

PDB|3QPM DN19197_c3_g1 Clustal Consensus

250

PDB|3QPM DN19197_c3_g1 Clustal Consensus (a)

99 AAY66580.1 I. scapularis

100 ABY76309.1 I. ricinus

61 TRINITY DN19197 c3 g1

52 XP 013776518.1 L. polyphemus

XP 023219033.1 C. sculpturatus

100 XP 013404705.1 L. anatina

ALU63747.1 H. madaka

34 KFM78263.1 S. mimosarum XP 015930286.1 P. tepidariorum

17 PRD32298.1 T. clavipes

71 QFS22431.1 R. microplus

0.2

(b) (c)

Figure 8: Sequence alignment, 3D modeling, and phylogenetic analysis of peroxiredoxin IV;. (a) A putative sequence sequence Trini- ty_DN19197_c3_g1 is aligned with a model PrxIV (PDB ID: 3QPM). At the bottom of columns, asterisk (∗) represent conserved positions, colons (:) represent conserved substitutions and points (.) represent nonconserved substitutions. Grey lines, green bends, blue-banded arrowheads, and red solenoids represent coils, turns, sheets, and helices, respectively. Different fragments are framed by red lines. (b) 3D modeling was simulated by the template PrxIV (PDB ID: 3QPM) in SWISS-MODEL and viewed in Discovery Studio 2016. 2e grey, green, blue, and red represent coils, turns, sheets, and helices, respectively. Different structures are indicated with red arrows. (c) Phylogenetic tree was constructed with sequence Trinity_DN19197_c3_g1, which is a putative PrxIV and 10 other sequences from different species, in MEGA 7, by the Neighbor-Joining method. 2e putative PrxIV is marked in a red dot, arthropoda in light blue dots, brachiopoda in a pink dot, and mollusca in a green dot. activity than the males [39]. Besides, the previous studies the history of traditional Chinese medicine, the Compen- also showed that sex-biased expression existed in the venoms dium of Materia Medica, written by Li Shizhen, a famous of other species like Centruroides hentzi, Anastatus disparis, medical scientist in Ming Dynasty. Overall, 50 kinds of Brachymeria lasus, and so on [10, 40, 41]. animal medicines were included in the main part of Chinese pharmacopoeia, version 2015, and these animal medicines involved 76 species of medicinal animals. 4.3. Sex is One Important Effect Factor in the Aerapeutic 2e sexes of the animals, which are used as medicines, Efficacy of Animal Medicine. As an important part of Chi- are considered not only in those ancient works but also in nese material medicine, animal medicines are widely used in Chinese pharmacopoeia. Tubiechong, which is the dry body clinical prescriptions and Chinese Patent Medicine. Al- of female Eupolyphaga sinensis Walker [12], has a wide though the varieties of animal medicines are less than those pharmacological effect, such as thrombolysis, anticoagulant, of phytomedicines, animal medicines still have great de- antitumor, antimutation, and hypoxia tolerance. Another velopment potential. In the oldest material medical book, animal medicine that also requires the female ones as the raw Shennong’s classic of material medical, the earliest known material is Oviductus Ranae, called Hamayou in Chinese. works of traditional Chinese Medicine, a total of 65 kinds of 2is kind of medicine is the processed products of female animal medicines were recorded. Also, 128 animal medi- Rana temporaria chensinensis David and is widely used in cines were included in the most complete medical book in antioxidant, antifatigue, antiaging, inflammation, and the Evidence-Based Complementary and Alternative Medicine 13 like [42, 43]. On the other hand, some male animals are available on request through Songyu Gao (e-mail: son- required as the sources of other animal medicines, such as [email protected]). Lurong, Lingyangjiao, and Shexiang. Cornu cervi panto- trichum was called Lurong in Chinese, and it was the antler Conflicts of Interest of male deer, which has the potential to invigorate the spleen, strengthen bone and skeletal muscles, and promote blood 2e authors declare that there are no conflicts of interest flow [44]. Lingyangjiao is the horn of male Saiga tatarica regarding the publication of this paper. Linnaeus and can be used to treat fever, eclampsia, and hemacelinosis [45]. Besides, Shexiang, also called artificial Authors’ Contributions moschus or musk, is the dried preputial secretion of male Songyu Gao, Feng Wu, and Xintong Chen contributed Moschus berezovskii Flerov, Moschus sifanicus Przewalski, or equally to this work. Moschus moschiferus Linnaeus. It has the potential to be used for treating the unstable angina pectoris and neurodegen- erative diseases [46]. Acknowledgments 2e results of this article showed that approximately 2is work was supported by the International Science and 39.53% unigenes of potassium channel toxins were upre- Technology Cooperation Programme of the Ministry of gulated in the samples of female scorpion, whereas only Science and Technology of China (2019YFE0116800), the 16.28% were downregulated. As for metalloproteinase, there Military Youth Training Program-Talent Project were 5 upregulated unigenes but only 1 downregulated in (18QNP016), and the general program of the National the female scorpions. For defensins, all the differentially Natural Science Foundation of China (81770329 and expressed unigenes were upregulated. Besides, the whole 81971824). ratio of differential expressed unigenes was much lower than the ratio in screened toxins, indicating that the sex-biased Supplementary Materials gene expression had more influence on the venoms than on the whole body. 2ere is no denying that there are indeed 2e toxin screening data are included. (Supplementary differences in toxin composition between male and female Materials) scorpions. 2is study is on the level of transcriptome, and omics will be an effective method in the study of this field. References [1] D. O. Rossato, D. Boligon, R. Fornel, M. R. Kronforst, 5. Conclusions G. L. Gonçalves, and G. R. P. 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