**3. Results**

#### *3.1. Samples Correlation Analysis*

The correlation of gene expression levels between samples performed in this study revealed a higher similarity of the expression patterns between samples. Among all the experimental units, biological samples collected from male mid-gut tissues were found to have the highest similarity (Figure 1). On the other hand, ovary samples have shown variability. The samples of testes have also shown similar expression patterns but remained between mid-gut and ovary samples.

#### *3.2. Sequence Assembly Characteristics of Two-Spotted Field Crickets*

In this study, the Illumina HiSeq platform was used to perform next-generation sequencing of two-spotted field crickets from di fferent organs including the ovaries, testes, and male and female mid-gut samples. The RNA-Seq led to an average of 50.04 million, 74.88 million, 63.90 million, and 64.63 million clean reads for the constructed full-length cDNA libraries of male mid-gut and female mid-gut, testes, and ovaries of two-spotted field crickets, respectively (Table 1). The Illumina platform generated an average of 7.9 G, 11.77 G, 10.07 G, and 10.07 G bases of outputs of the male mid-gut, female mid-gut, testes, and ovaries libraries, respectively (Table 1). The reads were deposited on the NCBI SRA under accession SRX8826426, SRX8826427, and SRX8826430 (mid-gut of Male two-spotted field crickets); SRX8826431, SRX8826432, and SRX8826433 (mid-gut of female two-spotted field crickets); SRX8826434, SRX8826435, and SRX8826436 (testes of male two-spotted field crickets); SRX8826437, SRX8826428, and SRX8826429 (ovaries of female two-spotted field crickets).

**Figure 1.** Correlations between cDNA libraries constructed from different parts including the ovaries, testes, female mid-gut, and male mid-gut of two-spotted field crickets. The correlation coefficient close to 1 represents the higher similarity of the expression patterns between samples.


**Table 1.** Summary of the Illumina Platform RNA-Seq Metrics for two-spotted field crickets.

#### *3.3. Classification and Functional Annotation of Two-Spotted Field Cricket Transcriptomes*

Each organ transcript assembly was completed by assembling all the three replicates of the reads and were archived into 158,658 UniGenes from the female mid-gut, 121,025 UniGenes from the male mid-gut, 157,036 UniGenes from the ovaries, and 72,216 UniGenes from the testes of two-spotted field crickets. Overall, the generated transcript sequence resources of all the studied organs were assembled into 233,172 UniGenes, which yielded approximately 163.58 million reads. Furthermore, the assembly comprised of 316.91 million reads of 325,568 transcripts. The length of the sequence reads in each case ranged between 201 to 32,390 base pairs.

The relationship of the genes assembled in this study was visualized by drawing a Venn diagram (Figure 2). Overall, 43,055 genes were common genes that were shared by all the biological samples. On the other hand, there were 15,787 unique genes from the male mid-gut, 22,369 unique genes from the female mid-gut, 31,418 unique genes from the ovaries, and 3261 unique genes from the testes that did not show any relationship.

**Figure 2.** Venn diagram showing the number of overlapping genes among all the libraries of two-spotted field crickets.

The assembly of cDNA libraries showed variations in identified and unidentified reads among di fferent organs. Among all the libraries, the highest reads (average of three male mid-gut libraries 22.52%) from the transcriptome of the male mid-gut were successfully identified, while the least reads (average of three Ovaries libraries 3.51%) were identified from the cDNA libraries constructed from the ovaries of two-spotted field crickets (Table 2). On the other hand, 10.28% (average of three female mid-gut libraries), and 12.40% (average of three testes libraries) assembled reads from the cDNA libraries of the female mid-gut and testes were identified, respectively. Interestingly, a major proportion of the identified reads matched with the Arthropoda, as can be seen in Table 2.

The transcriptomes were successfully mapped onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in order to reveal biochemical pathways operating in two-spotted field crickets. Overall, the KEGG analysis revealed the seven di fferent main functional processes (level 1), which were further composed of 48 GO terms as shown in Figure 3. Based on the analysis, a grea<sup>t</sup> number of genes mapped in this analysis were involved in the regulation of host defense mechanisms through signal transduction and immune system GO terms among all the cDNA libraries.


**Table 2.** Assembly metrics for two-spotted field crickets transcriptome sequencing.

**Figure 3.** GO mapping of the cDNA libraries of (**A**) female mid-gut, (**B**) male mid-gut, (**C**) testes, and (**D**) ovaries of two-spotted field crickets by the Kyoto Encyclopedia of Genes and Genomes, KEGG classification.

#### *3.4. Patterns of Di*ff*erential Gene Expression Levels*

Cluster analysis of gene expression patterns can intuitively reflect the level of gene expression and expression patterns in multiple samples. Overall, 2242 UniGenes were up-regulated, while 1805 were down-regulated between the male and female mid-gut samples. On the other hand, a comparatively a higher number of UniGenes were up-related (8921), and concurrently a higher number of genes were also down-regulated (8716) between teste and ovary samples of the two-spotted field crickets.

#### *3.5. Immune-Related Transcriptome of Two-Spotted Field Crickets*

The transcriptome of two-spotted field crickets assembled as a result of the current study revealed the identification of 492 different types of genes regulating the immune response mechanism under natural conditions (Supplementary Tables S1–S5). The immunity-related transcript analysis successfully identified a wide range of Pattern Recognition Receptors (62 genes), most importantly *PRPs*, *CTLs*, *GALE*, *GNBP*, *Immulectin*, *Beta-1*, *3-glucan-binding protein*, and *DSCAM*, which actually initiate the host immune defense mechanism (Table 3).


**Table 3.** Immune-related transcriptome of two-spotted field crickets.

We identified 57 genes amplifying the pathogen invasive signals through signal modulation. The representatives of these genes including *ATs*, *Ast*, *Ang*, *Chymotrypsin*, *Kazal domain-containing peptide*, *Kunitz-type protease inhibitor*, *Porin*, *proPO*, *Prostaglandin*, *SPs*, *Serpin*, and *Tetraspanin*, modulate a wide range of signaling pathways to combat pathogen invasion (Table 3 and Table S2). The activated signaling pathways including the JAK-STAT signaling Pathway, JNK pathway, Toll-like receptor (TLR) signaling pathways, Wnt signaling pathway, Notch signaling pathway, Hedgehog signaling pathway, Hippo signaling pathway, Immune Deficiency (Imd) pathway, and MAPK (Mitogen-activated protein kinases) signaling pathways are regulated through the identification of 214 genes (Table S3), involved in signal transduction (Table 3). The effectors expressed as a result of the immune mechanism among two-spotted field crickets revealed the identification of 36 genes (Table S4), encoding antimicrobial peptides and proteins. However, *Attacin*, *Bacteriocin*, *Carboxypeptidase*, *Cathepsin*, *Caspase*, *Lysozymes*, *Pyocin*, and *Thaumatin-like protein* were the most prominent genes identified in this study from two-spotted field crickets (Table 3). On the other hand, our transcriptome exploration study also revealed the identification of 123 genes categorized as other immunity-related genes because their role in immune mechanism is not ye<sup>t</sup> well categorized (Table S5).
