**1. Introduction**

The two-spotted field cricket, *Gryllus bimaculatus* (Orthoptera, Gryllidae, Gryllinae), is becoming a popular model insect in order to explore behavioral adaptations [1,2], evolutionary biology [3], and physiological [4] and developmental mechanisms [5,6]. The acceptance of two-spotted field cricket for research has mainly occurred because of its widespread abundance in different geographical regions, especially in Asia, Africa, and Europe, in addition to its ease of rearing under laboratory conditions. Furthermore, two-spotted field crickets are currently praised for their possible contribution to global food security by providing an alternate source of protein due to their intrinsic ability to efficiently utilize water and feed compared to traditional livestock [7]. In this regard, a recent study has successfully grown two-spotted field crickets on the by-products of the food industry in order to achieve the targets for the circular economy [7].

The *G. bimaculatus* is categorized as a sporadic pest due to its occasional outbreak under special circumstances at a specific time of the year. In the Kingdom of Saudi Arabia, the outbreak of two-spotted field crickets (black crickets) made its way to such western regions of the country as Makkah and Madinah, where it invaded human dwellings, including places of worship, in which chemical control approaches are not feasible. The residents of the regions were perplexed during their swarming in the last quarter of 2018 till the first quarter of 2019. The latest surveys (unpublished) revealed changing climatic conditions and vegetation covers providing an enormous breeding opportunity for their reproductive success to build sporadic outbreaks of two-spotted field crickets, especially in the Kingdom of Saudi Arabia.

The managemen<sup>t</sup> of such sporadic outbreaks has become very challenging in urban areas, as synthetic chemical pesticides cannot be applied due to potential threats to public health. Therefore, the use of effective biocontrol agents, which can quickly overcome the host immune defense mechanism, are gaining special attention [8,9]. In the meantime, the host has evolved a highly specialized immune response mechanism to combat the invading pathogens in their surroundings. Therefore, it is very important to document how sporadic pests that occasionally appear in swarms overcome surrounding natural fungal pathogens by fully exploiting their immune defense mechanisms. In the past, the transcriptome of immune response mechanisms of different pest species that appeared in the form of swarms have been well explored in insects such as ants [10,11], fall armyworms [12], hemipteran stinkbugs [13,14], potato leafhoppers [15], and termites [16,17]. The current study was aimed to fully explore the immune-related gene expression patterns evolved in the testes, ovaries, and mid-guts of the adults (males and females) of two-spotted field crickets during swarming to document for the first time a profound understanding of the highly specialized immune responsive combating strategy against natural pathogens that will be useful to develop high-quality reference transcriptomes of two-spotted field crickets for future research into the host–pathogen interactions.

#### **2. Materials and Methods**

#### *2.1. Collection, Maintenance, and Tissue Extraction of Two-Spotted Field Crickets*

The populations of two-spotted field crickets were directly collected from their outbreak areas located in the western part of the Kingdom of Saudi Arabia. The males and females of the adults of collected populations of two-spotted field crickets were separately kept at a photoperiod of 16 h light, 8 h dark under controlled temperature conditions (30 ± 0.50 ◦C). Male and female adults of two-spotted field crickets were separately dissected in saline in order to separate different tissues including male mid-gut, female mid-gut, male testes, and female ovaries. These target tissues were stored in 2 mL Eppendorf tubes pre-chilled with liquid nitrogen at −80 ◦C. Three biological replicates for each target tissue were prepared by separately extracting them from different two-spotted field crickets.

#### *2.2. Construction of cDNA Libraries of Two-Spotted Field Crickets*

The frozen tissues were separately ground using liquid nitrogen in a mortar and pestle. The cDNA libraries were constructed by following the previous methodology [16]. In brief, total RNA from each sample was separately extracted using TRIzol reagen<sup>t</sup> (Invitrogen, Waltham, MA, USA). The quality of the extracted total RNA was evaluated by electrophoresis (1% agarose gel).

The mRNA molecules were purified using an Oligotex mRNA Mini Kit (Qiagen, Hilden, Germany), which were used as templates to synthesize the first-strand cDNA using random hexamer primers, and ultimately to synthesize second-strand cDNA libraries. In this study, twelve cDNA libraries including three males mid-guts, three females mid-guts, three males testes, and three females ovaries were prepared to construct four cDNA libraries, each with three independent biological replicates that were separately prepared by sequencing through an Illumina HiSeqTM 4000 platform at MicroAnaly Gene Technologies Co., Ltd. (Wuhan, Hubei, China). These data have been made available at the NCBI Sequence Read Archive (SRA BioProject Acc. No. PRJNA647692).

#### *2.3. Sequence Assembly of the cDNA Libraries of Two-Spotted Field Crickets*

After filtering, the resulting clean reads were mapped to the *G. bimaculatus* genome on NCBI (Accession: PRJNA647692 submitted by the investigators) using the Hierarchical Indexing for Spliced Alignment of Transcripts, HISAT program. All reads were assembled by the Trinity assembly program (v2.8.6) in its genome-guided mode, and biological sequences were clustered to remove sequence redundancy in order to obtain the UniGene sequence set for subsequent analysis using the CD-HIT program [18].

#### *2.4. Analysis of Di*ff*erential Expression of Genes*

The DEGs between the paired comparisons (male mid-gut versus female mid-gut; male testes versus female ovaries) were analyzed with the Cu ffdi ff method. DEGs were considered between four libraries when the screening threshold of the p-value for False Discovery Rate (FDR) was less than 0.05, and an absolute value of log2Ratio FC (Fold Change) for a gene was greater than 1, using R language package DESeq2 (the screening threshold is FDR (false discovery rate) < 0.05, log2FC (fold change) for a gene > 1 or log2FC < −1) [19].

#### *2.5. Functional Annotation of the cDNA Libraries of Two-Spotted Field Crickets*

The new transcripts were annotated via BLAST searches against the NCBI non-redundant protein database. All identified genes were quantified in terms of the expected number of fragments per kb of transcript sequence per million base pairs sequenced (FPKM) with the software program Cu fflinks. The UniGenes were explored by performing annotations through the Kyoto Encyclopedia of Genes and Genomes (KEGG) [20], through a web-based interface. Immunity-related genes were accordingly classified into their main categories.
