**4. Discussion**

Our study to explore the molecular mechanisms of the immune system of two-spotted field crickets by next-generation sequencing of male mid-gut, female mid-gut, ovaries, and testes revealed the identification of novel genes contributing in the regulation of host defence. The characterized annotated transcriptome as a result of this study enabled us to sugges<sup>t</sup> that *G. bimaculatus* displayed a strong antimicrobial response in the form of e ffectors to defend naturally occurring pathogens. The current findings greatly help us to understand the molecular mechanisms of host–pathogen relationships.

The transcriptome sequencing of two-spotted field crickets performed in this study from tissues mainly aimed to explore for the first time the molecular mechanism of immunity in *G. bimaculatus.* Our methodology was successfully able to assemble the genome into 233,172 UniGenes, which yielded approximately 163.58 million reads. Furthermore, the assembly yielded 316.91 million reads of 325,568 transcripts. However, results revealed the variability, especially ovaries R3, and female mid-gut R3 biological replicates. The variations among few biological replicates evidenced in the current study and strengthened by previous investigation are quite obvious due to environmental and genetic di fferences because each sample was prepared by separately extracting from di fferent two-spotted field crickets [21]. Interestingly, the annotated sequences identified a huge number of genes (492) regulating the host defence mechanisms. The Pattern Recognition Receptors (PRRs) initiate the host immune defence system by recognising the receptors for pathogen-associated molecular patterns (PAMPs) [22]. Upon recognition of PAMPs, these PRRs can either mediate pathogen killing directly through phagocytosis and encapsulation or indirectly through intra-cellular signal transduction pathways. These pathways ultimately lead to the transcription of e ffector genes [23–26]. Our transcriptomic analysis revealed the identification of several di fferent classes of PRRs, and most importantly *GNBP1*, *beta-1,3-glucan-binding protein*, multiple isoforms of *apolipophorin*, *Ataxin*, *CTL*, *DSCAM*, *GALE*, and *Immulectin*. These recognition receptors are bounded with the components of the microbial cell wall, and it ultimately started a tug-of-war between the host and the invading pathogen [27,28].

Our two-spotted field cricket transcriptome analysis revealed the identification of five di fferent isoforms of the *apolipophorin family (apoLp).* The members of the *apoLp* family are known to be involved in multiple functions, especially in activating the immune response through binding with β-1,3-glucans of fungi, LPSs of Gram-negative bacteria, and lipoteichoic acid of Gram-positive bacteria [29,30]. The previous findings of *Galleria mellonella* already showed that *apoLp* not only binds to fungal conidia and beta-1,3-glucan, but also stimulates cellular encapsulation [31]. The *apoLp* of *G. mellonella* affects the fungal cell wall components and exhibits antibacterial activity against selected gram-positive and gram-negative bacteria *in vitro* [32,33]. Our results showed five *apoLp*, which might play an important role in microbial infection. The transcriptome analysis also revealed the identification of *C-type lectins*, *Immunolectin*, *Lectin-related*, and *Galectin*, which are known to be involved in the innate immune response. They recognize the chains of polysaccharide present on the surface of pathogens [34,35]. Another class of PRRs, such as *GNBP1*, which triggered the protease cascades by recognizing gram-positive bacteria, was identified in this study and ultimately causes the cleavage of Spaetzle [36]. Such a wide range of transcripts of PRRs revealed for the first time as a result of this study from two-spotted field crickets enabled us to sugges<sup>t</sup> that the host has well-developed weaponry mechanisms to recognize the invading natural pathogens prevailing in their surroundings.

Once the invading pathogen has been recognized, PRRs triggers the initiation of signal modulation genes that amplify the signals of pathogen invasion. These signals ultimately activate various lines of defence against the invasion of pathogens. In this transcriptome analysis, we identified 57 genes encoding proteins potentially involved in signal modulation. From our database, a number of signaling modulation genes were observed such as CLIP domain (CLIPs), *serine*, and *serpins*. Signal modulation genes, especially *serine proteases* (*SPs*), regulate several invertebrate defense responses, including hemolymph coagulation, antimicrobial peptide synthesis after toll signal-transduction pathway and activation of phenoloxidases (POs) [37]. Serine-type protease inhibitors (*Serpins*) and *Kazal* play an important role in inhibiting the protease cascades that activate toll and melanization reaction in *Drosophila* [23,38]. Serpin-like proteins have already been reported in many insects such as *H. cunea*, *A. melifera*, *A. gambiae*, *B. mori*, and *D. melanogaster* [39–41]. Interestingly, our transcriptome analysis successfully annotates genes modulating the immune mechanism.

Various pathways regulate the immune response of invertebrates against invading pathogens by transmitting signals from recognition receptors to the synthesis of AMPs and other effectors. The current exploration annotated 214 genes involved in various types of immune signaling pathways including JAK-STAT, JNK, Toll-like receptor (TLR), Wnt, Notch, Hedgehog, Hippo, Immune Deficiency (Imd), and MAPK (Mitogen-activated protein kinases) signaling pathways.

Members of the Ras superfamily identified in this transcriptome exploration are reported to be involved in the complex signaling pathways of *Drosophila* [42]. The Ras superfamily is the protein of small guanosine triphosphatases (GTPases) comprised of five major families, including *Arf*/*Sar*, *Rab*, *Ran*, *Ras*, and *Rho* [43,44]. The exact role of these genes in *G. bimaculatus* is unknown. However, *Ras* genes are known to be involved in the cellular immune response of beet armyworm, *S. exigua* [45]. Furthermore, Rojas., et al. [44] reviewed the role of *Ras* superfamily member genes and explained that they act as signaling nodes that regulate apoptosis, cell proliferation, and differentiation. Another important gene was the *Four-and-a-half LIM domain protein 1 isoform B.* These cysteine-rich LIM domain-containing genes are previously found to mediate signal transduction cascades. Their deficiency in mice resulted in delayed wound healing [46]. The presence of *Serine*/*threonine protein kinase (STK)* from the transcriptome of two-spotted field crickets is an important finding because these genes are well known to function as an important defence gene by mediating signal transduction pathways in plants [47]. Their role in the insect immune signaling pathway has been explored by Belvin and Anderson [48]. They suggested that *STK* is an important component of the Toll-Dorsal pathway responsible for the degradation of cactus proteins in *Drosophila*. The various isoforms of genes encoding *Zinc finger proteins* characterized in the current study promote the *Toll-like receptors* as depicted in the current study to trigger innate immune responses by pressing IκBα gene transcription as previously reported from human beings [49]. On the other hand, *Kruppel-like protein* characterized here is a transcriptional repressor associated with signal transduction and activator of transcription (STATs) in the immune response [50].

The transcriptome analysis showed the presence of *14-3-3* genes from the two-spotted field crickets. These signal transduction regulatory genes interact in a phospho-serine dependent manner. The members of this protein are involved in cellular and physiological processes. The recent findings suggested that these genes are important phagocytosis mediators that play a pivotal role in defending *S. aureus* attack on *Drosophila* and zebrafish [51]. Furthermore, they illustrated that the depletion of *14-3-3* expression reduced the ability to fight against microbial infection. However, this depletion does not compromise the production of antimicrobial peptides [51]. In addition to gene encoding *14-3-3* proteins, an important multifunctional group of genes encoding *COP9 signalosome complex subunits* was identified from two-spotted field crickets. These *COP9 signalosome complex subunits* are known to function as an important defence protein and dispensable for Toll/IL-1 activation in the fat body by mediating signal transduction pathways in *Drosophila* [52]. They suggested that *CSN5* is an important component of signaling pathways involved in Cactus and Dorsal regulation to mediate immune response in *Drosophila*.

The genes encoding *EF-hand domain-containing proteins* were also identified in this study. The members of this domain, including *calmodulins*, are known to regulate Ca2+ channel in vertebrates and invertebrates [53]. These genes are known to play an important role in immune responses by maintaining calcium levels. In addition to the above mentioned signal transduction genes, *Tubulin beta chain*, *Transgelin*, *Hedgehog protein*, *Hippo*, *Hippocampus abundant transcript 1 protein*, *IMD-like protein*, *JNK-interacting protein 1*, *Leucine-rich repeat-containing protein 68, Notch*, *Pelle*, *Relish*, *Renin*, *Spaetzle*, and *Wnt* identified in this study might help to understand their role in signal transduction cascades.

Our study revealed the identification of effectors among which *Attacin*, *Bacteriocin*, *thaumatin-like protein*, *C-type Lysozyme*, *I-type Lysozyme*, *Cathepsin D*, *Carboxypeptidase*, *Caspase*, and *Pyocin* were prominent. These molecules play an important direct role in combating pathogenic microorganisms. Generally, *thaumatin-like proteins* possessed 16 conserved cysteine residues that form eight disulfide bonds [54]. The antifungal activities of more than 20 isoforms of *thaumatin-like proteins* have been reported [55,56]. These proteins were first identified from the West African shrub *Thaumatococcus*

*daniellii* and is known to synthesize in plants against stress and microbial infection [57]. Later, these proteins were further identified from animals [58], and fungi [59]. More recently, these proteins have also been identified from insects including *Acyrthosiphon pisum*, *Coptotermes formosanus* Shiraki, and *Tribolium castaneum* [16,60].

The transcriptome analysis of two-spotted field crickets showed multiple isoforms of several genes encoding *c-type lysozymes*, *i-type-lysozymes*, and *lysozymes*. Generally, insect lysozymes are known to play a pivotal role in insect immunity [61–63]. In addition, *lysozymes* isolated from the eggs and salivary glands of *Reticulitermes speratus* showed a strong novel egg recognition activity [64]. Furthermore, they observed that the newly laid eggs are frequently coated with the saliva of the worker caste containing lysozyme through egg grooming. Along with multiple isoforms of *lysozymes*, numerous genes encoding various *cathepsins* were identified. These genes have shown to regulate multiple proteins facilitating bacterial killing [65]. These proteins are known to play an important role against infections because they are believed to be highly expressed in immune-related organs [66].

Glycine-rich proteins such as *Attacin* belong to an important antimicrobial peptide group, which was for the first time discovered from *Hyalophora cecropia* [67]. The latest review article revealed the identification of di fferent isoforms of *attacin* from di fferent insects including silkworms, tse-tse fly, housefly, tobacco budworm, cabbage looper, and wild silkworm [68]. Our transcriptome study for the first time identified *attacin* from two-spotted field crickets. These findings enabled us to sugges<sup>t</sup> the highly specialized broad spectrum host–antimicrobial response against invading pathogens.
