The complex (RISC), which then binds to one of

The Kudzu bug transcriptome database was
searched for the basic set of RNAi core genes including  dsRNA uptake (SID-1 and SID-2), dsRNA cleavage
(Dicer), dsRNA binding (R2D2 and Loquacious), target degradation by
endonuclease activity (Argonaute family, Aubergine), and signal amplification
by RNA-dependent RNA polymerase (RdRP) using homologous genes from T. castaneum (Table 2). Sequences
similarities representing RNAi Pathway in Kudzu bug were obtained in the established
transcriptome database (Table 2). However, R2D2, which function as a
cofactor of Dicer-2 in Drosophila was
not found. In this study, we identified sid-1 (4 contigs), however, we could
not find homologous sequences for sid-2. 
Moreso, sequences similarity were identified for dcr-1, dcr-2, and for
drosha. The searched for dsRNA binding factors found putative contig for
loquacious and not R2D2, the searches for endonucleases found sequences
similarity for argonaute-1, argonaute-2, argonaute-and aubergine (Table 2). A
phylogenetic tree was constructed for dsRNA uptake (Dicer), Argonaute proteins
and dsRNA-binding cofactors (Loquacious) and compared with corresponding
sequences of organisms of different taxonomic groups (Figs 3, 4 and 5). We used
these phylogenetic trees to confirm the sequences similarities of the
identified genes from the kudzu bug transcriptome, by comparing the identified
RNAi-related genes from the kudzu bug transcriptome with their homologs in
other species.

According to
literature review, it is well established in most arthropods that RNAi
machinery are made up of Argonaute endonucleases, Dicer enzymes and dsRNA
binding proteins (Jinek and Doudna 2009; Carthew and Sontheimer, 2009; Siomi and
Siomi; Moazed, 2009). Since the discovery of RNAi, significant efforts have
been made to describe the molecular mechanisms of RNAi (Aravin, et al., 2007).
Studies performed on several model insects, including D. melanogaster, T. castaneum and the silkworm, Bombyx mori whose genomes have been
sequenced revealed that the RNAi pathway is composed of two phases, the
initiator phase, and the effector phase, and together they result in
post-transcriptional gene silencing (PTGS) Vaucheret et al., 2001; Vaucheret
and Fagard, 2001; Castel and Martienssen, 2013). The initial step starts with
the introduction of long dsRNA into a cell. These long dsRNA molecules are cut
into small interfering RNAs (siRNA) by an enzyme called Dicer (Shreve et al.,
2013; Swevers et al., 2013; Hammond et al., 2000). Dicer encodes a multidomain
protein containing an ATP-dependent RNA helicase, PAZ domain, two tandem RNase III
domains, and a dsRNA-binding domain (Bernstein et al., 2001; Knight and Bass
2001). The 21- to 25-bp products of Dicer activity are referred to as short
interfering RNAs (siRNAs) (Hamilton and Balcombe, 1999). They are thought to
serve as “guides” to bring nuclease machinery to the target mRNA, each siRNA
associates with a protein complex called RNA-induced silencing complex (RISC),
which then binds to one of the strands in the RNA fragments, allowing it to
search for similar or identical mRNA (Hamilton and Balcombe, 1999). For this to
occur, the unincorporated sequence must be cleaved from the siRNA duplex by the
RNase H activity of an Argonaute (Ago) protein (Matranga et al., 2005; Rand et
al., 2005). If the incorporated sequence is the antisense strand, then it
guides RISC to its homologous target mRNA where the activity of the Ago protein
causes the destruction of the target mRNA (Hammond et al. 2001; Song et al.
2004).

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Interestingly, this
research found out that kudzu bug transcriptome lacked sequence similarity
encoding R2D2, a dsRNA-binding protein that functions as a cofactor of Dicer-2
in Drosophila. Liu et al. 2003; and Wang et al. 2006 reported in their result
that r2d2 is an essential gene for both RNAi and the innate immune response
against RNA viruses in Drosophila. Since RNAi has been reported to effective process
in hemipteran species after injecting or feeding dsRNA (Futahashi et al., 2010;
El-Shesheny et al. 2013; Hajeri et al., 2014).  These findings proved that R2D2 may not be
necessary required for this process in kudzu bug or could be software error. Moreso,
Swevers et al. (2013) reported that r2d2 was absent in the gut transcriptome of
the Colorado potato beetle, a species known to be very susceptible to RNAi
process after treating with dsRNA (Fu et al., 2016). Therefore, we assumed a
different dsRNA-binding cofactor may have compensated for the absence of R2D2
in kudzu bug. Moreover, BLASTX searches using Tribolium R2D2 as a query against the kudzu bug transcriptome
database showed Loquacious (Table 2). In Drosophila, it has been reported to
act as a cofactor for both Ago1- and Ago2-RISC (Tomari et al., 2007). Further research
need to work on the interaction of Loquacious in the RNAi pathway of kudzu bug.
The main components of RNAi are well conserved across phyla, however, the
mechanism and systemic spread of silencing are highly diverged and gene
regulation is mediated by a variety of RNA-based products (siRNAs, miRNA)
(Shreve et al., 2013; Swevers et al., 2013). RNAi is also likely to have
significant implications for the biological role of heterochromatin and genome
maintenance (Lippman and Martienssen 2004). Finally, miRNAs play important
roles in development and fundamental cellular processes, influencing the
expression of an estimated 30 percent of all protein coding genes (Ouellet et
al., 2006)