In the present study, DD-PCR revealed that several common bands were observed in both control and challenged samples (house keeping genes). Very few bands were recorded in control insects and disappeared in challenged ones (genes were turned off). On the other hand, many bands were induced as a result of bacterial-challenge at different time intervals post-infection. DD-PCR technique is considered a powerful genetic screening tool for complicated dynamic tissue processes, particularly when multiple, limited-sized samples are involved, because it allows for simultaneous amplification of multiple arbitrary transcripts . This technique has been developed as a tool to detect and compare altered gene expression in eukaryotic cells , to screen mRNAs, and to characterize differentially expressed mRNAs [17–20]. Many publications described the enhancement of the insect immune system and induction of AMPs due to stress and/or bacterial challenge [8–10, 12, 13, 21–24]. Although defensin genes were isolated from six insect orders; Lepidoptera, Diptera, Coleoptera, Hymenoptera, Hemiptera and Thysanura, the lepidopteran defensin-like genes included both the smallest (G. mellonella (216 bp), Acc# AAS19170) and the largest (S. frugiperda (306 bp), Acc# AAM96925) molecular-sized defensin genes isolated from class Insecta. Thus, the orf of Spli Def (300 bp) was comparable in size to that of other Spodoptera sp. defensin-like genes (303-306 bp).
Reconstruction of the phylogenetic trees of the Spli Def nucleotide seuquence and its deduced polypeptide resulted in two different topologies. In spite of constructing two different tree topologies, both trees clustered the Spli Def sequence with that of S. frugiperda to indicate that they descend from a common ancestor. The grouping of some lepidopteran and dipteran defensins (e.g. M. brassicae with A. aegypti and S. littoralis with D. melanogaster) in one sister clade indicated that they may be homologous or share some similarity. In addition, the lepidopteran defensin-like sequences were diverged in many sister clades as nucleotides but they were clustered in a monophyletic group as amino acids due to the difference in codon usage in the different insect species.
Although three signal peptide cleavage sites were detected, the most probable site is between positions 23-24. The detected glycosylation and glycation residues may serve for correct folding and stability of the protein. It was shown that the unglycosylated protein degrades quickly. Glycosylation may also play a role in cell-cell adhesion (a mechanism employed by cells of the immune system), as well . In addition, 15 phosphorylation sites were identified. Reversible phosphorylation of proteins (using kinases and phosphatases) is considered an important regulatory mechanism that occurs in both prokaryotic and eukaryotic organisms . It is very important in protein-protein interaction via recognition domains [27, 28], (i.e. many proteins and receptors are switched "on" or "off" by phosphorylation and dephosphorylation). It may also result in a conformational changes in the structure of many peptides, causing them to become activated, deactivated or degraded .
Spacing pattern of our anionic defensin revealed a possible frameshift mutation of at least six residues within the conserved cysteine motif of Spli Def (His77, Leu79, Lys81, Gly82, Tyr83 and Gly86) in comparison to (His73, Leu75, Lys77, Gly78, Tyr79, Gly82) of BmdefA . Numbers refer to the prepropeptide sequence of a defensin. The first two anionic defensins of Amblyomma hebraeum were reported by Lai et al. . Similar properties were reported by Wen et al.  for a novel anionic defensin peptide (PI: 4.12) isolated from B. mori. These results were contrary to almost all known defensins, which were described as cysteine-rich cationic AMPs [e.g. [11, 21].
Although most insect defensins are active against gram (+) bacteria, the purified mature Spli Def exhibited activity against both gram (+) and gram (-) bacteria. According to Shai-Matsuzaki-Huang (SMH) model, antibacterial activity was ascribed to the interaction between the positively charged AMPs and the negatively charged microbial membrane components, which include LPSs in gram (-) bacteria and polysaccharides in gram (+) bacteria . However, this model has difficulty explaining the behaviours of anionic defensins like Spli Def. For example, the Amblyomma defensin-2 contains a net negative charge with a PI value of 4.44, and exerts antimicrobial activity against the gram (-) bacterium E. coli and the gram (+) bacterium S. aureus . This suggests that anionic defensins might possess some novel antimicrobial mechanisms; although no convincing evidence is available until date. As the knowledge of anionic defensins is still poorly known, it is of interest to investigate the properties of the Spli Def.
Sequence motifs similar to the binding sites of TATA, CATT, IL-6-RE, and GATA transcription factors in mammals were found in promoter region of our sequence and almost in all genes that are up-regulated after immune challenge . TATA, CATT, IL-6-RE, and GATA boxes were found adjacent to each other indicating that they work cooperatively in the transcriptional activation of the Spli Def gene as previously described in cecropin A1 gene in Drosophila . Our results also showed that the relative transcription levels of Spli Def were up-regulated after bacterial-challenge, indicating the involvement of the Spli Def gene in immune responses of the Egyptian cotton leafworm, S. littoralis. The expression of Spli Def in haemocytes peaks at 48 h p.i. and gradually declines with time. These results were supported by the quantitative protein analysis which revealed the significant peak of increase at 48 h post-infcetion. Kaneko et al.  reported that the expression of BmDefensinB in fat body tissue of B. mori peaks at 8 h p.i. and declines with time. Ceraul et al.  demonstrated that the expression of two defensin isoforms in midgut and fat body tissues of the hard tick Dermacentor variabilis peak at 48 h p.i. with Rickettsia and gradually decline with time. The expression peak in fat body tissue may be retarded to be at 72 h p.i. in the case of defensin-2. Lopez et al.  found that the defensin expression pattern in fat body of bacteria-injected Rhodnius prolixus tissue is higher than in the intestinal tissue. This pattern may be due to the fact that the expression of defensin gene in deferent body tissues depends on the consequences of infection course of a pathogen (injected pathogen attacks haemocytes and fat body firstly and fed pathogen attacks alimentary canal firstly).