The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability

The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

The Dm14-3-3ε isoform shows partial redundancy with 14-3-3ζ isoform for RAS1 signaling in photoreceptor formation and animal viability [15].The Dm14-3-3ε is also responsible for controlling growth and apoptosis [16].Recently, the Dm14-3-3ε isoform was implicated in innate immunity by regulating the secretion of antimicrobial peptides from hemocyte to hemolymph in response to bacterial infection [7,17].In contrast, Dm14-3-3ζ was known to be involved in phagocytosis [18].In addition to Drosophila, 14-3-3ε and ζ isoforms have been cloned from other insects including Bombyx mori.These isoforms were shown to bind to Hsp60 in larval and adult tissues of B. mori and were suggested to work together with Hsp60 to achieve broad cellular functions [19].
The mealworm beetle, Tenebrio molitor is the most elegant and efficient model for an explorative study on the innate immune mechanisms in beetles.Several studies, including ours, are focused towards delineating the mechanisms of the Toll [20][21][22][23][24][25][26][27][28], prophenoloxidase cascade [29,30] and autophagy signaling pathways [31][32][33] in T. molitor with special reference to immunity against microorganisms.We have also taken advantage of the T. molitor model to show the requirement of apolipophorin-III in conferring immunity against an intracellular Gram-positive bacteria, Listeria monocytogenes [34,35].However, the understanding of antimicrobial peptide (AMP) secretion from hemocyte to hemolymph and the consequent bactericidal action is mostly unknown in this model insect.To gain further insights into the same, we cloned and characterized the Tm14-3-3ε from T. molitor.RNA interference (RNAi) knockdown techniques demonstrated that Tm14-3-3ε may play an important role in antimicrobial action in the hemocyte, affecting the secretion of AMPs to the hemolymph, and consequently increasing the larval mortality against Escherichia coli infection.

Insect Rearing, Microorganism Culture, and Challenge Experiments
T. molitor larvae were reared on an artificial diet (4.4 g of NeoVita, 0.5 g of chloramphenicol, 0.4 g of L-ascorbic acid, 0.5 g of sorbic acid, 0.5 mL of propionic acid, 2.2 g of yeast dry powder, 2.2 g of bean powder, 7.6 g of agar, 4.4 g of wheat powder and 73.3 g of wheat bran in 200 mL of distilled water; autoclaved at 121 • C for 15 min) in an insectary at 26 ± 1 • C, 60% ± 5% relative humidity and at dark condition.
Escherichia coli strain K12 and Staphylococcus aureus strain RN4220 were obtained from Pusan National University, Pusan, Korea and the mother culture of Candida albicans was procured from Hoseo University, Asan, South Korea.E. coli and S. aureus were cultured in Luria-Bertani (LB) broth and C. albicans was cultured in Sabouraud dextrose broth overnight at 37 • C. The cells were harvested, washed twice in phosphate-buffered saline (PBS), and were centrifuged at 3500 rpm for 10 min.The cultured cells were diluted with PBS to reach a concentration of 10 9 cells/mL of E. coli and S. aureus, and 5 × 10 7 cells/mL of C. albicans.

Full-Length cDNA Cloning and in Silico Analysis of the Putative Protein
Partial cDNA sequence of Tm14-3-3ε was screened from T. molitor expressed sequence tag (EST) and RNAseq database.The full-length cDNA sequence of Tm14-3-3ε was obtained by 5'-and 3'-Rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR).The cDNA templates for RACE-PCR were prepared using SMARTer TM RACE cDNA amplification kit (Clontech laboratories, Mountain View, CA, USA) according to manufacturer's instructions.The gene-specific primers, nested primers, and the RACE-PCR SMART universal primers were used for amplification (Table 1).The 5'-and 3'-end RACE amplification was conducted using the program: 30 cycles of denaturation at 94 • C for 30 s, annealing at 55 • C for 30 s, and extension at 72 • C for 30 s.The PCR products were purified, cloned into TOPO TA cloning vector (Invitrogen Corporation, Carlsbad, CA, USA), and subsequently transformed into competent E. coli DH5α cells.The transformants were screened by colony PCR and sequenced.
The gene-specific primers for qRT-PCR were designed with Primer 3 software and listed in Table 1.The T. molitor housekeeping gene, 60S ribosomal protein L27a (TmL27a) was used as an internal reference.Data from three independent observations were recorded and represented as the mean ± SE (n = 3).

Larval Mortality Assay
After the confirmation of Tm14-3-3ε gene silencing, the dsRNA injected groups (dsTm14-3-3ε and dsEGFP) were inoculated with E. coli suspension (10 6 cells per larva), and the larval mortality was monitored for a period of seven days.The study was conducted with 50 larvae, and the results represent an average of three biological replications.The cumulative survival rates were considered significant (p < 0.05) after conducting the Wilcoxon Mann Whitney test.

Antibacterial Activity Assay
Antibacterial activity of the hemolymph and hemocyte lysate against E. coli was assayed using the CFU count method [38].For the same, E. coli (10 7 cells per larva) were injected into dsTm14-3-3ε and dsTm14-3-3ζ treated larva, and allowed incubation within the insect larva for 12h.PBS and E. coli only (10 7 cells per larva) injected groups acted as negative and positive controls, respectively.The hemolymph from the larvae (pooled) was collected by cutting the proleg with sterile scissors and collected in a tube containing decoagulation buffer (DECO buffer; 15 mM sodium chloride, 30 mM trisodium citrate, 26 mM citric acid, 20 mM EDTA, pH 5.0).The hemolymph was centrifuged at 3500 rpm for 10 min at 4 • C to sediment the hemocytes.Hemocyte and hemolymph samples were boiled at 100 • C for 10 min, and centrifuged at 15,000 rpm for 10 min at 4 • C. The optical density at OD 220 of the supernatant was measured to estimate total protein.Bactericidal activity of hemolymph and hemocyte lysate was monitored using E. coli as an indicator bacterium.For the activity assay, the collected hemolymph and hemocyte lysate were diluted serially with PBS, and a portion (50 µg of peptides) of diluted samples was incubated with 10 6 E. coli cells in 200 µL of insect saline (130 mM NaCl, 5 Mm KCl, 1 mM CaCl 2 ) for 2 h at 37 • C. Subsequently, the mixture was diluted 2000-fold with insect saline and 100 µL aliquots were spread on LB agar plates.The plates were incubated for 16 h at 37 • C and the colony numbers on test and control plates were compared.

Statistical Analysis
All data are shown as means ± S.E.The difference between group means is assessed by one-way analysis of variance (ANOVA) and Tukey's multiple range tests at 95% confidence level (p < 0.05) using SAS 9.1.3for Windows (SAS Institute, Cary, NC, USA).

Molecular Cloning and Sequence Analysis of Tm14-3-3ε
We first retrieved a partial coding sequence of the 14-3-3ε gene from Tenebrio RNA-seq and EST database.After confirmation and validation of the full-length cDNA sequence of Tm14-3-3ε gene by cloning and sequencing, the information was submitted in GenBank under the accession number KP099937.The ORF of Tm14-3-3ε gene is composed of 771 nucleotides and encodes a protein of 256 amino acid residues.The Tm14-3-3ε gene shows a 5'-untranslated region (UTR) and a 3'-UTR of 105 bp and 209 bp, respectively.Furthermore, a polyadenylation signal (ACTAAA) was identified 22 nucleotides downstream of the stop codon.The deduced Tm14-3-3ε protein includes a conserved middle core region, called the 14-3-3 domain that participates as the main functional domain for interaction with partner proteins.In addition, a nuclear export signal (NES) sequence of 13 amino acids (N-LIMQLLRDNLTLW-C) has been identified in the 14-3-3ε protein of T. molitor that includes the presence of a few phosphopeptide-binding residues (Leu-219, Ile-220, Leu-223, Asn-227, Leu-230, and Trp-231).The NES signal sequence is found highly conserved in most of the identified 14-3-3 proteins and is critical for shuttling partner proteins in the nucleus [39].The nucleotide and deduced amino acid sequences of Tm14-3-3ε with the domain information are shown in Figure 1.
Genes 2016, 7, 53 5 of 12 and 209 bp, respectively.Furthermore, a polyadenylation signal (ACTAAA) was identified 22 nucleotides downstream of the stop codon.The deduced Tm14-3-3ɛ protein includes a conserved middle core region, called the 14-3-3 domain that participates as the main functional domain for interaction with partner proteins.In addition, a nuclear export signal (NES) sequence of 13 amino acids (N-LIMQLLRDNLTLW-C) has been identified in the 14-3-3ɛ protein of T. molitor that includes the presence of a few phosphopeptide-binding residues (Leu-219, Ile-220, Leu-223, Asn-227, Leu-230, and Trp-231).The NES signal sequence is found highly conserved in most of the identified 14-3-3 proteins and is critical for shuttling partner proteins in the nucleus [39].The nucleotide and deduced amino acid sequences of Tm14-3-3ɛ with the domain information are shown in Figure 1.A multiple sequence alignment analysis of Tm14-3-3ɛ isoform was conducted with 14-3-3ɛ proteins from insect orders to determine the evolutionary relationships and conservation within the critical 14-3-3 domain (Figure 2A).The evolutionary relationship of Tm14-3-3ɛ amino acid sequence with 14-3-3ɛ proteins from other insects shows well-spaced clusters.The 14-3-3ɛ proteins from the beetle partners, T. molitor and Tribolium castaneum were branched as separate cluster with 100% identity.The percentage identity analysis also shows a high (91%-94%) proximity to other insect 14-3-3ɛ proteins.Remarkably, Tenebrio 14-3-3ɛ shows a high (88%) identity to Dm14-3-3ɛ (Dm14-3-3ɛ) A multiple sequence alignment analysis of Tm14-3-3ε isoform was conducted with 14-3-3ε proteins from insect orders to determine the evolutionary relationships and conservation within the critical 14-3-3 domain (Figure 2A).The evolutionary relationship of Tm14-3-3ε amino acid sequence with 14-3-3ε proteins from other insects shows well-spaced clusters.The 14-3-3ε proteins from the beetle partners, T. molitor and Tribolium castaneum were branched as separate cluster with 100% identity.The percentage identity analysis also shows a high (91%-94%) proximity to other insect 14-3-3ε proteins.Remarkably, Tenebrio 14-3-3ε shows a high (88%) identity to Dm14-3-3ε (Dm14-3-3ε) suggesting functional redundancy of the protein isoform "ε" among the insects.Although this investigation did not include the analysis of the isoform-specific differences, it does show the putative occurrence of functional redundancy among the insect 14-3-3ε proteins.In any case, it was reported that the 14-3-3ε proteins from invertebrates group show domain sequence similarity with the non-epsilon isoforms from mammals highlighting their functional similarity with the ancestral protein [40].The phylogenetic tree and the percent identity analysis of the 14-3-3ε protein from T. molitor have been shown in Figure 2B,C, respectively.

Expression of Tm14-3-3ɛ Transcripts and Innate Immune Function in Hosts
The Tenebrio14-3-3ɛ transcript shows a consistent expression throughout development and was ubiquitously detected in all the larval and adult tissues.This finding is consistent to the previous report of Dm14-3-3ɛ expressed during development and also in almost all embryonic and larval tissues [7].The P. xylostella 14-3-3ɛ (Px14-3-3ɛ) protein also shows a constant expression in various tissue types and during the developmental stages of the moth [8].The broad expression patterns of 14-3-3ɛ proteins can be reflected in their regulatory function in modulating many cellular processes [12,41].These characters all need the T4 font encoding, which is provided by the fc package.* \m{v} and \m{V} are synonyms for \m{u} and \m{U}.
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Expression of Tm14-3-3ε Transcripts and Innate Immune Function in Hosts
The Tenebrio14-3-3ε transcript shows a consistent expression throughout development and was ubiquitously detected in all the larval and adult tissues.This finding is consistent to the previous report of Dm14-3-3ε expressed during development and also in almost all embryonic and larval tissues [7].The P. xylostella 14-3-3ε (Px14-3-3ε) protein also shows a constant expression in various tissue types and during the developmental stages of the moth [8].The broad expression patterns of 14-3-3ε proteins can be reflected in their regulatory function in modulating many cellular processes [12,41].
To study the putative function of Tenebrio 14-3-3ε protein in innate immunity, we studied the expression levels of the transcript in fat body, gut, and hemocyte after challenge with microorganisms such as E. coli, S. aureus, and C. albicans (Figure 3A).A significant rise in the Tm14-3-3ε mRNA levels was noticed in the hemocyte (nearly three-fold expression) in the case of E. coli infection.A small but significant rise in Tm14-3-3ε transcript level in the hemocyte was also noticed in the case of C. albicans infection.The increase in expression of Tm14-3-3ε mRNA by E. coli (Gram-negative bacteria) and C. albicans (fungus) and not by S. aureus (Gram-positive bacteria) in this study could be related to the specific interaction with cell surface pathogen receptors.As demonstrated before in Tenebrio and Drosophila, fungal and Gram-positive bacteria associated molecular patterns such as β-glucan and Lys-type peptidoglycan are recognized by Gram-negative binding protein 3 (GNBP-3) and PGRP-SA-GNBP-1, respectively.But unlike Drosophila, the monomeric DAP-type peptidoglycan (Gram-negative bacteria associated molecular patterns) induces the release of diptericin-like antimicrobial peptide in Tenebrio hemocytes [25].We suspect Tm14-3-3ε acts as an immune regulator and its expression on E. coli infection would influence the release of Drosophila diptericin-like antimicrobial peptides.Furthermore, it relates to the hemocyte-specific functions of Tm14-3-3ε transcripts.The expression level of the Tm14-3-3ε transcripts was measured after 12 h of inoculation into the host and statistically compared to the buffer-injected control.The Tm14-3-3ε mRNA expression analysis data provided leads to enquire about the hemocyte immune response in the E. coli infected larvae of the host.To date, only the Drosophila model system has been studied in detail regarding the role of 14-3-3ε in innate immunity [7].Considering an 88% sequence similarity between Tm14-3-3ε and Dm14-3-3ε proteins that suggests functional conservation, we hypothesized the requirement of Tm14-3-3ε in conferring immunity to the host against E. coli infection.For the same, we generated specific dsRNA to silence the Tm14-3-3ε transcripts in the host.We observed about 95% silencing (significant at the level of p < 0.05) of the transcript in the dsTm14-3-3ε injected group, when compared with the dsEGFP injected group (Figure 3B).Under this condition, we infected either the dsEGFP or Tm14-3-3ε silenced Tenebrio larvae, through the systemic route.The 12th instar larvae were challenged with a suspension of Gram-negative bacteria, E. coli.The surviving larvae were scored every day for the entire duration of the assay (for seven days, Figure 3C).We observed that the survival of the dsEGFP injected group was not significantly affected by E. coli infection (81% by seven days), but under Tm14-3-3ε silenced condition the survival of T. molitor larvae was dramatically reduced (18% by 7 days) when compared with the dsEGFP injected control group.This shows that Tm14-3-3ε silenced Tenebrio larvae are susceptible to death following Gram-negative bacterial challenge and that Tm14-3-3ε is necessary to ward off a possible infection by the pathogen in the host.The high mortality of Tenebrio larvae in Tm14-3-3ε silenced condition upon E. coli infection suggests a possible relationship of the Tm14-3-3ε transcript with immune system defects in the host.This has been elucidated succinctly in Dm14-3-3ε mutants that show decreased survival in response to Gram-negative E. coli and Gram-positive Micrococcus luteus infection when compared with wild-type strains [7].To study the putative function of Tenebrio 14-3-3ɛ protein in innate immunity, we studied the expression levels of the transcript in fat body, gut, and hemocyte after challenge with microorganisms such as E. coli, S. aureus, and C. albicans (Figure 3A).A significant rise in the Tm14-3-3ɛ mRNA levels was noticed in the hemocyte (nearly three-fold expression) in the case of E. coli infection.A small but significant rise in Tm14-3-3ɛ transcript level in the hemocyte was also noticed in the case of C. albicans infection.The increase in expression of Tm14-3-3ɛ mRNA by E. coli (Gram-negative bacteria) and C. albicans (fungus) and not by S. aureus (Gram-positive bacteria) in this study could be related to the specific interaction with cell surface pathogen receptors.As demonstrated before in Tenebrio and Drosophila, fungal and Gram-positive bacteria associated molecular patterns such as β-glucan and Lys-type peptidoglycan are recognized by Gram-negative binding protein 3 (GNBP-3) and PGRP-SA-GNBP-1, respectively.But unlike Drosophila, the monomeric DAP-type peptidoglycan (Gramnegative bacteria associated molecular patterns) induces the release of diptericin-like antimicrobial peptide in Tenebrio hemocytes [25].We suspect Tm14-3-3ɛ acts as an immune regulator and its expression on E. coli infection would influence the release of Drosophila diptericin-like antimicrobial peptides.Furthermore, it relates to the hemocyte-specific functions of Tm14-3-3ɛ transcripts.The expression level of the Tm14-3-3ɛ transcripts was measured after 12 h of inoculation into the host and statistically compared to the buffer-injected control.The Tm14-3-3ɛ mRNA expression analysis data provided leads to enquire about the hemocyte immune response in the E. coli infected larvae of the host.To date, only the Drosophila model system has been studied in detail regarding the role of 14-3-3ɛ in innate immunity [7].Considering an 88% sequence similarity between Tm14-3-3ɛ and Dm14-3-3ɛ proteins that suggests functional conservation, we hypothesized the requirement of Tm14-3-3ɛ in conferring immunity to the host against E. coli infection.For the same, we generated specific dsRNA to silence the Tm14-3-3ɛ transcripts in the host.We observed about 95% silencing (significant at the level of p < 0.05) of the transcript in the dsTm14-3-3ɛ injected group, when compared with the dsEGFP injected group (Figure . 3B).Under this condition, we infected either the dsEGFP or Tm14-3-3ɛ silenced Tenebrio larvae, through the systemic route.The 12th instar larvae were challenged with a suspension of Gram-negative bacteria, E. coli.The surviving larvae were scored every day for the entire duration of the assay (for seven days, Figure 3C).We observed that the survival of the dsEGFP injected group was not significantly affected by E. coli infection (81% by seven days), but under Tm14-3-3ɛ silenced condition the survival of T. molitor larvae was dramatically reduced (18% by 7 days) when compared with the dsEGFP injected control group.This shows that Tm14-3-3ɛ silenced Tenebrio larvae are susceptible to death following Gram-negative bacterial challenge and that Tm14-3-3ɛ is necessary to ward off a possible infection by the pathogen in the host.The high mortality of Tenebrio larvae in Tm14-3-3ɛ silenced condition upon E. coli infection suggests a possible relationship of the Tm14-3-3ɛ transcript with immune system defects in the host.This has been elucidated succinctly in Dm14-3-3ɛ mutants that show decreased survival in response to Gram-negative E. coli and Gram-positive Micrococcus luteus infection when compared with wild-type strains [7].

Tm14-3-3ɛ and Hemocyte Antimicrobial Activity
A high 14-3-3ɛ transcript level in the hemocyte of Tenebrio larvae and the reduced survival of Tm14-3-3ɛ silenced larvae to infection by E. coli suggests a role for the transcript in the activation of the hemocyte antimicrobial activity.This is consistent with previous reports showing the regulatory function of Dm14-3-3ɛ in the secretion of AMPs from hemocytes to hemolymph [7,17].To determine whether Tm14-3-3ɛ plays a similar role, we made a successful attempt in silencing the Tm14-3-3ɛ (93% silencing; p < 0.05; Figure 4A-I) and Tm14-3-3ζ (80% silencing; p < 0.05; Figure 4A-II) transcripts in two separate RNAi studies.Tm14-3-3ζ gene characterized in a separate study showed high degree of conservation at the amino acid sequence level with Tm14-3-3ε and ε isoforms from other insects.The phylogenetic analysis located 14-3-3ε and ζ isoforms of T. molitor to separate clusters of ε and ζ sequences (Supplementary Figure S1).RNAi knockdown of the 14-3-3ζ isoform in T. molitor was performed to examine its possible role in the antimicrobial response of hemocytes and hemolymph to Gram-negative E. coli infections.We had three groups of E. coli inoculation, including the Tm14-3-3ɛ and Tm14-3-3ζ silenced larval groups.A buffer injected group without E. coli injection was used as the negative control.We could find a significant increase (p < 0.05) of antimicrobial activity in hemocyte of Tm14-3-3ɛ silenced/E.coli injected group (lane 3; Figure 4B-I), in comparison with Tm14-3-3ζ silenced/E.coli injected group and E. coli only injected group (lane 2; Figure 4B-I).In the hemolymph, a significantly high (p < 0.05) reduction in colony-forming units (cfu) was noticed in Tm14-3-3ζ silenced/E.coli group (Lane 4; Figure 4B-II).This suggests that silencing of Tm14-3-3ɛ and not Tm14-3-3ζ transcripts significantly increase the antimicrobial activity in the hemocyte of the infected host, thereby putatively leading to a decreased secretion of AMPs from hemocyte into the hemolymph.We suspect the drop in secretion of AMP into the hemolymph would significantly affect the bactericidal action in the hemolymph.This potentially explains the multiplication of E. coli in the Tenebrio larvae and an increased mortality in Tm14-3-3ɛ silenced condition.The putative role of Tm14-3-3ε as an adaptor protein regulating the exocytosis of AMPs has been reported for Dm14-3-3ε through elegant studies.Drosophila mutant for 14-3-3ε show decreased survivability against bacterial

Tm14-3-3ε and Hemocyte Antimicrobial Activity
A high 14-3-3ε transcript level in the hemocyte of Tenebrio larvae and the reduced survival of Tm14-3-3ε silenced larvae to infection by E. coli suggests a role for the transcript in the activation of the hemocyte antimicrobial activity.This is consistent with previous reports showing the regulatory function of Dm14-3-3ε in the secretion of AMPs from hemocytes to hemolymph [7,17].To determine whether Tm14-3-3ε plays a similar role, we made a successful attempt in silencing the Tm14-3-3ε (93% silencing; p < 0.05; Figure 4A-I) and Tm14-3-3ζ (80% silencing; p < 0.05; Figure 4A-II) transcripts in two separate RNAi studies.Tm14-3-3ζ gene characterized in a separate study showed high degree of conservation at the amino acid sequence level with Tm14-3-3ε and ε isoforms from other insects.The phylogenetic analysis located 14-3-3ε and ζ isoforms of T. molitor to separate clusters of ε and ζ sequences (Supplementary Figure S1).RNAi knockdown of the 14-3-3ζ isoform in T. molitor was performed to examine its possible role in the antimicrobial response of hemocytes and hemolymph to Gram-negative E. coli infections.We had three groups of E. coli inoculation, including the Tm14-3-3ε and Tm14-3-3ζ silenced larval groups.A buffer injected group without E. coli injection was used as the negative control.We could find a significant increase (p < 0.05) of antimicrobial activity in hemocyte of Tm14-3-3ε silenced/E.coli injected group (lane 3; Figure 4B-I), in comparison with Tm14-3-3ζ silenced/E.coli injected group and E. coli only injected group (lane 2; Figure 4B-I).In the hemolymph, a significantly high (p < 0.05) reduction in colony-forming units (cfu) was noticed in Tm14-3-3ζ silenced/E.coli group (Lane 4; Figure 4B-II).This suggests that silencing of Tm14-3-3ε and not Tm14-3-3ζ transcripts significantly increase the antimicrobial activity in the hemocyte of the infected host, thereby putatively leading to a decreased secretion of AMPs from hemocyte into the hemolymph.We suspect the drop in secretion of AMP into the hemolymph would significantly affect the bactericidal action in the hemolymph.This potentially explains the multiplication of E. coli in the Tenebrio larvae and an increased mortality in Tm14-3-3ε silenced condition.The putative role of Tm14-3-3ε as an adaptor protein regulating the exocytosis of AMPs has been reported for Dm14-3-3ε through elegant studies.Drosophila mutant for 14-3-3ε show decreased survivability against bacterial infections due to the loss of exocytosis functions [17].From the present study, we also conclude that Tm14-3-3ζ does not show hemocyte antimicrobial function, suggesting the normal secretion of the AMPs and consequent bactericidal action.We could assume that the silencing of Tm14-3-3ζ could be implicated in the phagocytosis of microbes and would impede the survival of the larvae after E. coli infection.In an earlier report, Dm14-3-3ζ silencing had been attributed to compromised larval survival rate due to the phagocytosis of S. aureus [18].Herein, we argue that the loss of 14-3-3ε in Tenebrio larvae may putatively lead to reduced AMP in the hemolymph due to enhanced antimicrobial action in the hemocyte.A regulatory loss/reduction in AMP secretion may result in increased mortality when infected with E. coli.
Genes 2016, 7, 53 9 of 12 infections due to the loss of exocytosis functions [17].From the present study, we also conclude that Tm14-3-3ζ does not show hemocyte antimicrobial function, suggesting the normal secretion of the AMPs and consequent bactericidal action.We could assume that the silencing of Tm14-3-3ζ could be implicated in the phagocytosis of microbes and would impede the survival of the larvae after E. coli infection.In an earlier report, Dm14-3-3ζ silencing had been attributed to compromised larval survival rate due to the phagocytosis of S. aureus [18].Herein, we argue that the loss of 14-3-3ɛ in Tenebrio larvae may putatively lead to reduced AMP in the hemolymph due to enhanced antimicrobial action in the hemocyte.A regulatory loss/reduction in AMP secretion may result in increased mortality when infected with E. coli.

Conclusions
In this study, we show that the 14-3-3ɛ isoform cloned and characterized from the model insect T. molitor is required to control host viability under bacterial challenge.This crucial function in Tm14-3-3ɛ silenced larvae is possibly due to a reduction in AMP secretion from hemocyte to the Tm14-3-3ζ (II) transcripts in the whole-body of T. molitor larva six days after dsRNA injection.dsEGFP was used as a negative control.The data represent the mean ± S.E. of three independent biological replications; (B) Antimicrobial activity assay in hemocyte (I) and hemolymph (II) of Tm14-3-3ε and Tm14-3-3ζ silenced groups.Buffer injected and only E. coli injected groups is used as negative control and positive control, respectively.Bars represent mean ± standard error of three independent biological replications.One-way ANOVA analysis of variances should significant differences between group means (p < 0.05).Different subscripts over bars depict significant differences between means of groups.

Conclusions
In this study, we show that the 14-3-3ε isoform cloned and characterized from the model insect T. molitor is required to control host viability under bacterial challenge.This crucial function in Tm14-3-3ε silenced larvae is possibly due to a reduction in AMP secretion from hemocyte to the hemolymph.Lately, we have observed that the Tm14-3-3ε silenced larvae show a decreased survival in response to the Gram-positive bacterial and fungal challenge.We would be interested in providing necessary insights for the high mortality rate and the role of Tm14-3-3ε to elicit a common innate immune mechanism against the pathogens.

Figure 1 .
Figure 1.Tm14-3-3ε cDNA and deduced amino acid sequence information.The nucleotides are numbered from the first base of the translation start codon (ATG).The polyadenylation signal sequence in 3'-UTR is shown in grey text.The conserved 14-3-3 domain is shown in the grey shaded box.The direct peptide binding residues are boxed.The nuclear export sequence (NES) is shown underlined.

Figure 1 .
Figure 1.Tm14-3-3ε cDNA and deduced amino acid sequence information.The nucleotides are numbered from the first base of the translation start codon (ATG).The polyadenylation signal sequence in 3'-UTR is shown in grey text.The conserved 14-3-3 domain is shown in the grey shaded box.The direct peptide binding residues are boxed.The nuclear export sequence (NES) is shown underlined.

Figure 2 .
Figure 2. Multiple alignment, phylogenetic analysis, and percentage identity of 14-3-3ε.(A) Multiple alignment was conducted by Clustal X2 program and visualized by GeneDoc software; (B) Molecular phylogenetics of Tm14-3-3 sequence was used as outgroup.A bootstrap consensus tree is shown based on the Jones-Taylor-Thornton matrix model; (C) Percentage identity and distance were analyzed by Clustal X2 and MEGA6 program.

Figure 3 .
Figure 3. Expression analysis and RNA interference-based silencing of Tm14-3-3ε transcripts in T. molitor.(A) A qPCR analysis show the expression of Tm14-3-3ε transcripts in fat body, gut, and hemocytes of the host after the inoculation of Escherichia coli (Ec), Staphylococcus aureus (Sa), and Candida albicans (Ca).PBS injected larval group act as injection control.Total RNA was extracted from the tissues 12 h post-injection and profiled by qPCR.The housekeeping gene, TmL27a acts as an endogenous control.The experiments are conducted in three biological replications (n = 3).Bars represent mean ± standard error.* p < 0.05 (SAS, ANOVA); (B) (I).Silencing efficiency of Tm14-3-3ε transcripts in the whole-body of T. molitor larva after six days of dsRNA injection; (II).Timedependent survival of dsEGFP and dsTm14-3-3ε injected T. molitor larval groups after the inoculation of E. coli.For each group, at least n = 100 individual larvae were analyzed and results are shown as mean ± S.E.The percent mortality was recorded for at least seven days, and the experiment was conducted in three biological replications.Statistical significance was conferred by Wilcoxon-Mann-Whitney test (p < 0.05).

Figure 3 .
Figure 3. Expression analysis and RNA interference-based silencing of Tm14-3-3ε transcripts in T. molitor.(A) A qPCR analysis show the expression of Tm14-3-3ε transcripts in fat body, gut, and hemocytes of the host after the inoculation of Escherichia coli (Ec), Staphylococcus aureus (Sa), and Candida albicans (Ca).PBS injected larval group act as injection control.Total RNA was extracted from the tissues 12 h post-injection and profiled by qPCR.The housekeeping gene, TmL27a acts as an endogenous control.The experiments are conducted in three biological replications (n = 3).Bars represent mean ± standard error.* p < 0.05 (SAS, ANOVA); (B) (I).Silencing efficiency of Tm14-3-3ε transcripts in the whole-body of T. molitor larva after six days of dsRNA injection; (II).Time-dependent survival of dsEGFP and dsTm14-3-3ε injected T. molitor larval groups after the inoculation of E. coli.For each group, at least n = 100 individual larvae were analyzed and results are shown as mean ± S.E.The percent mortality was recorded for at least seven days, and the experiment was conducted in three biological replications.Statistical significance was conferred by Wilcoxon-Mann-Whitney test (p < 0.05).

Figure 4 .
Figure 4. Antimicrobial function of Tm14-3-3ɛ transcripts in T. molitor.(A) RNA interference and antimicrobial activity study of Tm14-3-3ε and Tm14-3-3ζ.Knockdown efficiency of Tm14-3-3ε (I) andTm14-3-3ζ (II) transcripts in the whole-body of T. molitor larva six days after dsRNA injection.dsEGFP was used as a negative control.The data represent the mean ± S.E. of three independent biological replications; (B) Antimicrobial activity assay in hemocyte (I) and hemolymph (II) of Tm14-3-3ε and Tm14-3-3ζ silenced groups.Buffer injected and only E. coli injected groups is used as negative control and positive control, respectively.Bars represent mean ± standard error of three independent biological replications.One-way ANOVA analysis of variances should significant differences between group means (p < 0.05).Different subscripts over bars depict significant differences between means of groups.

Figure 4 .
Figure 4. Antimicrobial function of Tm14-3-3ε transcripts in T. molitor.(A) RNA interference and antimicrobial activity study of Tm14-3-3ε and Tm14-3-3ζ.Knockdown efficiency of Tm14-3-3ε (I) andTm14-3-3ζ (II) transcripts in the whole-body of T. molitor larva six days after dsRNA injection.dsEGFP was used as a negative control.The data represent the mean ± S.E. of three independent biological replications; (B) Antimicrobial activity assay in hemocyte (I) and hemolymph (II) of Tm14-3-3ε and Tm14-3-3ζ silenced groups.Buffer injected and only E. coli injected groups is used as negative control and positive control, respectively.Bars represent mean ± standard error of three independent biological replications.One-way ANOVA analysis of variances should significant differences between group means (p < 0.05).Different subscripts over bars depict significant differences between means of groups.

Table 1 .
Primer sequences used in the present study.

Table 4 :
Non-ASCII Letters (Excluding Accented Letters) * * Not available in the OT1 font encoding.Use the fontenc package to select an alternate font encoding, such as T1.

Table 6 :
Punctuation Marks Not Found in OT1

Table 4
* ŋ \ng * * Not available in the OT1 font encoding.alternate font encoding, such as T1.