RNA interference mediated knockdown of an inhibitor of apoptosis protein induces apoptosis in Mythimna separata (Lepidoptera: Noctuidae)

Coordinated regulation of apoptosis is critical for development, homeostasis, and immunity in larvae of Metazoa. We determined the full nucleotide sequence of an inhibitor of an apoptosis protein in a lepidopteran insect Mythimna separata (Walker) (MsIAP) and carried out functional analyses of the MsIAP. The full-length cDNA of MsIAP was 1642 bp, which encoded 379 amino acid residues with a calculated molecular mass of 41,834 Da, and two BIR domains and one RING domain revealed using amino acid sequence analysis. In addition, the sequences of these domains were similar to Drosophila IAP1 and those of some other lepidopteran insects. We carried out a functional analyses of MsIAP related to apoptosis regulation using RNA interference. The effects of MsIAP knockdown on adhering hemocytes and non-adhering hemocytes as controls were examined using Hoechst33342/propidium iodide staining, effector caspase activity and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining. A signifi cantly higher number of propidium iodide and TUNEL-positive cells was recorded in adhering hemocytes from MsIAP knockdown larvae than from control larvae, but these differences were not recorded for non-adhering hemocytes. However, higher effector caspase activity was detected in both adhering and non-adhering hemocytes from MsIAP knockdown larvae compared to that in control larvae. These results indicate that the knockdown of MsIAP induces apoptosis in larval adhering hemocytes, which MsIAP negatively and non-redundantly regulate apoptosis, and that IAP function is conserved in M. separata and other insect species including Drosophila and several lepidopteran insects. * Present address: Biology Group, Discovery Health and Crop Sciences Research Laboratory, Sumitomo Chemical Co., Ltd, 4-2-1, Takarazuka 665-8555, Japan ** Corresponding and present address: Insect Genome Research and Engineering Unit, Division of Applied Genetics, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), 1-2 Owashi, Tsukuba, Ibaraki, 305-8634, Japan; e-mail: yokoi123@affrc.go.jp INTRODUCTION Apoptosis is a genetically programmed cellular process that removes unwanted, damaged, or infected cells by inducing “suicide” in these cells (Clarke, 1990). Thus, apoptosis has a crucial role in development, homeostasis and immunity (Vaux & Korsmeyer, 1999; Opferman & Korsmeyer, 2003). The molecular mechanism of apoptosis was initially investigated in the model nematode Caenorhabditis elegans (Maupas), which has a protease ced-3 and nuclease nuc-1 that function as positive and negative regulators of apoptosis, respectively (Horvitz, 2003; Riedl & Shi, 2004). Drosophila melanogaster (Meigen) and mammals have more complex cell death mechanisms. Multiple family ced-3 protease homologs, which are known as caspases, have central roles in regulating apoptosis. Caspases fall Eur. J. Entomol. 115: 223–231, 2018 doi: 10.14411/eje.2018.021


INTRODUCTION
Apoptosis is a genetically programmed cellular process that removes unwanted, damaged, or infected cells by inducing "suicide" in these cells (Clarke, 1990).Thus, apoptosis has a crucial role in development, homeostasis and immunity (Vaux & Korsmeyer, 1999;Opferman & Korsmeyer, 2003).The molecular mechanism of apoptosis was initially investigated in the model nematode Caenorhabditis elegans (Maupas), which has a protease ced-3 and nuclease nuc-1 that function as positive and negative regulators of apoptosis, respectively (Horvitz, 2003;Riedl & Shi, 2004).Drosophila melanogaster (Meigen) and mammals have more complex cell death mechanisms.Multiple family ced-3 protease homologs, which are known as caspases, have central roles in regulating apoptosis.Caspases fall systems).BLASTx search (URL: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch) confi rmed that the obtained DNA fragment encoded an amino acid sequence that is very similar to known insect IAPs.

3' and 5' RACE
For 3' RACE, the 1st strand cDNA synthesis was primed with a tagged oligo-(dT) primer (5'-CTACAGTCTGCTCA-CAGCATAGTATTTTTTTTTTTTTTTTTTTTTTTTTVN-3').The 3' region of MsIAP was amplifi ed with gene-specifi c primers (5'-ACGGAGGCTTGAAAGACTGGGAG-3') and a 3' anchor primer (5'-CTACAGTCTGCTCACAGCATAGTA-3') using thermal cycling conditions of initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 68°C for 30 s and elongation at 72°C for 1 min 30 s.For 5' RACE, a RNA oligo (5'-AAGCAGUGGUAU-CAACGCAGAGUGGG-3') was included in the reverse transcription reaction and the 3'-tagged 1st strand cDNA pool was subjected to RT-PCR with a 5' anchor primer (AAGCAGTG-GTATCAACGCAGAGT-3') and gene-specifi c primer (5'-GC-GAGCTTGAACCTCGGCAAT-3').The locations of the two gene-specifi c PCR primers used for the RACE procedures and degenerate primers are shown in Fig. 1.Thermal cycling conditions used were an initial denaturation step at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 68°C for 30 s and elongation at 72°C for 1 min 30 s.The amplifi cation products were fractionated by agarose gel electrophoresis and specifi cally-amplifi ed DNA fragments were recovered and sequenced directly.In some cases, the DNA fragments were sequenced directly with the same reagents and device described previously and the sequences assembled into the full MsIAP nucleotide sequence.The full MsIAP nucleotide sequence was deposited in GenBank (accession no.AB778567).

RNA interference
The materials and methods related to RNAi were the same as in Yokoi et al. (2013).Briefl y, the DNA templates for dsRNA synthesis were prepared using conventional PCR with the pairs of T7-tagged PCR primers for the respective factors, for which the locations in MsIAP are shown in Fig. 1.The sequences of the primers were: 5'-taatacgactcactatagggagaACTTTCCTTTCTG-GAGATGAACT-3' (sense) and 5'-taatacgactcactatagggagaT-CATCACCTTCTGGACGTACTC-3' (anti-sense).Synthesis of dsRNA was carried out using a MEGAscript RNAi kit (Ambion) with these templates.The dsRNA fragment was purifi ed according to the manufacturer's instructions, and the integrity and amount were assessed using agarose gel electrophoresis and spectrophotometry, respectively.For the production of dsRNA with unrelated sequences, enhanced green fl uorescent protein (EGFP) sequence-derived dsRNA was prepared in the same manner using the pEGFP-N1 plasmid (Clontech) as a PCR template.
For IAP gene function analysis, we prepared isolated abdomens using day 1 6th instar larvae.The detailed procedures for preparing and isolating abdomens and dsRNA injection are described by Yokoi et al. (2013).Briefl y, the larvae were constricted between the third thoracic segment and the fi rst abdominal segment with a nylon thread, and the posterior part minus two pairs of haematopoietic organs was used as an isolated abdomen.dsRNA was injected into isolated abdomens 24 h post ligation.We performed a preliminary assay for IAP knockdown effects on isolated abdomens.We found that when 3 μg dsRNA MsIAP was injected into an isolated abdomen and the isolated abdomen was incubated for 48 h, there was approximately a 60% reduction in MsIAP mRNA in adhering hemocytes compared to that in control hemocytes, but no signifi cant effects of MsIAP knockdown was recorded in nogaster initiator of caspase), drICE and DCP-1 (D. melanogaster effector caspases) (Kornbluth & White, 2005;Tenev et al., 2005).Most IAP family proteins have two important domains, a baculoviral IAP repeat (BIR) domain and really interesting new gene (RING) domain (Riedl & Shi, 2004;Vaux & Silke, 2005).The BIR domain is a Zn 2+binding domain and consists of approximately 70 amino acid residues.This domain interacts with effector or initiator caspases.The RING domain is a Zn 2+ -binding domain with E3 ubiquitin ligase activity and is involved in the negative regulation of apoptosis.
In several species of Lepidoptera, IAP orthologues function as negative regulators of apoptosis, which suggests that roles of IAPs are conserved in mammals, D. melanogaster and lepidopteran insects (Huang et al., 2000(Huang et al., , 2001;;Liao et al., 2002).We investigated whether the function of an IAP orthologue in the lepidopteran insect, Mythimna separata (Walker) (MsIAP), is conserved.Previously, we examined its sensitivity to RNAi in multiple tissues of M. separata (Yokoi et al., 2013).Using a partial sequence of MsIAP, we found that MsIAP mRNA was ubiquitously expressed and that signifi cant gene silencing of MsIAP was achieved only in adhering hemocytes from isolated abdomens treated with MsIAP double-stranded RNA (dsRNA).Here, we determined the full cDNA sequence of MsIAP and the predicted MsIAP amino acid sequence was analyzed.MsIAP function was determined in terms of cell viability, effector caspase activity and genomic DNA fragmentation.We found that MsIAP functioned as a negative regulator of apoptosis in adhering hemocytes, which indicates that the function of IAP as a negative regulator of apoptosis is conserved in M. separata.

Insect rearing and preparation of isolated abdomens
M. separata was reared as described in Suzuki & Tanaka (2007) and the method of preparing isolated abdomens is described in Yokoi et al. (2013).

First strand cDNA synthesis for RT-PCR
Total hemocyte RNA was used for 1st strand cDNA synthesis using PrimeScript reverse transcriptase (TAKARA) and oligo-(dT) 20 primer.The 1st strand cDNA library was used as a template for RT-PCR with a pair of degenerate primers.A pair of degenerate primers were designed based on the highly conserved regions identifi ed by alignment of several amino acid sequences of published insect IAPs using the Clustal W 1.83 program (Thompson et al., 1994).Sequences of the degenerate primer pair were: IAP-F1d, 5'-ATGAARACNTTYGARAARTGGC-3'; IAP-R1d, 5'-TCYTCNGCRWARCAIATYTTICA-3'.The locations of these degenerate primers in MsIAP are shown in Fig. 1.The RT-PCR reaction mixture was fi rst denatured at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 1 min and elongation at 72°C for 30 s.The PCR products were separated on a 1.8% agarose gel.The DNA bands of the expected sizes were excised and recovered using a QIAquick Gel Extraction Kit (QIAGEN).DNA fragments were checked for integrity and amount using gel electrophoresis, sub cloned into the pCR2.1 vector (Invitrogen), and sequenced with an ABI Prism Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and a DNA sequencer (Model 3130, Applied Bio-non-adhering hemocytes, which is similar to the results of Yokoi et al. (2013).Therefore, we used these conditions for MsIAP knockdown of hemocytes.

Hoechst33342 and propidium iodide staining
Hemocyte viability was assessed by plasma membrane integrity using double staining with Hoechst33342 and propidium iodide (PI).Hemolymph was diluted with 9 volumes of SF900 III medium (Thermo Fisher SCIENTIFIC) with 8% (v/v) saturated phenyltiourea (PTU), which inhibits spontaneous melanization of the hemolymph and incubated on a glass slide (Matsunami, S2441) for 15 min at room temperature.The glass slide was washed three times with the same medium and non-adhering hemocytes, consisting of spherulocytes, prohemocytes and oenocytoids (Lavine & Strand, 2002) were collected into a plastic tube and centrifuged at 600 g at 4°C for 10 min.Adhering hemocytes, which remained on the glass slide, mainly consisted of granulocytes and plasmatocytes (Lavine & Strand, 2002).The nonadhering hemocyte precipitate was resuspended in the same medium and the hemocyte suspension was put on a Mas-coated glass slide (Matsunami, S9441), which allows M. separata non-adhering hemocytes to adhere to the coated surface.Both adhering and non-adhering hemocytes on the glass surface were washed three times with PBS and double-stained with Hoechst33342 (10 mg/ mL in PBS) and PI (2 mg/mL in PBS) for 3 min.The samples were washed briefl y with PBS, immediately observed under a fl uorescent microscope (Olympus, Model BX41) and imaged.The numbers of Hoechst-positive (all) cells and PI-positive (dead or late apoptotic) cells were counted at 200 × magnifi cation in at least six randomly selected fi elds.

Caspase-3/7 activity assay
The activity of effector caspase-3/7 was determined in larval hemocyte preparations using a Caspase-Glo 3/7 kit (Promega).The hemolymph from larvae pretreated with MsIAP or EGFP dsRNA was mixed with an equal volume of SF900 III with 8% (v/v) PTU and aliquoted into a 96-well plate.Fifteen minutes later, non-adhering hemocytes were transferred to another well.Hemocyte preparations were lysed in the presence of substrate and caspase activity determined using a luminometer (Turner Designs, Model TD 20/20).The caspase activities in the IAP knockdown and control of adhering and non-adhering hemocytes were determined.First, data normality for each category was tested using the Kolmogorov-Smirnov test.If signifi cant normality of the two samples was detected, a Student's t test was used.If signifi cant normality of the two samples was not detected, a Mann-Whitney U test was used.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay
Adhering and non-adhering hemocyte samples on glass slides were prepared as above.Hemocytes were labelled according to the protocol of an In Situ Cell Death Detection Kit, AP (Roche), samples were observed under a fl uorescent microscope and imaged.The total numbers of cells and TUNEL-positive cells in both hemocyte samples were counted.

cDNA sequence determination and analyses of MsIAP
The full cDNA sequence of MsIAP was obtained by a combination of PCR-based approaches.The MsIAP cDNA has 1642 nucleotides and encodes a polypeptide of 379 amino acid residues with a calculated molecular mass of 41,834 Da (Fig. 1).

Effects of MsIAP dsRNA injection on hemocyte viability
Hemocytes were double-stained with Hoechst33342 and PI and observed using fl uorescence microscopy.We counted the number of hemocytes double-stained with Hoechst33342 and PI or stained only with Hoechst33342 (Table 1).Typical images of the fi ve hemocyte species are shown in Fig. 3A.Hemocyte spreading was observed in adhering hemocytes, such as granulocytes and plasmatocytes, but not in non-adhering hemocytes such as spherulocytes, prohemocytes and oenocytoids.Typical images of adhering and non-adhering hemocytes from isolated abdomens treated with EGFP or MsIAP dsRNA are shown in Fig. 3B.Adhering hemocytes consisted mainly of granulocytes and plasmatocytes, and non-adhering hemocytes consisted mainly of spherulocytes, prohemocytes and oenocytoids as indicated in the bright fi eld images in Fig. 3B.For adhering hemocytes from EGFP dsRNA injected larvae, only a few hemocytes were PI-positive (dead or late apoptotic) (Fig. 3B, top panels and Table 1).MsIAP knockdown increased the number of PI-stained adhering hemocytes but not the number of non-adhering hemocytes (Fig. 3B, the second panels from top and Table 1).The numbers of Hoechst33342-positive and PI-positive hemo-cytes in each sample are shown in Table 1.Thus, we confi rmed that injection of MsIAP dsRNA into isolated larval abdomens caused cell death of adhering hemocyte species.For non-adhering hemocytes there was no signifi cant difference between the MsIAP dsRNA treatment and control samples (Fig. 3B, the third from top and bottom panels, and Table 1).

dsRNA treatment and caspase activity
MsIAP dsRNA treatment disrupted plasma membrane integrity and caused cell death in adhering hemocytes (Fig. 3), which indicates that knockdown of MsIAP induced apoptosis in these cells.We determined the activity of two effector caspases, caspase-3 and caspase-7, in the apoptosis of hemocytes.Adhering and non-adhering hemocyte fractions were prepared from isolated larval abdomens treated with either EGFP or MsIAP dsRNA.Caspase activity in adhering hemocytes from MsIAP dsRNA-treated larvae was approximately 2-fold higher.A test of normality for the two samples was performed, and the P values of MsIAP dsR-NA-treated and control samples were 0.9977 and 0.8562, respectively.Therefore, we tested the difference in caspase activities using a Student's t-test.At the 10% signifi cance level, a signifi cant difference in caspase activities was recorded, which is consistent with the results of the Hoechst/ PI staining (Fig. 4).Surprisingly, MsIAP dsRNA treatment increased caspase activity in non-adhering hemocytes even though these hemocytes were PI-negative and a signifi cant RNAi knockdown was not achieved (Fig. 4).The test of normality of the results for the MsIAP and dsRNA-treated and control samples had P values of 0.6296 and 0.9804, respectively.Therefore, a Mann-Whitney U test was used because signifi cant normality in the MsIAP dsRNA-treated sample was not detected.A signifi cant difference in caspase activities between the two categories was detected at the 10% signifi cance level.

TUNEL staining
To further characterize the type of cell death caused by MsIAP knockdown, hemocytes treated with dsRNAs were subjected to TUNEL staining, which detects genomic DNA fragmentation.Typical images of TUNEL staining for each sample are shown in Fig. 5.The results were similar to those obtained using PI staining (Fig. 3).Only adhering hemocyte samples prepared from MsIAP dsRNA-treated  larvae had a high proportion of TUNEL-positive cells and other samples were TUNEL-negative.In addition, we counted the total and TUNEL-positive hemocyte numbers and present the ratio of TUNEL-positive hemocytes in Fig. 5.The ratio of the number of hemocyte cells to the number of TUNEL-positive cells in adhering hemocytes from dsEGFP-injected larvae and adhering hemocytes from dsMsIAP-injected larvae were 310/0 and 301/256, respectively.The ratio of the number of hemocyte cells to the number of TUNEL-positive cells in non-adhering hemocytes from dsEGFP-injected larvae and non-adhering hemocytes from dsMsIAP-injected larvae were 22/0 and 26/0, respectively.Thus, TUNEL-positive cells were recorded only in adhering hemocytes treated with MsIAP dsRNA.

DISCUSSION
We determined the full nucleotide sequence of MsIAP and whether it functioned as a negative regulator of apoptosis using RNAi.Sequence analysis revealed that MsIAP had two BIR domains and one RING domain.A phylogenetic tree indicated that the amino acid sequence of MsIAP is similar to that of other lepidopteran IAP amino acid sequences.The zinc-binding BIR domain serves as an interface for protein-protein interactions, especially with caspases (Salvesen & Duckett, 2002).The BIR1 and BIR2 domains of MsIAP both had the canonical pattern of three cysteines and one histidine, CX 2 CX 6 WX 9 HX 6 C (Deveraux & Reed, 1999), similar to that in other insect IAPs.The RING domain is also found in IAPs, and IAPs with RING domains have ubiquitin protein ligase (E3) activity.The MsIAP RING domain has a C3HC4 pattern, which is conserved in other insect IAPs.Binding caspases undergo ubiquitination by the C-terminal RING domain, which has ubiquitin ligase activity (Meier et al., 2000;Riedl & Shi, 2004;Vaux & Silke, 2005).In lepidopteran insects, a few IAPs have been investigated, and all of them have two BIR domains and one RING domain, except for Bombyx IAP2 that has three BIR domains (Seshagiri et al., 1999;Huang et al., 2000Huang et al., , 2001;;Vilaplana et al., 2007;Zhang et al., 2010).S. frugiperda and B. mori IAPs inhibit mammalian initiator caspase-9 but not effector caspase-3 or caspase-7 (Huang et al., 2000(Huang et al., , 2001)).In Drosophila, DIAP1 is essential for cell survival because the loss of DIAP1 leads to spontaneous initiation of apoptosis (Wang et al., 1999).DIAP1 also has two BIR domains, which are functionally distinct from each other.BIR1 mediates direct physical interaction with effector caspases, drICE and DCP-1, and BIR2 provides an interface for binding to the initiator caspase DRONC.Our sequence alignment and phylogenetic tree results reveal that MsIAP is similar to other lepidopteran IAPs, which indicates that MsIAP has the same target specifi city as the IAPs of S. frugiperda and B. mori.
Because of the reduced mRNA levels of MsIAP in adhering hemocytes from isolated abdomens treated with MsIAP dsRNA (Yokoi et al., 2013), we determined viability using Hoechst/PI staining.Dead cells or cells with damaged plasma membranes were frequent among the ad-hering hemocytes from MsIAP dsRNA-injected isolated larval abdomens, and less so in samples from controls, which indicates that knockdown of MsIAP induces cell death in adhering hemocytes.We determined the activity of caspase-3 and caspase-7 using an apoptosis-specifi c TUNEL assay.Contrary to our expectations, the activities of effector caspase-3 and caspase-7 were higher in both adhering and non-adhering hemocytes from MsIAP dsRNAtreated larvae compared to those from controls.Apoptotic pathways in mammals and Drosophila are well conserved (Riedl & Shi, 2004) so it is likely that the apoptotic pathways in M. separate are also conserved.Initiator caspase activation leads to effector caspase activation in mammals and Drosophila.Thus, the higher activities of caspase-3 and caspase-7 in adhering hemocytes from larvae treated with MsIAP dsRNA could be due to activation of the M. separata caspase-9 homologue, which is a potential target of MsIAP.The percentage of TUNEL-stained adhering hemocytes recorded for MsIAP dsRNA-injected larvae was comparable to that of PI-positive cells, which clearly indicates that knockdown of MsIAP in hemocytes induced apoptosis.These results indicate that MsIAP negatively regulates apoptosis and that the function of IAP is conserved in mice, Drosophila and lepidopteran insects.
In mice, gene disruption of XIAP results in no major cell death phenotypes because its effect is compensated for by the upregulation of the related genes, cIAP1 and cIAP2 (Harlin et al., 2001).In mutant DIAP-defi cient fl ies, cell death and death of embryos are reported (Martin, 2002), which indicates that DIAP is indispensable for cell survival.In the case of M. separata, knockdown of MsIAP induced apoptosis in adhering hemocytes, which indicates that MsIAP is a non-redundant factor that prevents unwanted apoptosis in this species.
Although injection of MsIAP dsRNA into isolated abdomens did not alter the amount of MsIAP mRNA in nonadhering hemocytes (Yokoi et al., 2013), a signifi cantly higher activity of caspase-3 and caspase-7 was recorded.Higher activation of caspases may be due to an immune reaction.Caspases are involved in innate immune reactions in mammals (Meunier et al., 2014).Fig. 5 shows that numerous apoptotic adhering hemocytes from MsIAP knockdown occurred in hemolymph from isolated abdomens treated with MsIAP dsRNA.The immune reaction in non-adhering hemocytes may be induced by these apoptotic hemocytes, which activates caspases in the immune reaction by an unidentifi ed mechanism.
In conclusion, we found an IAP orthologue in M. separata and the amino acid sequence of MsIAP is similar to that of other lepidopteran IAPs.In addition, we found that MsIAP functioned as a negative and non-redundant regulator of apoptosis, which indicates that the functions of IAP are conserved between Drosophila and lepidopteran insects including M. separata.
supported in part by JSPS KAKENHI Grant Numbers 23658047 and 25450486 to KM.

Fig. 1 .
Fig. 1. cDNA and predicted amino acid sequences of MsIAP.The full-length cDNA sequence was assembled from partial sequences obtained using RT-PCR with degenerate primers, 3' RACE and 5' RACE.Arrows with numerals 1 to 6 indicate the positions of primers used: 1 and 2, a pair of degenerate primers for RT-PCR; 3, gene-specifi c antisense primer for 5' RACE; 4, gene-specifi c sense primer for 3' RACE; 5 and 6, T7-tagged primer pair for in vitro transcription template preparation.An asterisk denotes the stop codon.Nucleotide and amino acid residue numbers are shown on the left and right sides of each line.

Fig. 2 .
Fig. 2. Amino acid sequence alignment (A) and phylogenetic tree (B) of MsIAP and known insect IAPs.The Spodoptera frugiperda IAP (SfIAP, accession no.AAF35285), Tricoplusia ni IAP (TnIAP, accession no.AF195528), Bombyx mori IAP (BmIAP, accession no.NP001037024) and Drosophila melanogaster DIAP1 (accession no.Q24306) amino acid sequences were aligned together with MsIAP sequence using the Clustal W algorithm.In (A), residue numbers are shown on both sides of each line.Gaps introduced are indicated by hyphens.Two BIR domains and one RING domain are marked by thick horizontal bars.The amino acid residues conserved in all (fi ve) sequences are shown in white letters on a black background while those found in three or four sequences are on a grey background.Asterisks indicate important residues in each domain.In (B), is the phylogenetic tree constructed based on sequence alignment data.The length of each branch represents the degree of difference between each sequence.

Fig. 3 .
Fig. 3. Hemocyte viability after dsRNA treatment.A -Images of M. separata hemocyte species show adhering hemocytes, such as granulocytes (GR) and plasmatocytes (PL), and non-adhering hemocytes, such as spherulocytes (SP), prohemocytes (PH) and oenocytoids (OE), under high magnifi cation with 10-μm scale bars.An adjacent SP is also present in the OE panel.B -Isolated larval abdomens were injected with dsRNA of either EGFP (top and third from top panels) or MsIAP (second from top and bottom panels).Forty-eight hours later, adhering and non-adhering hemocyte preparations were double-stained with Hoechst33342 and PI.Adhering hemocyte species with typical shapes are marked in the two upper left panels.Horizontal bars indicate a length of 100 μm.Note that dense PI staining of adhering hemocytes from MsIAP dsRNA-injected larvae was recorded.

Fig. 4 .
Fig. 4. Caspase activity in hemocytes treated with MsIAP or EGFP dsRNA.Isolated larval abdomens were treated as in Fig.3, and caspase-3/7 activity was determined in adhering and non-adhering hemocyte preparations 48 h post-dsRNA injection.There were 6-8 biological replicates in each sample.Each value is calculated relative to the average value of adhering hemocytes from EGFP dsR-NA-injected larvae, which was set to 1.Each vertical bar is a mean ± S.D. Asterisk and double asterisks indicate P = 0.09 using Student's t-test and P = 0.03 using Mann-Whitney U test, respectively.

Table 1 .Fig. 5 .
Fig. 5. TUNEL staining of hemocytes treated with MsIAP or EGFP dsRNA.Isolated abdomens of larvae were injected with either EGFP (top and third from top panels) or MsIAP dsRNA (second from top and bottom panels).Adhering and non-adhering hemocytes were prepared 48 h later and stained for fragmented genomic DNA using TUNEL.