An aquaporin mediates cell shape change required for cellular immunity in the beet armyworm, Spodoptera exigua

Abstract

Cellular immunity in insects is accompanied by change in hemocyte shape. This study hypothesizes that cytoskeletal rearrangement is accompanied by transmembrane water transport to change cell volume, thus changing cell shape. A water-transporting pore (=aquaporin:AQP) has been identified in the beet armyworm, Spodoptera exigua. Its expression was detected in all developmental stages and tissues, although its transcription levels were different between biotic and abiotic conditions. Heterologous expression of Se-AQP in Sf9 cells showed that Se-AQP was localized on cell membrane. RNA interference (RNAi) using double-stranded RNA effectively suppressed its transcript levels. Under different ionic concentrations, hemocytes of RNAi-treated larvae did not change cell volume presumably due to malfunction in water transportation. Se-AQP might participate in glycerol transport because up-regulation of hemolymph glycerol titer after rapid cold-hardening was prevented by RNAi treatment against Se-AQP expression. The inhibitory effect of RNAi treatment on change of cell shape significantly impaired cellular immune responses such as phagocytosis and nodule formation upon bacterial challenge. RNAi treatment also significantly interfered with immature development of S. exigua. These results indicate that Se-AQP plays a crucial role in cell shape change that is required for cellular immunity and other physiological processes.

Figure 1

Molecular characterization of S. exigua aquaporin (Se-AQP). (A) Transmembrane domain analysis of Se-AQP. Domains of Se-AQP were predicted using TMHMM^,. NPA domains are shown in red color while residues related to the Ar/R constriction region are shown in yellow color. (B) Organization of Ar/R constriction region. The structure depicted was from the extracellular side of the membrane. Classical NPA motifs are shown in red sphere. Ar/R selectivity residues regions (Phe-92, His-216, Ser-226, and Arg-231) are shown in blue balls and sticks. (C) Phylogenetic analysis of S. exigua aquaporin (Se-AQP, GenBank accession number: MH333284) with known insect AQPs. The analysis was performed using MEGA6. Bootstrapping values were obtained with 1,000 repetitions to support branching and clustering. Amino acid sequences of selected AQP genes were retrieved from GenBank. Accession numbers were added after species name.

Figure 2

Expression profile of Se-AQP. (A) Western blot analysis. Se-AQP was transfected into Sf9 cells. Protein size of recombinant Se-AQP was ~32 kDa. It was captured by V5 antibody. (B) Immunofluorescence assay for the detection of transient expression of Se-AQP in Sf9 cells. F-actin was specifically detected with Alexa Fluor 555 phalloidin while nucleus was stained with DAPI. To check transient expression, anti-V5-FITC antibody was used. (C) Expression patterns of Se-AQP in different developmental stages, including egg, first to fifth instar larvae (‘L1–L5’), pupa, and adult. (D) Expression patterns in indicated tissues of L5 larvae, including hemocyte (‘HC’), fat body (‘FB’), and gut (‘Gut’). A ribosomal gene RL32 was used as reference gene. Each treatment was replicated three times with independent tissue preparations. Different letters indicate significant differences among means at Type I error = 0.05 (LSD test).

Figure 3

Role of Se-AQP in water transport. (A) Function of Se-AQP in regulating hemocyte shape. Hemocytes treated with gene specific dsRNA (‘dsAQP’) were exposed to isotonic, hypertonic, or hypotonic solution for 10 min. Hemocytes were observed under a fluorescence microscope at 400× magnification. Spread, shrunk, and lysed cells were indicated with white arrows. Hemocytic F-actin filaments were specifically recognized by FITC-tagged phalloidin (green). (B) Quantitative representation of spread, shrunk, and lysed hemocytes after exposure to isotonic, hypertonic, and hypotonic solutions, respectively. A GFP gene was used as a control dsRNA (‘dsCON’). Each treatment was independently replicated three times. Asterisk mark (*) on bars indicates significant differences among means at Type I error = 0.05 (LSD test).

Figure 4

Role of Se-AQP in glycerol transport. (A) Chromatograms showing reduced glycerol titer in hemolymph of RNAi treated fifth instar larvae in response to exposure to 4 °C for 6 h. (B) Changes in glycerol content in fifth instar larval hemolymph in response to exposure to 4 °C for 6 h. The eluent was 400 mM NaOH at a flow rate of 0.4 mL/min. (C) Suppression of cold tolerance after RNAi treatment of Se-AQP. Each treatment was independently replicated three times. Each replicate used 10 larvae. Different letters and asterisk mark (*) indicate significant differences among means at Type I error = 0.05 (LSD test).

Figure 5

Role of Se-AQP in changing cell shape of hemocytes. For bacterial challenge, heat-killed (HK) E. coli (~3.2 × 10⁴ cells/larva) in 1 µL were injected into larvae at 24 h after dsRNA treatment. (A) Effect of dsAQP on F-actin growth in response to bacterial challenge. At 2 h PI, hemocytes were observed under a fluorescence microscope at 400× magnification. Hemocytic F-actin filaments were specifically recognized by FITC-tagged phalloidin (green) while the nucleus was stained with DAPI (blue). (B) Quantitative representation of hemocyte spreading assay. Each treatment was independently replicated three times. Different letters indicate significant differences among means at Type I error = 0.05 (LSD test).

Figure 6

Influence of RNAi treatment of Se-AQP on hemocyte phagocytosis. (A) Effect of dsAQP on FITC-labeled E. coli. One microliter of heat-killed (HK) E. coli (~3.5 × 10⁴ cells/larva) were injected into larvae at 24 h after dsRNA treatment. A GFP gene was used as a control dsRNA (‘dsCON’). (B) Quantitative representation of phagocytosis between dsCON and dsAQP treated hemocytes. Each treatment was independently replicated three times. Asterisk mark (*) on bars indicates significant differences among means at Type I error = 0.05 (LSD test).

Figure 7

Influence of RNAi treatment of Se-AQP on nodulation and phenoloxidase (PO) activity. (A) Inhibitory effect of dsAQP on hemocyte nodule formation in response to bacterial challenge. One microliter of heat-killed (HK) E. coli (~4.2 × 10⁴ cells/larva) were injected into larvae at 24 h after dsRNA treatment. At 8 h PI, numbers of nodules were assessed. (B) Inhibitory effect of dsAQP on PO activity in response to bacterial challenge. One microliter of heat-killed (HK) E. coli (~4.2 × 10⁴ cells/larva) was injected into larvae at 24 h after dsRNA treatment. At 8 h PI of bacteria, PO activity was measured. A GFP gene was used as a control dsRNA (‘dsCON’). Each treatment was independently replicated three times. Each replicate used 10 larvae. Different letters indicate significant differences among means at Type I error = 0.05 (LSD test).

Figure 8

Influence of RNAi treatment of Se-AQP on larval and pupal developmental processes. One µg of dsCON or dsAQP was injected into larvae (within an hour after emerging into L4 and L5) or pupae (<4 h old) using a microsyringe. (A) Effect of dsAQP on developmental period of larvae and pupa. (B) Effect of dsAQP in decreasing body weights of larvae and pupa. (C) Pupation percentage in larvae after treatment with dsRNA. (D) Percentage of successful adult emergence in larvae and pupa after RNAi. (E) Detrimental effect of dsAQP on pupa. A GFP gene was used as a control dsRNA (‘dsCON’). Each treatment was independently replicated three times. Each replicate used 10 larvae. Asterisk mark (*) on bars indicates significant differences among means at Type I error = 0.05 (LSD test).