Cellular uptake of extracellular dsRNA is tissue-dependent in insects
Xuekai Shi, Yaoming Liu, Xiaojian Liu, Mureed Abbas, Austin Merchant, Hans Merzendorfer, Zhangwu Zhao, Xuguo Zhou, Kun Yan Zhu, Jianzhen Zhang
TL;DR
This study shows that how insects take up RNA varies by tissue, which helps explain why RNAi works differently in different parts of their bodies.
Contribution
The study identifies tissue-specific dsRNA uptake mechanisms in insects using a combination of omics and phenotypic approaches.
Findings
dsRNA uptake is tissue-dependent in insects, involving multiple cell membrane receptors and pathways.
Clathrin-mediated endocytosis is the most conserved dsRNA uptake mechanism across insect species.
Different tissues use distinct uptake mechanisms, such as macropinocytosis in hemocytes and caveolin-mediated endocytosis in midgut cells.
Abstract
RNA interference (RNAi), a naturally occurring gene silencing mechanism found in almost all eukaryotic organisms, has proven to be an adaptable and powerful tool in therapeutics, bioengineering, and agriculture. Differential responses to RNAi, however, are a key limiting factor, in which cellular uptake of exogenous dsRNA in target organisms remains poorly understood. Here, to fill this knowledge gap, we integrated omics tools with phenotypic assays to characterize dsRNA uptake mechanisms across tissues in the migratory locust, Locusta migratoria (Orthoptera). Our findings clearly demonstrate that cellular uptake of dsRNA is tissue-dependent, involving multiple cell membrane receptors and pathways. In hemocytes, uptake is rapid and mediated by clathrin-mediated endocytosis and macropinocytosis. Epidermal cells utilize clathrin- and caveolin-mediated endocytosis, while midgut cells…
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Figure 6- —the National Natural Science Foundation of China
- —Fundamental Research Program of Shanxi Province
- —C. W. Kearns, C. L. Metcalf, and W. P. Flint Endowed Chair Professorship, University of Illinois
- —the National Key R&D Program of China
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Taxonomy
TopicsInsect Resistance and Genetics · Studies on Chitinases and Chitosanases · RNA Interference and Gene Delivery
Background
RNA interference (RNAi) is an ancient gene silencing mechanism found in all eukaryotic organisms, including protozoa, invertebrates, vertebrates, fungi, algae, and plants [1–4]. Since its initial discovery in plants (Petunia hybrida) and nematodes (Caenorhabditis elegans), RNAi has become an effective tool for analysis of gene functions [1, 5]. Because of its selectivity and specificity, this RNA-based biotechnology has flourished in therapeutics, bioengineering, and agriculture as a targeted, personalized, and precise alternative drug [6–9]. In the USA, there are at least 21 U.S. Food and Drug Administration (FDA) approved RNAi-based therapies, including 6 siRNA drugs, and over 131 RNA-based therapies are currently being tested in clinical trials. However, the development and use of nucleic acid-based drugs can be challenging, given that double-stranded RNAs (dsRNAs) must be specifically, efficiently, and safely delivered to the desired tissues and cells of the target organism [10].
While dsRNA delivery is of paramount importance for nucleic acid drug efficacy, molecular mechanisms of dsRNA uptake in different tissues or organs are grossly understudied. For pest management, commercial applications of RNAi-based biopesticides include production of dsRNA in transgenic plants and sprayable formulations. In 2017, transgenic maize expressing an insecticidal dsRNA targeting DvSnf7 was commercialized under the trade name SmartStax® PRO, becoming the world’s first RNAi trait against the western corn rootworm, Diabrotica virgifera virgifera [11]. Six years later, Ledprona (trade name Calantha™), the first sprayable RNAi-based biopesticide, was deregulated by the United States Environmental Protection Agency (US-EPA) [11]. During continuous laboratory screening, however, several insect pests, including D. virgifera virgifera, Leptinotarsa decemlineata, and Plagiodera versicolora, have already developed resistance to RNAi-based biopesticides, with impaired cellular uptake of dsRNA contributing to this resistance [12].
dsRNA uptake process involves two steps in insects. First, when dsRNA is delivered to hemolymph, specific proteins bind with the dsRNA and interact with cell membrane receptors. Second, the naked dsRNA/dsRNA-protein complex is internalized by cells. In animal serum, apolipophorins (ApoLp) are carrier proteins that bind and transport lipids via the blood circulatory system. Various ApoLp receptors, such as scavenger receptor (SR), ApoLp receptor (LPR) and lipophorin receptor-related protein (LRP), have been reported in mammals [13]. Saleh et al. have shown that dsRNA entry requires cell surface receptors in Drosophila S2 cells, polyinosinic acid and fucoidan, the ligands of the SR family, inhibit both binding and uptake of dsRNA, suggesting that SR is involved in dsRNA uptake in S2 cells [14]. However, whether ApoLp can carry dsRNA and/or ApoLp receptors can mediate dsRNA uptake remains unknown. After cells take up dsRNA, two different dsRNA uptake pathways have been documented in invertebrates. One involves transmembrane systemic interference defective (SID) channels, which have been investigated in C. elegans. Sid-1 and Sid-2, dsRNA transport proteins, are required for the uptake and spreading of RNAi silencing signals [15–18]. Sun et al. reported that human SIDT1 mediates dsRNA uptake in HEK293T cells [19]. However, fungi and most insect species do not possess Sid orthologues [20, 21]. In some insect species, Sid-like genes are involved in dsRNA uptake, such as Leptinotarsa decemlineata, Diabrotica virgifera virgifera, and Nilaparvata lugens, while others do not participate [22–24].
Another major pathway for dsRNA uptake is receptor-dependent endocytosis, which is the cells’ basal mechanism for internalizing extracellular material and maintaining homeostasis [25, 26]. Clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis are the major endocytic pathways. Among them, clathrin-mediated endocytosis is considered a primary mechanism of dsRNA internalization [19, 21, 27, 28]. In addition, macropinocytosis has been implicated in cellular uptake of dsRNA in Anthonomus grandis [29]. In plant cells, due to having both a cell wall and plasma membrane, dsRNA delivery is blocked. Bennett et al. reported that transfection agents were required for dsRNA delivery to plant cells, which resulted in local but not systemic silencing of target genes in plant species, except in 16C Nicotiana benthamiana [30]. The lack of knowledge and research on the uptake mechanisms of dsRNA in different tissue types severely hampers its potential application in therapeutics, bioengineering, and agriculture.
Previously, we showed that the stability of dsRNA is different among tissue types in the migratory locust, Locusta migratoria. Specifically, incubation in locust midgut fluid resulted in rapid degradation of dsRNA, whereas dsRNA persisted when incubated with hemolymph [31]. Given that stability and degradation of extracellular dsRNAs are tissue-specific, we hypothesized that the corresponding mechanisms for dsRNA uptake are tissue dependent as well. To test our overarching hypothesis, we (1) examined the uptake efficiency of extracellular dsRNA; (2) characterized factors associated with dsRNA uptake, including membrane receptors, endocytic pathways, and Sid-like channels; and systematically (3) traced the dsRNA cellular uptake process across different tissue types.
Results
Uptake efficiency of extracellular dsRNA across different tissue types
To determine dsRNA uptake efficiency in different tissue types, including the integument, midgut, fat body, testis, ovary, and hemocytes, dsRNA was quantified by reverse transcription-quantitative PCR (RT-qPCR). Calibration for dsGFP quantification was based on serial dilutions of known amounts of dsGFP (Additional file 1: Fig. S1). The amount of dsGFP in different tissue types was determined in vivo after injection of dsGFP into the hemocoel. Results showed significant differences in the efficiency of dsRNA uptake among various tissues. The tissue type that contained the highest amount of dsGFP was the hemocytes, followed by the fat body, integument, ovary, testis, and midgut (Fig. 1A).Fig. 1. Tissue-specific dsGFP uptake; and apolipophorin-mediated dsRNA transport, protein upregulation, and modulation of RNAi efficiency in different tissues of L. migratoria.** A** Relative content of dsGFP across different tissue types, including integument (IN), midgut (MG), fat body (FB), testis (TE), ovary (OV), and hemocyte (HC). The relative content of dsGFP in midgut is set to 1.0. Error bars represent standard errors of three replicates, and different letters indicate statistically significant differences (p < 0.05; Tukey test). B Differentially expressed proteins (upregulated) analyzed by LC–MS. C The abundance of peptides in 17 upregulated proteins. D LmApoLp-III protein bound to the streptavidin-bio-dsGFP complex was separated by SDS-PAGE and detected by western blotting using LmApoLp-III polyclonal antibodies. E Relative content of dsGFP in the hemocytes, integument, and midgut after silencing of LmApoLp-II/I or LmApoLp-III. Error bars represent standard errors of three replicates, and asterisk(s) on the error bar indicate that dsGFP content in the treatment is significantly different from that in the control for the same tissue type (*, p < 0.05; ***, p < 0.001; Student’s t test). F The effect of RNAi against Apolipophorin (LmApoLp-II/I, LmApoLp-III) on RNAi efficiency against the marker gene (LmLgl) across different tissue types as determined by RT-qPCR. Results are presented as means ± SE of three biological replicates. Different letters indicate significant differences using ANOVA followed by Tukey’s test (p < 0.05)
Factors associated with dsRNA uptake
ApoLp
To further investigate whether there are proteins that can bind and transport dsRNA in hemolymph, we first isolated the hemolymph from L. migratoria and incubated the supernatant of the hemolymph with streptavidin-bio-dsGFP complex. We then performed mass analysis by using proteins extracted from the streptavidin-bio-dsGFP complex. Compared to the control, 17 proteins (fold changeompared tp < 0.05) were detected in the treatment group (Fig. 1B and Additional file 1: Table S1). Among a total of 424 peptides obtained from the 17 proteins, 358 peptides matched ApoLp-II/I (Fig. 1C), implying that ApoLp might play a predominant role in binding dsRNA. We then utilized transcriptome databases from our lab to search for ApoLp in L. migratoria and identified an LmApoLp-III. The open reading frames (ORFs) of the LmApoLp-II/I and LmApoLp-III genes consist of 10,140 and 543 nucleotides and encode proteins of 3380 and 180 amino acid residues, respectively. The deduced LmApoLp-II/I protein contains four dsRNA binding motifs whereas LmApoLp-III contains only one (Additional file 1: Fig. S2A). Both genes were highly expressed in the integument, fat body, testis, ovary, and hemolymph, but only slightly expressed in the foregut, midgut, and hindgut (Additional file 1: Fig. S2B). Because of its large molecular size, with an estimated molecular mass of approximately 371.8 kDa, it is difficult to express the ApoLp-II/I protein and perform its dsRNA binding assay in vitro. Thus, we chose LmApoLp-III to employ an in vitro binding assay to examine whether LmApoLp-III can efficiently bind to dsRNA. We found that the LmApoLp-III antibody detected a 37.3-KDa band after LmApoLp-III protein was incubated with the streptavidin-bio-dsGFP complex, but not with the control sample (Fig. 1D), indicating that LmApoLp-III is able to bind dsGFP.
To determine possible roles of LmApoLp-II/I and LmApoLp-III in the internalization of dsRNA in various tissue types from L. migratoria, we detected relative amounts of dsGFP after silencing LmApoLp-II/I or LmApoLp-III in the hemocytes, integument, and midgut. The approaches used are described in Additional file 1: Fig. S3. Results showed that dsRNA uptake efficiency was highest in hemocytes, followed by the integument, and lowest in the midgut (Fig. 1A). When LmApoLp-II/I was silenced in each of these three tissue types, we found that the relative content of dsGFP decreased significantly. When LmApoLp-III was silenced, the relative content of dsGFP in the hemocytes and integument decreased significantly, but the amount of dsGFP in the midgut increased compared to the control (Fig. 1E and Additional file 1: Fig. S4).
We then used RNAi of RNAi experiments to ascertain if ApoLp affects RNAi efficiency. LmLgl (lethal giant larvae gene) was chosen as a reporter gene due to its broad expression in different developmental stages and tissue types [32]. Results showed that silencing of LmApoLp-II/I significantly diminished RNAi efficiency of dsLmLgl in the hemocytes, integument, and midgut. In addition, silencing of LmApoLp-III significantly decreased RNAi efficiency of dsLmLgl in the hemocytes and integument, however, enhanced RNAi efficiency of dsLmLgl in the midgut (Fig. 1F). These results suggest that LmApoLp-II/I is involved in the transport of dsRNA in the hemocytes, integument, and midgut, whereas LmApoLp-III is involved in dsRNA uptake in the hemocytes and integument, but not in the midgut. However, the dsRNA uptake pathway in the midgut may be different from that in the hemocytes and integument.
Receptors participate in dsRNA uptake
Based on the involvement of Apo-Lp in dsRNA uptake in L. migratoria, we hypothesized that apolipoprotein receptors might be involved in the internalization of dsRNA. Twenty genes encoding putative receptors of LmApoLp, which include SR family (LmSRA, LmSRB1, LmSRB2, LmSRB3, LmSRB4, LmSRB5, LmSRB6, LmSRB7, LmSRB8, LmSRB9, LmSRB10, LmSRC), LPR family (LmLPR1, LmLPR2, LmLPR3), and LRP family (LmLRP1, LmLRP2, LmLRP3, LmLRP4, LmLRP6) were revealed by searching the L. migratoria transcriptomic databases from our lab. The domain architectures of their deduced proteins are summarized in Additional file 1: Fig. S5. To determine relative expression levels of all the identified genes in different tissues, including the integument, foregut, midgut, hindgut, fat body, testis, ovary, and hemolymph, mRNA was prepared from each of these tissues dissected from the 4-day-old third-instar (N3D4) nymphs of L. migratoria and subjected to RT-qPCR. The tissue-specific expression profiles revealed that the majority of the genes potentially involved in dsRNA uptake exhibited high expression in various tissues. Specifically, 7 genes including* LmSRB1*/SRB2/SRB4/SRB6/SRB9, LmLPR, and LmLRP6 were highly expressed in the ovary. In contrast, the remaining 15 genes showed the highest expression level in the remaining tissues: three (LmSRB10/SRC, LmLRP3) in the hemolymph, four (LmSRA/SRB7/SRB8, LmLRP4) in the fat body, three (LmSRB3, LmLRP1, LmLRP2) in the hindgut, and one (LmSRB5) in the integument (Additional file 1: Fig. S6-1, S6-2).
To investigate potential receptors required for dsRNA internalization in L. migratoria, the genes encoding different receptors were silenced in the hemocytes, integument, and midgut, respectively. When putative membrane receptors of LmApoLp-II/I or LmApoLp-III were silenced, we found that with the exceptions of LmSRB3, LmSRB4, and LmLRP1 in the hemocytes, the expression of the receptor genes could be suppressed after injection of their corresponding dsRNAs (Additional file 1: Fig. S4). These results indicate that LmLPR,* LmLRP1*,* LmLRP2*,* LmLRP6*,* LmSRA*, LmSRB2, LmSRB4, LmSRB6, LmSRB7, LmSRB9, and LmSRC are all involved in dsRNA uptake. Specifically, dsGFP uptake was blocked after silencing LmLPR,* LmLRP2*, LmSRA, LmSRB2, LmSRB9, and LmSRC separately in the hemocytes. Furthermore, dsGFP uptake was significantly reduced after knockdown of LmLRP1, LmLRP2,* LmLRP6*,* LmSRA*, LmSRB6, or LmSRB7 in the integument. Finally, the silencing of LmLRP6, LmSRB4, and LmSRB6 led to decreased dsGFP uptake in the midgut (Fig. 2A, B).Fig. 2. Effects of RNAi-mediated silencing of dsRNA uptake-related membrane receptors on the internalization of dsGFP across different tissue types. Data are the relative content of dsGFP (fold change ± SE) after silencing of LmLPR,* LmLRP* (A) or LmSR (B) in hemocytes, integument, and midgut. Error bars represent standard errors of three replicates, and asterisks denote significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001; Student’s t test)
Endocytic pathways
Three types of common endocytic pathways, including clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis are illustrated, and the key genes involved in each pathway are indicated (Fig. 3A). To examine putative endocytic pathways mediating dsRNA uptake in hemocytes, integument, and midgut, we used a pharmacological approach. Chlorpromazine (CPZ), genistein, and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were employed to block each possible endocytic pathway by preventing the assembly of clathrin-coated pits at the plasma membrane, broad-spectrum receptor tyrosine kinase activity, and Na^+^/H^+^ exchanger (necessary for the formation of membrane ruffles in macropinocytosis), respectively [28, 33, 34]. We tested various concentrations of the endocytosis inhibitors and determined the survival rate of 2-day-old third-instar (N3D2) nymphs. Only the effective concentrations of the inhibitors with the lowest toxic effects were chosen for the subsequent experiments (Additional file 1: Fig. S7). The results showed that CPZ strongly inhibited dsRNA uptake in hemocytes and integument; genistein treatment resulted in a significant decrease in dsGFP uptake in integument and midgut; and EIPA treatment caused a significant reduction in dsGFP uptake in hemocytes (Fig. 3B).Fig. 3. Endocytic pathways and their roles in cellular uptake of dsRNA evidenced from pharmacological inhibitions and targeted gene silencing.** A** A schematic drawing showing endocytosis-related genes and their projected involvement in dsRNA uptake. The top section of the figure depicts clathrin-mediated endocytosis, the middle section depicts caveolin-mediated endocytosis, and the bottom section depicts macropinocytosis. B Relative content of dsGFP (fold change ± SE) after administration of each of three inhibitors at three doses in the hemocytes, integument, and midgut. The top section of the figure depicts relative content of dsGFP (fold change ± SE) after administration of CPZ at three doses in three tissue types, the middle section depicts relative content of dsGFP (fold change ± SE) after administration of genistein at three doses in the three tissue types, and the bottom section depicts relative content of dsGFP (fold change ± SE) after administration of EIPA at three doses in the three tissue types. Error bars represent standard errors of three replicates, and asterisk(s) on the error bars indicate statistically significant differences in dsGFP content among three concentrations of the same inhibitor (p < 0.05; Tukey test). C Relative amounts of dsGFP (fold change ± SE) after silencing of endocytosis-related genes in the hemocytes, integument, and midgut. The top section of the figure depicts relative content of dsGFP (fold change ± SE) after silencing of clathrin-mediated endocytosis-related genes in the three tissue types, the middle section depicts relative content of dsGFP (fold change ± SE) after silencing of clathrin and caveolin-mediated endocytosis-related genes in the three tissue types, and the bottom section depicts relative content of dsGFP (fold change ± SE) after silencing of macropinocytosis-related genes in the three tissue types. Error bars represent standard errors of three replicates, and asterisk(s) on the error bars indicate that dsGFP content in the treatment is significantly different from that in the control (*, p < 0.05; **, p < 0.01; Student’s t test)
To validate the pathway for dsRNA uptake and evaluate critical players involved in dsRNA internalization, RNAi-mediated knockdown of specific genes was carried out. A total of 7 genes for components of the endocytic pathway were identified. These genes included those involved in clathrin-mediated endocytosis (LmChc, LmAP50, and LmArrestin2), those implicated in both clathrin-mediated and caveolin-mediated endocytosis (LmDYM, LmDYML1, and LmDYML2), and one functioning as a regulator of macropinocytosis (LmSid-3(cdc42)) [35]. The domain architectures of their deduced proteins are summarized in Additional file 1: Fig. S5. To determine relative expression levels of all the identified genes in different tissues of L. migratoria, the tissue-specific expression profiles revealed that four genes including LmChc, LmAP50, LmDYM, and LmDYML1 were highly expressed in the ovary, the other three genes (LmArrestin2, LmDYML2, LmSid-3(cdc42)) in the hemolymph (Additional file 1: Fig. S6-2). Furthermore, our experiments revealed that LmChc, LmAP50, LmArrestin2, LmDYM/LmDYML1/LmDYML2, and LmSid-3(cdc42) are involved in dsRNA uptake in all three tissue types (Fig. 3C). Specifically, RNAi-mediated knockdown of LmChc, LmAP50, LmArrestin2, LmDYM, LmDYML1, and LmSid-3(cdc42) reduced dsGFP uptake in the hemocytes; knockdown of LmChc, LmArrestin2, or LmDYM reduced dsGFP uptake in the integument; and silencing of LmDYML2 significantly reduced dsRNA uptake in the midgut (Fig. 3C). Taken together, our results indicate that both clathrin-mediated endocytosis and macropinocytosis contribute to dsRNA internalization in the hemocytes, and both clathrin-mediated and caveolin-mediated endocytosis participate in dsRNA uptake in the integument. Caveolin-mediated endocytosis is involved in dsRNA uptake in midgut.
Sid-like dsRNA transporter
To confirm the function of LmSid-like, we first focused on the effects of silencing LmSid-like on the efficiency of dsGFP uptake in the hemocytes, integument, and midgut, and found that the expression of the LmSid-like gene could be suppressed after injection of dsLmSid-like (Additional file 1: Fig. S4). Silencing of LmSid-like led to decreased dsGFP uptake in the midgut (Fig. 4A). We further analyzed the N-terminal extracellular domain (ECD) and 11 C-terminal transmembrane domains of LmSid-like (Fig. 4B), followed by an in vitro dsRNA binding assay*.* After the specificity of LmSid-like ECD polyclonal antibodies was validated (Fig. 4C), immunohistochemistry was performed. Our results showed that LmSid-like was localized at the plasma membrane of the midgut cells (Fig. 4D), and LmSid-like ECD can bind dsRNA according to our dsRNA binding assay (Fig. 4E). These results suggest that Sid-like channels are involved in dsRNA uptake in the midgut.Fig. 4. Functional analysis and localization of LmSid-like in dsRNA internalization across L. migratoria tissues. A** Effects of silencing LmSid-like on the internalization of dsGFP in the hemocytes, integument, and midgut. Error bars represent standard errors of three replicates, and asterisk(s) on the error bars indicate that dsGFP content in the treatment is significantly different from that in the control (, p < 0.05; Student’s t test). B Domain structure of LmSid-like. C Validation of LmSid-like polyclonal antibodies and localization of LmSid-like protein by immunohistochemistry using the antibodies in L. migratoria. Individual and merged immunohistochemical images of the midgut showing the locations of cell membranes (red, stained with CM-dil) and LmSid-like (green, stained with antibodies). Suppression of LmSid-like gene resulted in the loss of the immunohistochemical signal for LmSid-like protein, indicating the specificity of the antibodies against its protein (LmSid-like). Scale bar: 20 μm. D Localization of LmSid-like in the midgut as revealed by immunohistochemistry; the green signal represents LmSid-like. Scale bar: 20 μm. E LmSid-like extracellular domain protein bound to the streptavidin-bio-dsGFP* complex was separated by SDS-PAGE and detected by western blotting using LmSid-like polyclonal antibodies
Tracing the sequential process of dsRNA cellular uptake in the hemocytes
To trace dsRNA uptake in hemocytes, fluorescein-labeled dsGFP (dsGFP-F) was injected into the hemocoel of N3D2 nymphs after validation of LmSRC and LmAP50 polyclonal antibody specificity (Additional file 1: Fig. S8). Based on previously reported morphology of hemocytes in L. migratoria [36], we found that both LmSRC and LmChc were localized to plasmocytes and granulocytes, while LmAP50 was detectable in plasmocytes, granulocytes, and oenocytes (Additional file 1: Fig. S9). In this study, plasmocytes were chosen to visualize the process of dsRNA entry. As soon as 10 s after the injection, a large amount of dsGFP-F was enriched at the cell surface. After 30 s, the dsGFP-F signal moved to the cytoplasm, and after 60 s, dsGFP-F appeared to aggregate inside the hemocytes (Fig. 5A). Further, we examined the roles of membrane receptor LmSRC, endocytosis-related protein LmChc, and adaptor protein LmAP50. After the knockdown of LmSRC, LmChc, and LmAP50 separately, only a very faint fluorescence signal was observed, suggesting that very little dsGFP-F was taken up by the hemocytes (Fig. 5A). Quantitative analysis of the integrated density of dsGFP-F showed no significant difference in the amount of dsGFP-F at the cell surface at 10 s after silencing of LmChc or LmAP50 as compared with the control; however, the quantity of dsGFP-F detected significantly decreased after knockdown of LmSRC. After 30 and 60 s, a large amount of dsGFP-F enriched in the cell, but a few dsGFP-F was detected in the cell after the knockdown of LmSRC, LmChc, or LmAP50 separately (Fig. 5B). Furthermore, we observed that dsGFP-F co-localized with the immunosignals of LmSRC and found that LmChc and LmAP50 appeared at the cell membrane at 10 s and in the cytoplasm at 30 s post injection (Fig. 5C and Additional file 1: Fig. S10). The co-localization of LmAP50 and LmSRC/LmChc suggests that LmAP50 might interact with LmSRC and LmChc in hemocytes (Fig. 5D).Fig. 5. Roles of LmSRC, LmChc, and LmAP50 in dsRNA uptake and RNAi efficiency in L. migratoria hemocytes.** A** Effect of silencing LmSRC, LmChc, or LmAP50 on dsGFP-Fluorescein uptake in the hemocytes of L. migratoria. The localization of dsGFP in hemocytes was analyzed by immunocytochemistry. dsGFP was labeled with Fluorescein (green) and injected into the hemocoel of L. migratoria. Hemocytes were collected at four different time points (0, 10, 30, and 60 s) after injection of dsGFP. The cell membrane was stained with CM-dil (red); nuclei were stained with DAPI (blue). Scale bar: 2 μm. B Quantitative analyses of the integrated density of dsGFP after silencing LmSRC, LmChc, or LmAP50 as compared with the control, as measured using ImageJ software. C Co-localization of LmSRC, LmChc, and LmAP50 with dsGFP-Fluorescein in hemocytes of L. migratoria was analyzed by immunocytochemistry. dsGFP was labeled with Fluorescence (green) and injected into hemocoel of L. migratoria. Hemocytes were collected at 10 s after injection of dsGFP. The red signals represent LmSRC, LmChc, or LmAP50. Nuclei were stained with DAPI (blue). Scale bar: 2 μm. D Co-localization of LmSRC or LmChc with LmAP50 in the hemocytes. The green signal represents LmAP50, and the red represents either LmSRC or LmChc. Scale bar: 20 μm. E Effects of silencing LmSRC, LmChc, or LmAP50 on RNAi efficiency against the marker gene LmLgl. Error bars represent standard errors of three replicates, and different letters on the error bars indicate statistically significant differences in RNAi efficiency against the marker gene (p < 0.05; Tukey test)
We then performed small RNA sequencing to determinate the fate of dsRNA after silencing the dsRNA-uptake-related genes. Knockdown of LmSRC, LmChc, or LmAP50 prior to dsLmLgl injection sharply reduced the amount of dsLmlgl-derived siRNAs in hemocytes compared with the control group. In addition, silencing LmSRC, LmChc, or LmAP50 sharply reduced this ratio from 2.3% in the control group to 0.1–0.3% in the RNAi-treated groups (Additional file 1: Fig. S11 and Table S2). Finally, an RNAi of RNAi experiment was performed to determine if silencing of LmSRC, LmChc, and LmAP50 can affect RNAi efficiency using a marker gene (LmLgl). We found that suppression of LmSRC, LmChc, or LmAP50 prior to dsLmLgl injection reduced the silencing efficiency of LmLgl by 16.5%, 35.3%, or 35.3%, respectively, in hemocytes (Fig. 5E). Taking it together, our results suggest that clathrin-mediated endocytosis and macropinocytosis jointly contribute to dsRNA internalization in the hemocytes of L. migratoria.
dsRNA uptake pathways among different insect species
To investigate dsRNA uptake pathways in evolutionarily distant insect species, we also examined T. castaneum (coleopteran) and Ostrinia furnacalis (lepidopteran). We injected a defined amount of dsGFP into the hemocoel of T. castaneum and O. furnacalis and determined the amounts of dsGFP present in different tissue types. Because the 16-day-old larvae of T. castaneum were too small, it is difficult to obtain enough hemolymph for extracting RNA and detecting the content of dsGFP. Therefore, only the integument and midgut were chosen to detect the content of dsGFP. The dsGFP amounts differed in the following orders: integument > midgut in T. castaneum and hemolymph > integument > midgut in O. furnacalis (Additional file 1: Fig. S12). These results were consistent with those observed in L. migratoria. To identify the endocytic pathways involved in dsRNA uptake in T. castaneum, Ly294002, which prevents PI3K activity (necessary for the completion of actin-dependent macropinocytosis) [37], was used in place of EIPA. The pharmacological assays showed that CPZ and genistein strongly inhibit dsRNA uptake in both the integument and midgut of T. castaneum, as assessed by the amounts of dsGFP taken up. In contrast, there was no significant reduction in the amounts of dsGFP in the integument and midgut when T. castaneum larvae were treated with Ly294002 (Additional file 1: Fig. S12A), suggesting that clathrin-mediated and caveolin-mediated endocytosis are involved in dsRNA uptake in both the integument and midgut of T. castaneum, but not macropinocytosis.
To determine putative endocytic pathways for dsRNA uptake in O. furnacalis, we used the endocytosis inhibitors described above in L. migratoria. The pharmacological assay using CPZ demonstrated that clathrin-mediated endocytosis is involved in dsRNA uptake in the hemocyte of O. furnacalis, whereas caveolin-mediated endocytosis was implicated in dsRNA uptake in the midgut (Additional file 1: Fig. S12B). However, the mechanism of dsRNA uptake in the integument of O. furnacalis is still unknown and requires further investigation. Taken together, our results indicate that clathrin-mediated endocytosis is an important pathway for dsRNA internalization and such a mechanism is conserved in L. migratoria, T. castaneum, and O. furnacalis (Additional file 1: Table S3). Furthermore, our results point out the occurrence of diverse pathways in dsRNA internalization in various tissue types in insects.
Discussion
Since its discovery, RNAi has been applied across numerous fields, including gene therapy and pest management [6–9]. Achieving an effective RNAi response requires successful delivery of dsRNA into a sufficient number of responsive cells within the target organism. However, the mechanisms governing cellular uptake of dsRNA remain poorly understood, limiting our ability to enhance RNAi efficiency across diverse species.
Efficiency of cellular uptake of dsRNA varies among tissue types
Previous studies on dsRNA cellular uptake have mainly focused on whole organisms or exclusively on cells, without comparing different tissue types. In mammals, dsRNA uptake mechanisms are typically examined using cell lines derived from target tissues [19]. In insects, research has largely focused on whole organisms or on the midgut, with little attention to other specific tissues involved in RNAi [22, 28]. In plants, Bennett et al. reported that when dsRNA was applied directly to the vasculature of 16C N. benthamiana and other species, it entered the apoplast and xylem but not the phloem [30]. In fungi, Wytinck et al. found that dsRNA was abundantly internalized in thinner, younger hyphae compared with thicker, more mature, and vacuolated hyphae [27]. Despite these efforts, dsRNA uptake mechanisms have never been comparatively investigated across different tissue types in any organism.
Our work extends this understanding by examining dsRNA uptake across insect tissues. Notably, dsRNA uptake efficiency varies markedly among tissue types in L. migratoria. Hemocytes and fat body cells take up dsRNA at very high levels (> 1000 ×), consistent with their central roles in immune defense against pathogens such as viruses [38]. In both L. migratoria and O. furnacalis, the efficiency of dsRNA uptake is highest in hemocytes, followed by the integument and midgut. These findings suggest the presence of conserved dsRNA uptake pathways across tissue types and help explain why dsRNA uptake efficiency is higher in immune-relevant tissues than in other tissues.
Although dsRNA can be administered directly by injection in L. migratoria and remain effective without a carrier, our previous work showed that dsRNA is unstable in the gut lumen. Rapid degradation by LmdsRNase2 in the midgut contributes significantly to the low RNAi efficiency observed with oral delivery [31]. Ma et al. demonstrated that star polycation (SPc) enhances dsRNA delivery in Spodoptera frugiperda by activating clathrin-mediated endocytosis [39]. In our team, we found that chitosan, used as a biological nanocarrier, markedly improves dsRNA stability in midgut fluid, enhances delivery and RNAi efficiency, and increases locust mortality [40]. Thus, future research could focus on developing small-molecule RNase inhibitors or nano-encapsulated dsRNA pesticides designed for oral delivery, which may provide practical and effective strategies for agricultural pest control.
Multiple factors associated with dsRNA uptake
ApoLp
This is the first time that we have identified ApoLp-II/I by LC–MS analyses and confirmed that ApoIII can directly bind dsRNA by employing an in vitro binding assay. Furthermore, we found that ApoLpII/I can transport dsRNA in the hemolymph and induce cellular uptake of dsRNA in three tissue types, while LmApoLp-III assists dsRNA into cuticle epidermal cells and hemocytes, but not into midgut. Both genes were highly expressed in the fat body. In our previous work, we found that LmApoLp-III functions as a key carrier that facilitates the initial transport of dsRNA from the hemolymph to the fat body cell membrane. In contrast, silencing LmApoLp-II/I did not affect dsRNA uptake in the fat body [41]. This study fills an important knowledge gap regarding the roles of ApoLp genes in insects, as ApoI and ApoIII have previously been identified only in the silk moth and the desert locust [42, 43]. In mammals, ApoB100 and other lipid-binding proteins have been reported to participate in nucleic acid binding and innate immunity [44], suggesting that ApoLp genes may play analogous roles across insects and mammals.
Receptors participate in dsRNA uptake
Three ApoLp receptor families, including SR, LPR, and LRP, have been reported in mammals [13, 45], whereas only LPR in L. migratoria and SRC (an SR-family member) in Drosophila have been characterized at the cellular level for their roles in endocytosis [46, 47]. By screening transcriptome databases from our laboratory, we identified 20 putative ApoLp receptor homologs in L. migratoria, including 12 in the SR family, 3 in the LPR family, and 5 in the LRP family. Our findings demonstrate for the first time that all three types of receptors are involved in cellular uptake of dsRNA. Notably, different types of receptors mediate dsRNA entry in distinct tissue types. Specifically, SRA, SRB2, SRB9, SRC, LPR, and LRP2 function in hemocytes; SRA, SRB6, SRB7, LRP1, LRP2, and LRP6 function in the integument; and Sid-like, SRB4, SRB6, and LRP6 contribute to dsRNA uptake in the midgut. These results provide new insights into the diversity of membrane receptors and their roles in dsRNA uptake across tissues.
Endocytic pathways
In the current study, we carried out a comprehensive analysis on the internalization of dsRNA through diverse pathways in different tissue types of L. migratoria. RNAi experiments coupled with the application of pharmacological inhibitors showed that dsRNA enters the cells of different tissue types through diverse pathways. Notably, different protein components involved in endocytic pathways include Chc, AP50, Arrestin2, DYM, DYML1, DYML2, and Sid-3(cdc42). Based on our findings, we propose a model for the mechanisms of cellular uptake of dsRNA in L. migratoria (Fig. 6). We found that clathrin-mediated endocytosis and caveolin-mediated endocytosis participated in dsRNA uptake in the integument, caveolin-mediated endocytosis and Sid-like channels contribute to dsRNA uptake in the midgut, and clathrin-mediated endocytosis and macropinocytosis jointly mediate dsRNA uptake in hemocytes. Furthermore, to determine whether the mechanisms of dsRNA uptake are conservative or not in diverse organisms, we also examined the distantly related species T. castaneum and O. furnacalis. Results testified that clathrin-mediated endocytosis is a conserved pathway for dsRNA uptake in these species, as has been demonstrated previously in other organisms, including humans, fungi, and other insects [19, 21, 27, 28].Fig. 6. Diversity of cellular mechanisms underlying dsRNA uptake across hemocytes, epidermis, and midgut of L. migratoria. The cellular uptake mechanisms are highly diverse in different tissues as summarized below. ApoLp-II/I, ApoLp-III, and other unknown proteins bind dsRNA, and the resulting complex is internalized into the cells of different tissues through receptor-mediated endocytosis. (1) In hemocytes, SRA, SRB2, SRB9, SRC, LPR, and LRP2 were involved in dsRNA uptake, and both clathrin-mediated endocytosis and macropinocytosis function cooperatively in dsRNA uptake. The internalization of dsRNA through clathrin-mediated endocytosis in hemocytes is outlined as follows: (a) ApoLp or an unknown protein-dsRNA complex binds to receptor proteins (e.g., SRC) on the plasma membrane of a hemocyte; (b) ApoLp or an unknown protein-dsRNA complex bound to the receptor protein complexes interact with adaptor protein 50 (AP50/Arrestin2) and clathrin to form a clathrin-coated pit on the inner surface of the plasma membrane; (c) the pit separates from the plasma membrane via Dynamin protein and reaches the cytoplasm to form a clathrin-coated vesicle; (d) the vesicle is uncoated and fuses with an early endosome. Macropinocytosis is also implicated in the uptake of dsRNA in hemocytes, which involves the formation of macropinosomes that fuse with the early endosome. (2) In epidermal cells, SRA, SRB6, SRB7, LRP1, LRP2, and LRP6 were involved in dsRNA uptake, and both clathrin-mediated endocytosis and caveolin-mediated endocytosis participate in dsRNA uptake. (3) In midgut cells, Sid-like, SRB4, SRB6, and LRP6 were involved in dsRNA uptake. Sid-like mediated transport and caveolin-mediated endocytosis contribute to dsRNA uptake
Sid-like dsRNA transporter
Previous studies have reported that Sid-1 homolog or Sid-like proteins mediate dsRNA uptake in different organisms, including humans, mice, L. decemlineata, D. v. virgifera, N. lugens, and Apis mellifera [19, 22–24, 48]. However, several studies have reported that Sid-like proteins do not function in cellular uptake of dsRNA in some insects, including T. castaneum, L. migratoria, and S. gregaria [49–51]. In this study, we found that Sid-like channels contributed to dsRNA uptake in the midgut of L. migratoria by using a quantitative analysis method, binding assay, and immunostaining. Hence, Sid-like plays a role in cellular uptake of dsRNA in the midgut of L. migratoria.
Tracing the sequential process of dsRNA cellular uptake across different tissue types
Currently, the rate of dsRNA internalization in the cell is still unclear. In this study, we found that a substantial amount of dsGFP-F accumulated on the cell surface within 10 s and subsequently entered the cytoplasm within 30 s, suggesting that the hemolymph, as immune tissue, responds very quickly to dsRNA. To our knowledge, this is the first report demonstrating that dsRNA uptake occurs rapidly after delivery into the hemolymph. We also observed aggregation of dsGFP-F with a strong fluorescence signal at 60 s. It has similarly been reported that clathrin-mediated endocytosis of virus is generally a rapid process, the surface-bound viruses enter cell within minutes after attachment to the membrane followed by transport to early endosomes within 1–2 min [52]. Thus, the observed aggregation of dsGFP-F in early endosomes may imply that dsRNA could be trapped in early endosomes within 60 s after injection. This finding highlights potential strategies for enhancing RNAi efficiency by promoting endosomal escape, allowing dsRNA to rapidly engage in the RNAi pathway. Small RNA sequencing results indicate that the observed decrease in RNAi efficiency is primarily due to impaired dsRNA uptake rather than enhanced processing or degradation. This method could provide a more specific measure of dsRNA uptake and processing, offering further validation of our findings. However, conventional sRNA-seq may capture heterogeneous small RNAs, including non-Dicer-derived and non-specific dsRNA degradation products, which are expected in the insects examined in this study. In contrast, RISC-bound sRNA-seq enables the identification of mature siRNAs processed from dsRNA, thereby excluding non-specific small RNAs and capturing bona fide Dicer-generated siRNA sequences [53, 54]. In future work, we plan to apply RISC-bound sRNA-seq to quantify mature siRNA abundance and to characterize the corresponding cleavage events.
Conclusions
In this study, we provide a comprehensive, tissue-resolved analysis of dsRNA uptake mechanisms in insects, addressing a critical knowledge gap that has constrained the broader application of RNA interference. By systematically comparing multiple tissues in L. migratoria and validating key findings in T. castaneum and O. furnacalis, we demonstrate that dsRNA uptake efficiency and mechanism are highly tissue dependent, with immune-related tissues such as hemocytes and fat body exhibiting significantly enhanced uptake relative to other tissues.
Our results reveal that dsRNA uptake is a multistep process involving coordinated actions of lipid-associated carriers (ApoLp proteins), diverse membrane receptors, and multiple endocytic pathways (Fig. 6). We identify distinct, tissue-specific combinations of ApoLp receptors and internalization routes, including clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis, and Sid-like channels. Importantly, we show that Sid-like proteins contribute to dsRNA uptake in the midgut of L. migratoria, resolving prior inconsistencies in the literature and underscoring the context-dependent roles of dsRNA transporters.
By tracing dsRNA internalization in real time, we further demonstrate that dsRNA uptake occurs extremely rapidly following delivery into the hemolymph, with surface binding, internalization, and early endosomal accumulation occurring within seconds to minutes. These findings highlight endosomal escape as a key bottleneck for RNAi efficiency and suggest promising avenues for intervention. Consistent with this, our small RNA analyses indicate that reduced RNAi efficiency primarily reflects impaired dsRNA uptake rather than enhanced degradation or altered processing.
Collectively, this work establishes a unified framework linking molecular dsRNA uptake mechanisms to functional RNAi outcomes across tissues and species. Beyond advancing fundamental understanding of insect RNAi biology, our findings provide a mechanistic foundation for the rational design of next-generation RNAi-based pest control strategies, including tissue-targeted delivery systems, carrier-assisted formulations, and approaches that enhance endosomal escape. Although the precise molecular interactions among ApoLp proteins, their receptors, and endocytic machinery remain to be fully elucidated, our results highlight clear priorities for future investigation. In particular, the rapid degradation of dsRNA in the locust gut lumen underscores the need for improved protective and delivery strategies. Future integration of RISC-bound small RNA sequencing and nanocarrier technologies will further refine these approaches, enhancing RNAi efficiency in both dietary and intravenous systems and accelerating the translation of RNAi into robust, sustainable agricultural and biomedical applications.
Methods
Uptake efficiency of extracellular dsRNA across different tissue types
Colony maintenance
Eggs of L. migratoria were acquired from Cangzhou, Hebei Province, China, and maintained at 32 ± 2 ℃, 40–60% humidity under a photoperiod of 14 h light and 10 h dark. After hatching, nymphs were fed daily with fresh wheat seedlings. The O. furnacalis eggs were acquired from Keyun, Henan Province, China, and incubated at 28 °C, 70% relative humidity, and a photoperiod of 16 h light and 8 h dark. The larvae were fed on an artificial diet. The Georgia-1 (GA-1) strain of T. castaneum was reared on whole-wheat flour containing 5% (w/w) brewers’ yeast in a growth chamber under standard laboratory conditions of 30 °C, 65% RH and 14:10 h (L:D).
Quantification of dsRNA in different tissue types
To quantify dsRNA across different tissue types in L. migratoria, 20 μg of dsGFP was injected into the abdominal segment of N3D2 nymphs. After 1 h, different tissue types, including integument, midgut, fat body, testis, ovary, and hemocytes, were dissected from the nymphs and washed three times with PBS to remove any excess dsGFP from the surface except for hemocytes, which were collected by centrifugation at 4 °C. Finally, total RNA was extracted, and RT-qPCR analysis was carried out according to previously published protocols [53]. The quantification of dsGFP was carried out using RT-qPCR based on a calibration curve of known amounts of dsGFP. First, dsGFP was synthesized using the T7 RiboMAX Express RNAi System (Promega, Madison, WI, USA), then, dsGFP concentrations were determined and adjusted to 2 μg/μL and 2 μg dsGFP, and the samples were denatured at 75 °C for 10 min prior to cDNA preparation by reverse transcription using a PrimeScriptTM 1 st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) with specific primers for dsGFP; finally, the cDNA of dsGFP was diluted in a gradient to establish a calibration curve. The standard curve of dsGFP is shown in Additional file 1: Fig. S1. The primers of dsGFP are shown in Additional file 1: Table S5. Each experiment was replicated three times, each with two technical replicates.
In addition, 1 μg of dsGFP was injected into the abdominal segment of L5D2 larvae of O. furnacalis to determine the amount of dsGFP in three tissue types (hemolymph, integument, and midgut). Furthermore, 400 ng of dsGFP was injected into the abdominal segment of 16-day-old T. castaneum larvae to determine the amount of dsGFP in the integument and midgut. dsRNA quantification was performed as described above.
To determine possible roles of dsRNA uptake-related genes in the internalization of dsRNA in various tissue types in L. migratoria, N3D2 nymphs were injected with either 5 μg of dspEASY-Blunt Zero (vector control) or 5 μg of dsRNA. After 24 h, each nymph was re-injected with either 20 μg of dsGFP to assess its content in the integument and midgut, or 5 μg of dsGFP to evaluate its content in the hemocytes. Quantification of dsRNA was conducted as previously described [55].
Factors associated with dsRNA uptake
LC–MS analysis
Hemolymph was collected from N3D4 nymphs of L. migratoria and centrifuged to obtain the supernatant. The supernatant was immediately mixed with Buffer I containing 1 mM EDTA to inhibit melanization (Buffer I: 10 mM Tris–HCl, pH 7.5; 1 mM EDTA; 2 M NaCl; adjusted to a final volume of 250 µL with DEPC-treated water). Biotinylated dsRNA targeting GFP (bio-dsGFP) was synthesized using the HiScribe™ T7 RNA High Yield RNA Synthesis Kit (New England BioLabs, Ipswich, MA, USA) and biotin-11-uridine-5′-triphosphate (Bio-11-UTP; Ambion, Austin, TX, USA) according to the manufacturer’s instructions. A total of 7.4 µg bio-dsGFP was incubated with 250 µL streptavidin magnetic beads (2 µg/µL; Thermo Fisher Scientific) at 4 °C for 2 h to generate the streptavidin–bio-dsGFP complex, as previously described [56]. The beads were washed three times with Buffer I using magnetic separation to remove unbound bio-dsGFP, followed by overnight incubation at 4 °C with 1 mg hemolymph supernatant. As a control, equal amounts of supernatant were incubated with streptavidin magnetic beads alone under identical conditions to control for non-specific binding. Each experiment was performed with two biological replicates. Proteins bound to the streptavidin–bio-dsGFP complex were subjected to LC–MS analysis. Each sample was separated by capillary high-performance liquid chromatography (HPLC) and then subjected to mass spectrometry analysis using a Q Exactive mass spectrometer (Thermo Scientific). Parameter settings are as follows: run time: 90 min; polarity: positive; scan range: 300–1400 m/z; microscan: 1; resolution: 70,000; AGC target: 3e6; Maximum IT: 60 ms; and dynamic exclusion: 15.0 s. The heatmap was generated by Pheatmap software (R version 3.6.3). Peptide and protein identification, as well as statistical analyses of differentially expressed proteins, were carried out by FanXing BoAo Biolab (China).
Gene acquisition and bioinformatics analysis
Complementary DNA (cDNA) sequences for apolipophorin, various receptors, and endocytosis-related genes were searched for in the locust transcriptomic databases from our lab. The identity of each candidate gene was validated by BLAST searching GenBank (https://blast.ncbi.nlm.nih.gov). ExPASy-translational tool (https://web.expasy.org/translate/) was used to obtain protein sequences of each candidate gene. Domain architecture of proteins was predicted using SMART (http://smart.embl.de/). Guevara et al. reported that the motifs ΦΦΦ -K/R(Φ, hydrophobic residue), R/K-X-R/K-X-R/K, R/K-XX-R/K, and K/R-XXX-K/R serve as the nucleic acid-binding regions [44]. Based on this study, these motifs were searched for within the amino acid sequence of ApoLp. The GenBank accession numbers of all candidate genes are provided in Additional file 1: Table S4.
Analysis of tissue-specific gene expression
Eight different tissue types, including integument, foregut, midgut, hindgut, fat body, testis, ovary, and hemolymph, were dissected from N3D4 nymphs of L. migratoria. All tissue samples were collected in three biological replicates, each from four nymphs. Total RNAs were isolated, cDNAs were synthesized, and RT-qPCR reactions were performed as previously described [31]. The primers of each candidate gene are shown in Additional file 1: Table S5.
Binding assay of LmApoLp-III/LmSid-like protein and dsRNA
A pair of primers containing HindIII or BamHI restriction sites was designed to amplify the open reading frame (ORF) encoding the full-length amino acid sequence of LmApoLp-III and to clone it into the pET-32a vector linearized with the same restriction enzymes (Table S5). The PCR amplification product and the pET-32a vector were both digested with HindIII and BamHI, respectively, and subsequently ligated using T4 DNA ligase to generate the recombinant plasmid pET-32a-LmApoLp-III, and subsequently into Escherichia coli BL21 (DE3) competent cells. Recombinant LmApoLp-III protein was expressed and purified from bacterial extracts as previously described [57]. Briefly, the protein was purified using Nirified from bacteria. The His-tag fused to recombinant LmApoLp-III was retained for all subsequent analyses.
Based on previous reports suggesting that the extracellular domain of LmSID-like may bind dsRNA [19], we analyzed the domain structure of LmSID-like and identified an N-terminal extracellular domain (ECD; amino acids 1–197). Primers containing NotI or BamHI restriction sites were designed to amplify the ORF corresponding to LmSID-like-ECD (aa 1–197) and to subclone it into the pET-32a vector digested with the same enzymes (Table S5). The PCR product and vector were digested with NotI and BamHI, respectively, ligated using T4 DNA ligase, and transformed into E. coli BL21 (DE3) cells to generate pET-32a-LmSid-like-EDC (Table S5). The PCR produced as described above. The empty pET-32a vector was used as a negative control. An equivalent amount of protein expressed from the empty pET-32a vector was incubated with the streptavidin-bio-dsGFP complex to control for non-specific interactions.
Polyclonal antibodies to LmApoLp-III peptide (RPDAAGQVNIAEAVQ) and LmSid-like peptide (AEQWPENKNLSTIIL) were produced by ChinaPeptides Co., Ltd (Shanghai, China) in rabbit and mouse, respectively. bio-dsGFP was synthesized as described above. Then, 2 µg of bio-dsGFP was incubated with 50 µL of streptavidin beads (Thermo Fisher Scientific) to obtain streptavidin-bio-dsGFP complex as previously described [56]. For the treatment group, either 300 µg of LmApoLp-III or 100 µg of LmSid-like ECD supernatant protein was incubated overnight at 4 °C with a streptavidin-bio-dsGFP complex. Then, the western blotting experiment was performed with anti-ApoLp-III/Sid-like polyclonal antibodies. The empty pET32a vector was used as a negative control. The same amount of protein that produced by empty pET32a vector was incubated with streptavidin bio-dsGFP complex as a control, which was directly used for western blotting.
dsRNA and RNAi synthesis experiments
To assess possible roles of genes involved in dsRNA uptake in L. migratoria, RNAi experiments were performed. Briefly, primers for dsRNA synthesis were designed using the E-RNAi webservice (http://www.dkfz.de/signaling/e-rnai3/). Primers are provided in Additional file 1: Table S5. The dsRNA was synthesized the same way using the T7 RiboMAX Express RNAi System (Promega, Madison, WI, USA) as previously described [50]. Finally, dsRNA concentrations were determined and adjusted to 2 μg/μL.
To optimize RNAi efficiency, a specific dose (5 μg) of dsRNA was injected into the abdomen of each N3D1 nymph using a microsyringe (Ningbo, China). The same amount of dsGFP was injected as a control. Each group consisted of 3 nymphs as a biological replicate, and each control or treatment was performed in five biological replicates. The silencing efficiency in nymphs was evaluated 1 day after injection of dsRNA by using RT-qPCR. The expression of apolipophorin, different receptors, and endocytosis-related genes was normalized to the expression of the reference gene LmEF1a.
RNAi of RNAi experiments
For testing the effect of five genes (LmApoLp-II/I, LmApoLp-III, LmSRC, LmChc, and LmAP50) on RNAi of LmLgl in the hemolymph, integument, and midgut, each of the N3D2 nymphs was injected with 5 μg of dsGFP in the negative control and 5 μg of each of five dsRNAs in the treatment. After 24 h, each nymph was re-injected with 400 ng of dsGFP in the negative control and 400 ng of LmLgl in the treatments. After 24 h, the hemolymph, integument, or midgut was collected from five nymphs to determine relative LmLgl transcript levels by RT-qPCR. Each control and treatment were performed in three biological replicates, each with two technical replicates.
Pharmacological inhibitors and viability assay in immature insects
The inhibitors CPZ and genistein were purchased from Sigma-Aldrich (St. Louis, MO, USA), whereas Ly294002 and EIPA were purchased from Selleck (Shanghai, China). CPZ was dissolved in water, whereas the other inhibitors were resuspended in 20% (w/v) dimethylsulfoxide (DMSO). Viability assays of nymphs after treatment with inhibitors were carried out as follows: Different doses of inhibitors were injected into N3D2 nymphs of L. migratoria, 2-day-old fifth-instar larvae (L5D2) of O. furnacalis, and 16-day-old larvae of T. castaneum. Twenty-four hours after this treatment, nymphal/larval survivorship was recorded.
Immunostaining
To detect the subcellular localization of LmSid-like in the midgut, an immunocytochemistry analysis was performed. Paraffin slides (3-μm thickness) from the midgut of N3D2 nymphs of L. migratoria were prepared, which were deparaffinized, dehydrated, and blocked with 10% (w/v) goat serum albumin for 1 h at 37 °C. Subsequently, the slides were incubated with anti-LmSid-like (1:100) at 4 °C. After overnight incubation, the slides were treated with Alexa Fluor 488 goat anti-mouse IgG (1:200; Invitrogen, Carlsbad, CA, USA) for 1 h in the dark at 37 °C. The cell nuclei were stained with 4–6-diamidino-2-phenylindole (DAPI) (1 μg/mL) (Thermo Fisher Scientific) for 5 min and image acquisition was carried out under an LSM 880 confocal fluorescence microscope (Zeiss, Jena, Germany).
Polyclonal antibodies against the LmSRC peptide (RDRKRTRPYRCPPLS) were generated in mouse by ChinaPeptides Co., Ltd. (Shanghai, China). To generate antibodies against LmChc and LmAP50, truncated recombinant proteins (LmChc^rec and LmAP50^rec) were expressed and purified from bacterial extracts as previously described [57]. Primer sequences are provided in Additional file 1: Table S5. LmChc^rec was used as the antigen for mouse immunization, and LmAP50^rec was used for rabbit immunization; antibody production was conducted by Zhongding Biotechnology Co., Ltd. (Nanjing, China). The specificity of the LmChc polyclonal antibody has been validated previously [57]. Specificity of the LmAP50 and LmSRC polyclonal antibodies was confirmed by immunohistochemistry in hemocytes using negative serum as a control.
To detect the subcellular localization of LmSRC, LmChc, and LmAP50 in hemocytes, the hemolymph was extracted from N3D2 nymphs with a micropipette, then spread onto slides, fixed with 4% paraformaldehyde (diluted in PBS), and permeabilized with 0.2% (v/v) Triton X-100. The immunohistochemical staining was performed as described above. The slides were incubated with anti-LmSRC, anti-LmChc, or anti-LmAP50 antibodies (1:100, diluted in blocking reagent), then the secondary antibody, either Alexa Fluor 488 mouse IgG (1:200, green) for detecting LmSRC and LmChc or Alexa Fluor 488 rabbit IgG (1: 200, green) for detecting LmAP50 (Molecular Probes, Jackson Immuno Research, West Grove, PA, USA), was added and the slides were incubated for 1 h at 37 °C. The cell membranes were stained with CM-dil (Thermos Fisher Scientific) for 30 min at 37 °C and the cell nuclei were stained with DAPI for 5 min. To co-localize LmSRC/LmChc and LmAP50, the hemocytes were prepared as above, but the first incubations were performed with anti-LmSRC/anti-LmChc and anti-LmAP50 antibodies (1:100) for 1 h at 37 °C. The secondary antibodies, Alexa Fluor 594 mouse IgG (1:200, red) for detecting LmSRC/LmChc or Alexa Fluor 488 rabbit IgG (1:200, green) for detecting LmAP50, were added and incubated for 1 h at 37 °C. The subsequent procedures were performed as described above. Co-localization of LmSRC, LmChc, or LmAP50 with fluorescein-labeled dsGFP (dsGFP-F) in hemocytes was examined by immunofluorescence staining. For detection of LmSRC and dsGFP-F co-localization, slides were incubated with anti-LmSRC primary antibody for 1 h at 37 °C, followed by incubation with Alexa Fluor 594-conjugated mouse IgG secondary antibody (1:200) for 1 h at 37 °C. Slides were then processed as described above. Co-localization of LmChc or LmAP50 with dsGFP-F was performed using the same procedure.
Tracing the sequential process of dsRNA cellular uptake across different tissue types
Cellular uptake of dsGFP-Fluorescein in hemocytes
dsGFP with fluorescein (dsGFP-F) was synthesized using Fluorescein RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN, USA), incorporating fluorescein-labeled UTP. The sequence of dsGFP is shown in Additional file 1: Table S6. N3D2 nymphs were injected with either 5 μg of dsGFP as a control or 5 μg of the respective dsRNA (dsLmSRC, dsLmChc, dsLmAP50). After 24 h, each nymph in all control and treatment groups was re-injected with 5 μg of dsGFP-F. Hemocytes were spread onto slides at different time points (0, 10, 30, and 60 s) after dsGFP-F injection. The sample at time 0 was not injected with dsGFP-F and the derived hemocytes acted as background control. The immunohistochemical staining was performed as described above. Based on the report by Jia et al., the hemocytes of L. migratoria comprise four types: protocytes, cytopoietic cells, granular cells, and chromatin-like cells, with granulocytes being the most abundant [36]. Therefore, granulocytes were selected for image acquisition in the co-localization experiments, including the co-localization of LmSRC/LmChc and LmAP50, as well as the co-localization of LmSRC/LmChc/LmAP50 with dsGFP-F in hemocytes. Scanned images were processed using ZEN software (Zen2012, Jena, Germany). The integrated density of dsGFP-F was subsequently measured using ImageJ software (National Institutes of Health).
Sequencing of siRNA after knockdown LmChc, LmAP50, or LmSRC
Each N3D2 nymph was injected with 5 μg of dsGFP as a negative control or 5 μg of each of dsRNA (dsLmChc,* LmAP50*, or LmSRC) in the treatment group. After 24 h, each nymph was re-injected with 400 ng of dsLmLgl. Twenty-four hours later, hemolymph was collected from five nymphs for total RNA isolation, and small RNA sequencing was performed using a BGISEQ-500 analyzer at BGI (Shenzhen, China).
Statistical analysis
Relative contents of dsGFP after injection of dsGFP among different tissue types in L. migratoria or relative expressions of all genes (apolipophorin, various receptors, and endocytosis-related genes) were compared using an ANOVA followed by Tukey’s test. Relative content of dsGFP in each tissue type after RNAi-mediated silencing of each candidate gene potentially involved in cellular uptake of dsRNA or after administration of each endocytosis inhibitor was compared against its corresponding control using Student’s t test. SPSS statistics software was used to perform statistical analyses (SPSS Inc., Chicago, IL, USA).
Supplementary Information
Additional file 1.Additional file 2.
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