The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.
Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects’ feeding behavior. A convenient method that will be useful for research on insects’ saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.
Rice stripe virus (RSV), a negative-stranded RNA virus in the genus Tenuivirus, causes severe diseases in rice production in East Asia1,2,3. Transmission of RSV from infected rice plants to healthy ones depends on insect vectors, mainly Laodelphax striatellus, which transmits RSV in a persistent-propagative manner. SBPH acquires the virus after feeding on RSV-infected plants. Once inside the insect, RSV infects the midgut epithelial cell one day after feeding and then passes through the midgut barrier to penetrate the hemolymph. Subsequently, RSV spreads into different tissues via the hemolymph and then propagates. After a latent period of about 10-14 days post-acquisition, the virus inside the salivary gland can be transmitted to the healthy host plants via the secreted saliva while SBPH sucks sap from the phloem4,5,6,7,8,9,10. An efficient feeding process and various factors in the saliva are essential for the spread of RSV from the insect to the host plant.
Insect saliva secreted by salivary glands is believed to mediate insects, viruses, and host plants.Hemipteran insects usually produce two types of saliva: gelling saliva and watery saliva11,12,13. Gelling saliva is mainly secreted into the apoplasm to sustain the movement of the stylet among host cells and is also related to overcoming plant resistance and immune responses14,15,16,17. At the probing stage of feeding, insects intermittently secrete gelling saliva that immediately gets oxidized to form a surface flange. Then, single or branched sheaths encase the stylet to reserve a tubular channel18,19,20. The surface flange on the epidermis is presumed to facilitate penetration of the stylet by serving as an anchor point, while the sheaths around the stylet may provide mechanical stability and lubrication16,21,22,23. Nlshp was identified as an essential protein for salivary sheath formation and successful feeding of brown planthopper (Nilaparvata lugens, BPH). Inhibition of the expression of the structural sheath protein (SHP) secreted by the aphid Acyrthosiphon pisum reduced its reproduction by disrupting feeding from host sieve tubes24. Moreover, in some insect species, gel saliva factors are supposed to trigger plant immune responses by forming so-called herbivore-associated molecular patterns (HAMPs). In N. lugens, NlMLP, a mucin-like protein related to sheath formation, induces plant defenses against feeding, including cell death, the expression of defense-related genes, and callose deposition 25,26. Also, some gel saliva factors in aphids have been proved to trigger plant defense responses via gene-to-gene interactions similar to pathogen-associated molecular patterns12,15,27.
For studying the saliva factors essential for insect feeding and/or pathogen transmission, it is necessary to analyze secreted saliva. Here, artificial feeding and collection methods to obtain sufficient amounts of saliva are described for further analysis. Using a medium containing only a single nutritional element, many saliva proteins were collected and analyzed by silver staining and western blotting. This method will be helpful in further research on factors in saliva that are essential for RSV transmission by SBPH.
1. SBPH maintenance
2. Preparation of feeding chamber and artificial diet
3. Collection of SBPH saliva
4. Concentration of the collected saliva
5. Silver staining of saliva proteins
6. Protein detection by western blotting
7. Detection of LssgMP expression pattern in SBPH
Schematics of artificial feeding installation and saliva collection
Figure 1A depicts the glass cylinder (15 cm x 2.5 cm) used as a feeding chamber to collect the saliva. Firstly, the SBPH larvae were starved for several hours to improve the collection efficiency and then immobilized by chilling for 5 min. After the insects were transferred into the glass cylinder, both open ends of the chamber were covered with stretched Paraffin membrane. At one end, 200 µL of 5% sucrose was sandwiched between two layers of Paraffin membrane extended to about double its original area (Figure 1B). The chamber was covered with foil, but the end with the artificial diet was exposed to light. Because SBPH displays phototropic behavior, the starved insects gathered at the end were exposed to light and fed on the artificial diet solution through the stretched inner Paraffin membrane. Based on it, saliva could be released into the artificial diet, which was collected each day. The Parafilm-diet device was replaced with a new one every day. In this way, the artificial diet was collected for 5 days to 2 weeks, and then the whole sample was concentrated to a final volume of 100 µL using a 10 KD centrifugal filter (Figure 1C). During the collection of saliva, the survival rate of SBPH feeding the 5% sucrose was counted. In the first 4 days, more than 80% SBPH survived. However, from the 5th day onward, mortality increased quickly to 40%, and less than half SBPH survived on the 7th day (Figure 1D). To collect sufficient saliva, fresh SBPH was suggested to supply on the 4th day.
Verification of collected saliva proteins
For assessing the effectiveness of this collection method, the saliva sample was subjected to protein analysis. Firstly, proteins were separated by SDS-PAGE, and then detected by silver staining (Figure 2A). Compared with the negative control (5% sucrose), the concentrated saliva samples from RSV-infected SBPH saliva contained many proteins that could be further analyzed, for example, by mass spectrometry. As the primary insect vector of RSV, SBPH transmits the virus to plants via its sucking-piercing feeding process. The successful release of RSV is related to saliva secretion, and RSV is considered to be an essential factor in viruliferous insects' saliva. Here, saliva was tested after collecting non-infected and viruliferous insects with an antibody against RSV and successfully detected the RSV coat protein (CP) in the viruliferous sample (Figure 2B). Another study found that mucin proteins are essential gel saliva proteins in hemipteran insects to meditate the formation of a sheath for feeding23. For confirming that SBPH also produces a mucin protein, the whole open reading frame of a putative mucin-encoding gene was amplified from RNA extracted from the saliva gland of SBPH. Its sequence was used as a query in BLAST analyses against the genome sequence of SBPH28. A gene consisting of 2175 bp, named LssgMP, was identified. An antibody against this protein was prepared previously and was used to detect the protein in the collected sample by western blot analysis. A 78 KD protein was detected in non-infected and viruliferous samples, demonstrating that LssgMP is a saliva protein (Figure 2C). Next, the transcript levels of LssgMP in different tissues (salivary gland, gut, and remaining body) were determined by qRT-PCR. The results showed that the gene transcript level was 20-fold higher in the salivary gland than in the gut and other body parts (Figure 2D), confirming the specific expression of LssgMP in the salivary gland.
Figure 1: Diagram of feeding chamber with Paraffin membrane sandwich to allow feeding of SBPH on artificial diet. (A) Illustration of artificial feeding chamber. The cylinder is 15.0 cm long and 2.5 cm in diameter. At one end of the chamber is the Paraffin membrane sandwich; the other end and the cylinder wall covered with tin foil paper (slanted lines) represent the device covered with tin foil paper. The light source was set to attract SBPH to feed on the artificial diet. (B) Diagram of Paraffin sandwich containing artificial diet. 200 µL of 5% sucrose aqueous solution was sandwiched between two layers of Paraffin membranes stretched to about double its original area. (C) The concentration of collected saliva using a 10 KD centrifugal filter. (D) The survival rate of SBPH feeding on 5% sucrose. Mean and SEM was calculated from four biological replicates with three technical replicates. Please click here to view a larger version of this figure.
Figure 2: Verification of collected saliva proteins. (A) Saliva proteins detection by silver staining. The 5% sucrose is negative control. (B) Detection of rice stripe virus coat protein (CP) in collected saliva by western blot analysis. (C) Western blotting to confirm the presence of LssgMP in collected saliva. (D) Specific expression of LssgMP in the salivary gland of L. striatellus. ef2: translation elongation factor 2 of SBPH. Mean and SEM was calculated from four biological replicates with three technical replicates. Please click here to view a larger version of this figure.
Successful rearing of insects on artificial diets was first reported in 1962 when Mittler and Dadd described the Paraffin membrane technique to hold an artificial diet29,30. And this method has been explored in many aspects of insect biology and behavior, for example, nutrient supplement, dsRNA feeding, and virus acquisition. Based on the requirements of saliva analysis, 5% sucrose is used as the general artificial diet to collect saliva of SBPH in this study. For successful saliva collection, several critical steps are worth noting here. Firstly, starving the experimental insects before introducing them into the chamber is necessary to ensure the efficiency of saliva collection. Secondly, to imitate the stylet environment, a two-layer Paraffin sandwich is made. When SBPH feeds on the artificial diet, the salivary sheath forms at the inside end facing the diet, and the watery saliva is secreted subsequently. Thirdly, an artificial medium should be collected and exchanged in time to reduce microbial contamination in the collected sample. Finally, anesthetizing the insects at 4 °C for 5 min is essential to avoid the loss of insects while changing the artificial diet.
In contrast to most artificial diets which contain amino acids, vitamins, and carbohydrates, the advantages of 5% sucrose medium are noteworthy. First, it is easy to prepare. Second, the simple composition of the diet means few substances to interfere with further analysis of the various factors in saliva. Nevertheless, the survival ratio of the SBPH declined in the last days of the feeding period using 5% sucrose as the general artificial diet (Figure 1D). For overcoming this flaw, fresh SBPH should be supplied in time for enough saliva. And for further research of the salivary function on feeding behavior or virus transmission, the saliva collection duration should be limited to the accurate time before insects' mortality increased owing to innutrition. It seems to be a common limitation of the artificial medium. Some other studies found that the survival of N. lugens reared on the chemically defined diet D-97 was inferior to that of those raised on the susceptible rice variety TN1, implying that the original host supplies more than just food vector insects. And several studies have focused on optimizing chemically defined diets for continuous feeding of insects to improve the rearing efficiency.
Transcriptome analysis of the salivary gland is a traditional method for saliva protein identification. And different saliva proteins have been detected in the same species of A. pisum and M. persicae14,31, and the abundance of saliva proteins determines the frequency of their detection32. However, a valid method to investigate the suspected protein in the saliva was lacking. This Paraffin membrane sandwich method provides an innovative manner to confirm a suspected saliva protein identified by transcriptome analysis, which is further proved by detecting LssgMP (Figure 2C). Moreover, protein analysis confirmed that abundant proteins are present in the collected saliva (Figure 2A), which is a sufficient quantity for further analysis by, for example, mass spectrometry. Proteomics analysis of collected saliva will be a direct and effective method for identifying secreted factors involved in the feeding stage of SBPH, minimizing the risk of detecting false-positive redundant proteins.
Saliva works as the virus carrier from insect to host plants and is an essential component of the vector-virus-plant interaction14,33,34. The titer of virus released from insect vectors is a crucial factor for the infection to the host. Compared with indirect methods that test the viral titer in infected plants, this protocol can directly detect RSV released by viruliferous SBPH (Figure 2B). Supported by this Paraffin membrane sandwich method, further comparative analyses of saliva proteins collected from RSV-free and RSV-infected insects may also reveal potential candidates involved in the virus-plant-vector interaction.
The authors have nothing to disclose.
This work was supported by the National Key Research and Development Program of China (No. 2019YFC1200503), by the National Science Foundation of China (No. 32072385), and by Youth Innovation Promotion Association CAS (2021084).
10-KD centrifugal filter | Merck Millipore | R5PA83496 | For concentration |
10x Protein Transfer Buffer(wet) | macGENE | MP008 | Transfer buffer for western blotting |
10x TBST buffer | Coolaber | SL1328-500mL×10 | Wash buffer for western blotting |
Azure c600 biosystems | Azure Biosystems | Azure c600 | Imaging system for western blotting and silver staining |
Color Prestained protein ladder | GenStar | M221-01 | Protein marker for western blotting |
ECL western blotting detection reagents | GE Healthcare | RPN2209 | Western blotting detection |
Enchanced HRP-DAB Chromogenic Kit | TIANGEN | #PA110 | Chromogenic reaction |
Horseradish peroxidase-conjugated goat anti-rabbit antibodies | Sigma | 401393-2ML | Polyclonal secondary antibody for western blotting |
Immobilon(R)-P Polyvinylidene difluoride membrane | Merck Millipore | IPVH00010 | Transfer membrane for western blotting |
iTaq Universal SYBR Green Supermix | Bio-Rad | 1725125 | For quantitative real-time PCR (qRT-PCR) |
KIT,iSCRIPT cDNA SYNTHES | Bio-Rad | 1708891 | For Reverse-transcriptional PCR (RT-PCR) |
Millex-GP Filter, 0.22 µm | Merck Millipore | SLGP033RB | For filtration |
Mini-PROTEAB TGX Gels | Bio-Rad | 4561043 | For SDS-PAGE |
NanoDrop One | Thermo Scientific | ND-ONEC-W | Detection of protein concentration |
Nylon membrane | PALL | T42754 | Membrane for dot-ELISA |
Parafilm M Membrane | Sigma | P7793-1EA | Making artifical diet sandwichs |
Rabbit anti-LssgMP polyclonal antibody against LssgMP peptides | Genstript | Rabbit primary anti-LssgMP polyclonal antibody for western blotting | |
Rabbit anti-RSV polyclonal antibody | Genstript | Rabbit primary anti-RSV polyclonal antibody for western blotting and dot-ELISA | |
RNAprep pure Micro Kit | TIANGEN | DP420 | For RNA Extraction |