Isolation, Identification, and Bioinformatic Analysis of Antibacterial Proteins and Peptides from Immunized Hemolymph of Red Palm Weevil Rhynchophorus ferrugineus

Red palm weevil (Rhynchophorus ferrugineus Olivier, 1791, Coleoptera: Curculionidae) is a destructive pest of palms, rapidly extending its native geographical range and causing large economic losses worldwide. The present work describes isolation, identification, and bioinformatic analysis of antibacterial proteins and peptides from the immunized hemolymph of this beetle. In total, 17 different bactericidal or bacteriostatic compounds were isolated via a series of high-pressure liquid chromatography steps, and their partial amino acid sequences were determined by N-terminal sequencing or by mass spectrometry. The bioinformatic analysis of the results facilitated identification and description of corresponding nucleotide coding sequences for each peptide and protein, based on the recently published R. ferrugineus transcriptome database. The identified compounds are represented by several well-known bactericidal factors: two peptides similar to defensins, one cecropin-A1-like peptide, and one attacin-B-like protein. Interestingly, we have also identified some unexpected compounds comprising five isoforms of pheromone-binding proteins as well as seven isoforms of odorant-binding proteins. The particular role of these factors in insect response to bacterial infection needs further investigation.


Introduction
Currently, there is a worldwide rapid increase in the number of pathogenic bacteria with resistance to the array of available antibiotics, which poses a growing threat to human and animal health [1,2]. To overcome this problem, antimicrobial peptides/proteins (AMPs) have come to the forefront as potential antibiotic surrogates with robust killing activity against a wide spectrum of bacterial species, including drug-resistant strains [3]. They exert an antimicrobial effect mainly by disrupting the microbial membrane, which makes microbes unable to easily develop resistance against these compounds [4][5][6]. Additionally, they often display positive immunomodulatory functions, such as modulation of cytokine production, chemotactic activity or promote wound healing [7,8]. molecular and proteomic studies on RPW. In the present paper, we isolated and identified AMPs that function in the hemolymph of RPW. Because majority of insect AMPs are produced by the fat body tissue and excreted into the hemolymph in response to infection or injury, the studied AMPs were isolated from hemolymph of beetles immunized previously by injection of a mixture of live E. coli and M. luteus cells. After isolation of AMPs, verification of their bactericidal activity and identification by N-terminal sequencing or by mass spectrometry, we performed bioinformatic analysis of obtained sequences based on the aforementioned transcriptome database. Obtained results provide new insights into the defense response in Coleoptera, particularly in pests.

Insect Sampling and Immunization
Adult RPW beetles, without sex indication, were collected in June 2019 from infested Canary Island date palm (Phoenix canariensis) fields in El Kassasin District, Ismailia Governorate in Egypt (northeastern part of the country). The insects were reared for a short time in sugar cane-containing plastic boxes in the dark at room temperature. Each beetle was identified as Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae: Dryophthorinae: Rhynchophorini) with the use of all morphology identification keys [27,29,30]. In total, 79 individuals were immunized by injection into the hemocoel of 2 µL of liquid phosphate buffered saline (PBS) containing a suspension of live Escherichia coli K12/ATCC 10,798 and Micrococcus luteus ATCC 4698 cells (2 × 10 7 CFU/mL). As a non-immunized control, 12 individuals were only pierced on the thorax with a sterilized needle. After 24 h of the immune challenge, 5 adults died and another 2 individuals were not analyzed. All individuals in the control group survived.

Hemolymph Collection
Hemolymph samples were collected into Eppendorf tubes containing an equal volume of PBS with 0.1% (w/v) phenylthiourea (PTU, melanization inhibitor), mixed, and deprived of cellular components by two subsequent centrifugation steps: at 5000× g for 15 min and then at 20,000× g for 20 min, both at 4 • C. The clear supernatant was frozen and stored for further analyses.

Chromatographic Separations
All reversed-phase high-pressure liquid chromatography (RP-HPLC) separations were performed using Ultimate 3000 apparatus (Thermo Scientific, Waltham, MA, USA) equipped with a Discovery Bio Wide Pore C18 4.6 × 250 mm column (Sigma, St. Louis, MI, USA). Two solvents were applied: A-0.1% (v/v) trifluoroacetic acid (TFA) in water and B-0.07% TFA, 80% acetonitrile (both v/v) in water. The spectrophotometric detection at 220 and 280 nm was carried out at a flow rate of 1 mL/min. The following linear gradient steps were designed: -Total 0-100% B for 20 min for fast comparative analyses of hemolymph shown in Supplementary Materials Figure S1; -Total 0-75% B for 40 min for separation of hemolymph shown in Figure 1A; -Total 30-55% B for 40 min for separation of fractions 1-5 shown in Figure 1B Figure 1G.
Before each separation, the column was equilibrated at the starting percentage of solvent B. After completion of the gradient, the column was regenerated for 5 min at 100% B. During separations, the fractions and subfractions were collected manually into plastic tubes, evaporated in a vacuum centrifuge, and dissolved in water for further analyses.

Antibacterial Radial Diffusion Assay
The bactericidal or bacteriostatic activity of collected fractions was evaluated using the radial diffusion assay. Two subsequent 5 µL portions of relevant solutions were pipetted onto tryptic soy (TSB) plates solidified with 0.75% agarose (low EEO grade, Sigma, St. Louis, MI, USA) containing a 200× diluted overnight culture of Escherichia coli K12/ATCC 10,798 or Staphylococcus intermedius ATCC 29663. Following overnight incubation at 37 • C, the antibacterial activity was evaluated visually and non-quantitatively: compounds causing clear inhibition zones were presumed as bactericidal, while compounds causing partial clearance were regarded as bacteriostatic ones.

Bioinformatic Techniques
The cDNA sequences of the Rhynchophorus ferrugineus transcriptome were obtained from the National Center for Biotechnology Information (NCBI) Nucleotide database (accession numbers from JR467464 to JR494080). All analyses were carried out in Jupyter Notebook environment release 6.0.1 [44,45]. The sequences were searched for fragments coding for the analyzed peptides using a translated BLAST tool (tblastn, BLAST+, v. 2.9.0) [46] at the default E-value threshold of 10. Using in-house Python scripts utilizing Pandas library [47], the search results were filtered to contain only hits of 100% identity to the query peptide sequences. Each BLAST hit location was extended both downstream to a stop codon (TAA, TAG, TGA) or upstream up to the furthest possible initiation codon (preferentially ATG or TTG/CTG if ATG was not present) to uncover the putative coding sequence (CDS). The products of the coding sequences were searched in CLC Main Workbench (8.1.3, Qiagen) using a plugin for SignalP 4.1 [48] to determine the presence and the extent of putative signal peptides. The identity of CDS products was determined using translated BLAST as described above to search the non-redundant NCBI Nucleotide database obtained from the NCBI FTP server (ftp://ftp.ncbi.nlm.nih.gov/blast/db/). The results were filtered accordingly to select hits with the highest statistical significance, i.e., those with the lowest E-value. The information on CDS locations, cross-referenced accession numbers of the protein/peptide product, and the source organisms was obtained directly from respective NCBI Nucleotide database entries using in-house Python scripts and NCBI E-utilities.

Results
RP-HPLC chromatography is a convenient technique facilitating effective separation and quantitation of different immune polypeptide factors present in various insect hemolymph extracts [49][50][51][52]. This technique was also applied in this study to identify bactericidal or bacteriostatic peptides and proteins excreted by adult RPW individuals into the hemolymph. During preliminary experiments, we compared the chromatographic profiles of hemolymph samples collected from both control (healthy) insects and infected beetles 24 h after injection of a mixture of Gram-negative and Gram-positive bacteria (Escherichia coli and Micrococcus luteus) into the hemocoel. The RP-HPLC profiles were determined for hemolymph sampled from four control and six immunized insects, but the chromatograms obtained did not allow unambiguous identification of peaks whose intensity increase could be attributed only to immunization (Supplementary Materials Figure S1). However, the antibacterial radial diffusion assay performed on fractions collected during separation of immunized hemolymph distinguished a group of peaks (dashed square in Figure 1A) that indicated bactericidal or bacteriostatic activity towards the standard bacterial strains used: E. coli and Staphylococcus intermedius. The compounds eluted in this region were further separated, and the successive antibacterial tests indicated five main fractions with activity towards bacteria (peaks 1-5 shown in Figure 1B). All these peaks were individually collected and fractionated in a series of different individually optimized gradient separations ( Figure Figure 2) were identified by determination of their N-terminal amino acid sequences by direct chemical sequencing using Edman degradation. The analyses were performed using bands excised from the PVDF membrane. In all cases, it was possible to determine the sequence of 15 to 25 residues (Table 1). However, sequencing was impossible in the case of subfractions 3.2, 4.1, and 4.7, proving that these proteins bear a chemical blockade at the alpha-amino group of the N-terminal amino acid. These three compounds were identified with the bottom-up proteomic approach, using mass spectrometry. The results allowed determination of a 12-24 residue-long amino acid sequence of 2 to 5 internal peptides from each subfraction (Table 1). In the case of fraction 3.4, which contained a protein of molecular mass of approx. 15 kDa, both sequencing and mass spectrometry identification attempts failed. The bioinformatic analyses revealed that all determined amino acid sequences have 100% identity to those encoded in corresponding nucleotide sequences identified several years ago in the first large-scale full-length cDNA sequencing project for R. ferrugineus [33] and in different individual nucleotide sequences of particular mRNA obtained independently from R. ferrugineus. Furthermore, we also showed significant sequence similarities for corresponding bactericidal peptides and proteins found in other insects. In sum, the identified factors are two peptides similar to defensins, one cecropin-A1-like peptide, one attacin-B-like protein, five isoforms of pheromone-binding proteins, and seven isoforms of odorant-binding proteins. All essential information about the identified proteins and peptides is collected in Table 1, which also includes the determined amino acid sequences, accession numbers of respective cDNA sequences of R. ferrugineus transcriptome, theoretical molecular masses of the encoded mature proteins or peptide products, and data on the most similar proteins and peptides found in other insect species or in other studies of R. ferrugineus. More detailed information concerning the bioinformatic parameters of the analyzed sequences can be found in Supplementary Materials Table S1.
In the case of subfraction 1.1, the determined 15-mer N-terminal amino acid sequence allowed to identify of three coding sequences (CDS) in the R. ferrugineus transcriptome for this particular peptide motif: JR492050.1, JR485705.1, and JR485629.1. These sequences encode three similar proteins of molecular mass of approx. 12 kDa. All of them share approx. 55% of identical residues with attacin-B-like protein from Dendroctonus ponderosae.
The 19-mer N-terminal amino acid sequence of the peptide from subfraction 4.2 was identical to that encoded in nucleotide sequence JR486084.1 found in the R. ferrugineus transcriptome, which is a 4.97 kDa and 44-mer-long peptide demonstrating 73.91% of sequence identity to a hypothetical antimicrobial peptide from Sitophilus zeamais. This peptide also shares some degree of similarity with the predicted sequence of cecropin-A1-like peptide of Dendroctonus ponderosae.
For subfractions 3.1, 4.3, 4.4, 4.8, and 4.9, we were able to determine 20-25 amino acid-long N-terminal sequences which all are identical to motifs present in two 13,187 and 12,840 Da proteins encoded in two respective cDNA sequences, namely sequence JR470869.1 and JR489305.1, from the R. ferrugineus transcriptome. The protein product of the JR470869.1 sequence, i.e., a protein from subfraction 3.1, has over 74% of identical residues with pheromone-binding protein 14 from Cyrtotrachelus buqueti. In turn, the product of the JR489305.1 gene (corresponding to proteins from subfractions 4.3, 4.4, 4.8, and 4.9) has over 79% identity with pheromone-binding protein 10, also from C. buqueti. Moreover, the presence of four different protein subfractions with the same N-terminal sequences encoded by the same gene indicates that subfractions 4.3, 4.4, 4.8, and 4.9 contain isoforms of the same protein trimmed proteolytically at the C-terminus or bearing different posttranslational modifications.

Discussion
The well-known insect bactericidal factors identified in our study were represented by one attacin-B-like protein, two peptides similar to defensins, and one cecropin-A1-like peptide. The attacin-B-like protein was found in subfraction 1.1. Attacins are glycine-rich, bacteriostatic, medium-size (ca. 20 kDa) proteins which interact with lipopolysaccharides; hence, their action is directed mainly at defense against Gram-negative bacteria, with the greatest effectiveness against Escherichia coli [5,53,54]. These proteins have a negligible hemolytic effect on red blood cells; therefore, they are considered as potential alternatives to antibiotics [5,55,56]. They are synthesized as pre-pro-peptides with an N-terminal signal sequence, a P domain, an attacin domain, and two glycine-rich (G1 and G2) domains at the C-terminus. Attacins were first discovered in the Lepidoptera [57,58], but they are also common in other insect orders [5]. We were able to detect a single attacin-like protein in the immunized hemolymph of RPW; however, in the current version of the R. ferrugineus transcriptome, three different CDS code for products that contain the motif found in the N-terminal sequence of the isolated protein. These sequences encode three proteins of the same molecular mass of approx. 12 kDa. Most probably, only one of them is expressed and detected in the hemolymph of RPW. All these proteins share relatively small sequence similarity to the gene of the predicted attacin-B-like protein (unpublished, NCBI reference sequence No XP_019761640.1) identified in mountain pine beetle Dendroctonus ponderosae during whole genome shotgun sequencing. However, in the case of the putative RPW attacin found in subfraction 1.1, the low sequence similarity to other known attacins (only 55% of identical residues) necessitates further research on the function and role of this protein in the R. ferrugineus immune response.
Defensins are a family of small cationic peptides with potent antimicrobial activity and a molecular mass of about 4 kDa (approx. 40 amino acids in length), usually containing six cysteines forming three disulfide bridges [59]. They are found in various living organisms, including humans, other mammals, birds, reptiles, fish, mollusks, arthropods, many different groups of insects, plants, and fungi. The structures and mechanisms of the antimicrobial action of defensins have been broadly described, and possible applications of these peptides have been comprehensively discussed by many authors [60][61][62][63]. However, the structure of the insect defensin molecule is quite characteristic, as it usually consists of an N-terminal loop, an α-helix, and anti-parallel β-sheets at the C-terminus, where two disulfide bridges connect the helix with the first β-sheet and the third bridge connects the loop with the second β-sheet. Defensins are most active against Gram-positive bacteria, including such human pathogens as Staphylococcus aureus [5,64,65]. As demonstrated earlier, we discovered three isoforms of these peptides (63-, 65-, and 44-amino acid-long) in the RPW hemolymph, with pronounced similarities to defensin in grain weevil Sitophilus zeamais. The weevil defensin has not been characterized at the protein level to date, but its genes were identified by Anselme et al. in a study of the immune response of the weevil to mutualistic endosymbiotic intracellular bacteria [23]. The authors of this study hypothesize that the weevil defensin limits infections by endosymbionts only to the specialized bacteria-bearing tissue of the insect host.
Cecropins are alpha-helical linear peptides without cysteines, containing 31-42 amino acid residues and having individual names, depending on the taxa in which they were detected. They were discovered for the first time in the Lepidoptera and have been described in other insect orders as well, including Coleoptera and Diptera. Cecropins exhibit a broad spectrum of antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi [63]. Cecropins also exhibit a number of other properties, e.g., immunomodulatory [66,67] and toxic activity against tumor cells [67]. The RPW peptide found in subfraction 4.2 has a similar sequence to the hypothetical antimicrobial peptide of Sitophilus zeamais identified during the aforementioned studies of weevil tolerance of endosymbionts [23]. However, the sequence of this hypothetical peptide is highly similar to numerous cecropins from different insects. The sequence of peptide 4.2 is also similar to the sequence of putative cecropin-like protein A1 found in the genome of mountain pine beetle Dendroctonus ponderosae (unpublished, NCBI reference sequence No XP_019757573.1).
Interestingly, in the immunized RPW hemolymph, we unexpectedly identified some other bactericidal factors as well. These were five isoforms of pheromone-binding proteins and seven isoforms of odorant-binding proteins. The term "odorant-binding proteins" (OBPs) usually describes proteins that are unique in terms of their number, abundance, and diversity in the olfactory system of various insects and which are able to bind odorous substances [68]. The first insect OBP was found as a sex pheromone-binding protein in an antennal extract of the giant silk moth Antheraea polyphemus [69,70]. Soon, mammalian OBPs were discovered. They have similar function but a completely different structure than insect OBPs [71,72]. The vertebrate proteins belong to the lipocalin superfamily and therefore represent a structurally different class to the OBPs of Hexapoda [73,74]. Within the Hexapoda, the number of genes encoding OBPs is highly variable among species [75]. For example, within the Entognatha, a larger number of genes coding for OBPs have been reported for Collembola in comparison to Protura and Diplura [76].
The odorant-binding proteins are relatively small (10 to 30 kDa in size), water-soluble, and uniquely expressed in the olfactory tissue of insects and vertebrates [72,77]. They can be grouped into general odorant-binding proteins, pheromone-binding proteins, and antennal binding protein X [69,78]. The insect OBPs contain mainly α-helical domains, which define the hydrophobic binding cavity and are divergent across and within species [74,79,80]. The structure of OBP molecules is stabilized by three disulfide bridges between the conserved pattern of six cysteines [81][82][83]. The family of OBPs includes members with a smaller (C-minus OBPs) or higher number (C-plus OBPs) of cysteines and atypical OBPs containing additional domains [84,85]. Alongside other proteins, such as chemosensory proteins, odorant receptors, ionotropic receptors, sensory neuron membrane proteins, and odorant degrading enzymes, OBPs belong to the main proteins of the peripheral olfactory system in insects [78,86]. This family has been found mainly in Lepidoptera (butterflies and moths) but also in other insect orders, and the genomic analysis of Drosophila and other insect species, e.g., Anopheles gambiae, Apis mellifera, Bombyx mori, and Tribolium castaneum, has revealed that the OBP genes significantly differ between species [87][88][89].
The odorant-binding proteins are mostly and abundantly expressed in the antennae [90][91][92], including the taste system and chemosensory organs [93][94][95]. They are also present in reproductive organs [96] and are produced in the sperm and transferred to females during mating [97][98][99][100]. They are also known to be ectopically expressed in such tissues as the gut [101]. Functionally, the insect OBPs are involved in the detection of both general odorants and sex pheromones; they capture and transport them to receptor neurons [70,89,102,103]. Some OBPs are hypothesized to hasten odor response termination by extracting odorant molecules from the sensillar lymph or from receptors themselves [104]. It has also been hypothesized that OBPs are part of the molecular coding of odors and pheromones by forming specific complexes with odorant molecules that could ultimately stimulate olfactory receptors to trigger the olfactory transduction cascade [105]. However, an increasing body of evidence reveals a much broader role for this family of proteins [68,78,106], and OBPs are thought to have multiple roles, besides olfaction, in reproduction, egg laying, and anti-inflammatory responses [96]. Quite recently, Bianchi et al. have shown that vertebrate OBPs exhibit antimicrobial activity against Candida albicans, Pseudomonas aeruginosa, and several other bacterial and yeast strains and suggested that this activity may be related to scavenging several compounds important for bacteria, such as quorum-sensing peptide-pheromones, N-acyl-homoserine lactones, furanones, hormones, quinolones, and fatty acids [107]. The authors of the aforementioned study hypothesized about the role of OBPs in the anti-infective immunity of vertebrates, because OBPs are synthesized in all tracts of the respiratory apparatus and are secreted at millimolar levels into the mucus layer of the epithelium. In our study, we have identified for the first time OBPs in the immunized hemolymph of the adult Rhynchophorus ferrugineuss. This finding confirms that also insect OBPs have antibacterial activities and that their level increases in the hemolymph after immunization. However, the detailed role of OBPs in anti-infective insect immunity and their mechanism of action toward pathogenic bacteria need further separate and detailed verification.
The second surprising group of new antibacterial factors found in this study in the immunized RPW hemolymph is the pheromone-binding proteins. We have found these proteins in as many as seven isolated subfractions. They are most probably proteolytically trimmed protein products of three different R. ferrugineus CDS. The pheromone-binding proteins are a subtype of odorant-binding proteins mediating the early stages of detection of volatiles in both insects and vertebrates, with the major function of pheromone binding [69,75,86,[108][109][110]. They represent a family of proteins related to insect sex pheromone recognition identified in many species representing different insect orders [110][111][112]. They are small (14-20 kDa), water-soluble, extracellular proteins of around 130-150 amino acids, containing six or seven alpha helices that form a conical binding cavity and six cysteine residues that form three disulfide bonds stabilizing the three-dimensional structure [83,113]. The pheromone-binding proteins are located in the male antennal long olfactory trichoid sensilla among several proteins involved in insect olfactory recognition [86,91]; however, lower levels of expression of these proteins have also been found in female antennae [110]. Besides the antennae, pheromone-binding proteins were also identified in other appendages, e.g., the proboscis, labial palps, and legs [114,115] and in the sex pheromone gland of some Lepidoptera moths [116,117]. The pheromone-binding proteins are synthesized by two olfactory accessory cells: trichogen and tormogen cells and are secreted abundantly into the sensillum lymph of trichoid sensilla [118][119][120].
Studies on insect pheromone-binding proteins indicate that they are multifunctional: they act as solubilizers and carriers of hydrophobic pheromones in the aqueous sensillum lymph, concentrate odorants in the sensillum lymph, protect pheromones from enzymatic degradation, and serve as cofactors in the activation of pheromone receptors. Furthermore, they are involved in the postulated odorant molecule deactivation and thereby in facilitation of their transport to the receptor neurons, enhancing the sensitivity of olfactory receptors to sex pheromones [106,121]. Hence, pheromone-binding proteins play important roles in the information exchange between insect sexes, specifically in the process of transporting fat-soluble odor molecules from the external environment to olfactory receptors through the olfactory sensillum lymph. The functions of pheromone-binding proteins in this process may explain the sex pheromone identification mechanism used by insects, laying a theoretical foundation for the prevention and control of pests by interfering with olfactory recognition. A study conducted by McKenna et al. [91] suggests the possibility that pheromone-binding proteins are members of a larger class of proteins, extending beyond the olfactory system. In weevils, pheromone-binding proteins have been well studied in Cyrtotrachelus buqueti [112,122,123]. In this beetle, the pheromone-binding protein has dual roles in the processes of sensing sex pheromones and plant volatiles [123]. The phylogenetic analysis showed that C. buqueti pheromone-binding proteins are similar to pheromone-binding proteins of other insects, for example, the similarity to pheromonebinding proteins from Coleoptera reaches 38.47% [112]. On the other hand, the identified amino acid sequences of the bactericidal proteins from immunized hemolymph of Rhynchophorus ferrugineus show a high degree of similarity to the C. buqueti pheromonebinding proteins. In fact, these weevil species are closely related phylogenetically, representing the same subtribe Rhynchophorina [124].
In Rhynchophorus ferrugineus, which is a widely distributed and highly destructive pest of palms, the pheromone-binding proteins play an especially important role in the process of olfactory recognition of plants. In this study, we have also proved that these proteins have antibacterial properties and their level increases in the hemolymph after immunization. To the best of our knowledge, this is the first report of the bactericidal properties of this class of proteins and, as in the case of OBPs, this phenomenon needs separate detailed verification, especially in terms of the mechanism of bactericidal activity and the role in anti-infective response. Since pheromone-binding proteins and OPBs are closely functionally and phylogenetically related, one may expect that their role in insect immunity is similar. Moreover, the fact that both OBPs and pheromone-binding proteins were found in the immunized hemolymph, beyond the typical tissues in which they are expressed, suggests that these proteins may have acquired new functions along evolution. One can also speculate that they have adopted an alternative gene expression control system in comparison with regular OBPs or pheromone-binding proteins. Since pheromone-binding proteins are strongly male-specific [110], both their level and the role in the hemolymph should also be verified in the context of the sex of the insects as well as the sexual maturity status.

Conclusions
The present work is the first comprehensive study focused on identification of bactericidal proteinaceous factors produced by red palm weevil Rhynchophorus ferrugineus-a troublesome pest of palms. In immunized hemolymph of this beetle, we found both well-known families of insect bactericidal peptides and proteins, such as attacins, defensins and cecropins, as well as two groups of proteins, which were earlier not known in Insecta to serve functions related to anti-infective response: odorant-and pheromone-binding proteins. Indeed, some recent studies suggest that vertebrate odorant-binding proteins are able to exhibit antimicrobial activity by scavenging selected low-molecular compounds important for bacteria. On the other hand, at present, nothing is known about the mechanism of the bactericidal action of pheromone-binding proteins. However, both odorant-and pheromone-binding proteins have similar functions and phylogeny; therefore, one may expect that their role in insect immunity is similar. The present work provides a base for further detailed studies of the particular role of both protein families in insect response to bacterial infections.
Supplementary Materials: The following are available online at https://www.mdpi.com/2218 -273X/11/1/83/s1, Figure S1: Comparison of RP-HPLC elution profiles of four control and six immunized cell-free hemolymph samples, Figure S2: Bactericidal or bacteriostatic activities of peptides and proteins isolated from Rhynchophorus ferrugineus hemolymph, Table S1: Detailed information concerning the bioinformatic parameters of bactericidal proteins and peptides isolated from Rhynchophorus ferrugineus immunized hemolymph.