Antimicrobial peptides expressed in the fat body of Spodoptera frugiperda (Lepidoptera: Noctuidae) in response to the microbial challenge

Spodoptera frugiperda (Lepidoptera: Noctuidae) is the key pest in maize crops and has a major impact on world agriculture. Several studies on the biological control of this species have been carried out, and these demonstrate resistance to biological control and pesticides. However, the humoral immune system of S. frugiperda is not yet completely known in relation to the innate immune response to biological control agents. The aim of this study was to evaluate the expression of Gloverin , Sf-gallerimycin, and Attacin genes in the fat body of larvae at the 6th instar of development. Our work confirmed the expression of Sf-gallerimycin in response to the bacterial challenge, 24 h post-inoculation, and revealed the expression of Gloverin and Attacin for the first time for both bacterial challenge and entomopathogenic fungus Beauveria bassiana challenge. Gloverin and Attacin genes were upregulated under the conditions analyzed. In addition, we revealed the presence of two probable antimicrobial peptides in the hemolymph, induced 24 h post challenge with the microorganisms. The 4.7 kDa band b is probably a defensin-like peptide, and the 6.1 kDa band a is a peptide not yet reported in S. frugiperda.


Introduction
Food cultivated in the world suffers attacks from pests, which cause approximately one third of food production losses during the process of growth, harvesting, and storage. Insects are a prominent group of organisms that attack crops, in addition to other small animals (Sarmento, et al., 2002). The attack of pests is intensified due to the biological imbalance caused by the elimination of natural enemies (Ribeiro, et al., 2016).
The corn culture (Zea mays) that has economic and social prominence (Melo, et al., 2006) suffers attacks during almost the entire period of its development (Peterlini, et al., 2020), causing direct damage to plants (Wangen, et al., 2015).
Although biological agents can help to control these insect pests, insecticides are now essential for effective and economical pest control on a large scale. The non-target effect of some pesticides is partly due to their effects on insect immunity, which is necessary for insect survival in natural environments (Casanova & Goodrich, 2013). In the defense against pathogens, insects depend mainly on their immune system (Viljakaínen, 2015).
In insects, the innate immune system is the first line of defense against invading pathogens. Although innate immune responses are nonspecific, they are widely distributed throughout the insect, allowing them to play a crucial role in maintaining homeostasis and preventing disease and infection (Sheehan, et al., 2018). The immune response depends on several humoral and cellular factors, occurring both locally and systemically (Destoumieux, et al., 2009).
Antimicrobial peptides (AMPs) are short proteins with antimicrobial activity (Pinto et al., 2015), widely distributed among living organisms (Duvic et al., 2012). Insect AMPs (humoral responses) play an important role in eliminating pathogens and parasites. These peptides are synthesized in specific tissues, such as the fat body and hemocytes; after their production, AMPs are rapidly excreted into the insect's hemolymph, performing an antimicrobial function (Bulet, et al., 1999). They can potentially be applied in medicine and agriculture instead of traditional antibiotics, as they are easy to synthesize due to their relatively small size, have a fast and efficient action against pathogens, provide a wide range of antimicrobial activity, and have low toxicity to cells of vertebrates (Bang, et al., 2012).
Most of these AMPs have been identified from insect hemolymphs using molecular and proteomic methods, such as mass spectrometry or cDNA cloning (Silva, et al., 2010;Koehbach, 2017). Some researchers have used techniques to challenge insects in order to enhance the innate immune response (Charles & Killian, 2015). Research, Society andDevelopment, v. 11, n. 13, e81111335263, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i13.35263 The insect model of the study is the cartridge caterpillar, Spodoptera frugiperda (Lepidoptera: Noctuidae), which is the main pest of the corn culture in Brazil. The S. frugiperda caterpillar has been studied using entomopathogenic fungi for biological control (Thomazoni, et al., 2014); however, knowledge of the innate immune response of this species to entomopathogenic fungi is still scarce.
Recently the complete genome of this species has been elucidated (Kakumani, et al., 2014), becoming a model organism for molecular studies, which allows the use of molecular techniques to analyze the expression of AMPs.
The aim of this work was to evaluate the expression of the genes of AMPs in S. frugiperda: Gloverin, Sf-Gallerimycin and Attacin. For the first time, the Gloverin and Attacin are revealed in vivo in corn borer larvae challenged by non-pathogenic bacteria and the fungus Beauveria bassiana. In addition, two peptides were revealed in the hemolymph in response to the septic challenge.

Maintenance of cultivation, immune challenge and sample collection
Larvae of S. frugiperda were kept on an artificial diet at 27 ± 1 °C and 75 ± 5% relative humidity with a 16L:8D photoperiod. The entomopathogenic fungus Beauveria bassiana was maintained according to Thomazoni et al., (2013), and the bacteria Escherichia coli ATCC 11229 and Bacillus subtilis ATCC 6623 were used in the challenge bacteria mix.
Sixth instar larvae were distributed in three groups: unchallenged (n=30), challenged with a mix (Gram-/Gram+) of bacteria (n=30), and challenged with Beauveria bassiana (n=30). The septic injury was performed by perforating the integument with a dental needle immersed in the pellets of the microorganisms. Samples were collected after 24 hours post-inoculation (24 h pi) at a controlled temperature of 25 ± 1 °C (Silva et al., 2010).
Larvae were cleaned with 70% ethanol, and the hemolymphs were collected in microtubes containing phenylthiurea crystals (C7HN2S). After the material was centrifuged at 200 xg (1450 rpm) for 5 min at 4 °C, the supernatant was transferred to another tube, which was centrifuged at 20,000 xg (14,500 rpm) for 15 min at 4 °C, and the samples were kept at -20 °C until use.
The fat body was dissected in 0.9% saline solution containing RNAse inhibitor and stored in microtubes with 250 µL of RNA latter® solution (Ambion) at 4 °C. The collected material was centrifuged at 400 xg (2,900 rpm) for 5 min at 4 °C. The supernatant was discarded, and the material was frozen at -20 °C.

Sample extraction, dosage and RT-PCR
The proteins from the hemolymph were extracted according to Silva et al., (2010), and the dosage was performed according to the method of Bradford (Bradford, 1976).
TRIzol® reagent (Invitrogen®, EUA) was used to extract the total RNA from the fat body. The total RNA obtained was quantified by spectrophotometry at 260 nm, and the samples were then subjected to treatment with DNAse I, according to the manufacturer's protocol.
The cDNA synthesis was performed according to the instructions in the ImProm-II kit (Promega®). The primers for amplification of the PAM genes were synthesized by the company IDT DNA Technologies (Iowa/USA) by semi-quantitative RT-PCR and are shown in Table 1. (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i13.35263 The Sf-Gallerimycin primer was used as described by Volkof et al., (2003), and the primer for Actin was used as a control of gene expression (Kakumani et al., 2014). The PCR reaction was performed with the Taq polymerase enzyme, and the PCR products were visualized on a 1.5% agarose gel stained with ethidium bromide.

Analysis of samples by electrophoresis and quantification of expression
The visualization of the RT-PCR amplicons was performed on 1.5% agarose gel, and the electrophoresis of protein samples was performed in Tricine SDS-PAGE 16% (Schägger, 2006).
The ImageJ software (free software) was used for the densitometric quantification of the bands of differential expression in RT-PCR and the protein bands of the gels of Tricine SDS-PAGE. In the RT-PCR, we used the 100 bp Ludwig standard (0.1 µg/µL), using the 500 bp band (90 ng/5 µL) as a reference to determine the concentration of the AMP bands. In the Tricine SDS-PAGE, we used the area measurement of the bands to produce a correlation of the AMP expressions.

AMPs expressed in the fat body of S. frugiperda challenged by septic injury
The fat body is the organ that produces AMPs in response to the septic challenge (Bulet et al., 1999). In our work, the study of PAM expression was carried out in sixth instar larvae challenged by microorganisms. The result of the entire process of extracting total RNA from the fat body and expression of the AMP genes by semi-quantitative RT-PCR can be seen in Figure 1. In lane 3, we have the Gloverin gene with 210 bp amplicon, where the increased expression is seen in Figure 1-B compared to the actin control in Figure 1 In lane 5, we have the amplicon of the Attacin gene (397 bp), where we can see an increase in expression in 1-B and 1-C compared to 1-A (not challenged). In assay 1-B, the Attacin gene was 1.45x or 45% more expressed than the Actin (1-B control); in assay 1-C, it was 2.20x or 45.40% downregulated compared to the Actin (1-C control).

AMPs in the S. frugiperda hemolymph challenged by septic injury
We analyzed the presence of AMPs in the hemolymph, 24 h after the septic challenge. In Figure 2, the presence of AMPs was observed in samples 1N (native/unchallenged), 2B (challenged bacteria mix), and 3F (challenged by B. bassiana). In this assay, an increase in the concentration of AMPs was detected in the challenged samples compared to the native sample. The bands with molecular mass below 6.5 kDa are indicated by the arrow in Figure 2-A. The band a had a relative molecular mass of 6.1 kDa, and band b 4.7 kDa; both calculated through relative migration in the gel compared to the molecular mass standard.

Discussion
One of the first lepidopterans to have its immune system studied was the moth Hyalophora cecropia. In H. cecropia, glycine rich AMPs were identified (Hultmark et al., 1983;Axén, et al., 1997). From these initial findings, AMPs of this class were found in several other Lepidoptera, highlighting Bombyx mori (Tanaka et al., 2008).
In Spodoptera exigua, it was reported that Gloverin is important for resistance against Bacillus thuringiensis, and Gloverin expression was induced in a septic challenge with Serratia marcescens (Hwang & Kim, 2011;De Mandal, et al., 2020).
In 2003, the presence of AMPs similar to the class of defensins in S. frugiperda were identified (Volkoff et al., 2003), and Gloverin-3 expression was recently reported in S. frugiperda challenged by Ascovirus (Zaghloul, et al., 2020). However, in the septic challenge of S. frugiperda by non-pathogenic bacteria and the entomopathogenic fungus B. bassiana, no Gloverin has been reported to date. In our work, although we have basal expression in native larvae, we have shown the induction of Gloverin expression with the septic challenge using the mix of bacteria and B. bassiana.
Attacins are mainly active against bacteria (Gram-) and have been described in S. exigua (Bang et al., 2012). The expression of Attacin and Gloverin were suppressed by infection with Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) in S. exigua (Choi et al., 2012). In our work, we observed the induction of Attacin in the challenge with fungus and the mix of bacteria (Gram+/Gram-), indicating that this AMP is an important effector of the innate immune system. Sf-Gallerimycin was induced by the challenge of the mix of bacteria and suppressed in the challenge with B. bassiana after 24 h of inoculation. This AMP is active against bacteria and fungi, as reported by Volkof et al., (2003), and our work confirmed that induction by the fungus is a later response.
The peptides present in the hemolymph are products of the fat body and hemocytes, in response to the immune challenge. Our work revealed the presence of AMPs that were induced; however, they were not identified with the applied methodologies. The 4.7 kDa band b is probably a defensin-like peptide, which has a molecular mass of 4 kDa and is active against bacteria and fungi (Marshall & Arenas, 2003). Based on its molecular mass, the 6.1 kDa band a is a peptide not yet reported in S. frugiperda. These are PAM candidates that still need to be isolated and identified to understand their role in the immune response of S. frugiperda.

Conclusion
In our work, it was shown that microbial challenges induced the humoral immune response of the S. frugiperda larvae as observed in the RT-PCR assay for the analyzed genes. The induction of low molecular weight antimicrobial peptides was demonstrated in the hemolymph of challenged larvae as shown on SDS-PAGE. This study shows for the first time the induction of the Attacin and Gloverin genes in microbial challenge, in addition to pointing out possible antimicrobial peptides in the hemolymph of S. frugiperda.
However, the results of this article indicate that a research effort is needed in this area of insect immunology to understand its biology and then possibly we will have better perspectives for the control of insects and agricultural pests.