Diversity of bacterial communities associated with the gut of the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in Eastern India

Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) is an invasive alien pest native to the Americas, and it was introduced in the state of Karnataka, India in the year 2018. They cause severe economic damage to the maize crop, which significantly decreases the quality and quantity of the crop’s yield. The microbiota of fall armyworm could play important roles in their growth, development and environmental adaptation to their host plants or animals and not much is known about the microbiota of FAW in India. Even though bacterial communities in S. frugiperda are inadequately studied, therefore, a study was undertaken on the microbial communities associated with the gut of S. frugiperda collected randomly from twelve different locations in the eastern part of India under laboratory conditions. The results revealed that the two bacterial phyla, namely, proteobacteria and firmicutes, were predominantly present in the larval gut of S. frugiperda. However, analyses at the genus level revealed that despite the high genus-level diversity between samples, there were 9 different genera observed. Interestingly, we found two bacterial genera, i.e., Kluyvera and Yokenella, which may be new findings from the eastern part of India and were not reported earlier from any other countries. On the other hand, analyses at the species level revealed that a total of thirty-three (33) species were found from the 12 samples of S. frugiperda collected from different locations. Among the 33 species, Enterococcus group genera were most abundant across the FAW gut samples collected, followed by Klebsiella sp. and Enterobacter sp. and a small proportion of Raoultella, Citrobacter, Leclercia and Pantoea.


Introduction
The fall armyworm (FAW), Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) is a highly destructive pest of a number of economically important agricultural crops such as corn, soybeans, sorghum, peanuts, bermudagrass and cowpeas (Yu et al., 2003). The fall armyworm is native to the Americas and is also found in other countries like Mexico, Brazil, Argentina and USA (Prowell et al., 2004;Clark et al., 2007). The report of the fall armyworm as an invasive alien pest was first reported from Africa in 2016 (Goergen et al., 2016), where it causes havoc on the maize crop and is reported as "the hungry caterpillar threatening a global food crisis". Nowadays, S. frugiperda has reached the major maize-growing areas (Deole & Paul, 2018;Sisodiya et al., 2018) as an invasive pest and was first reported from India on maize in 2018 (Sharanabasappa et al., 2018). The existence of two subpopulations, namely the "Rice" and "Corn" strains, that are preferentially associated with smaller grasses (such as rice and Bermuda grasses) or larger grasses (such as sorghum and maize), respectively (Nagoshi & Meagher, 2004) has been supported earlier through genetic characterization.
The microbial community residing in the insect gut plays various roles in the growth and reproduction of the insect. The various challenges faced by the insects, such as toxins, food sources, environmental conditions, parasites and pathogen attacks (Ugwu et al., 2021), inhabiting various diverse sets of niches, are solved through symbiotic associations of insects with microbes (Douglas, 2015). Herbivorous insects encounter diets that may be difficult to digest, have reduced nutrient value, and possess defences that prevent optimal nutrient extraction. Insects overcome these difficulties related to the substrates through various multi-modal strategies, and one such strategy is the co-option of microbiota (Manson et al., 2018). These insects have co-opted microbes through horizontal gene transfers (HGTs). Microbes may change insect biology, metabolism, and behaviour, thus influencing plant-insect interactions significantly. These microorganisms play an important and diverse role in the growth and development of many insect species. The symbiotic associations between the insects and their gut bacteria have been studied in detail in some insects, such as termites and aphids (Breznak, 1982;Chen & Purcell, 1997) that feed on wood and plant phloem content. But little is known about the microbial associations of insects that feed on foliage crops.
And very little is known about lepidopteran insects and their gut microbial associations. The lepidopteran larvae are alkaline in nature (pH > 8), hence they are in extreme environments for the microorganisms (Harrison, 2001). The sources of gut microbiota in lepidopterans have received little attention; understanding the factors that influence bacterial community composition may shed light on symbiont-host co-adaptation and the strategies insects use to acquire their microbial partners. Previous studies on the lepidopteran insects reveal that they harbour midgut bacteria, thus suggesting that these microorganisms provide essential nutrients or play some role in important biochemical functions and also help in regulating the metabolism of the insects to improve digestion for the extraction of maximum energy from the ingested foods (Campbell, 1989;Kaufman & Klug, 1991;McKillip et al., 1997). There are various symbiotic associations of bacteria within the insect gut (Hooper & Gordon, 2001). Although research into microbial diversity and its relationship with plants and insects has been conducted, the precise role of these microbial communities in interacting with plants and herbivorous insects remains unknown. However, the advanced sequencing methods and molecular technologies are a boon for us in understanding the role of these microbes in plant-insect interactions at the molecular level. In order to study the microbial associations inside the insect gut, both culture-dependent (Apte-Deshpande et al., 2012;Arias-Cordero et al., 2012) and culture-independent methods (Colman et al., 2012;Jones et al., 2013) are available. The culture-independent method, which is based on 16 S rRNA gene analyses, gives a clear picture of the bacterial communities and a more precise understanding of the microbes living inside the insect gut.
As a result, a thorough understanding of the bacterial communities of insect guts is critical for a complete understanding of its hosts' biology and ecology, as well as for the development of a novel pest management strategy. The microbiome associated with S. frugiperda could play a role in the insects' survival and adaptability. However, bacterial communities in S. frugiperda remain poorly studied. Characterizing the pest-associated microbiomes would be a useful tool for studying the insect-microbiome interactions, which could be exploited to improve control strategies. Hence, in the present study used 16 S rDNA sequence profiling to characterize the diversity of bacteria associated with populations of S. frugiperda in different parts of eastern India.

Insect collection and gut dissection
The larvae of the fall armyworm, S. frugiperda, were collected randomly from infested maize fields at the vegetative stage from twelve different locations in eastern India from July 2020 to May 2022, and the locations were presented in Table 1; Figs. 1 and 2. Larval species identification was confirmed using Petersen (1962) morphological keys, while the remaining larvae were kept in ethanol 70% at 4 o C and cultured in the laboratory, Department of Entomology, Bihar Agricultural University, Sabour. The cultures were maintained at 25 ± 2 o C temperature, relative humidity 75% and provide untreated maize leaves as a food.
The isolation process was done inside the laminarair flow with proper sanitation. Larvae were surfacesterilized with 70% ethanol for 1 min and 5% sodium hypochlorite for 1 min, after which they were washed in sterile 10 mM phosphate-buffered saline (PBS) before dissection in a laminar flow hood. Five fullgrown (5th instar) larvae from the same geographical location were selected for gut isolation by the rupturing of the epithelium wall and isolating the midgut, where microbe associations are intensively present and help in the breakdown of complex organic foods. To avoid contamination from other tissues, midguts were aseptically dissected from the larvae and pooled midgut was transferred into a 2 ml NucleoSpin R Bead Tube Type D (Snyman et al., 2016).

DNA extraction
Genomic DNA was extracted from the whole larva using the MN Insect Kit (Germany) according to the manufacturer's protocol. The pooled midgut was homogenized in NucleoSpin R Bead Tube Type D (100 µl Elution Buffer BE, 40 µl Lysis Buffer MG, 10 µl of Liquid Proteinase K). The homogenised sample was agitated on Vertex-Genie2 for approximately 20-25 min. In order to obtain optimum rDNA yield, a complete disruption of the sample material is essential.
To clean the lid, the homogenised mixture was centrifuged for 1 min at 11,000 rpm (revolutions per minute) (higher rpm g-force may damage the DNA quality and break the NucleoSpinR Bead Tubes). To adjust the conditions for DNA binding, added 600 µl of MG-Buffer and vertex mix 3-4 times before centrifuging to sediment steel bread and cell debris. Furthermore, the supernatant (500-600 µl) was transferred into the NucleoSpin R DNA Insect Column, which was placed in a 2 ml collection tube. The collection tube was discarded after centrifugation, and the column was placed in a new collection tube. The silica membrane of the column was washed with both 500 µl buffers BW and B 5 . Furthermore, the silica membrane was dried for 5 min to remove the residual effect of the wash buffer. Finally, the NucleoSpin R DNA insect column was placed into a 1.5 ml nuclease-free tube and 100 µl of elution buffer BE was added to the column. It was incubated at room temperature for 1 min., followed by centrifugation for 1 min. at 11,000 rpm. The concentration of DNA was checked on an illuminated UV screen (NanoDrop, Thermo Scientific), followed by gel electrophoresis on 0.8% agarose at 100 V for 20 min. Depending on the concentration, the DNA samples were used for PCR (Polymerase Chain Reaction) purification.

PCR amplification and 16 S rRNA sequencing
Polymerase Chain Reaction (PCR) amplicon size (1500 bp) nearly full-length 16S rRNA genes were amplified from the DNA extracted from bacterial isolates by using the primer set 16SrRNA (27F: 5'-AGA GTT TGA TCC TGG CTC AG-3') and 16 S rRNA (1392R: 5'-GGT TAC CTT GTT ACG ACT T-3') and by using PCR conditions. The primer pair 16F27/16R1392 was capable of retrieving 16 S rRNA genes from a phylogenetically and taxonomically wide range of bacterial genera (Weisburg et al., 1991).The PCR reaction mixture was prepared using 6.0 µl genomic DNA, 1.5 µl of each primer, 12 µl of Premix Ex Taq II Green PCR Master Mix (Takara Bioscience Inc.) and 7 µl dH2O). The PCR cycling parameters were one denaturation cycle of 94 o C for 5 min, followed by 40 cycles of 94 o C for 1 min, 52 o C for 1 min, 72 o C for 1.40 min, followed by a final extension at 72 o C for 10 min. PCR amplification products were separated by electrophoresis in a 1.5% agarose gel in TAE buffer (40 mMTris-acetate [pH 8.0], 1 mM EDTA) at 100 V for 30 min. The gel was then visualized under ultraviolet light, and final images were taken for a gel documentation system.

Data analysis of sequencing results
The raw sequencing results obtained from a sequencer were checked for quality parameters. After trimming the unwanted sequences from the original paired-end Fig. 2 Damage symptoms caused by FAW and gut isolation in laminar air flow hood data, a consensus region sequence was constructed using BioEdit version 7.2.5 for Windows (Hall, 1999). Then applied multiple filters, viz., conserved region filter, the spacer filter, and the mismatch filter and the highest quality sequences were taken for various downstream analyses, and the sequences were submitted to NCBI GenBank. The pair-wise alignment done through cluster W and the phylogenetic tree were constructed using MEGA11 software version 11.0.11 with neighbor-joining (NJ) with complete gap detection resampled and the alignment of maximum composite likelihood with 1000 bootstrap replications (Tamura et al., 2004(Tamura et al., , 2021. The phylogenetic tree was generated using the ribosomal RNA gene sequences from the NCBI gene bank database. The relative abundance of the bacterial species communities was subjected to statistical analysis (Fig. 5). Representatives of that bacterial community closely assembled with isolates of the gut of S. frugiperda are presented in Fig. 6.

Results
The experimental findings revealed the composition of bacterial communities present in the larval gut of S. frugiperda and grouped them into each taxonomic category from phyla to species level. The abundance of major bacterial groups in each taxonomic category was given in Table 2. Altogether, only two bacterial phyla were mostly abundant across the 12 samples, i.e., proteobacteria (59.32%), and firmicutes (40.68%) contributing their roles as insect habits and habitats. Bacilli and Gammaproteobacteria were found in operational taxonomic units (OTUs) of the FAW gut population, according to class-level identification. As far as analyses at the genus level are concerned, despite the high genus-level diversity between samples (Fig. 3), there were nine (09) different genera observed in the collected samples from different locations. For example, there was a very high proportion of Enterococcus in the samples from Begusarai, Purnea, Ktihar, Birbhum and Kaimur, followed by Klebsiella in larval samples from Rohtas, Sahebganj and Banka, while Khagaria, Bhagalpur, Samastipur and Munger samples have a group of bacterial communities. Interestingly, two new genera of bacteria, i.e., Kluyvera and Yokenella, were recorded from the gut of S. frugiperda in the present study, which have not been reported to date in any of the other countries mentioned in Table 3, and these may be new findings from samples collected in different locations in the eastern part of India.
Apart from that, at the species level identification revealed that there is a huge diversity of bacteria associated with the gut of S. frugiperda. A total of 33 species were found from the 12 samples of S. frugiperda collected from different locations. Among the 33 species, the highest relative abundance was recorded with Klebsiella pneumoniae, followed by Enterococcus hirae, Enterococcus mundtii, and Klebsiella oxytoca species, which were mostly abundant across the FAW gut samples and covered 50% of the total bacterial community richness, while a small proportion of Raoultella, Citrobacter, Leclercia, and Pantoea were contributing their roles in the gut of S. frugiperda (Figs. 4 and 5), respectively, to the bacterial community richness and diversity (Fig. 6),

Discussions
The symbiotic microbiomes associated with the gut of S. frugiperda play a critical role in insect lifecycles, and the insect gut microbiome is influenced by many factors such as host diet and taxonomy, which jointly affect the gut bacterial community composition and also play diverse roles in the growth and development of many insect species (Zheng et al., 2021;Li et al., 2022). However, bacterial communities in S. frugiperda remain poorly studied. Hence, the symbiotic associations between the insects and their gut bacteria have been studied, and it was revealed that Firmicutes and proteobacteria were the most dominant groups in the larval gut of S. frugiperda, according to phyla-level bacterial community analysis. The present finding is similar to the proportion reported in reports of phytophagous lepidopteran insects (Xia et al., 2013(Xia et al., , 2017Landry et al., 2015;Ramya et al., 2016;Snyman et al., 2016;Strano et al., 2018;Chen et al., 2018 Gichuhi et al., 2020;Rozadilla et al., 2020;Ugwu et al., 2020). In the same way, seven of the isolated bacterial genera namely, Enterococcus, Pseudomonas, Comamonas, Stenotrophomonas, Eshcerichia-Shigella, Acinetobacter andCarnobacterium, have been recorded using a similar methodology in the beet armyworm, S. exigua (Gao et al., 2019). The microbial diversity richness varies from larval to adult stages of S. frugiperda; in the larvae, i.e., the 6th larval instar, the highest microbial diversity was found rather than from the egg to the adult stage of males and females. Firmicutes were the most abundant bacterial community at the late larval stage, while the proteobacteria population was more abundant in the egg and adult stages. At the genus and species level, the community composition of Enterococcaceae and Enterobacteriaceae is significant (Li et al., 2022). These findings revealed that some bacterial genera are often associated with lepidopteran insects, although it is difficult to define a core microbiota for such a diverse insect order. The result was obtained despite the significant bacterial diversities in OTU composition between larvae from different sites. This was most likely caused by complex biological and environmental factors in the diverse agro-ecological zones that were sampled. Polyphagous feeding habits also play a vital role in influencing the microbiome of lepidopterans (Strano et al., 2018;Sittenfeld et al., 2002;Priya et al., 2012;Montagna et al., 2016), though in this study all samples were collected from maize plants. Therefore, the observed compositional differences are not likely to be caused solely by diet. It is interesting that many of the detected bacterial genera, such as Enterococcus, Klebsiella, Raoultella, Enterobacter, Citrobacter, Pantoea, Leclercia, Kluyvera and Yokenella were found in larval life stages, which suggests that gut bacterial community members are transmitted across developmental stages. The bacterial communities that continually pass on across developmental stages (early instar to late larval instar) may develop a mutualistic relationship with their hosts (Moran, 2006). Hence, studies should examine the effects of these microbes on host fitness and investigate the extent to which they are vertically transmitted from parents to offspring. In contrast, Citrobacter and Sphingobacterium were observed to be differentially abundant in larvae than in adults, an additional indicator that these two genera may be part of the fraction of bacterial communities that are lost during the transition of S. frugiperda into the adult stage.   On the other hand, the described taxonomic and functional profile of bacteria associated with the gut of S. frugiperda larvae plays an important role in the insect's fitness, is beneficial for its abilities of invasiveness and adaptation, and is an invaluable tool for identifying new pest control strategies. Therefore, it is of great importance to systematically understand the microbial dynamics of S. frugiperda (Li et al., 2022). Nevertheless, there are extremely few reports on the functionally active profile of the gut bacteria that influence different physiological aspects, especially in lepidopteran insects (Rozadilla et al., 2020;Xia et al., 2017;Kyritsis et al., 2019;Meng et al., 2019). The phylum Firmicutes and Proteobacteria mostly play a defensive role in the lepidopteran insect gut, such as carbohydrate metabolism, nitrogen metabolism, genetic information processing, essential nutrient provisioning, followed by digestion and detoxification, and the development of resistance, except for the genus Pantoe, which is mostly toxic increase susceptibility to toxins by affecting midgut epithelial permeability (Broderick et al., 2009;Mason et al., 2011;Jing et al., 2020). In the case of the lepidopterans and coleopterans, the anal droplet enzymes degrading plant secondary metabolites (PSMs) showed the digestion routes of cellulose, starch, trehalose, sucrose, pectin, arabinan, galactan, xylan, chitin, etc. (Jing et al., 2020).

Conclusion
The gut bacterial communities in S. frugiperda larvae samples randomly collected from several locations in eastern India, finding some important differences and similarities with the previous report, have been published across the country in relation to other studies on this species. Almost all the bacterial genera under the phylum Firmicutes and Proteobacteria play a defensive role in the lepidopteran insect gut, such as carbohydrate metabolism, nitrogen metabolism, genetic information processing, detoxification, and the development of resistance, except the genera Pantoe, which are mostly toxic and increase susceptibility to toxins by affecting midgut epithelial permeability. The result was obtained despite the significant bacterial diversities in OTU composition between larvae from different sites. This was most likely caused by complex biological and environmental factors in the diverse agro-ecological zones that were sampled. Polyphagous feeding habits also play a vital role in influencing the microbiome of lepidopterans. From this study, two new genera of bacteria, i.e., Kluyvera and Yokenella, were recorded from the gut of S. Fig. 6 Neighbor-joining tree of bacteria OUT detected in S. frugiperda samples from Eastern India. Similar sequences are labeled with their accessions number followed by genus, species were reported. Branches with a bootstrap value less than 50 are collapsed. A sequence from a species 0.01 frugiperda, which had not been reported so far from any other countries.