Gut microbiome of Oecophylla smaragdina: potential functions in host and environment

doi:10.21203/rs.3.rs-3250723/v1

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Gut microbiome of Oecophylla smaragdina: potential functions in host and environment

https://doi.org/10.21203/rs.3.rs-3250723/v1

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Oecophylla smaragdina (red weaver ant) is widely consumed either in the form of protein and nutrient rich diet or as a source of medicine provided by traditional healers in various parts of the world. Similar to other animals, ants also form mutualistic relationship with the microbes that live inside them. These endosymbionts perform various crucial functions in their host such as nutritional upgrading, digestion, nitrogen recycling and pathogen defence. However, not much literature is available on the role of gut specific microbes and their functions in the ant host. In this study, we have determined the microbiome profile of O. smaragdina colonies using 16S rRNA sequencing and biochemical characterization for understanding the role of both culturable and non-culturable microbes present in ant gut. While, a number of microbes are reported for the first time, Enterobacteriaceae and Staphylococcaceae were found to be the most dominant families found in O. smaragdina. Some endosymbionts were similar to human gut microbiota depicting the diversity of microbes inside an ant gut. We also focused on the bioactive compounds produced by endosymbionts, which may find an application in the treatment of various ailments in humans.

Endosymbionts

antimicrobial

defence

ants

metabolism

bioactive compounds

sequencing

Ants (family: Formicidae) perform various functions in terrestrial ecosystem including soil aeration, seed dispersal, plant protection against insect pests and pathogens as well as a source of food for other organisms (Heil 2008; Rajagopal et al. 2019). They are especially well-known for their division of labour and well-developed social organization (Offenberg 2001; Peng and Christian 2004; Wernegreen et al. 2003). Amongst ants, Oecophylla smaragdina is of special importance as it has been used in modern medicine as well as by traditional healers for their nutritional and medicinal value (Chellappandian et al. 2014; Langthasa et al. 2017; Van Huis 2021; Vidhu and Evans 2015). Its larvae and pupae are known to have a high nutritive value because of which they are being used as delicacies in Australia, Thailand, Philippines, Myanmar, and some parts of India (Chakravorty et al. 2016; Pattarayingsakul et al. 2017; Roy and Rao 1957; Srivastava et al. 2009).

Throughout their lifetime, ants form various associations with different organisms which range from mutualistic to parasitic (Davidson et al. 2003; Eilmus and Heil 2009; Hölldobler and Wilson 1990; Kautz et al. 2013). Among these associations, their interaction with microbes, especially endosymbionts, is vital for their life processes (Currie et al. 1999; Lanan et al. 2016). Within class insecta, ants are known to harbour most abundant microbiota in their gut (Hosmath and Timmappa 2019). These symbiotic microbes associated with ants play an important role in the development, nutrition, reproduction and survival of their host (Gil et al. 2003; Sauer et al. 2000; Werren 1997). Moreover, symbiotic association between ant and microbes is also known to play crucial role in the evolutionary success of the ants (Russell et al. 2009). It has been inferred by Anderson et al. (2012) that ant symbionts play a significant ecological role as they have co-evolved, one of the mechanisms behind which could be phoresis or trophallaxis. The earliest known endosymbiont, Blochmannia sp., was reported from the Camponotus genus of ants (Blochmann 1892; Wernegreen et al. 2009).

There has been an increasing interest in determining the contribution of endosymionts in the host metabolism. For example, Blochmannia plays an important role in enhancing competitive ability, nutritional upgrading, improving immune defence as well as pheromone production in its ant host (de Souza et al. 2009; Degnan et al. 2004; Feldhaar et al. 2007; Sauer et al. 2000). Asaia, a gram negative bacteria is known to possibly contribute in utilizing sugar rich substrate and nitrogen fixation in Cephalotes varians (Kautz et al. 2013). Endosymbionts present in the herbivorous giant turtle ant (Cephalotes atratus) are known to supplement the nitrogen-rich nutrients which are absent in their diet (Kelly et al. 2019). Symbionts such as Enterobacter, Citrobacter, Klebsiella and Pantoea play various roles in nitrogen fixation and lignocellulose degradation in ant cultivated gardens (Aylward et al. 2012; Mueller et al. 2005; Pinto-Tomás et al. 2009). Some gut microbes such as Wolbachia and Spiroplasma are known for influencing sex ratios in host and reproductive manipulation by male killing (Pontieri et al., 2017). Another important function of Spiroplasma is protecting its host of genus Myrmica from natural enemies, as this ant is susceptible to parasitic infections (Witek et al. 2014).

Studies have revealed that gut microflora play a crucial role in host defence against pathogens by the production of bioactive compounds (Agarwal et al. 2022; Kellner et al. 2015; Zakalyukina et al. 2019). These bioactive compounds, with potential therapeutic properties, were reported in the methanolic extract of ants (Natarajan et al. 2019). Ants also produce a number of antimicrobial peptides as well as elicitor molecules from their exocrine glands that can be helpful in pathogen recognition and defence (Schluns and Crozier 2009). Similarly, ant endosymbionts are also capable of antibiotic production. One such example is of Psuedonocardia known for antibiotic production in higher attine ants against variety of fungal and bacterial microbes (Mueller et al. 2010; Oh et al. 2009). It has also been reported that chemicals derived from ants and their microbiome have a great potential to combat plant pathogens (Offenberg et al. 2022). Some ant endosymbionts are also known to produce bioactive compounds that are found to be beneficial in treating human diseases (Zakalyukina et al. 2019). For example, endosymbionts including Serratia and Chromobacterium violaceum produces a number of secondary metabolites which have different types of biological activities (Suryavanshi et al. 2017; Suryawanshi et al. 2015).

Hence, the study of microbial population present in the ant gut is important as it will help in identification of microbial diversity and functions in the host. 16S rRNA gene amplicon sequencing is a well-known technique that can be used for the identification of novel culturable and non-culturable bacteria present in the ant gut (Clarridge III 2004). However, there is a dearth of literature available on the functions of the gut microbiota present in O. smaragdina. In this study, we identified the gut microbiome of O. smaragdina workers through biochemical and genomic studies. Worker ants were selected because of their importance in traditional medicine and their restorative properties (Gahukar 2016). We also discusses the role of gut microflora in the growth, development and overall fitness crucial to their host ant as well as their potential therapeutic applications in humans.

Sample collection:

O. smaragdina colonies were collected from the leaf nest on mango tree from Khizirsarai, Gaya, Bihar, India located between the 24º 46' 48.0360'' N latitudes and 84º 58' 54.5772'' E longitudes. Ant colonies were kept in polypropylene insect cage (21.5×21.5×21.5 cm) and were provided with a mango twig, which was changed every 4th day. The cage was maintained at temperature of 27 ± 2^º C and relative humidity of 40%. The ants were provided with 50% honey solution twice a week (He et al. 2014). After rearing for one month, the gut of worker ants was extracted in sterile environment.

Dissection of ant gut:

Fifteen worker ants were surface sterilized by 70% ethanol and placed in a petri-plate containing double distilled water. The whole gut was dissected out from ant gaster (abdomen) using sterilized fine-tip forceps and scissors He et al. (2014). The guts of all workers were extracted and placed in sterilized 1 ml microcentrifuge tube (1 gut per tube) individually and stored in -80º deep freeze for DNA extraction.

Microbial isolation:

The guts of ten lab-raised worker ants were pooled in one sample in a microcentrifuge tube for bacterial isolation. Microbial isolation was performed as per (He et al. 2014) with slight modifications. Fifty microliters of 10 mM Phosphate Buffered Saline (PBS) was added to each sample. Samples were homogenized with a sterile micro pestle and then serially diluted up to 10^− 2, through ten-fold serial dilution. These diluted gut samples were then inoculated on Tryptic soy agar (TSA), Luria-Bertani agar (LB), Reasoner’s 2 agar (R2A) and Nutrient agar media (NAM). Plates were kept in incubator at 28 and 37°C to obtain maximum number of culturable endosymbionts from ant gut.

Biochemical characterization of microbial isolates:

Microbial growth on different media plates was observed after 24 hours for their Colony Forming Units (CFUs) and morphological features such as colour, texture, margin and elevation (Table 1). Biochemical characterization of the obtained microbes were performed as per Bergey’s manual for Gram staining, citrate utilization, lysine decarboxylase, lactose, glucose, sucrose, DNase, Sulphur Indole motility (SIM) media, MacConkey agar assay, catalase and Voges Prosker Test (VP) (Bergey 1994; Sneath et al. 1986).

Table 1

Biochemical tests for the isolation of culturable microbes from *Oecophylla* gut
Organism	Gram staining	DNase	EMB	MacConkey	Citrate Utilization	Lysine Decarboxylase	Triple sugar iron	Sulphur Indole	Motility	Voges Proskauer	Colour	Texture and Margin	Form	Elevation
Pseudomonas aeruginosa	-	+	+	+	+	-	Non-fermenter	-	+	+	Yellow	Smooth with entire margin	Circular	Raised
Serratia marcescens	-	+	+	+	+	-	Glucose fermenter	+	+	-	Cream	Smooth with undulate margin	Irregular	Flat
Proteus mirabilis	-	-	-	-	-	-	Glucose and Lactose fermenter	-	+	+	transparent	Rough with entire margin	Circular	Flat
Enterococcus faecalis	+	-	-	-	-	-	Glucose fermenter	+	-	+	Yellow	Smooth with entire margin	Circular	Flat
Salmonella sp.	-	-	-	-	-	-	Glucose and Lactose fermenter	-	+	+	off white	Smooth with entire margin	Circular	Raised
Staphylococcus epidermis	+	-	-	-	-	+	Glucose and Lactose fermenter	-	+	+	Cream	Rough with undulate margin	Irregular	Flat
Staphylococcus epidermis	+	-	-	-	-	+	Glucose and Lactose fermenter	-	+	+	White	Smooth with undulate margin	Punctiform	Flat
Shigella sp.	-	-	-	-	-	-	Non-fermenter	-	-	+	off white	Smooth with entire margin	Circular	Raised
E. coli	-	-	-	-	-	-	Glucose and Lactose fermenter	+	+	+	White	smooth with undulate margin	Punctiform	Raised
Proteus mirabilis	-	-	-	-	-	-	Glucose and Lactose fermenter	-	+	+	off white	Smooth with undulate margin	Irregular	Raised
Enterobacter sp.	-	+	+	+	+	-	Glucose and Lactose fermenter	-	+	+	Cream	Smooth with undulate margin	Irregular	Umbonate
Staphylococcus aureus	+	+	+	-	-	-	Lactose fermenter	-	-	-	Cream	Smooth with undulate margin	Irregular	umbonate

16S rRNA sequencing analysis

For DNA extraction, five biological replicates were pooled for one sample. Genomic DNA was extracted by using Xpress DNA™ Tissue/Cell Line DNA Extraction Kit (MagGenome® Technologies Pvt. Ltd., India) as per manufacturer’s instructions. The DNA isolation was performed in three replicates. The concentration and purity of the extracted DNA sample was analysed using Thermo Scientific µDROP™ Plate (Thermo Fisher Scientific, USA).

Raw reads were generated for the samples using V3 and V4 amplicon sequencing. Quality check was done on raw fastq files for checking the base quality, base composition and GC content using FASTQC toolkit which also excluded low quality reads from the analysis. The forward and reverse reads were overlapped and stitched together to form longer reads of greater than 400 bp using FLASH programme. Stitched reads were pre-processed using VSEARCH for the identification of unique sequences and chimera removal was done using uchime (Edgar et al. 2011) utility from VSEARCH. Further analysis was performed using QIIME 1.

Sequences with a similarity of 97% were grouped together using a closed_reference method under a single operational taxonomic unit (OTU represents species richness) for classification of the whole data (Rideout et al. 2014). Any OTU that had a count of 1 in a single sample (identified as singletons) were filtered out as these may have been created as experimental artifact. OTU for microbes were obtained using RDP classifier after mapping the final reads.

For taxonomic classification one representative sequence was picked from each OTU and classified using RDP classifier against Silva-132 database at 97% similarity. The taxonomy classification and relative abundance were done using QIIME1 and the classification was done at phylum, class, order, genera and species level (16S rRNA sequencing was performed by Barcode Biosciences, India).

Microbial isolation:

Our study revealed 12 different strains of culturable microbes in the gut of O. smaragdina. Biochemical characterization analysis showed that eight gram negative and four-gram positive bacteria have been reported inside the gut of this ant. Microbial profile included Staphylococcus epidermis, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Enterobacter sp., Proteus mirabilis, Pseudomonas aeruginosa, Salmonella sp., Shigella sp. and Serratia marcescens. Table 1 provides gut microbial profile of O. smaragdina based on morphological characteristics and different biochemical tests performed. Table 2 describes the microbial density in terms of colony forming unit (CFU).

Table 2

Microbial density obtained in colony forming unit (CFU) in different agar media
Media	Dilution 10^− 1	Dilution 10^− 2	CFU/ml of different dilutions	No. of Morphologically different colonies
TSA	0	0	0	2
R2A	61	4	3.13×10⁴	5
NAM	252	0	4×10³	5

16S rRNA sequencing analysis

Sequencing of 16S libraries produced 4,81,823 raw reads, which have revealed a mean phred score (Cock et al. 2010) of 36.25 displaying 99% accuracy with 0.1% sequencing error rate. The obtained sequenced reads were then overlapped and stitched to form longer reads greater than 400 bp using FLASH program which has given 4,59,154 combined pairs. After de-replication and chimera removal 1,76,298 high quality reads were obtained. Over 0.17 million quality filtered reads were clustered to obtain 80 families.

Microbial profile obtained through 16S rRNA analysis contains both culturable and non-culturable microbes. The characterization of microbial community that inhabits O. smaragdina by sequencing of 16S rRNA genes led to the identification of 12 phyla, 27 classes, 56 orders, 80 families, 130 genus and 204 species. Proteobacteria, Firmicutes and Actinobacteria were the three most abundant phyla constituting almost 70% of the bacterial communities (Fig. 1). Out of the 27 classes reported, Gammaproteobacteria was the most abundant (36%), followed by Bacilli (23%) and Actinobacteria (9.4%), respectively. Among the obtained orders, Bacillales was the most abundant order (22.9%) followed by Enterobacteriales (22.8%) and Clostridiales (8.6%). Out of the 80 families found, Pseudonocardiaceae was the most abundant family and the remaining were either mostly unidentified or least abundant. The families with maximum relative abundance are given in Table 3. The two most abundant genera were Staphylococcus and Enterobacter with relative abundance of 17% and 8.8% in terms of OTU. Other abundant microbes reported were Megamonas sp., Blautia, Stenotrophomonas, Stenotrophomonas, Prevotella-9, Bifidobacterium longum, Staphylococcus warneri, Cornybacterium-1 and Pseudomonas. The genera with maximum abundance are provided in Fig. 2.

Table 3

Most abundant families found in the O. smaragdina microbiome

S.No.

Family

Relative abundance

Enterobacteriaceae

22.8%

Staphylococcaceae

19.7%

Lachnospiraceae

7.8%

Bacteroidaceae

4.1%

Burkholderiaceae

3.7%

Veillonellaceae

3.7%

Xanthomonadaceae

3.4%

Propionibacteriaceae

2.8%

Prevotellaceae

2.8%

10.

Moraxellaceae

2.5%

11.

Pseudomonadaceae

2.3%

12.

Rhodobacteraceae

2.2%

13.

Bifidobacteriaceae

2.16%

14.

Corynebacteriaceae

2.13%

15.

Bacillaceae

1.56%

16.

Sphingomonadaceae

1.47%

17.

Coriobacteriaceae

1.43%

18.

Planococcaceae

1.27%

19.

Micrococcaceae

1.02%

The sequencing and microbial isolation studies on the workers of O. smaragdina revealed that different bacterial taxa exist inside the gut of this ant. Microbial communities obtained in our study were different from the earlier study conducted by (Chua et al. 2018) in Malaysia, which may imply that geographical isolation or habitat play an important role in the variation of microbial diversity within the same species. Out of the 80 obtained families, Entomoplasmataceae, Moraxellaceae, Enterobacteriaceae, Lactobacillaceae and Acetobacteraceae were similar to the available report on O. smaragdina gut microbiome (Chua et al. 2018). The two most abundant families amongst these are Enterobacteriaceae and Staphylococcaceae in both 16S rRNA analysis and microbes isolated in culturable conditions. The microbes that were common in both culturable and non-culturable analysis were Pseudomonas, Klebsiella, Bacillus, Enterobacter, Shigella and Staphylococcus. The microbes reported in this study are known to play crucial roles in the host as well as in the environment. These bacterial mutualists play a crucial role in nutrient upgradation, metabolism and nitrogen recycling in their ant host (Chua et al. 2020; Feldhaar et al. 2007; Zientz et al. 2006).

Amongst the two most abundant families, the genus reported under Enterobacteriaceae were Enterobacter, E. hormaechei subspecies, Klebsiella, Buttiauxella, Escherichia and Shigella. In contrast, the species diversity in Staphylococcaceae was lesser in our study as only one species was reported (Staphylococcus warneri). Out of the commonly detected microbes, Enterobacter is known to be involved in nitrogen, starch and sucrose metabolism as well as helps in the biosynthesis of various amino acids (Aylward et al. 2012). Other microbial species such as Pseudomonas, Bacillus and Burkholderia reported in the present study are known for nitrogen fixation and also provides essential nutrients to ants (Van Borm et al., 2002; Aylward et al., 2012; Oliveira et al., 2016). Pseudomonas is also used as a potential biocontrol agent against soil borne pathogens (Haas and Défago 2005). Microbes such as Sphingobium, Flavobacterium, Acidobacterium and Burkholderia belonging to other families are also known to help in metabolism and nitrogen fixation in ant Liometopum apiculatum and might play a beneficial role in Atta cephalotes and Acromyrmex echinatior (González-Escobar et al. 2018; Zhukova et al. 2017). It has been reported that Bacillus, Klebsiella and Burkholderia isolated from the legs of the ants is known to inhibit pathogenic strains of Pseudomonas sp. and Escherichia coli in ant mutualist plant (González-Teuber et al. 2014). A recent study also suggests that chemicals derived from ant gut microbes have more antagonistic activity against plant pathogens in comparison to microorganisms from other sources (Offenberg et al. 2022). However, the potential of endosymbionts is not just limited to their functions in the host. They also play a crucial role in the production of antimicrobial secondary metabolites that can be used further for the development of antibiotics.

S. warneri produces bacteriocins such as Warnericin RB4 and S. warneri RK bacteriocin, which are known to inhibit the growth of both pathogenic and non-pathogenic bacteria namely, Legionella pneumophila and Alicyclobacillus acidoterrestris (Héchard et al. 2005; Minamikawa et al. 2005). Another common microbe found in our 16S rRNA study was Bacillus altitudinis, which has earlier shown potential antimicrobial activity against Micrococcus luteus (Ngalimat et al. 2019). Contrary to B. altitudinis, endosymbiotic E. hormaechei subsp. is known to carry genes that confers resistance to β-lactam and other antibiotics (Soliman et al. 2020; Yang et al. 2018). Ant endosymbionts are known to spread through nosocomial infection to humans hence, hospital transmission of such antibiotic resistant genes is possible (Moreira et al. 2005; Samuels et al. 2013; Teixeira et al. 2009). One such example is Corynebacterium striatum, which is a part of normal skin microbiota, and is an aetiologic agent of multidrug resistance via nosocomial infection, but is also identified as a component of O. smaragdina gut microbiota in our study (Silva-Santana et al. 2021; Souza et al. 2015). However, besides nosocomial infection, entomophagy plays a notable role in the transmission of such antibiotic resistant bacteria. Ants, especially O. smaragdina, is being consumed as delicacy in various countries due to their high nutritive value (Chakravorty et al. 2016; Pattarayingsakul et al. 2017; Roy and Rao 1957; Srivastava et al. 2009) which suggests that people eating them might get resistant to various antibiotics.

Furthermore, some human gut microbes are also reported in the 16S rRNA analysis of O. smaragdina such as Megamonas, Bacteroides NLAE-zl G24, Fusicatenibacter sp., Achromobacter xylosoxidans, Butyricicoccus sp., Alloprevotella, Lachnoclostridium, Bacteroides thetaiotaomicron, Blautia obeum, Haemophilus haemolyticus and Bifidobacterium. Most of these microbes are known for their anti-inflammatory properties (Eeckhaut et al. 2013; Hatziioanou et al. 2017; Ng et al. 2022; Riedel et al. 2006). Hence, these microbes may serve as potential drug candidates against various inflammatory diseases.

Additionally, we report, for the first time, Bifidobacterium longum, Blautia obeum, Collinsella, Dialister, Haemophilus haemolyticus, Prevotella-9 and bacteria belonging to Euzebyaceae family in the ant gut. Bifidobacterium longum is normally found in human intestine and few strains of this microbe are known to cure infectious diseases including cancer (Yazawa et al. 2000). Euzebyaceae members are found in dry valleys in Antarctica. However, very little is known about their functional role in their environment (Rego et al. 2019). Species of Prevotella-9, Collinsella and Dialister have been reported in the gut of myrmecophagus animals till date, indicating the possibility that they get transferred through ant specific diet (Delsuc et al. 2014).

Our study suggests that habitat variation plays a key role in microbial diversity. Hence, there is a scope to explore in terms microbial community existing in different body parts of these ants. However, detailed studies maybe conducted to identify specific therapeutic role of the metabolites produced by these microbial symbionts. It is still unclear that whether the metabolites are produced by gut microbiota or the ant itself releases metabolites with bioactive potential due to the microbe intervention. Further research on the ant gut microbial extracts in-vitro, in-silico and through biochemical analysis may provide better understanding of the potential of such microbes in treating various health ailments.

Conflict of interest:

The authors have no competing interests to declare that are relevant to the content of this article.

Authors Contribution:

SA and VM conceived and designed research. SA conducted the experiments. SA and GS analysed the sequencing data. SA wrote the whole manuscript. All authors have read and approved the manuscript.

Acknowledgment:

The authors would like to acknowledge the support by University Grants Commission (UGC), India for granting Junior Research Fellowship to SA for pursuing her PhD. The authors would also like to thank Ms. Kavita Verma (PhD scholar, Animal Plant Interactions Lab) for her assistance during sequencing result analysis.

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Gut microbiome of Oecophylla smaragdina: potential functions in host and environment

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Abstract

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Introduction

Material And Methods

Sample collection:

Dissection of ant gut:

Microbial isolation:

Biochemical characterization of microbial isolates:

16S rRNA sequencing analysis

Results

Microbial isolation:

16S rRNA sequencing analysis

Discussion

Declarations

Conflict of interest:

Authors Contribution:

Acknowledgment:

References

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Version 1