DOI: 10.31038/MGJ.2022512

Abstract

The complete mitochondrial genome (mitogenome) of the comma butterfly, Polygonia c-aureum (Lepidoptera: Nymphalidae) is determined in this study. It is a circular molecule of 15,208 bp, containing 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and an A+T-rich region, which is a common feature of lepidopteran mitogenomes. Based on nucleotide sequences of 13 protein-coding genes, we reconstructed the phylogenetic relationships among 87 species of the family Nymphalidae using Bayesian Inference (BI) and Maximum Likelihood (ML) methods, and calculated the divergence times using multiple fossil calibrations. The phylogenetic analyses supported the sister-group relationship between the subfamilies Nymphalinae and Cyrestinae. Moreover, monophyly of the Nymphalidae was strongly supported. The results were highly consistent with the traditional relationships within the Nymphalidae from morphological data. For the first time, our results suggest that the genus Polygonia diverged from the common ancestor of the rest of Nymphalinae at 45.64 Ma. In addition, the first divergence time in the Nymphalidae is in the Early Cretaceous, about 89.72 Ma.

Keywords

Mitochondrial genome, Molecular phylogeny, Divergence time, Nymphalidae, Polygonia c-aureum

Introduction

The Nymphalidae is the largest family of butterflies, including 7,200 species belonging to 600 genera and 12 subfamilies [1-6]. Consequently, it has been the subject of intense studies [7-9]. Nymphalidae is the first taxa that helped us to begin to understand the complex relationships between insects and their host plants [10], the effects of habitat fragmentation on the population dynamics of endangered species [11], and the genetic mechanisms behind the developmental pathways of morphological features [12], and the coevolutionary interactions between organisms in mimicry rings and aposematic coloration [13,14]. Especially the butterflies of the subfamily Nymphalinae [5] have extensively contributed to our knowledge on ecological and evolutionary processes [15-18]. However, the phylogenetic relationships among the different subfamilies and tribes have been chaotic because of the variable shapes and life cycles, it made them become the argue focus for taxonmists [6,19-21]. There are still several competing classification schemes based on different data sets and researchers [5,21,22]. With the development of sequencing technologies and increasing number of molecular data set, more and more researches investigated the phylogenetic relationships of butterflies. For example, [23] using the wingless gene, and [5] using COI, EF-1α and wingless genes, both including good taxonomic coverage of the Nymphalidae, showed that many of the traditional subgroups are monophyletic. [7] inferred a robust phylogenetic hypothesis based on 10 genes and 235 morphological characters.

Meanwhile, there are many difficulities in the research of orgin and evolution in most of the families, in view of the lack of fossils data. [7] used a surprisingly good fossil record for the Nymphalinae to estimate the ages of diversification major lineages using Bayesian relaxed clock methods, suggesting that the age of Nymphalidae is older than 70 million years. [24] explored the divergence time in butterflies using the sequences of ultraviolet-sensitive (UVRh), blue-sensitive (BRh), long-wavelength sensitive (LWRh) opsins, EF-1α and COI obtained from 27 taxa representing the five major butterfly families.

The comma butterfly, Polygonia c-aureum, is a major defoliator leaf pest on the scandent hostlant Humulus scandens (Lour.) Merr., which is used for medicine in China [25]. Here, we sequenced the complete mitochondrial genome (mitogenome), which could can be used to develop molecular markers for phylogenetics and, identification, and also to examine the evolution of Nymphalidae. In addition, we hope our study would be useful for the prevention and control of insect pests.

In this study, based on the complete mitogenome sequences of P. c-aureumy and additional homologous sequences of 86 species downloaded from GenBank, we estimated the divergence times of Nymphalidae, to enhance our understanding of the origin and evolution of this family, and to provide a relative accurate results for estimating divergence times of butterflies.

Materials and Methods

Sample Collection and DNA Extraction

The adult specimens of P. c-aureum were collected from Nanjing, Jiangsu Province in China. After an examination of external morphology for identification, the fresh adult specimens were directly frozen and maintained at -80°C until DNA extraction. Total genomic DNA was extracted from adult butterfly tissues, typically thorax or abdomen, using the Wizard Genomic DNA Purification Kit (Promega, Beijing, China) according to the manufacturer’s instruction. The extracted DNA was stored at -20°C and used for PCR amplification of the complete mitogenome.

Primers Design, PCR Amplification and DNA Sequencing

In order to amplify the complete mitogenome of P. c-aureum, nineteen pairs primers were designed and synthesized. Among them, four pairs are lepidoptera universal primers [26,27], twelve pairs specific primers for this study were designed using Primer Premier 5.0 software [28] and the remainder of three pairs primers were the combination of universal primers and specific primers. Detailed information about primers used in this study are shown in Table 1.

Table 1: Primers used for amplification of the Polygonia c-aureum mitogenome

a: Primers modified from Simon et al. (1994) up to this mtgenome
b: Primers from Simon et al. (2006)
c: Primers newly designed for this genome

Some PCR reactions (the target fragments <2 kb) were performed in a 25 μL volume with 0.2 μL rTaq (TaKaRa Co., Dalian, China), 1 μL of DNA, 2.0 μL dNTPs, 2.0 μL 25 mM MgCl₂, 2.5 μL 10× rTaq buffer (Mg²⁺ free), 0.5 μL each primer and 16.3 μL sterile distilled H₂O. The PCR amplification was performed using the following cycling protocol: an initial denaturation for 5 min at 94°C, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C~59°C (depending on primer pairs) for 30 seconds, extension at 72°C for 1~2 min, with a subsequent 10 min final extension at 72°C. Besides, the other PCR reactions (the target fragments ≥ 2 kb) were carried out with 25 μL reaction volume containing 0.2 μL of LATaq (TaKaRa Co., Dalian, China), 1 μL of DNA, 4.0 μL dNTPs, 2.5 μL 10×Taq buffer (Mg²⁺ plus), 16.3 μL sterile distilled H₂O and 0.5 μL each primer. The fragments were amplified under the following cycling protocol: 5 min of initial denaturation at 94°C, followed by 35 cycles of denaturation for 30 seconds at 94°C, annealing at 50°C~59°C (depending on primer pairs) for 30 seconds, extension at 72°C for 1~2 min, with additional 5 seconds for each cycle, and a final extension for 10 min at 72°C.

Products were examined by electrophoresis on 1% agarose gel. All the PCR fragments were directly sequenced from both strands by Jin Si Rui Company, Nanjing, China and Sheng Gong Company, Shanghai, China with the PCR primers.

Sequence Assembling and Annotation

The raw sequences files were proofread and assembled manually using the SeqMan module of the Lasergene 8.0 software (DNASTAR, Madison, WI, USA) [29]. The probable locations of the sequences were confirmed by BLAST search function on the NCBI website and comparison with the other lepidopteran sequences which can be obtained in GenBank. By using MEGA7.0, we determined the translation of 13 PCGs open reading frames [30]. The base composition of nucleotide sequences was described by skewness and measured according to the formulas (AT skew = [A−T]/[A+T], GC skew = [G−C]/[G+C]) [31]. 22 tRNA were confirmed using the program tRNAscan-SE. The proposed cloverleaf secondary structures within these tRNA genes and anticodon sequences were calculated using the tRNAscan-SE Search Server available online (http://lowelab.ucsc.edu/tRNAscan-SE/) [32]. We drew the secondary structure of tRNA by using the RNA structure program DNASIS MAX v.3.0 [33]. The secondary structure of the tRNA^{Ser (AGN)} was developed as proposed by [34]. Annotation was checked by comparison with tRNA determined for other lepidopteran species. Ribosomal RNA genes (rRNAs) were identified by NCBI Internet BLAST search.

Phylogenetic Analyses

To further probe into the phylogenetic relationship of Nymphalidae, a total of 84 complete mitogenomes and three uncomplete mitogenomes were chosen for the phylogenetic analyses based on the concatenated set of amino acid from 13 protein coding genes. The GenBank accession numbers used in this study were listed in Table 2. Among the 87 species, Coreana raphaelis (DQ102703.1), Japonica lutea (KM655768.1), Eurema hecabe (KC257480.1), Colias erate (KP715146.1), Curetis bulis (JX262888.1), Papilio bianor (KF859738.1), P. machaon (HM243594.1) and Leptidea morsei (JX274648.1) were selected as outgroups (Table 2). The PCG sequences of 87 species were aligned by using MEGA7.0 [30]. Sites with more than 90% gaps were excluded from the analysis. We chose two analysis approaches, Bayesian Inference (BI) and Maximum Likelihood (ML) to reconstruct phylogenetic relationships. We used the MrModeltest 2.3 [35] to select the best model for the ML and BI analyses. Thirteen datasets were established to calculate the best model for each PCG. According to the Akaike information criterion, the GTR + G model was selected as the most model appropriate for ND4L, and the GTR+I+G model was selected for other genes. The BI analysis was performed using MrBayes vers. 3.1.2 [36] under both of the models. The analysis were run twice simultaneously for 10,000,000 generations with every 1000 trees sampled. We discarded the first 1000,000 generations (1000 samples) as burn-in (based on visual inspection of the convergence and stability of the log likelihood values of the two independent runs). The ML analysis were performed using the program MEGA7.0 [30] with the same model. The bootstrap analysis were performed with 1000 replicates. Resulting tree files were inspected in FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).

Table 2: Taxonomy, GenBank accession numbers, and mitogenome sizes of 87 the mitochondrial genomes used for the phylogenetic analysis, sourced from GenBank databases.

Divergence Time Estimation

The analyses were performed based on sequences of 13 PCGs from 87 species, including eight outgroups. The program BEAST 2 [37] was used to estimate divergence times, with calibrations using five fossils nodes. Three fossils of Vanessa amerindica, Prodryas persephone and Lithopsyche styx were found in the Florissant formation in Colorado, which were formed in the early Oligocene and were thought to be related to the extant genus Hypanartia about 34 Ma in age. The fourth fossil is a hind wing that has been assigned to the extant genus Aglais, which was found in the Karagan deposits from the Miocene and has been dated at 14 Ma [38]. The last fossil is Dynamine alexaen deposits from the Miocene [39]. In addition, we used the results from Wahlberg et al. as secondary calibration point to calibrate the age of the first split in Nymphalidae at 90 Ma [7] and Papilionoidea at 104 Ma [40]. According to the result of our study, the Bayesian relaxed clock analyses were carried out with the program BEAST 2 [37]. The XML file for the beast analysis was created using BEAUti (in the BEAST package) with the following non-default settings and priors: the site model was set to the GTR +Γ distribution with default parameters, the clock model was set to a relaxed clock with uncorrelated rates, the tree model was set to a Yule process of speciation. The Markov chain Monte Carlo (MCMC) analyses were run for 100 million generations, sampling every 2000 generations and the first 25% discarded as burn-in. We used Tracer v1.5 to assess whether the likelihood traces of the four runs had converged to a stable equilibrium and that ESS values were above 200 for all parameters.

Results and Discussion

Genome Organization, Gene Arrangement, and Base Composition

The mitogenome of P. c-aureum (GenBank accession no. KX096653) is a closed circular molecule of 15,208 bp in size and similar to a typical insect mitogenome. The organization of the skipper mitogenome was shown in Figure 1. It contains the complete set of 37 genes, including 13 protein-coding genes (ND1-6, ND4L, COI-III, Cytb, ATP6, ATP8), 2 rRNA genes (12S and 16S), 22 putative tRNA genes, and an A+T-rich region (Figure 1). Similar to many insect mitogenomes, the majority (J) strand encodes more genes (9 PCGs and 14 tRNAs), whereas the minority (N) strand encodes lesser genes (4 PCGs, 8 tRNAs and 2 rRNAs) (Table 3). The order of genes and the orientation of the mitogenome of P. c-aureum are consistent with those sequenced lepidopteran mitogenomes. The nucleotide composition of the mitogenome of P. c-aureum is: A = 40.08%, T = 40.56%, G = 7.44% and C = 11.92% (Table 4). A + T content is 80.64%. Like other lepidopterans, the nucleotide composition of the P. c-aureum mitogenome is also biased toward A or T. This value is well in the range of the lepidopteran mitogenome, from 77.84 to 82.66%, which show a remarkable variability. Nucleotide skew statistics for the complete majority strand of P. c-aureum is AT-skew = −0.06 and GC-skew = −0.23 (Table 4), indicating slight A or T skews. A similar trend has been observed in many previously sequenced lepidopteran mitogenomes that the value of AT-skew varies from −0.031 (Eriogyna pyretorum) to 0.059 (Bombyx mori) and the GC-skew is always negative ranging from −0.318 (Ochrogaster lunifer) to −0.178 (Adoxophyes honmai) [41].

Figure 1: Map of the circular mitochondrial genome of Polygonia c-aureum. Different colors represent different regions. The abbreviations for the genes are as follows: COI-III stands for cytochrome oxidase subunits, Cytb for cytochrome b, and ND1-6 for NADH dehydrogenase Components. tRNAs are indicated by one-letter symbol according to the IUPAC-IUB single letter amino acid codes

Table 3: Annotation and gene organization of the Polygonia c-aureum mitogenome. Strands of the genes are presented as J for majority and N for minority strand. IN, negative numbers indicate that adjacent genes overlap, positive numbers indicate that intergenic sequences

Table 4: Composition and skewness of Polygonia c-aureum mitogenome regions. # = position

Protein-coding Genes

The PCGs of the P. c-aureum mitogenome include 7 NADH dehydrogenase subunits, 3 cytochrome c oxidase subunits, 2 ATPase subunits, and one cytochrome b gene. The PCGs of the mitogenome consists of 3,715 codons in total, except the termination codons. The start and stop codons of the 13 PCGs in the P. c-aureum mitogenome are shown in Table 3. Seven PCGs share the start codon ATG (COII, ATP6, COIII, ND4, ND4L, Cytb and ND1), four genes start with ATT (ND2, ATP8, ND5 and ND6), ND3 gene starts with ATA, and COI starts with CGA (Table 3). Among 13 PCGs, nine genes (ND2, COI, COII, ATP8, ATP6, COIII, ND3, ND6, Cytb) are coded on the majority strand, while the rest (ND5, ND4, ND4L, ND1) are coded on the minority strand. Three PCGs (COI, COII and ND4) have incomplete stop codons consisting of a T- or TA- nucleotide, two PCGs (ND5, ND1) stop with standard terminal codon (TAT) and the other PCGs stop with standard terminal codon (TAA) (Table 4). A recent study has used expressed sequence tag to explain that COI may start with CGA [42]. COI and COII usually have an incomplete stop codon in lepidopteran species, such as in A. honmai [43], M. sexta [44], Artogeia melete [45], Phthonandria atrilineata [46], O. lunifer [47], Hyphantria cunea [48] and A. emma [49]. Between ATP8 gene and ATP6 gene of the P. c-aureum mitogenome, we found seven overlapping nucleotides which is a common feature for all lepidopteran mitogenomes known to date (Table 3).

The A+T contents of three codon positions of the PCGs were calculated and were showed in Table 5. The second position has a relatively high A +T content (80.42%), while the first and the third positions have 78.36 % and 79.02 % respectively. In addition, both the positions have negative AT-skew and postive GC-skew. Relative Synonymous Codon Usage (RSCU) for the P. c-aureum mitogenome is showed in Table 5. The results show that RSCU has a distinct bias towards T/A for 13 PCGs. Among the 64 available codons, the four most used codons are Phenylalanine (F, UUU, 11.20%), Leucine (L, UUA, 8.17%), Isoleucine (I, AUU, 8.14%), and Methionine (M, AUA, 4.77%).

Table 5: Codon usage of the protein-coding genes in Polygonia c-aureum

A total of 3,733codons were analyzed.
RSCU, relative synonymous codon usage.
*= termination codon.

Transfer RNA and Ribosomal RNA Genes

The P. c-aureum mitogenome contains the set of 22 tRNA genes as shown in Figure 1, in which 14 tRNAs are coded on the J-strand and eight on the N-strand (Table 3). All the tRNAs have the typical clover-leaf structure, except for the tRNA^Ser (AGN) that lacking the Dihydrouridine (DHU) arm of which forms a simple loop (Figure 3). In addition, all their anticodons are similar to those found in lepidopteran insects. We can not find a complete typical clover-leaf structure of tRNA^{Ser (AGN)} by using tRNAscan-SE, as in some animal mitogenomes [50], especially in insects. The P. c-aureum mitogenome is as most of lepidopteran mitogenomes though the feature is not very conserved in the animal mitogenomes. However, there are two exceptions, showing typical clover leaf secondary structures, appeared in the tRNAs of lepidopteran insects, i.e. Diaphania pyloalis [41] and A. honmai [43]. The 22 tRNA molecules varied between 62 bp (tRNA^Cys) and 71 bp (tRNA^Lys) in length (Table 3), showing a highly A+T content of 80.97% and exhibiting positive AT-skew (0.006) (Table 4).

Figure 3: Predicted secondary clover-leaf structure for the 22 tRNA genes of Polygonia c-aureum. The tRNAs are labeled with the names of their corresponding amino acids. The minus sign (-) indicates Watson-Crick base pairing and the plus sign (+) indicates unmatched base pairing.

The A+T-rich Region

The A+T-rich region of P. c-aureum is 351 bp long (Table 3) with 94.02% A+T content and locates between the 16S and tRNA^Met (Figure 1). This shorter region is similar to 458 bp A+T-rich region of Papilio protenor [51]. Some conserved structures found in other Nymphalidae mitogenomes were also observed in the A+T-rich region of P. c-aureum mitogenome, shown in Figure 2. It contains the motif ATAGA followed by a 19 bp poly-T stretch and contains a relatively conservative microsatellite (AT)n element (n=25). However, we did not find a poly-A (in majority strand) which is often located upstream of tRNA^Met  in some lepidopteran insects.

Figure 2: a) Alignment of the initiation codons of COI genes of 29 species in the study. The arrow shows the initial direction of COI genes. B) Alignment of overlapping region between ATP8 and ATP6 across Nymphalidae. C) The features present in the A+T-rich region of Polygonia c-aureum.

Phylogenetic Relationships

Different optimality criteria and dataset compilation techniques have been applied to find the best method of analyzing complex mitogenomic data [52-54]. A total of 87 available mitogenomes, including the newly sequenced mitogenome, were applied to the phylogenetic analysis (Table 1). The results of the BI and ML analyses revealed the relationships of 11 Nymphalidae subfamily lineages (Biblidinae, Apaturinae, Nymphalinae, Cyrestidinae, Limenitidinae, Heliconiinae, Satyrinae, Charaxinae, Calinaginae, Danainae and Libytheinae) with very high nodal supports, shown in Figures 4 and 5.

Figure 4: Phylogenetic relationship of Nymphalidae. Phylogenetic tree inferrd from nucleotide sequences of 13 PCGs using Bayesian Inference (BI) method. Number at each node show bootstrap values. The branches are coloured and their content indicated at the subfamily level.

Figure 5: Inferred phylogenetic relationship among 87 species based on mitogenome sequences of 13 PCGs using Maximum Likehood (ML) method. Number at each node show bootstrap values. The branches are coloured and their content indicated at the subfamily level.

The phylogenetic analyses by BI method showed the relationships of the subfamilies of Nymphalidae, i.e. (((((Biblidinae + Apaturinae) + (Nymphalinae+ Cyrestidinae)) + (Limenitidinae + Heliconiinae)) + (((Satyrinae + Charaxinae)+ Calinaginae)+ Danainae)) + Libytheinae), with well high nodal supports. The result was consistent with the [7] whose phylogenetic analyses were based on ten nuclear genes.

Within the Nymphalidae, almost all nodes were supported by more than 0.80 supports in the BI tree. Our results showed clearly the relationships that Limenitidinae and Heliconiinae are sisters, with quite well supported by both BI (posterior probabilities =1) and ML (bootstrap =100) analyses. The results were identical to [23] and [7]. Moreover, the relationships (Calinaginae + (Charaxinae + Satyrinae)) were strongly supported by borh BI and ML trees. In addition, we found the subfamily Libytheinae located at the base of the phylogenetic tree of the Nymphalidae, which is the same as most previous hypotheses based on adult morphological studies [55-57] and molecular phylogenetic studies [7,58,59].

Though the supports were high in this study, the future studies need more samples and data to build a more powerful phylogenetic framework for Nymphalidae.

Divergence Time Estimation

The estimated divergence times among the Nymphalidae were shown in Figure 6. Our result suggested the first divergence in Nymphalidae occureded during the Cretaceous, at 89.72 Ma, and most clades appeared to have been diverged during the Cretaceous, at 86.9 Ma. The conclusion consisted with the previous result based on fossils and historical biogeography events by [38]. Besides, the Nymphalinae seems to be diverged from the group ((Biblidinae + Apaturinae) + Cyrestidinae) during the Cretaceous, at 75 Ma. These results were similar with the report of [24], and more accurated than the result of [38].

Figure 6: Estimated times of divergence for the family of Nymphalidae. The bootstrap values are shown at branching point. The time scale shows ages in million years (My) before present.

In this study, we found that the Heliconiinae clade and the Limenitidinae clade appeared to be approximately the same age about 70 Myrs. This result is consistent with the recent studies [24,40]. Our results situate the split between Limenitidinae and Heliconiinae about 69-76 Ma, which is consistent with the results of [24] who estimated this split to have occurred at 55.0-93.1 Ma. Moreover, the split between Satyrinae and Charaxinae at 66.03–72.98 Ma. We estimated that the Danainae diverged from the group (Calinaginae+ (Charaxinae + Satyrinae)) to be situated between 85–75 Ma, consistent with [24]. The Libytheinae arised as basal to the Nymphalidae diverged from the other subfamilies of Nymphalidae at 87.92 Ma. This is also consistent with [24]. In addition，for the first time, our analyses suggest that the genus Polygonia began to diversify, with the other lineage off from the common ancestor of the rest of Nymphalinae, at about 45.64 Ma.

Conclusions

In summary, we have shown that a complete mitogenome of the Asian comma butterfly, P c-aureum. The formerly identified conserved elements of Lepidoptera mitogenomes, i.e. the motif ‘ATAGA’ and poly-T stretch in the A+T-rich region, the long intergenic spacer upstream of ND2 and the 7 bp overlapping between ATP8 and ATP6, are present in P. c-aureum, only with some subtle differences in both of the size of genes and of the intergenic regions. The phylogenetic relationships based on nucleotide sequences of 13 PCGs by using BI and ML methods clarified the taxonomic status of Nymphalidae with a robust support. Furthermore, our results indicated that the complete mitogenome can be as an effective molecular marker to resolve the relationships of subfamilies within a family of butterflies. Our research is consistent with previous studies on the phylogenetic relationships of Nymphalidae. For the first time, we found that the genus Polygonia began to diversify at about 45.64 Ma. In addition, as in previous molecular studies, the subfamilies within Nymphalidae maybe diverged from each other in the Early Cretaceous, at about 90 Ma. We hope our results would be useful for the further phylogenetic analyses of insects and for the prevention and control of insect pests as well. Consequently, excellent phylogenetic resolution will come from larger integrated datasets. Predicatively, greater integration of nuclear and mitogenome studies is necessary to further our understanding for insect evolution.

Acknowledgments

This work was supported by grants from the Natural Science Foundation of China (No. 31572246) to Guo-Fang Jiang.

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DOI: 10.31038/MGJ.2022511

Abstract

The unique ultrafast easy and consistent bio synthesis process was established for the synthesis of nanoparticles using haloalkaliphilic bacteria. The strain used was Halomonas organivorans, which was characterized by 16S rRNA sequencing having the NCBI Accession No. JQ906721. This strain accumulates the compatible solutes – ectoine in response to high salinity. In the present study ectoine based silver nanoparticles was obtained within seconds which showed rapid synthesis of nanoparticles using sunlight. Synthesis of the silver (Ag) nanoparticles is aimed at the innovative probable artistic applications in the field of Bio-nanotechnology. Sunlight was converged using convex lens into the sample containing AgNO3, compatible solute – ectoine. Rapid change in colour was observed in 3-5 seconds which designated the formation of silver nanoparticles and was further characterized by UV-visible spectroscopy, FTIR and AFM.

Keywords

Ectoine, Halomonas organivoarans, UV-Vis. and AFM

Introduction

Development of consistent technology to produce nanoparticles is an important aspect of nanotechnology. Biological synthesis provides a wide range of environmentally suitable procedure, low-cost production with minutest time required. Biosynthesis of silver-based nanoparticles fascinated the attention of the Scientists for the ultrafast green synthesis. Due to the unique physicochemical properties and their potential applications in biotechnology, medical biotechnology, material science, chemistry and physics have led to the revolution of synthesizing novel generation miniscule particles with noble properties. The thirst among the researchers for the new approaches for the synthesis of silver nanoparticles was observed for the antimicrobial activity. Several biological methods employed for the synthesis are eco-friendly in nature and are nontoxic to environment, whereas chemical methods are dangerous to environment [1]. There are substantial reports suggesting the use of biological material for the synthesis of nanoparticles by bacteria, fungi, plant extract and yeast. Recently Scientists have used microorganisms and their intracellular and extracellular preparations for the synthesis of silver nanoparticles and whole cells were also known to reduce Ag+ ions to produce silver nanoparticles.

In our present investigation, marine isolate Halomonas organivornas accumulates higher quantity of compatible solutes in response to counter balance the extracellular NaCl concentration. Compatible solute based ultrafast green synthesis of silver nanoparticles was carried out by convergence of the irradiated sunlight into the solution resulting in the formation of silver nanoparticles within (3-5) seconds.

Materials and Methods

Bacterial Culture

The strain Halomonas organivorans was isolated in our laboratory and deposited in (NCBI Accession No. JQ906721) cultivated in Halophilic media containing 10 g Peptone, 10 g Yeast extract, 20 g MgSO_4, 2 g KCl, 3 g tri-sodium citrate, incubated at 37°C for 24 h. H. organivorans was then inoculated into 100 ml Halophilic broth and incubated at 37°C in a shaking incubator. The bacterial biomass obtained was centrifuged at 10,000 rpm for 10 min at 4°C and was used for the extraction of the osmolytes.

Purification of the Compatible Solute-ectoine

50 ml of the bacterial culture was centrifuged at 10,000 rpm for 10 min at 4°C cell pellet obtained was suspended in the double distilled sterile water for 20 min. The extract was separated by repeating the centrifugation step and the supernatant was collected. The extract obtained was filtered through 0.2 µ filter and finally a colourless extract was obtained and scanned by UV-VIS for the detection of synthesis of nanoparticles (UV region 210-230 nm) and stored at 4°C until further use.

Biosynthesis of Silver Nanoparticles

In a typical biosynthesis of silver nanoparticles, 1 ml of compatible solute-ectoine was mixed with 20 ml aqueous solution of 1 mM silver nitrate (AgNO3). The sunlight was converged using the convex lens into the sample mixture then within 3-5 seconds change in colour of the solution to brown was observed, thus, this indicated the formation of silver nanoparticles.

Optimization of Reaction Time

Silver nanoparticle synthesis was assessed at different reaction time of 2, 3, 4, 5 and 60 Sec using UV–Vis spectroscopy which showed the colour changes of silver nanoparticles in different reaction. Sunlight irradiation time was accurately monitored, increase in the time of exposure led to the formation of particle agglutination and thus rapid change in colour was measured indicating the formation of silver nanoparticles.

Characterization of Nanoparticles UV-VIS Visible Analysis

The reduction of silver ions was monitored by visual observation, actual reduction and formation of nanoparticles were examined by the UV-VIS spectroscopy scan from 200 nm to 800 nm.

Fourier Transform Infrared Spectroscopy (FTIR) Analysis

FTIR is highly diverse molecular spectroscopy technique employed for the analysis of biological and chemical properties. AgNPs are reduced with different biomolecules in the reaction mixture, hence understanding the precise functional group of biomolecules produced and FTIR spectra shed more information on nature of the AgNPs synthesized and were well documented in various studies. FTIR System used in this study was Shimadzu FTIR-8201 PC instrument and it was run in the diffuse reflectance mode at a resolution of 4 cm^-1 in KBR pellets.

Atomic Force Microscope (AFM) Studies

The samples were diluted to 10 times with distilled water and then dropped onto the glass slides, followed by vacuum drying at 30°C for 24 h. The measurements of the height of nanoparticles were observed by AFM image analysis software. The surface properties of the biosynthesized nanoparticles were visualized by an atomic force microscope, under the normal atmospheric conditions. Explorer atomic force microscope was in tapping mode, using high-resonant-frequency for the analysis, the study was carried out at central instrumentation center, Karnataka University, Dharwad. AFM imaging in UHV and in ambient air, the dynamic mode (DM-AFM), sometimes also termed the ‘‘tapping mode’’ is the method of choice for imaging surfaces.

Scanning Electron Microscope (SEM) Studies

The bacterial cells of Halomonas organivorans were obtained by centrifuging at 5,000 rev/min and the cells were washed twice with potassium phosphate buffer (50 mM, pH 7.0). Bacterial cells were then fixed with immersion in 2.5% glutaraldehyde in potassium phosphate buffer (50 mM, pH 7) overnight at 4°C. The specimens were washed twice with buffer and dehydrated with an ethanol series (v/v) ranging from 30, 40, 50, 60, 70, 80, 90 and 100% and stored in 100% ethanol. For SEM, the specimens were dried to critical point, coated with gold and examined with an S-200C scanning electron microscope.

Transmission Electron Microscopy (TEM) Studies

Transmission electron micrographs reveals the morphology of the nanoparticles under examination, substantial morphology of the silver nanoparticles synthesized were spherical in shape, appeared in bulk and this was carried out at SAIF, Cochin. TEM studies were performed using an electron microscope operating at an accelerating voltage of 90 kV. The dimensions of the silver nanoparticles were carried out by TEM by adding a drop of the solution containing the particles and was placed on a copper grid covered with amorphous carbon. Allow the film to stand for 2 min and the extra solution was removed by means of blotting paper and the grid was allowed to drying before using in the microscope. The nanoparticle films were also formed on carbon coated copper grids (40 μm × 40 μm mesh size) and transmission electron microscopy (TEM) images of the films were scanned on a JEOL 1200 EX instrument operated at an accelerated voltage of 120 kV.

Results and Discussion

In the present study synthesis of AgNPs from compatible solute – Ectoine was produced by Halomonas organivorans (No. JQ906721) and the phylogenetic tree as shown in Figure 1. this study is reported for the first-time using convergence of the irradiated sunlight as per our knowledge as shown in Figure 2. We are contributing a simple ultrafast green biosynthesis of AgNPs using converged photon irradiation. AgNPs have appeared as a promising candidate in the field of medical because of their physically illustrious size and shape, nanoparticles exhibit different properties when compared with the bulk material.

Figure 1: Phylogenetic tree of Halomonas organivorans (No. JQ906721)

Figure 2: Synthesis of silver nanoparticles with photon irradiation

Synthesis of the nanoparticles was carried out by chemical, biological and radiation methods, but there is always a scope for the development of easy, swift, safe and green synthesis of Nanoparticles. Hence, our existing study proves to be a noteworthy ultrafast, easy economic step in the of biological synthesis of nanoparticles.

UV-VIS Visible Analysis

This technique is based on the Surface Plasmon Resonance phenomenon (SPR), the change in colour of the nanoparticles has its derivation in collective electron excitation when related to external excitation which arises when external electromagnetic wave focuses on the particle. This creates an oscillation and each wavelength produces different oscillations in the electron cloud, which may be resonant or non-resonant. The specific wavelength of the electromagnetic radiation converted into thermal energy is the SPR peak obtained.

In the current work extracellular biosynthesis of AgNPs using the compatible solute ectoine extract and 20 ml of 1 mM AgNO3 were used. Colour changed from white to dark-brown within 3-5 sec after exposure to the condensed sunlight using convex lens. The UV–Vis spectrophotometer analysis showed the absorbance peak at round 400 nm as shown in Figure 3 which was specific for the synthesis of AgNPs. These findings are similar to the report of [2], in which the Capsicum annuum L. extract reacted with aqueous silver ions, the reaction mixture containing AgNPs showed the absorption peak at about 410 nm due to the excitation of longitudinal plasmon resonance vibration.

Figure 3: UV Spectral Characterization of nanoparticles

The maximum absorbance band at 412 nm [3] showed the surface plasmon resonance band for silver colloid thus the peak confirms the formation of spherical structures of the silver nanoparticles [4]. The intensity of the absorbed peak for silver nanoparticle exhibits the peculiar surface plasmon resonance peak between 400 to 430 nm was well known phenomenon for AgNP characterization in various studies. Most of the studies use UV Visible to identify the presence and production of Nanoparticles as well identify the size and shape of the NP [5,6].

Fourier Transform Infrared Spectroscopy (FTIR)

The biomolecule responsible for the reduction and formation of silver nanoparticles was identified by evaluating and interpreting the FTIR data analysis. The IR bands observed at 3,427 cm^-1 indicated the O-H stretch of carboxylic acid, or phenols, band at 2924 cm^-1 corresponding to C-H stretch and the bands at 1,626 cm^-1 corresponding to primary and secondary amides [7,8]. And 1384 cm^-1 indicated the presence of methyl group, the bands in the fingerprint region at 1102, 1023 cm^-1 is as shown in Figure 4 which represented the involvement of C-N aliphatic amine assessment from the bands indicated the presence of peptide bound AgNPs. Hence the conceivable reduction with the ectoine molecules in the compatible solute occurred.

Figure 4: FTIR analysis of silver nanoparticles

Atomic Force Microscopy (AFM) Studies

Atomic force microscopy (AFM) documents the imaging of the surface of samples on a nanometer scale in ultrahigh vacuum (UHV), ambient air, and liquids. The measurement of very small structures by AFM in air implies a relatively constant environment in terms of temperature and humidity. The slightest air drift or temperature shift during the measurement can cause a drift in the image. Therefore, an isolating box was built around the microscope which maintains a constant humidity inside and keep the temperature fluctuations to minimum. In addition, the box containing the microscope was put on top of an active damping table to prevent vibration. Humidity was reduced by placing silica gel inside the box; humidity and temperature were monitored by a Lab-view programme. Imaging was done in the non-contact dynamic mode at ambient conditions with humidity of 30−40% and all the images have been processed for better quality.

Atomic force microscopy has been used to study the silver nanoparticles morphology and surface topology as shown in Figure 5. AgNP’s are spherical in shape and exhibit smooth surface which was observed by atomic force microscope under tapping mode. The silver nanoparticles prepared from the compatible solute solution showed smooth topology as recorded by [9]. AFM Surface morphology of the formulated nanoparticles studied under AFM are displaying spherical shape of nanoparticles with smooth surface, without any pinholes or cracks.

Figure 5: The topology of the Ectoine silver nanoparticles by AFM

Scanning Electron Microscopy (SEM) studies

The interpretation of scanning electron microscopy shows the size of the particles and they were nanosized as spherical in shape ranging from 1.63 to 1.85 µm as shown in Figure 6. Predominantly most of the structures are spherical and the silver nanoparticles synthesized from the reaction of silver ions and compatible solutes were stable even after 1-2 months of storage. [10] the size of the nanoparticle synthesised were closely relevant with extracellular silver nanoparticles from Azadirachta indica (Plant) 50–100 nm. Extracellular nanoparticles from Colletotrichum sp. with 20–40 nm was reported by [11] and extracellular silver nanoparticles of Nitrate reductases from Fusarium oxysporum, was 10–35 nm.

Figure 6: Scanning Electron Microscopy of silver nanoparticles

Transmission Electron Microscopy

Transmission Electron micrographs reveals the morphology of the nanoparticles which showed the shape and size of Nanoparticles of 30-55 nm with spherical shape and appeared in bulk as shown in Figure 7. This was similar to the result observed by Bacillus lichiniformis mediated silver nanoparticles reported by [12]. The Synthesis of nanoparticles outside the cell extracellularly has many applications and the microbial synthesis of the metal nanoparticles depends upon the localization of the reductive components of the cell. When the cell wall reductive enzymes or soluble secreted enzymes are involved in the reductive process of metal ions then it is obvious to find the metal nanoparticles. It is one of the simple and eco-friendly method when compared to the chemical and physical method as it is cost effective and there are no side effects.

Figure 7: TEM of AgNPs biosynthesis from compatible solute

Conclusion

• The present investigation demonstrates the rapid biosynthesis of silver nanoparticles from the compatible solutes ectoine using converged photon irradiation, an eco-friendly, green and ultrafast protocol for the biosynthesis of AgNP’s.
• Halomonas organivorans (No. JQ906721) stores ectoine intracellularly to encounter the high concentration of NaCl extracellularly. The reduction of silver nitrate to AgNPs is facilitated by the presence of the ectoine, a pyrimidine carboxylic acid present in the compatible
• The Ectoine molecule present in the reaction mixture, in presence of high photon energy was reduced to ectoine–bonded Silver Nanoparticles and was detected at the UV absorbance peak at 400 nm. The intensity of the absorbed peak for silver nanoparticle exhibits the peculiar surface plasmon resonance peak between 400 to 430
• This is the first report for the biosynthesis of AgNPs using converged photon irradiation using the biological method in just few seconds (3-5). There are no reports about the use of converged light for the synthesis of AgNPs, from the available literature, comparing to the current method of the synthesis, this method is very precise, swift and
• The O-H stretch of carboxylic acid, or phenols bands in the fingerprint region at 1102, 1023 cm^-1 represented the involvement of C-N aliphatic amine assessment, from the bands indicated the presence of peptide bound AgNPs which were confirmed by (FTIR).
• AgNP’s are spherical in shape and exhibit smooth surface which was observed by atomic force microscope under tapping mode. AFM Surface morphology of the formulated nanoparticles studied under AFM are displaying spherical shape of nanoparticles with smooth surface, without any pinholes or
• The interpretation of scanning electron microscopy (SEM) shows the size of the particles and they were nanosized as spherical in shape ranging from 1.63 to 1.85 µm. Predominantly most of the structures are spherical and the silver nanoparticles synthesized from the reaction of silver ions and compatible solutes were stable even after 1-2 months of storage.

Transmission Electron micrographs reveals the morphology of the nanoparticles which showed the shape and size of Nanoparticles of 30-55 nm with spherical shape and appeared in bulk. The increase in polydispersity and broad size distribution with increase in metal ion concentration was evident from TEM image. It consisted of almost uniformly sized spherical nanoparticles of average size of 15 nm with diameter ranging from 7 to 25 nm.

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