Introduction

Lepidopteran insects are basically phytophagous in nature. The larvae of these lepidopteran insects are voracious. Hence, they have become major pests of various crops [₁,₂,₃,₄]. Intra-specific conservation and variations have been reported using ITS2 region collected from diverse geographic locations that are used for phylogenetic studies [₅, ₆, ₇, ₈]. The internal transcribed spacers (ITS) are located between the repeating array of nuclear 18S, 5.8S and 28S ribosomal RNA genes, a locus that has 100–200 copies per genome. The ITS spacers are versatile genetic markers and have been used for phylogenetic analysis, evaluation of the evolutionary process, as well as for determination of taxonomic identities [₉]. The lepidopteran insects selected for the present study were: Helicoverpa punctigera, Luehdorfia longicaudata, Luehdorfia chinensis, Luehdorfia puziloi puziloi, Luehdorfia puziloi yessoensis, Heliconius charithonia, Heliconius hecale, Heliconius melpomene.

Mature rRNAs are produced by the processing of a large precursor from which different transcribed spacer regions are sequentially removed through an elaborate pathway of cleavage steps [₁₀]. In eukaryotes, transcribed spacer regions may represent a very substantial fraction of the length of the primary transcript. Although these transcribed spacer regions are obvious candidates for important roles in the control of ribosome biogenesis, elucidation of their biological function and of the molecular mechanisms involved in their accurate excision still remain a major challenge. Recent functional analyses performed on yeast ribosomal RNA genes clearly show that the structural integrity of the transcribed spacer regions is an essential prerequisite for correct processing of mature rRNA and biogenesis of active ribosomal subunits [₆, ₁₁]. The derivation of reliable secondary structure models for each transcribed spacer region would undoubtedly represent a major step towards a detailed understanding of their biological role. The comparative sequence analysis provides the most powerful tool for identifying the biologically relevant folding pattern of an RNA molecule, i.e. its native structure within the cellular context [₆]. However, an essential prerequisite for its effective utilization is the availability of a collection of sequences exhibiting a substantial number of nucleotide differences while remaining similar enough for unequivocal sequence alignments. Due to the high rate of sequence variation of transcribed spacers, this may exhibit dramatic size variation and extensive sequence divergence even among moderately distant species [₁₂, ₁₃]. Nevertheless, the presence of phylogenetically conserved secondary structure elements in the 5' externally transcribed spacer was recently revealed by the comparative analysis of a limited set of vertebrate sequences [₁₄].

The present study is focused on internal transcribed spacer, ITS2, which interrupts the eukaryotic large subunit rRNA molecule and has no prokaryotic equivalent [₁₅]. The sequence and secondary structures of ITS2 of selected geographically variant Lepidopteran insect species were comprehensively investigated. Such case studies are relevant in broader phylogenetic contexts and for analyzing the function in ribosome biogenesis. Since the secondary structures of ITS region are more conserved than the nucleotide sequences their analysis helps in understanding molecular evolution and increases the number of structural characters. Thus the structure models developed in this study can be used for future phylogenetic analyses.

Materials and Methods

Data set

Sequence alignments

Secondary structure prediction

RNA fold

Phylogenetic analysis

Results

Sequence analysis

The length of ITS2 elements of eight selected lepidopteran species ranged in size between 644 and 699 bp. eight dispersed but unambiguously conserved sequence segments encompassing about a third of the ITS2 length have been identified. They were interspersed with variable regions and gaps where size variations accumulate. The characteristics of the sequences for each species are shown in Table 1. The length variations were observed with maximum length being 699 bp and minimum of 644 bp for Heliconius charithonia and Helicoverpa punctigera respectively. The G+C contents for the two regions of rDNA (5.8S and ITS2) of all species ranged from 54% to 69%. For ITS2 regions the sequence identities ranges, with maximum 99% similarity between Luehdorfia longicaudataand Luehdorfia chinensis; 95% between Luehdorfia longicaudata and Luehdorfia puziloi puziloi whereas the minimum being the 2% between Luehdorfia puziloi puziloi and Heliconius hecale. Alignment of ITS2 region is shown in the Figure: 1 Simple tandem repeats were present at various locations along the ITS2. The sequence similarity is more towards the 5' end and with dispersed conserved ness in the middle than towards the 3' end.

Figure 1

Table 1: Lengths (in mers); G+C content (in %); GC, AU, GU base pairs (in numbers); No: stems and energies (in kcal) of secondary structures of the second internal transcribed spacer (ITS-2) rDNA sequences of the species.

Secondary structure

Discussion

The selected 8 Lepidopteran insect species occur worldwide and they are pests of various crops. In the present investigation, the ITS2 sequences reflected the trend observed in the phylogeny. The more distantly related the less was the convergence at the ITS2 level (Figure 1 &2).

Figure 2

Figure 1a: Alignment between species ITS2 regions

Figure 3

Figure 4

Figure 1b: Phylogenetic tree of 8 selected lepidatperan species.

Figure 5

Figure 2: A secondary structural fold found in common to all 8 Lepidateran species. Identical bases are marked.

However accumulated substitutions in the ITS sequence leading to length variation also had a profound effect on the conservedness among the structures. The length variation observed was may be due to insertions effected by many factors including genetic drift, the relative number and size of repeats, rates of unequal crossover, gene conversion, immigration and the number of the loci [₂₄]. But still high level of sequence conservation was found between some species like Luehdorfia longicaudata and Luehdorfia chinensis i, Luehdorfia longicaudata and Luehdorfia puziloi puziloi, [₂₅] even the simple tandem repeats were found to be conserved to a large extent among them. This conservation was further reflected at secondary structural and energy level. The predicted features of ITS2 using RNADRAW are given in Table 1. The stems (double stranded paired regions) stabilize RNA secondary structures and the number of stems present in each ITS2 is given (Table 1). ITS2 RNA structures from Heliconius hecale and Heliconius melpomene the highest negative free energy (-263.21Kcal and -256.14 Kcal) followed by Luehdorfia puziloi puziloi (-253.31), Heliconius charithonia (-252.85) Luehdorfia chinensis (-252.52) and then by Luehdorfia longicaudata (-251.67), Luehdorfia puziloi yessoensis (-249.39) and Helicoverpa punctigera (-229.83). Visual comparison shows that this is related to the trend in the cladogram given in Figure 1A.This convergence at secondary structural level among few species from different geographic isolates is may be due to the evolutionary pressure on ITS2 to maintain the RNA secondary structure involved in post-transcriptional processing of rRNA [₇]. Secondary structure predictions for the ITS2 region indicate that these domains base pair to form a core region central to several stem features implying that conserved ness is more important for the proper rRNA folding pattern [₂₆]. In contrast to the present observation with respect to Class I, Barker in his study found that [₂₇] ITS 2 is unique in the 16 populations, of Rhipicephalus and Boophilus species, with considerable nucleotide variation among species and genera. However intraspecific variation of ITS2 sequences was also found in the populations. Analysis of ITS spacers of T. rangeli and T. cruzi allowed the distinction of two distinct groups, revealing a low-level similarity between them [₂₈]. Figure 3 shows the distribution of different types of loops (hairpin, bulge, multi branched, interior and exterior) among different isolates. The segments of the ITS2 having score ? 50 are further probed carefully for target site to assess the likelihood of unpaired segments. Interestingly, the observed phylogenetic trend was identified with respect to the target accessibility sites for the eight Lepidopteron isolates. The order of preference is interior loop, bulge loop, multiple branched loop, hairpin loop and exterior loops in the all the isolates.

Figure 6

Figure 3: Distribution of different types of loops [Hairpin (H), Bulge (B), Multi branched (M), Interior (I) and Exterior (E)] among different isolates of Lepidopteron species.

These results suggest that the differences and conserved ness observed between ITS-2 of different species are not “neutral” and are not simple accumulated random nucleotide changes, but bear a significant functional load. In the previous study of three related mosquito genera (Aedes, Psorophora, and Haemogogus), [₂₆] it was found that intra spacer variable regions appear to co-evolve and that ITS-2 variation is constrained to some extent by its secondary structure. Further studies on yeast [₂₉] have demonstrated that the ITS-2 is essential for the correct and efficient processing and maturation of certain ribosomal units. Furthermore, information for the efficient removal of ITS-2 from its RNA precursor is dispersed through the entire ITS-2 region, and indels that effect secondary structure differentially alters rRNA processing. Critical changes in the rRNA folding pattern brought about by sequence evolution in the ITS spacer regions may thus have an important influence on the kinetics of precursor rRNA formation, and ultimately on the efficient functioning of the rDNA cluster.

Conclusion

The present study shows two contrasting aspects of ITS2 regions i.e. the general trend of variability among the species as well as the conservedness between few species. Surprisingly, the species displaying the conservedness belong to different geographical locations with diverse climatic and ecological conditions. Our study implies that the ITS2 regions though have less selective pressure than the ribosomal regions but still evolve slower than the intergenic spacers, indicating that some selective pressure do exists on them, probably from the constraint to maintain the RNA secondary structure required for post-transcriptional processing and are more species specific than geographically influenced. Several common structural folds were shared among the selected lepidopteran insects for maintaining functional equivalents. Identifying the homologous regions and reconstructing their evolution increases the traits available for the phylogenetic analysis. Construction of an evolutionary tree using more isolates of Lepidopteron will provide an understanding for their functional selection. The present study indicates the phylogenetic relationship among the selected Lepidopteron species belonging to diverse geographical locations.

Correspondence to

Prof. Poduri Nagaraja Rao, Department of Zoology, Osmania University College for Women, Koti, Hyderabad, Andhra Pradesh, India. e-mail: nagarajaraop@yahoo.com