Genome Organization and Comparative Evolutionary Mitochondriomics of Brown Planthopper, Nilaparvata lugens Biotype 4 Using Next Generation Sequencing (NGS).

Nilaparvata lugens is the main rice pest in India. Until now, the Indian N. lugens mitochondrial genome has not been sequenced, which is a very important basis for population genetics and phylogenetic evolution studies. An attempt was made to sequence two examples of the whole mitochondrial genome of N. lugens biotype 4 from the Indian population for the first time. The mitogenomes of N. lugens are 16,072 and 16,081 bp long with 77.50% and 77.45% A + T contents, respectively, for both of the samples. The mitochondrial genome of N. lugens contains 37 genes, including 13 protein-coding genes (PCGs) (cox1-3, atp6, atp8, nad1-6, nad4l, and cob), 22 transfer RNA genes, and two ribosomal RNA (rrnS and rrnL) subunits genes, which are typical of metazoan mitogenomes. However, both samples of N. lugens mitogenome in the present study retained one extra copy of the trnC gene. Additionally, we also found 93 bp lengths for the atp8 gene in both of the samples, which were 60-70 bp less than that of the other sequenced mitogenomes of hemipteran insects. The phylogenetic analysis of the 19 delphacids mitogenome dataset yielded two identical topologies when rooted with Ugyops sp. in one clade, and the remaining species formed another clade with P. maidis and M. muiri being sisters to the remaining species. Further, the genus Nilaparvata formed a separate subclade with the other genera (Sogatella, Laodelphax, Changeondelphax, and Unkanodes) of Delphacidae. Additionally, the relationship among the biotypes of N. lugens was recovered as the present study samples (biotype-4) were separated from the three biotypes reported earlier. The present study provides the reference mitogenome for N. lugens biotype 4 that may be utilized for biotype differentiation and molecular-aspect-based future studies of N. lugens.

Unfolding the mitochondrial genome structure of green semilooper (Chrysodeixis acuta Walker): An emerging pest of onion (Allium cepa L.).

Onion is the most important crop challenged by a diverse group of insect pests in the agricultural ecosystem. The green semilooper (Chrysodeixis acuta Walker), a widespread tomato and soybean pest, has lately been described as an emergent onion crop pest in India. C. acuta whole mitochondrial genome was sequenced in this work. The circular genome of C. acuta measured 15,743 base pairs (bp) in length. Thirteen protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and one control region were found in the 37 sequence elements. With an average 395 bp gene length, the maximum and minimum gene length observed was 1749 bp and 63 bp of nad5 and trnR, respectively. Nine of the thirteen PCGs have (ATN) as a stop codon, while the other four have a single (T) as a stop codon. Except for trnS1, all of the tRNAs were capable of producing a conventional clover leaf structure. Conserved ATAGA motif sequences and poly-T stretch were identified at the start of the control region. Six overlapping areas and 18 intergenic spacer regions were found, with sizes ranged from 1 to 20 bp and 1 to 111 bp correspondingly. Phylogenetically, C. acuta belongs to the Plusiinae subfamily of the Noctuidae superfamily, and is closely linked to Trichoplusia ni species from the same subfamily. In the present study, the emerging onion pest C. acuta has its complete mitochondrial genome sequenced for the first time.

High Taxonomic and Functional Diversity of Bacterial Communities Associated with Melon Fly, Zeugodacus cucurbitae (Diptera: Tephritidae).

The next generation sequencing (NGS) approach has facilitated the investigations of gut microbiota with high throughput and resolution. The present study was focused on the taxonomic and functional characterization of bacterial community associated with different developmental stages of melon fly, Zeugodacus cucurbitae using 16S ribosomal RNA (rRNA) gene amplicons metagenomics. Z. cucurbitae is considered an invasive and most staid polyphagous pest of cucurbitaceous and other related crops. The taxonomic analysis of highly variable V3-V4 region of bacterial 16S rRNA gene sequencing indicated that the bacterial community associated with Z. cucurbitae consists of a total of 23 bacterial phyla (including unclassified and unassigned bacteria), comprising 32 classes, 69 orders, 99 families and 130 genera. Proteobacteria, Firmicutes, Actinobacteria and Tenericutes were dominant phyla of which family, Enterobacteriaceae was the most abundant in the larval and adult female stages, whereas Mycoplasmataceae was the dominant in the pupal stage. In larval stages of Z. cucurbitae, genus Providencia and Comamonas were the most abundant. However, genus Candidatus-Bacilloplasma and Klebsiella were the most dominant in pupae and adult females of Z. cucurbitae, respectively. PICRUSt analysis conducted for prediction of metabolic activities revealed that associated microbiota were involved in membrane transport, carbohydrate metabolism, amino acid metabolism, energy metabolism, replication and repair processes as well as cellular processes and signalling. The higher number of OTUs was annotated for phosphoglycerate mutase and transketolase in adult females followed by larval stages, which may support the digestive function of the microbiota in larvae and adult females. Our findings provide insights about the high variation in microbiota across developmental stages and basis for microbiota-based management strategies of fruit flies.

Developmental stage-associated microbiota profile of the peach fruit fly, Bactrocera zonata (Diptera: Tephritidae) and their functional prediction using 16S rRNA gene metabarcoding sequencing.

The different developmental stage-associated microbiota of the peach fruit fly, Bactrocera zonata (Diptera: Tephritidae), was characterized using 16S rRNA gene (V3-V4 region) metabarcoding on the Illumina HiSeq platform. Taxonomically, at 97% similarity, there were total 16 bacterial phyla, comprising of 24 classes, 55 orders, 90 families and 134 genera. Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the most abundant phyla with Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, Bacteroidia and Bacilli being the most abundant classes. The bacterial genus Enterobacter was dominant in the larval and adult stages and Pseudomonas in the pupal stage. A total of 2645 operational taxonomic units (OTUs) were identified, out of which 151 OTUs (core microbiota) were common among all the developmental stages of B. zonata. The genus Enterobacter, Klebsiella and Pantoea were dominant among the core microbiota. PICURSt analysis predicted that microbiota associated with B. zonata may be involved in membrane transport, carbohydrate metabolism, amino acid metabolism, replication and repair processes as well as in cellular processes and signalling. The microbiota that was shared by all the developmental stages of B. zonata in the present study could be targeted and the foundation for research on microbiota-based management of fruit flies.

Complete mitochondrial genome of Idioscopus nitidulus (Hemiptera: Cicadellidae).

The complete mitogenome of Idioscopus nitidulus (Cicadellidae) was sequenced. It comprises 15,287 base pairs (bp), including 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and a control region. The phylogenetic analyses based on concatenated thirteen protein-coding genes of mitogenomes recover the monophyly of Auchenorrhyncha (Fulgoromorpha + Cicadomorpha) and Sternorrhyncha as a sister group to Auchenorrhyncha. The complete mitogenome sequence of Idioscopus nitidulusis available in the GenBank with accession number: KR024406.

Genetic analysis of Bactrocera zonata (Diptera: Tephritidae) populations from India based on cox1 and nad1 gene sequences.

The peach fruit fly, Bactrocera zonata, is among the most serious and polyphagous insect pest of fruit crops in many parts of the world under genus Bactrocera. In the present study, the genetic structure, diversity and demographic history of B. zonata in India were inferred from mitochondrial cytochrome oxidase 1 (cox1) and NADH dehydrogenase 1 (nad1) sequences. The efficiency of DNA barcodes for identification of B. zonata was also tested. Genetic diversity indices [number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π) and average number of nucleotide differences (k)] of B. zonata populations across India maintain high level of genetic diversity without isolation by distance among the geographic regions. Non-significant negative correlation between pairwise Fst and geographic distance suggests a high level of gene flow among studied populations of B. zonata. The possibility of sudden expansion of B. zonata revealed through mismatch distribution analysis as well as negative Tajima's D and Fu's Fs values further supported by star-like network of haplotypes. DNA barcoding analysis suggests that B. zonata specimens can be clearly differentiated from other species with 100% accuracy of identification. Therefore, cytochrome oxidase 1 (cox1) barcode sequences generated in the present study could be a valuable source for the rapid identification and global population genetic study of B. zonata.

Genetic analysis of oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae) populations based on mitochondrial cox1 and nad1 gene sequences from India and other Asian countries.

The study examined the genetic diversity and demographic history of Bactrocera dorsalis, a destructive and polyphagous insect pest of fruit crops in diverse geographic regions of India. 19 widely dispersed populations of the fly from India and other Asian countries were analysed using partial sequences of mitochondrial cytochrome oxidase I (cox1) and NADH dehydrogenase 1 (nad1) genes to investigate genetic diversity, genetic structure, and demographic history in the region. Genetic diversity indices [number of haplotypes (H), haloptype diversity (Hd), nucleotide diversity (π) and average number of nucleotide difference (k)] of populations revealed that B. dorsalis maintains fairly high level of genetic diversity without isolation by distance among the geographic regions. Demographic analysis showed significant (negative) Tajimas' D and Fu's F S with non significant sum of squared deviations (SSD) values, which indicate the possibility of recent sudden expansion of species and is further supported through distinctively star-like distribution structure of haplotypes among populations. Thus, the results indicate that both ongoing and historical factors have played important role in determining the genetic structure and diversity of the species in India. Consequently, sterile insect technique (SIT) could be a possible management strategy of species in the regions.

The mitochondrial genome of the peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae): Complete DNA sequence, genome organization, and phylogenetic analysis with other tephritids using next generation DNA sequencing.

Mitochondrial genome can provide information for genomic structure as well as for phylogenetic analysis and evolutionary biology. The complete 15,935 bp mitochondrial genome of Bactrocera zonata (Diptera: Tephritidae), is assembled from Illumina MiSeq read data. The mitogenome information for B. zonata was compared to the homologous sequences of other tephritids. Annotation indicated that the structure and orientation of 13 protein coding genes (PCGs), 22 tRNA and 2 rRNA sequences were typical of, and similar to, the ten closely related tephritid species. The nucleotide composition shows heavily biased toward As and Ts accounting 73.34% and exhibits a slightly positive AT skew, which is similar to other known tephritid species. All PCGs are initiated by ATN codons, except for cox1 with TCG and atp8 with GTG. Nine PCGs use a common stop codon of TAA or TAG, whereas the remaining four use an incomplete termination codon T or TA likely to be completed by adenylation. All tRNAs have the typical clover-leaf structure, with an exception for trnS((AGN)). Four short intergenic spacers showed high degree of conservation among B. zonata and other ten tephritids. A poly(T) stretch at the 5' end followed by [TA(A)]n-like stretch and a tandem repeats of 39 bp has been observed in CR. The analysis of gene evolutionary rate revealed that the cox1 and atp6 exhibits lowest and highest gene substitution rates, respectively than other genes. The phylogenetic relationships based on Maximum Likelihood method using all protein-coding genes and two ribosomal RNA genes confirmed that B. zonata is closely related to Bactrocera correcta, Bactrocera carambolae, Bactrocera papayae, and Bactrocera philippinensis and Bactrocera dorsalis belonging to B. dorsalis species complex forms a monophyletic clade, which is in accordance with the traditional morphological classification and recent molecular works.