The role of insect gut microbiota in host fitness, detoxification and nutrient supplementation.

Insects are incredibly diverse, ubiquitous and have successfully flourished out of the dynamic and often unpredictable nature of evolutionary processes. The resident microbiome has accompanied the physical and biological adaptations that enable their continued survival and proliferation in a wide array of environments. The host insect and microbiome's bidirectional relationship exhibits their capability to influence each other's physiology, behavior and characteristics. Insects are reported to rely directly on the microbial community to break down complex food, adapt to nutrient-deficit environments, protect themselves from natural adversaries and control the expression of social behavior. High-throughput metagenomic approaches have enhanced the potential for determining the abundance, composition, diversity and functional activities of microbial fauna associated with insect hosts, enabling in-depth investigation into insect-microbe interactions. We undertook a review of some of the major advances in the field of metagenomics, focusing on insect-microbe interaction, diversity and composition of resident microbiota, the functional capability of endosymbionts and discussions on different symbiotic relationships. The review aims to be a valuable resource on insect gut symbiotic microbiota by providing a comprehensive understanding of how insect gut symbionts systematically perform a range of functions, viz., insecticide degradation, nutritional support and immune fitness. A thorough understanding of manipulating specific gut symbionts may aid in developing advanced insect-associated research to attain health and design strategies for pest management.

Reference genes for qPCR expression in black tiger shrimp, Penaeus monodon.

BACKGROUND: Gene expression profiling via qPCR is an essential tool for unraveling the intricate molecular mechanisms underlying growth and development. Identifying and validating the most appropriate reference genes is essential for qPCR experiments. Nevertheless, there exists a deficiency in a thorough assessment of reference genes concerning the expression of the genes in the research in the context of the growth and development of the Black Tiger Shrimp, P. monodon. This popular marine crustacean is extensively raised for human consumption. In this study, we assessed the expression stability of seven reference genes (ACTB, 18S, EF-1α, AK, PK, cox1, and CLTC) in adult tissues (hepatopancreas, gills, and stomach) of small and large polymorphs of P. monodon. METHODS AND RESULTS: The stability of gene expressions was assessed utilizing NormFinder, BestKeeper, and geNorm, and a comprehensive ranking of these genes was conducted through the online tool RefFinder. In the overall ranking, 18S and CLTC emerged as the most stable genes in the hepatopancreas and stomach, while CLTC and AK exhibited significant statistical reliability in the gills of adult P. monodon. The validation of these identified stable genes was carried out using a growth-associated gene, insr-1. CONCLUSION: The results indicated that 18S and CLTC stand out as the most versatile reference genes for conducting qPCR analysis focused on the growth of P. monodon. This study represents the first comprehensive exploration that identifies and assesses reference genes for qPCR analysis in P. monodon, providing valuable tools for research involving similar crustaceans.

Transcriptome analysis of Corvus splendens reveals a repertoire of antimicrobial peptides.

Multidrug resistance has become a global health problem associated with high morbidity and mortality. Antimicrobial peptides have been acknowledged as potential leads for prospective anti-infectives. Owing to their scavenging lifestyle, Corvus splendens is thought to have developed robust immunity to pathogens found in their diet, implying that they have evolved mechanisms to resist infection. In the current study, the transcriptome of C. splendens was sequenced, and de novo assembled to identify the presence of antimicrobial peptide genes. 72.09 million high-quality clean reads were obtained which were then de novo assembled into 3,43,503 transcripts and 74,958 unigenes. About 37,559 unigenes were successfully annotated using SwissProt, Pfam, GO, and KEGG databases. A search against APD3, CAMPR3 and LAMP databases identified 63 AMP candidates belonging to more than 20 diverse families and functional classes. mRNA of AvBD-2, AvBD-13 and CATH-2 were found to be differentially expressed between the three tested crows as well as among the tissues. We also characterized Corvus Cathelicidin 2 (CATH-2) to gain knowledge of its antimicrobial mechanisms. The CD spectroscopy of synthesized mature Corvus CATH-2 peptide displayed an amphipathic α-helical structure. Though the synthetic CATH-2 caused hemolysis of human RBC, it also exhibited antimicrobial activity against E. coli, S. aureus, and B. cereus. Docking simulation results revealed that this peptide could bind to the LPS binding site of MD-2, which may prevent LPS from entering the MD-2 binding pocket, and trigger TLR4 signaling pathway. The Corvus CATH-2 characterized in this study could aid in the development of novel therapeutics.

Transcriptome Profiling of Rhipicephalus annulatus Reveals Differential Gene Expression of Metabolic Detoxifying Enzymes in Response to Acaricide Treatment.

Ticks are hematophagous ectoparasites of economic consequence by virtue of being carriers of infectious diseases that affect livestock and other sectors of the agricultural industry. A widely prevalent tick species, Rhipicephalus (Boophilus) annulatus, has been recognized as a prime vector of tick-borne diseases in South Indian regions. Over time, the use of chemical acaricides for tick control has promoted the evolution of resistance to these widely used compounds through metabolic detoxification. Identifying the genes related to this detoxification is extremely important, as it could help detect valid insecticide targets and develop novel strategies for effective insect control. We performed an RNA-sequencing analysis of acaricide-treated and untreated R. (B.) annulatus and mapped the detoxification genes expressed due to acaricide exposure. Our results provided high-quality RNA-sequenced data of untreated and amitraz-treated R. (B.) annulatus, and then the data were assembled into contigs and clustered into 50,591 and 71,711 uni-gene sequences, respectively. The expression levels of the detoxification genes across different developmental stages of R. (B.) annulatu identified 16,635 transcripts as upregulated and 15,539 transcripts as downregulated. The annotations of the differentially expressed genes (DEGs) revealed the significant expression of 70 detoxification genes in response to the amitraz treatment. The qRT-PCR revealed significant differences in the gene expression levels across different life stages of R. (B.) annulatus.

Long-read genome sequence data of pomegranate bacterial blight pathogen Xanthomonas citri pv. punicae 119.

Bacterial blight of pomegranate caused by Xanthomonas citri pv. punicae (Xcp) is a major constraint on pomegranate production in India. Multiple incidences of 60-80% yield losses have been reported from major growing areas. The molecular mechanism of pomegranate susceptibility to this disease is yet to be unraveled. A complete genome sequence is required to identify the pathogen's virulence factors. The available genomes of the pathogen are not complete. We have used the PacBio platform to obtain long reads and assembled the Xcp-119 genome and it consists of 15 contigs with an N50 value of 1.65 Mb. The assembled genome is 5.4 Mb in size; further, our annotation analysis yielded 5088 genes, 58 RNAs, and 7 Transcription activator-like effectors  (TALEs). The assembled genome is suitable for identifying the virulence factors of this pathogen. The data presented here will be helpful to the scientific community for various comparative genome analyses and to decipher the mode and factors of pathogenesis.

Exploring variation in the fecal microbial communities of Kasaragod Dwarf and Holstein crossbred cattle.

The gut microbiota and its impact on health and nutrition in animals, including cattle has been of intense interest in recent times. Cattle, in particular indigenous varieties like Kasaragod Dwarf cow, have not received the due consideration given to other non-native cattle breeds, and the composition of their fecal microbiome is yet to be established. This study applied 16S rRNA high-throughput sequencing of fecal samples and compared the Kasaragod Dwarf with the highly prevalent Holstein crossbred cattle. Variation in their microbial composition was confirmed by marker gene-based taxonomic analysis. Principle Coordinate Analysis (PCoA) showed the distinct microbial architecture of the two cattle types. While the two cattle types possess unique signature taxa, in Kasaragod Dwarf cattle, many of the identified genera, including Anaerovibrio, Succinivibrio, Roseburia, Coprococcus, Paludibacter, Sutterella, Coprobacillus, and Ruminobacter, have previously been shown to be present in higher abundance in animals with higher feed efficiency. This is the first report of Kasaragod Dwarf cattle fecal microbiome profiling. Our findings highlight the predominance of specific taxa potentially associated with different fermentation products and feed efficiency phenotypes in Kasaragod Dwarf cattle compared to Holstein crossbred cattle.

TAL effectors and the predicted host targets of pomegranate bacterial blight pathogen Xanthomonas citri pv. punicae.

The molecular mechanism of pomegranate susceptibility to bacterial blight, a serious threat to pomegranate production in India, is largely unknown. In the current study, we have used PacBio and Illumina sequencing of Xanthomonas citri pv. punicae (Xcp) strain 119 genome to identify tal genes and RNA-Seq analysis to identify putative host targets in the susceptible pomegranate variety Bhagwa challenged with Xcp119. Xcp119 genome encodes seven transcription activator-like effectors (TALEs), three of which are harbored by a plasmid. RVD-based phylogenetic analysis of TALEs of Xanthomonas citri pathovars indicate the TALEs of Xcp as evolutionarily and functionally close to Xanthomonas citri pv. malvacearum and Xanthomonas citri pv. glycines. Comparative RNA-Seq of Xcp and mock-inoculated leaf tissues revealed Xcp-induced pomegranate transcription modulation. The prediction of TALE binding elements (EBEs) in the promoters of up-regulated genes identified a set of TALE-targeted candidate genes in pomegranate-Xcp interaction. The predicted candidate susceptibility genes include two oxoglutarate-dependent dioxygenase gene, ethylene-responsive transcription factor and flavanone 3-hydroxylase-like gene, and the further characterization of these would enable blight resistance engineering in pomegranate.

Draft genome sequence, annotation and SSR mining data of Oryctes rhinoceros Linn. (Coleoptera: Scarabaeidae), the coconut rhinoceros beetle.

The coconut rhinoceros beetle (CRB), Oryctes rhinoceros Linn. (Coleoptera: Scarabaeidae), is one of the major pests of coconut causing severe yield losses. The adult beetles feed on unopened spear leaf (resulting in the typical 'V'-shaped cuts), spathes, inflorescence, and tender nut leading to stunted palm growth and yield reduction. Moreover, these damages serve as predisposing factors to the entry of other fatal enemies on palms, viz., red palm weevil and bud rot disease, causing yield loss as high as 10%. CRB attacks juvenile palms through the collar region, affecting the growth and initial establishment of the juvenile palms. While the immature stages of CRB sustain on organic debris, the adult beetles are ubiquitous pests on coconut and other palms. The discovery of a new invasive haplotype of CRB from Guam and other Pacific Islands, insensitive to Oryctes rhinoceros nudivirus (OrNV), a potent biocontrol agent, has raised serious concerns. The draft genome sequence and simple sequence repeat (SSR) marker data for this important pest of coconut are presented here. A total of 30 Gb of sequence data from an individual third instar larva was obtained on an Illumina HiSeq X Five platform. The draft genome assembly was found to be 372 Mb, with 97.6% completeness based on Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment. Functional gene annotation predicted about 16,241 genes. In addition, a total of 21,999 putative simple sequence repeat (SSR) markers were identified. The obtained draft genome is a valuable resource for comprehending population genetics, dispersal patterns, phylogenetics, and species behavior.

Exploring prevalence of potential pathogens and fecal indicators in geographically distinct river systems through comparative metagenomics.

Microbial communities are considered as vital members to reflect the health of a riverine system. Among them, pathogenic and fecal indicators imply health risks involved with potability of river water. The present study explores the diverse microbial communities, distribution pattern of potential pathogens, and fecal indicators between the geographically distinct Himalayan and Peninsular river systems of India. It also inquires into the environmental factors associated with community variance and distribution pattern of microbial indicators. The application of high-throughput amplicon sequencing approach unveiled significant demarcation (p < 0.004, Anosim R = 0.62) of samples suggesting unique microbial diversities in these two river sediments. Random forest analysis revealed Desulfobulbulus, PSB_M_3, and Opitutus in Himalayan, while DA101, Bacillus, and Streptomyces in the Peninsular as significant contributors to develop overall dissimilarity between the river systems. Permutational multivariate analysis of variance and co-occurrence network analysis were used to study the relationships between microbial taxa and environmental factors. Amongst the various studied environmental parameters, pH, K, Ca, Mg, Ba, and Al in the Himalayan and salinity, Na, temperature, and Th in the Peninsular significantly influenced shaping of distinct microbial communities. Furthermore, the potential pathogenic genera, including Flavobacterium, Clostridium, Arcobacter, Pseudomonas, and Bacillus were highly prevalent in both the river systems. Arcobacter, Clostridium, Acinetobacter, Bacteroides, and Caloramator were the prominent fecal indicators in these river systems. Our findings provide salient information about the crucial role and interplay between various environmental factors and anthropogenic influences in framing the microbiome of the distinct river systems in India. Moreover, assessing potential pathogenic and fecal indicators suggest the public health risk associated with untreated sewage discharge into these water sources. The detection of various F/S indicators and potentially pathogenic bacteria in Himalayan and Peninsular river systems emphasize the urgent need for future monitoring and management of major riverine systems in India.

Dataset of de novo assembly and functional annotation of the transcriptome of certain developmental stages of coconut rhinoceros beetle, Oryctes rhinoceros L.

The coconut rhinoceros beetle, Oryctes rhinoceros L. (Insecta: Coleoptera: Scarabaeidae: Dynastinae) is one of the world's most important endemic and incessant pests of coconut (particularly in India and Southeast Asia), causing an estimated 10% yield loss in the crop. Various management strategies formulated and implemented to control this pest include bioagents, insecticide sprays, liquid formulations, pheromone traps, and botanical formulations. Also, potential microbial bioagents viz., Oryctes rhinoceros nudivirus (OrNV) and Metarhizium anisopliae have been implemented as biological control agents and this has led to a beneficial reduction of the pest population unless significant immigration occurs. To date, research and development activities are still on-going for the successful management of the pest; yet advances in understanding at the molecular level have been limited because basic genomic information is lacking for this cosmopolitan pest. Transcriptome approach has been proved extremely useful in finding potential genes for pest control. Transcriptome analysis aids in gaining insights into the transcriptional changes which occur during different developmental stages of an organism. We have performed RNA sequencing of certain different developmental stages of O. rhinoceros viz., early instar larva, late instar larva, pupa, and adult, in an Illumina HiSeq™ 2500 platform. Due to the unavailability of O. rhinoceros genome, the RNA-seq data generated were assembled de novo using Trinity and annotated following redundancy removal. A dataset of 87,451 transcripts, which resulted after redundancy removal, were annotated using the NCBI non-redundant (nr) protein and Uniprot databases. The data furnished could be used by others working in the development of pest management strategies, especially the identification of molecular targets for effective pest control. This information allows a better understanding of O. rhinoceros biology which would contribute to outlining a new generation of stage-specific, environmentally friendly pest management techniques.

Role of the Gut Microbiome in Autism Spectrum Disorders.

Autism spectrum disorder (ASD) is a severe neurodevelopmental or neuropsychiatric disorder with elusive etiology and obscure pathophysiology. Cognitive inabilities, impaired communication, repetitive behavior pattern, and restricted social interaction and communication lead to a debilitating situation in autism. The pattern of co-occurrence of medical comorbidities is most intriguing in autism, compared to any other neurodevelopmental disorders. They have an elevated comorbidity burden among which most frequently are seizures, psychiatric illness, and gastrointestinal disorders. The gut microbiota is believed to play a pivotal role in human health and disease through involvement in physiological homoeostasis, immunological development, glutathione metabolism, amino acid metabolism, etc., which in a reasonable way explain the role of gut-brain axis in autism. Branded as a neurodevelopmental disorder with psychiatric impairment and often misclassified as a mental disorder, many experts in the field think that a therapeutic solution to autism is unlikely to emerge. As the pathophysiology is still elusive, taking into account of the various symptoms that are concurrent in autism is important. Gastrointestinal problems that are seen associated with most of the autism cases suggest that it is not just a psychiatric disorder as many claim but have a physiological base, and alleviating the gastrointestinal problems could help alleviating the symptoms by bringing out the much needed overall improvement in the affected victims. A gut disorder akin to Crohn's disease is, sometimes, reported in autistic children, an extremely painful gastrointestinal disease which is named as autistic enterocolitis. This disturbed situation hypothesized to be initiated by dysbiosis or microbial imbalance could in turn perturb the coordination of microbiota-gut-brain axis which is important in human mental health as goes the popular dictum: "fix your gut, fix your brain."

Gut Microbial Dysbiosis in Indian Children with Autism Spectrum Disorders.

Autism spectrum disorder (ASD) is a term associated with a group of neurodevelopmental disorders. The etiology of ASD is not yet completely understood; however, a disorder in the gut-brain axis is emerging as a prominent factor leading to autism. To identify the taxonomic composition and markers associated with ASD, we compared the fecal microbiota of 30 ASD children diagnosed using Childhood Autism Rating Scale (CARS) score, DSM-5 approved AIIMS-modified INCLEN Diagnostic Tool for Autism Spectrum Disorder (INDT-ASD), and Indian Scale for Assessment of Autism (ISAA) tool, with family-matched 24 healthy children from Indian population using next-generation sequencing (NGS) of 16S rRNA gene amplicon. Our study showed prominent dysbiosis in the gut microbiome of ASD children, with higher relative abundances of families Lactobacillaceae, Bifidobacteraceae, and Veillonellaceae, whereas the gut microbiome of healthy children was dominated by the family Prevotellaceae. Comparative meta-analysis with a publicly available dataset from the US population consisting of 20 ASD and 20 healthy control samples from children of similar age, revealed a significantly high abundance of genus Lactobacillus in ASD children from both the populations. The results reveal the microbial dysbiosis and an association of selected Lactobacillus species with the gut microbiome of ASD children.

A massive expansion of effector genes underlies gall-formation in the wheat pest Mayetiola destructor.

Gall-forming arthropods are highly specialized herbivores that, in combination with their hosts, produce extended phenotypes with unique morphologies [1]. Many are economically important, and others have improved our understanding of ecology and adaptive radiation [2]. However, the mechanisms that these arthropods use to induce plant galls are poorly understood. We sequenced the genome of the Hessian fly (Mayetiola destructor; Diptera: Cecidomyiidae), a plant parasitic gall midge and a pest of wheat (Triticum spp.), with the aim of identifying genic modifications that contribute to its plant-parasitic lifestyle. Among several adaptive modifications, we discovered an expansive reservoir of potential effector proteins. Nearly 5% of the 20,163 predicted gene models matched putative effector gene transcripts present in the M. destructor larval salivary gland. Another 466 putative effectors were discovered among the genes that have no sequence similarities in other organisms. The largest known arthropod gene family (family SSGP-71) was also discovered within the effector reservoir. SSGP-71 proteins lack sequence homologies to other proteins, but their structures resemble both ubiquitin E3 ligases in plants and E3-ligase-mimicking effectors in plant pathogenic bacteria. SSGP-71 proteins and wheat Skp proteins interact in vivo. Mutations in different SSGP-71 genes avoid the effector-triggered immunity that is directed by the wheat resistance genes H6 and H9. Results point to effectors as the agents responsible for arthropod-induced plant gall formation.

Comparative genome scan detects host-related divergent selection in the grasshopper Hesperotettix viridis.

In this study, we used a comparative genome scan to examine patterns of population differentiation with respect to host plant use in Hesperotettix viridis, a Nearctic oligophagous grasshopper locally specialized on various Asteraceae including Solidago, Gutierrezia, and Ericameria. We identified amplified fragment length polymorphism (AFLP) loci with significantly elevated F(ST) (outlier loci) in multiple different-host and same-host comparisons of populations while controlling for geographic distance. By comparing the number and identities of outlier loci in different-host vs. same-host comparisons, we found evidence of host plant-related divergent selection for some population comparisons (Solidago- vs. Gutierrezia-feeders), while other comparisons (Ericameria- vs. Gutierrezia-feeders) failed to demonstrate a strong role for host association in population differentiation. In comparisons of Solidago- vs. Gutierrezia-feeding populations, a relatively high number of outlier loci observed repeatedly in different-host comparisons (35% of all outliers and 2.7% of all 625 AFLP loci) indicated a significant role for host-related selection in contributing to overall genomic differentiation in this grasshopper. Mitochondrial DNA sequence data revealed a star-shaped phylogeny with no host- or geography-related structure, low nucleotide diversity, and high haplotype diversity, suggesting a recent population expansion. mtDNA data do not suggest a long period of isolation in separate glacial refugia but are instead more compatible with a single glacial refugium and more recent divergence in host use. Our study adds to research documenting heterogeneity in differentiation across the genome as a consequence of divergent natural selection, a phenomenon that may occur as part of the process of ecological speciation.