Entomopathogenic nematode Steinernema carpocapsae surpasses the cellular immune responses of the hispid beetle, Octodonta nipae (Coleoptera: Chrysomelidae).

The Nipa palm hispid, Octodonta nipae (Maulik) is an important invasive pest of palm trees particularly in Southern China. How this beetle interacts with invading pathogens via its immune system remains to be dissected. Steinernema carpocapsae is a pathogenic nematode that attacks a number of insects of economic importance. The present study systematically investigates the cellular immune responses of O. nipae against S. carpocapsae infection using combined immunological, biochemical and transcriptomics approaches. Our data reveal that S. carpocapsae efficiently resists being encapsulated and melanized within the host's hemolymph and most of the nematodes were observed moving freely in the hemolymph even at 24 h post incubation. Consistently, isolated cuticles from the parasite also withstand encapsulation by the O. nipae hemocytes at all-time points. However, significant encapsulation and melanization of the isolated cuticles were recorded following heat treatment of the cuticles. The host's phenoloxidase activity was found to be slightly suppressed due to S. carpocapsae infection. Furthermore, the expression levels of some antimicrobial peptide (AMP) genes were significantly up-regulated in the S. carpocapsae-challenged O. nipae. Taken together, our data suggest that S. carpocapsae modulates and surpasses the O. nipae immune responses and hence can serve as an excellent biological control agent of the pest.

The Symbiotic Bacteria-Xenorhabdus nematophila All and Photorhabdus luminescens H06 Strongly Affected the Phenoloxidase Activation of Nipa Palm Hispid, Octodonta nipae (Coleoptera: Chrysomelidae) Larvae.

Symbiotic bacteria form a mutualistic relationship with nematodes and are pathogenic to many insect pests. They kill insects using various strategies to evade or suppress their humoral and cellular immunity. Here we evaluate the toxic effects of these bacteria and their secondary metabolites on the survival and phenoloxidase (PO) activation of Octodonta nipae larvae using biochemical and molecular methods. The results show P. luminescens H06 and X. nematophila All treatments caused significant reductions in the number of O. nipae larvae in a dose-dependent manner. Secondly, the O. nipae immune system recognizes symbiotic bacteria at early and late stages of infection via the induction of C-type lectin. Live symbiotic bacteria significantly inhibit PO activity in O. nipae whereas heat-treated bacteria strongly increase PO activity. Additionally, expression levels of four O. nipae proPhenoloxidase genes following treatment with P. luminescens H06 and X. nematophila All were compared. We found that the expression levels of all proPhenoloxidase genes were significantly down-regulated at all-time points. Similarly, treatments of O. nipae larvae with metabolites benzylideneacetone and oxindole significantly down-regulated the expression of the PPO gene and inhibited PO activity. However, the addition of arachidonic acid to metabolite-treated larvae restored the expression level of the PPO gene and increased PO activity. Our results provide new insight into the roles of symbiotic bacteria in countering the insect phenoloxidase activation system.

The Entomopathogenic Nematodes H. bacteriophora and S. carpocapsae Inhibit the Activation of proPO System of the Nipa Palm Hispid Octodonta nipae (Coleoptera: Chrysomelidae).

Entomopathogenic nematodes are biocontrol agents of invasive insect pests in soil and cryptic habitats. Nipa palm hispid, Octodonta nipae, is a pest of palm trees in Sothern China. To address its increasing damage, environmentally friendly control methods are required. This study aimed to test efficacy of Heterorhabditis bacteriophora and Steinernema carpocapsae on O. nipae and investigated the influence of secondary metabolites, nematodes, and their isolated cuticles on the activation of O. nipae's prophenoloxidase system using qPCR analysis. Our data revealed that O. nipae were less susceptible to H. bacteriophora than S. carpocapsae and penetrations of infective juveniles were higher with S. carpocapsae treatment than H. bacteriophora. Moreover, expression levels of the serine protease P56, prophenoloxidase activation factor 1, PPO and serine protease inhibitor 28 upon S. carpocapsae and H. bacteriophora infections were generally downregulated at all times. However, upon heating, the cuticles lost their inhibitory effects and resulted in upregulation of the PPO gene. Similarly, the addition of arachidonic acid reversed the process and resulted in the upregulation of the PPO gene compared to the control. Further work is needed to identify toxic substances secreted by these EPNs to evade O. nipae's immune system.

Pyrosequencing Uncovers a Shift in Bacterial Communities Across Life Stages of Octodonta nipae (Coleoptera: Chrysomelidae).

Bacterial symbionts of insects affect a wide array of host traits including fitness and immunity. Octodonta nipae (Maulik), commonly known as hispid leaf beetle is a destructive palm pest around the world. Understanding the dynamics of microbiota is essential to unravel the complex interplay between O. nipae and its bacterial symbionts. In this study, bacterial 16S rRNA V3-V4 region was targeted to decipher the diversity and dynamics of bacterial symbionts across different life stages [eggs, larvae, pupae, and adult (male and female)] and reproductive organs (ovaries and testis) of O. nipae. Clustering analysis at ≥97% similarity threshold produced 3,959 operational taxonomic units (OTUs) that belonged to nine different phyla. Proteobacteria, Actinobacteria, and Firmicutes represented the bulk of taxa that underwent notable changes during metamorphosis. Enterobacteriaceae and Dermabacteraceae were the most abundant families in immature stages (eggs, larvae, and pupae), while Anaplasmataceae family was dominated in adults (male and female) and reproductive organs (ovaries and testis). The genus Serratia and Lactococcus were most abundant in eggs, whereas Pantoea and Brachybacterium represented the bulk of larvae and pupae microbiota. Interestingly the genus Wolbachia found positive to all tested samples and was recorded extremely high (>64%) in the adults and reproductive organs. The bacteria varied across the developmental stages and responsible for various metabolic activities. Selection choice exerted by the insect host as a result of its age or developmental stage could be the main reason to ascertain the shift in the bacteria populations. Maternally inherited Wolbachia was found to be an obligate endosymbiont infecting all tested life stages, body parts, and tissues. These outcomes foster our understanding of the intricate associations between bacteria and O. nipae and will incorporate in devising novel pest control strategies against this palm pest.

Exploring the Role of Relish on Antimicrobial Peptide Expressions (AMPs) Upon Nematode-Bacteria Complex Challenge in the Nipa Palm Hispid Beetle, Octodonta nipae Maulik (Coleoptera: Chrysomelidae).

The humoral immune responses of the nipa palm hispid beetle Octodonta nipae involves the inducible expression of the genes coding for antimicrobial peptides (AMPs) which are mediated by immune deficiency signaling pathways. In insects, the nuclear factor-κB (NF-κB) transcription factor, Relish, has been shown to regulate AMP gene expressions upon microbial infections. Here, we dissect the expression patterns of some AMPs in O. nipae during infections by entomopathogenic nematodes (EPNs) and their symbionts, before and after Relish knock down. Our results indicate that, prior to gene silencing, the AMPs attacin C1, attacin C2, and defensin 2B were especially expressed to great extents in the insects challenged with the nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora as well as with their respective symbionts Xenorhabdus nematophila and Photorhabdus luminescens. The study also established the partial sequence of OnRelish/NF-κB p110 subunit in O. nipae, with an open reading frame coding for a protein with 102 amino acid residues. A typical Death domain-containing protein was detected (as seen in Drosophila) at the C-terminus of the protein. Phylogenetic analysis revealed that in O. nipae, Relish is clustered with registered Relish/NF-κB p110 proteins from other species of insect especially Leptinotarsa decemlineata from the same order Coleoptera. Injection of OnRelish dsRNA remarkably brought down the expression of OnRelish and also reduced the magnitude of transcription of attacin C1 and defensin 2B upon S. carpocapsae and H. bacteriophora and their symbionts infections. Altogether, our data unveil the expression pattern of OnRelish as well as that of some AMP genes it influences during immune responses of O. nipae against EPNs and their symbionts.