Efficient biosynthesis of creatine by whole-cell catalysis from guanidinoacetic acid in Corynebacterium glutamicum.

Creatine is a naturally occurring derivative of an amino acid commonly utilized in functional foods and pharmaceuticals. Nevertheless, the current industrial synthesis of creatine relies on chemical processes, which may hinder its utilization in certain applications. Therefore, a biological approach was devised that employs whole-cell biocatalysis in the bacterium Corynebacterium glutamicum, which is considered safe for use in food production, to produce safe-for-consumption creatine. The objective of this study was to identify a guanidinoacetate N-methyltransferase (GAMT) with superior catalytic activity for creatine production. Through employing whole-cell biocatalysis, a gamt gene from Mus caroli (Mcgamt) was cloned and expressed in C. glutamicum ATCC 13032, resulting in a creatine titer of 3.37 g/L. Additionally, the study employed a promoter screening strategy that utilized nine native strong promoters in C. glutamicum to enhance the expression level of GAMT. The highest titer was achieved using the P1676 promoter, reaching 4.14 g/L. The conditions of whole-cell biocatalysis were further optimized, resulting in a creatine titer of 5.42 g/L. This is the first report of successful secretory creatine expression in C. glutamicum, which provides a safer and eco-friendly approach for the industrial production of creatine.

Transcriptomic and Functional Analyses of Phenotypic Plasticity in a Higher Termite, Macrotermes barneyi Light.

Eusocial termites have a complex caste system, which leads to the division of labor. Previous studies offered some insight into the caste differentiation in lower termites; however, few studies were focusing on the molecular mechanisms of higher termites with sophisticated societies. Comparative transcriptomic analyses of five immature castes of a higher termite, Macrotermes barneyi Light, suggest that phenotypic plasticity is modulated by an array of transcriptional changes, including differentially expressed genes (e.g., caste-biased genes Vtg and TnC), co-expression networks (e.g., genes associated with nymph reproduction), and alternative splicing (e.g., events related to muscle development in presoldiers). Transcriptional (RT-PCR and RT-qPCR) and functional (in vivo RNAi) validation studies reveal multiple molecular mechanisms contributing to the phenotypic plasticity in eusocial termites. Molecular mechanisms governing the phenotypic plasticity in M. barneyi could be a rule rather than an exception in the evolution of sociality.

Downregulation of Orco and 5-HTT Alters Nestmate Discrimination in the Subterranean Termite Odontotermes formosanus (Shiraki).

Nestmate discrimination allows social insects to recognize nestmates from non-nestmates using colony-specific chemosensory cues, which typically evoke aggressive behavior toward non-nestmates. Functional analysis of genes associated with nestmate discrimination has been primarily focused on inter-colonial discrimination in Hymenopterans, and parallel studies in termites, however, are grossly lacking. To fill this gap, we investigated the role of two genes, Orco and 5-HTT, associated with chemosensation and neurotransmission respectively, in nestmate discrimination in a highly eusocial subterranean termite, Odontotermes formosanus (Shiraki). We hypothesized that knocking down of these genes will compromise the nestmate recognition and lead to the antagonistic behavior. To test this hypothesis, we carried out (1) an in vivo RNAi to suppress the expression of Orco and 5-HTT, respectively, (2) a validation study to examine the knockdown efficiency, and finally, (3) a behavioral assay to document the phenotypic impacts/behavioral consequences. As expected, the suppression of either of these two genes elevated stress level (e.g., vibrations and retreats), and led to aggressive behaviors (e.g., biting) in O. formosanus workers toward their nestmates, suggesting both Orco and 5-HTT can modulate nestmate discrimination in termites. This research links chemosensation and neurotransmission with nestmate discrimination at the genetic basis, and lays the foundation for functional analyses of nestmate discrimination in termites.

The Influence of Allogrooming Behavior on Individual Innate Immunity in the Subterranean Termite Reticulitermes chinensis (Isoptera: Rhinotermitidae).

Insect societies have evolved a series of disease defenses against pathogens, including social sanitary behavior and individual innate immunity. However, whether sanitary behavior can affect individual innate immunity remains unknown. Here, we exposed the termite Reticulitermes chinensis Snyder to the entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin(Ascomycota: Hypocreales), and then measured their allogrooming behavior, conidia load, infection mortality, antifungal activity and immune gene expressions . Our results showed that most of the fungal conidia were fast removed from the cuticles of the grouped termites by intensive allogrooming behavior, resulting in low mortality. The antifungal activity and immune gene expressions (termicin and transferrin) in grouped exposed termites were significantly lower than those in single exposed termite but not significantly different from those in unexposed treatments. These results suggest that allogrooming behavior can fast remove fungal conidia from termite cuticles and then decrease their physiological investment in individual innate immunity.

Bioactivity of diatomaceous earth against the subterranean termite Reticulitermes chinensis Snyder (Isoptera: Rhinotermitidae).

The effects of diatomaceous earth (DE) on the penetrating behavior, tunneling behavior, mortality, and body surface characteristics of the subterranean termite Reticulitermes chinensis were investigated in this study. Our results show that the workers of R. chinensis were able to penetrate 1- and 2-mm layers of dry DE but not 3-mm layers. After treatment with dry DE for 6 h, the mortality of termites reached 100%, which was significantly higher than in the treatment with DE with a 10 and 25% moisture content and treatment with sand of three different moisture contents. The tunneling distances of workers in DE with 10, 25, and 50% moisture contents were all significantly shorter than those in sand with the same moisture contents (10, 25, and 50%), indicating that DE has a good suppressing effect on the tunneling behavior of workers. After treatment with dry DE for different times (1, 3, and 6 h), many DE particles adhered to the bodies of workers, whereas no particles adhered to the body of workers in the case of treatment with dry sand. The treatment with dry DE for 6 h resulted in the death of all workers, which presented conspicuous abdominal shrinkage, whereas workers treated with sand had no significant mortality and no obvious abdominal shrinkage. In summary, we suggest that dry DE has ideal insecticidal activity against the subterranean termite R. chinensis and can be further exploited for controlling termites inside houses.

Physiological profiles associated with ceasing growth of unfertilized eggs produced by unmated queens in the subterranean termite Reticulitermes chinensis.

In Reticulitermes chinensis, a close relative of R. speratus with asexual queen succession, unfertilized eggs can be produced but do not hatch as larvae. To explain this phenomenon, we analyzed the physiological differences between unfertilized eggs/unmated queens and fertilized eggs/mated queens. Fertilized eggs had significantly lower quantities of five amino acids (Cys, Met, Ile, Leu and Tyr), Ca, protein and cholesterol during development. The higher levels of four trace elements (Na, K, Zn and Fe) in fertilized eggs and their lower levels in mated queens indicated that mated queens might transfer these trace elements to fertilized eggs to aid development. The higher levels of Mn, triglycerides and serotonin in mated queens and higher levels of Mn and glucose in fertilized eggs suggested that these substances are very important for normal ovarian and embryonic growth. The different expression of three reproductive genes (vtg 1, rab 11 and JHE 1) suggested that they might be involved in the regulation of ovarian and embryonic growth. Overall, changes in these physiological indices may substantially affect ovarian and embryonic growth and inhibit development of unfertilized eggs in R. chinensis.

Experimental verification and molecular basis of active immunization against fungal pathogens in termites.

Termites are constantly exposed to many pathogens when they nest and forage in the field, so they employ various immune strategies to defend against pathogenic infections. Here, we demonstrate that the subterranean termite Reticulitermes chinensis employs active immunization to defend against the entomopathogen Metarhizium anisopliae. Our results showed that allogrooming frequency increased significantly between fungus-treated termites and their nestmates. Through active social contact, previously healthy nestmates only received small numbers of conidia from fungus-treated individuals. These nestmates experienced low-level fungal infections, resulting in low mortality and apparently improved antifungal defences. Moreover, infected nestmates promoted the activity of two antioxidant enzymes (SOD and CAT) and upregulated the expression of three immune genes (phenoloxidase, transferrin, and termicin). We found 20 differentially expressed proteins associated with active immunization in R. chinensis through iTRAQ proteomics, including 12 stress response proteins, six immune signalling proteins, and two immune effector molecules. Subsequently, two significantly upregulated (60S ribosomal protein L23 and isocitrate dehydrogenase) and three significantly downregulated (glutathione S-transferase D1, cuticle protein 19, and ubiquitin conjugating enzyme) candidate immune proteins were validated by MRM assays. These findings suggest that active immunization in termites may be regulated by different immune proteins.

Characterization of head transcriptome and analysis of gene expression involved in caste differentiation and aggression in Odontotermes formosanus (Shiraki).

BACKGROUND: The subterranean termite Odontotermes formosanus (Shiraki) is a serious insect pest of trees and dams in China. To date, very little is known about genomic or transcriptomic data for caste differentiation and aggression in O. formosanus. Hence, studies on transcriptome and gene expression profiling are helpful to better understand molecular basis underlying caste differentiation and aggressive behavior in O. formosanus. METHODOLOGY AND PRINCIPAL FINDINGS: Using the Illumina sequencing, we obtained more than 57 million sequencing reads derived from the heads of O. formosanus. These reads were assembled into 116,885 unique sequences (mean size  =  536 bp). Of the unigenes, 30,646 (26.22%) had significant similarity with proteins in the NCBI nonredundant protein database and Swiss-Prot database (E-value<10(-5)). Of these annotated unigenes, 10,409 and 9,009 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. In total, 19,611 (25.52%) unigenes were mapped onto 242 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). A total of 11,661 simple sequence repeats (SSRs) were predicted from the current transcriptome database. Moreover, we detected seven putative genes involved in caste differentiation and six putative genes involved in aggression. The qPCR analysis showed that there were significant differences in the expression levels of the three putative genes hexamerin 2, ß-glycosidase and bicaudal D involved in caste differentiation and one putative gene Cyp6a20 involved in aggression among workers, soldiers and larvae of O. formosanus. CONCLUSIONS: To our knowledge, this is the first study to characterize the complete head transcriptome of a higher fungus-cultivating termite using high-throughput sequencing. Our study has provided the comprehensive sequence resources available for elucidating molecular basis underlying caste differentiation and aggressive behavior in O. formosanus.