Botulinum neurotoxins are considered as one of the most potent toxins and are produced by Clostridium botulinum. It is crucial to have a rapid and sensitive method to detect the bacterium Clostridium botulinum in food. In this study, a rapid detection assay of C. botulinum in food using loop-mediated isothermal amplification (LAMP) technology was developed. The optimal primers were identified among three sets of primers designed specifically based on the partial ntnh gene encoding nontoxic-nonhaemagglutinin (NTNH) for rapid detection of the target DNA in plasmids. The optimal temperature and reaction time of the LAMP assay were determined to be 64 °C and 60 min, respectively. The chemical kit could be assembled based on these optimized reaction conditions for quick, initial high-throughput screening of C. botulinum in food samples. The established LAMP assay showed high specificity and sensitivity in detecting the target DNA with a limit of 0.0001 pg/ul (i.e., ten times more sensitive than that of the PCR method) and an accuracy rate of 100%. This study demonstrated a potentially rapid, cost-effective, and easy-operating method to detect C. botulinum in food and clinical samples based on LAMP technology.

High-frequency hearing is regarded as one of the most functionally important traits in laryngeally echolocating bats. Abundant candidate hearing-related genes have been identified to be the important genetic bases underlying high-frequency hearing for laryngeally echolocating bats, however, extensive metabolites presented in the cochleae have not been studied. In this study, we identified 4,717 annotated metabolites in the cochleae of two typical laryngeally echolocating bats using the liquid chromatography–mass spectroscopy technology, metabolites classified as amino acids, peptides, and fatty acid esters were identified as the most abundant in the cochleae of these two echolocating bat species, Rhinolophus sinicus and Vespertilio sinensis. Furthermore, 357 metabolites were identified as significant differentially accumulated (adjusted p-value <0.05) in the cochleae of these two bat species with distinct echolocating dominant frequencies. Downstream KEGG enrichment analyses indicated that multiple biological processes, including signaling pathways, nervous system, and metabolic process, were putatively different in the cochleae of R. sinicus and V. sinensis. For the first time, this study investigated the extensive metabolites and associated biological pathways in the cochleae of two laryngeal echolocating bats and expanded our knowledge of the metabolic molecular bases underlying high-frequency hearing in the cochleae of echolocating bats.

Rickett’s big-footed bat, Myotis pilosus, which belongs to the family Vespertilionida, is the only known piscivorous bat in East Asia. Accurate whole genome and transcriptome annotations are essential for the study of bat biological evolution. The lack of a whole genome for M. pilosus has limited our understanding of the molecular mechanisms underlying the species’ evolution, echolocation, and immune response. In the present work, we sequenced the entire transcriptome using error-corrected PacBio single-molecule real-time (SMRT) data. Then, a total of 40 GB of subreads were generated, including 29,991 full-length non-chimeric (FLNC) sequences. After correction by Illumina short reads and de-redundancy, we obtained 26,717 error-corrected isoforms with an average length of 3018.91 bp and an N50 length of 3447 bp. A total of 1528 alternative splicing (AS) events were detected by transcriptome structural analysis. Furthermore, 1032 putative transcription factors (TFs) were identified, with additional identification of several long non-coding RNAs (lncRNAs) with high confidence. Moreover, several key genes, including PRL-2, DPP4, Glul, and ND1 were also identified as being associated with metabolism, immunity, nervous system processes, and auditory perception. A multitude of pattern recognition receptors was identified, including NLR, RLR, SRCR, the antiviral molecule IRF3, and the IFN receptor subunit IFNAR1. High-quality reference genomes at the transcriptome level may be used to quantify gene or transcript expression, evaluate alternative splicing levels, identify novel transcripts, and enhance genome annotation in bats.

The Great Himalayan Leaf-nosed bat (Hipposideros armiger) is one of the most representative species of all echolocating bats and is an ideal model for studying the echolocation system of bats. An incomplete reference genome and limited availability of full-length cDNAs have hindered the identification of alternatively spliced transcripts, which slowed down related basic studies on bats’ echolocation and evolution. In this study, we analyzed five organs from H. armiger for the first time using PacBio single-molecule real-time sequencing (SMRT). There were 120 GB of subreads generated, including 1,472,058 full-length non-chimeric (FLNC) sequences. A total of 34,611 alternative splicing (AS) events and 66,010 Alternative Polyadenylation (APA) sites were detected by transcriptome structural analysis. Moreover, a total of 110,611 isoforms were identified, consisting of 52% new isoforms of known genes and 5% of novel gene loci, as well as 2112 novel genes that have not been annotated before in the current reference genome of H. armiger. Furthermore, several key novel genes, including Pol, RAS, NFKB1, and CAMK4, were identified as being associated with nervous, signal transduction, and immune system processes, which may be involved in regulating the auditory nervous perception and immune system that helps bats to regulate in echolocation. In conclusion, the full-length transcriptome results optimized and replenished existing H. armiger genome annotation in multiple ways and offer advantages for newly discovered or previously unrecognized protein-coding genes and isoforms, which can be used as a reference resource.