Novel Exploration of Composition and Functional Profiling of Microbiota Across the Life Cycle of the Medically Important Blow Fly, Hemipyrellia ligurriens (Diptera: Calliphoridae)

The blow fly, Hemipyrellia ligurriens (Diptera: Calliphoridae), plays a key role in medical and forensic science, acting as a mechanical vector for various pathogens and thriving in pathogen-rich environments. While its developmental stages have been studied, the microbiota throughout its life cycle remains underexplored. This study aimed to elucidate on the structural composition and metabolic functions of the microbiota in all life stages (eggs, first-, second- and third-stage larvae, prepupae, pupae and adults) across the entire body of H. ligurriens using 16S rRNA gene amplicon sequencing. Adults from laboratory derived and natural strain flies were compared. The result showed significant differences in microbial communities across life stages. The dominant bacterial phyla identified were Proteobacteria, Firmicutes, and Bacteroidetes. At the genus level, Commensalibacter and Asaia were prevalent in most adult stages in the laboratory strains, whereas Acinetobacter, Proteus, Pseudomonas, and Myroides were abundant in the natural strains. Eggs were colonized primarily by Acinetobacter, then first-stage larvae by Myroides, and later larval stages by Ignatzschineria. No significant differences were observed between male and female microbiota. Metabolic profiling indicated that microbiota evolves alongside the host, thus contributing to essential physiological functions such as immune regulation and pathogen protection. This study highlighted the dynamic microbial interactions within H. ligurriens, and underscored the importance of microbiota in its development and health. The Hemipyrellia ligurriens colony used in this study from Naresuan University. Adults in the natural strain were collected by using a sweeping net in open areas of Naresuan University, Muang district, Phitsanulok, Phitsanulok province, Thailand. All of the fly samples (eggs, first-, second- and third-stage larvae, prepupae, pupae and adults) were inactivated by exposure to -20 degree Celsius for 10 minutes. Then, the remaining samples, except for the pork liver, were rinsed with sterile water and 70% ethanol to eliminate surface contaminants. Fresh and tainted liver from the rearing box were collected as control.DNA from each frozen sample was extracted using the NucleoSpin tissue kit. DNA concentration and purity were measured using a NanoDrop Spectrophotometer. Polymerase chain reaction (PCR) amplification of the V3-V4 region of the 16S rRNA gene was performed using specific primers 341F (5'-CCT AYG GGR BGC ASC AG-3') and 806R (5'-GGA CTA CNN GGG TAT CTA AT-3'). The PCR products (400-450 bp) were generated. These PCR products with proper size were evaluated by 2% agarose gel electrophoresis and used for library preparation. The DNA fragments were end-repaired and A-tailed then further ligated with Illumina adapters. Library quality was evaluated using the Qubit 2.0 Fluorometer and the Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated.