A Mass Spectrometric Approach: VOC Emissions, Fatty Acid Profile, and Bacteriota Identification of Spider Communities

This study presents the first integrated analysis of volatile organic compounds (VOCs), fatty acids, and cultivable bacteriota in whole‐body spider specimens, combining HS‐SPME‐GC/MS, GC/MS, and MALDI‐TOF‐MS. A total of 100 spiders from five synanthropic species, Steatoda opiliones , S. bipunctata , S. triangulosa , Scytodes thoracica , and Parasteatoda tepidariorum , were analyzed. Twenty‐one VOCs were identified, including aliphatic hydrocarbons, terpenes (farnesene, camphene), short‐chain fatty acids (SCFAs), and volatile sulfur compounds (dimethyl disulfide and dimethyl trisulfide), showing pronounced species‐specific profiles. S. triangulosa and Sc. thoracica exhibited a high relative abundance of SCFAs, supporting a microbial contribution to key components of spider‐associated volatilomes. Fatty acid profiling revealed 10 compounds dominated by oleic acid in most species; in contrast, Sc. thoracica displayed an inverted saturation pattern with stearic acid predominance and the presence of branched‐chain fatty acids. Cultivation on four media (tryptone soy agar, triple sugar iron agar, anaerobic agar, and blood agar supplemented with 7% horse blood) followed by MALDI‐TOF‐MS identification revealed species‐associated bacterial assemblages. Lysinibacillus fusiformis dominated S. opiliones ; Acinetobacter johnsonii and Enterococcus durans were characteristic of S. bipunctata ; Aerococcus viridans , Corynebacterium singulare , and Lysinibacillus sphaericus were isolated from S. triangulosa ; Lysinibacillus boronitolerans from Sc. thoracica ; and Bacillus altitudinis , together with Sphingomonas parapaucimobilis , from P. tepidariorum . The observed association between antimicrobial VOCs (SCFAs and sulfur compounds) and distinct bacterial communities suggests chemically mediated host–microbe interactions that may be advantageous in anthropogenic microhabitats. Overall, this work establishes a methodological framework for investigating spider chemical ecology across the volatilome–lipidome–microbiome axis.