Microbial mass spectrometry imaging (MSI) is a powerful tool used to generate biological hypotheses about the roles of metabolites in microbial competition based upon their twodimensional spatial distribution. The most commonly used ionization method for microbial MSI is matrix-assisted laser desorption ionization (MALDI). However, medium components and microbial metabolites influence the adhesion of agar samples to the MALDI target, limiting the applicability of MALDI MSI to microbes grown on specific media. Here, we describe a three-step process using a robotic sprayer for a matrix application that improves the adherence of agar samples to the MALDI target, enabling the use of different media for microbial growth and an MSI analysis of larger sample surface areas.

The MBRA-2 with media rails provides an accessible system for investigators to culture heterogenous, suspended biofilms under constant flow.

The strategic allocation of resources into immunity poses a unique challenge for individuals, where infection at different stages of development may result in unique trade‐offs with concurrent physiological processes or future fitness‐enhancing traits. Here, we experimentally induced an immune challenge in female Gryllus firmus crickets to test whether illness at discrete life stages differentially impacts fitness. We injected heat‐killed Serratia marcescens bacteria into antepenultimate juveniles, penultimate juveniles, sexually immature adults, and sexually mature adults, and then measured body growth, instar duration, mating rate, viability of stored sperm, egg production, oviposition rate, and egg viability. Immune activation significantly impacted reproductive traits, where females that were immune challenged as adults had decreased mating success and decreased egg viability compared to healthy individuals or females that were immune challenged as juveniles. Although there was no effect of an immune challenge on the other traits measured, the stress of handling resulted in reduced mass gain and smaller adult body size in females from the juvenile treatments, and females in the adult treatments suffered from reduced viability of sperm stored within their spermatheca. In summary, we found that an immune challenge does have negative impacts on reproduction, but also that even minor acute stressors can have significant impacts on fitness‐enhancing traits. These findings highlight that the factors affecting fitness can be complex and at times unpredictable, and that the consequences of illness are specific to when during an individual's life an immune challenge is induced.