Blanking on blanks: few insect microbiota studies control for contaminants

Research on insect-microbe relationships is booming, with DNA sequencing being the most commonly used method to describe insect microbiota. However, sequencing is vulnerable to contamination, especially when the sample has low microbial biomass. Such low-biomass samples are common across insect taxa, developmental stages, and tissue types. Identifying putative contaminants is essential to distinguish between true microbiota and introduced contaminant DNA. It is therefore important that studies control for contamination, but how often this is done is unknown. To investigate the status quo of contamination control, we undertook a systematic literature review to quantify the prevalence of negative control usage and contamination control across the literature on insect microbiota (specifically bacterial communities) over a 10 year period. Two-thirds of the 243 insect microbiota studies evaluated had not included blanks (negative controls), and only 13.6% of the studies sequenced these blanks and controlled for contamination in their samples. Our findings highlight a major lack of contamination control in the field of insect microbiota research. This result suggests that a number of microbes reported in the literature may be contaminants as opposed to insect-associated microbiota and that more rigorous contamination control is needed to improve research reliability, validity, and reproducibility. Based on our findings, we recommend the previously developed guidelines outlined in the RIDE checklist, with the addition of one more guideline. We refer to this as the RIDES checklist, which stands for Report methodology, Include negative controls, Determine the level of contamination, Explore contamination downstream, and State the amount of off-target amplification.IMPORTANCEOur systematic review reveals a major lack of methodological rigor within the field of research on insect-associated microbiota. The small percentage of studies that control for contamination suggests that an unknown but potentially considerable number of bacteria reported in the literature could be contaminants. The implication of this finding is that true microbiota may be masked or misrepresented, especially in insects with low microbial biomass.