Evaluation of Sample Preservation Approaches for Better Insect Microbiome Research According to Next-Generation and Third-Generation Sequencing

The microbial communities associated with insects play critical roles in many physiological functions such as digestion, nutrition, and defense. Meanwhile, with the development of sequencing technology, more and more studies begin to focus on broader biodiversity of insects and the corresponding mechanisms of insect microbial symbiosis, which need longer time collecting in the field. However, few studies have evaluated the effect of insect microbiome sample preservation approaches especially in different time durations or have assessed whether these approaches are appropriate for both next-generation sequencing (NGS) and third-generation sequencing (TGS) technologies. Here, we used Tessaratoma papillosa (Hemiptera: Tessaratomidae), an important litchi pest, as the model insect and adopted two sequencing technologies to evaluate the effect of four different preservation approaches (cetyltrimethylammonium bromide (CTAB), ethanol, air dried, and RNAlater). We found the samples treated by air dried method, which entomologists adopted for morphological observation and classical taxonomy, would get worse soon. RNAlater as the most expensive approaches for insect microbiome sample preservation did not suit for field works longer than 1 month. We recommended CTAB and ethanol as better preservatives in longer time field work for their effectiveness and low cost. Comparing with the full-length 16S rRNA gene sequenced by TGS, the V4 region of 16S rRNA gene sequenced by NGS has a lower resolution trait and may misestimate the composition of microbial communities. Our results provided recommendations for suitable preservation approaches applied to insect microbiome studies based on two sequencing technologies, which can help researchers properly preserve samples in field works.