Differences in the Diversity and Structure of the Gut Microbiome in Different Life Stages of the American Cockroach ( Periplaneta americana )

Gut microbes play critical roles in host nutrition, physiology, and behavior. Periplaneta americana is a famous urban pest which is widely distributed in the tropics and subtropics, but very few information is available on the gut microbiome of Periplaneta americana , particularly in its different life stages. Here, we characterized the diversity and structure of gut microbiome in eggs, nymph and adult life stages of Periplaneta americana using high-throughput 16S rRNA genes sequencing. Both the results of Alpha- and Beta-diversity analysis showed the diversity and structure of gut microbiome were significant different among the eggs, nymph and adult stages. The result of species distribution showed the predominant phyla in three life stages were Bacteroidetes , Firmicutes and Proteobacteria , but the relative abundances of these bacteria were significant different among each life stage. 1,169 operational taxonomic units were shared by three stages, which indicating the gut microbiome may be inherited to offspring from parents of Periplaneta americana . According to the prediction of functional genes in metabolic pathways, most of them were distributed in the metabolic pathways of basic physiology such as nutrition, growth, development and immunity, etc. The relative abundances of functional genes in metabolic pathways were significant different among life stages of Periplaneta americana , indicating the gut microbiome might play an important role in the physiology across its different life stages. This study revealed the diversity and structure of gut microbiome in different life stages of Periplaneta americana , which may contribute to us to understand it’s physiology and behaviors. have been few reports comparing diversity and structure of gut microbiome associated with successive life stages. This study is the first to discuss the differences in the diversity and structure of gut microbiome across the eggs, nymph and adult life stages of Periplaneta americana. The results in this study reveals the diversity and structure of gut microbiome in eggs, nymph and adult life stages of Periplaneta americana, and suggests the gut microbiome will


Introduction
The American cockroach, Periplaneta americana, is one of the most diverse and abundant pests in the world (Nasirian 2018;Li et al 2018). Similar to other hemimetabolous insects, its life cycle can be divided into egg, nymph, and adult stages (Mullins 2015;Bell and Adiyodi 1981). Periplaneta americana can survive in a wide variety of habitats due to its strong environmental adaptability, and it prefers moist, shady, and warm areas so that it is famous for its household name "Waterbug".
Periplaneta americana is also a serious urban pest due to its ability to act as a carrier and transfer many disease-causing organisms, such as bacteria, viruses and parasites (Baggio-Deibler et al 2018; control program suite QIIME (v1.8.0) (Caporase et al 2010). Sequences that were less than 200 bp in length or that contained homopolymers longer than 8 bp were removed. The chimeric sequences were detected by comparing tags with the references database (RDP Gold database) and removed using the UCHIME algorithm. The effective sequences were obtained and used in the final analysis.
Operational taxonomic units (OTUs) were clustered on the basis of 97% similarity sequence identity using the clustering program UCLUST (v1.2.22) (Edgar 2010). Then OTUs were taxonomically classified to different levels of phylum, class, order, family, genus, and species by the Ribosomal Database Program (RDP) classifier.
Alpha-diversity analysis (i.e., Observed species, ACE, Chao 1, Shannon and Simpson indices) were calculated by QIIME (v.1.8.0) using for richness and diversity indices of the gut bacterial community in each sample. Beta-diversity analysis was calculated by unweighted UniFrac and non-metric multidimensional scaling (NMDS). A one-way analysis of similarity (ANOSIM) was performed to determine the differences in gut microbiome structures among eggs, nymph and adult stages. Linear discriminant analysis (LDA) effect size (LefSe) analysis was performed to reveal the significant differences in ranking of abundant among eggs, nymph and adult stages (Segata et al 2011). The functional biomarkers were discriminated using a size-effect threshold of 4.0 on the logarithmic LDA score. The functional gene content in the gut microbiome was predicted by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUst).
Alpha-diversity indices are presented as the means ± SD. The differences in Alpha-diversity indices and relative abundances between groups at the phyla and genus levels were calculated by use of the clean tags and 173,205 (Mean 238,758 ± 33,191)  The rarefaction curves for the OTUs indicated that the quantity of observed species increased as the sequencing depth increased at the beginning, and decreased while the rarefaction curves tapered off at the end. The rarefaction curves tended to approach the saturation plateau revealed that the quantity of sequencing in this study was adequate. (Fig. 2b). The Good's Coverage can reflect the database coverage rate of each sample, and its value in this study was close to 99%, indicating that all samples reached sufficient sampling depth.
Differences of the gut microbiome diversity among the eggs, nymph and adult of Periplaneta americana The richness and diversity of the gut microbial communities from all samples were assessed using the Alpha-diversity indexes, including the Ace, Chao 1, Simpson and Shannon estimator (Fig. 3 The difference in bacterial communities between groups were reflected by the Beta-diversity analysis. The Venn diagram of the eggs, nymph and adult samples showed that 1,169 OTUs were shared by the three groups and only a few OTUs (ranging 8-31) were unique present in one life stage of Periplaneta americana gut sample (Fig. 4). The NMDS plot showed that the microbial communities of eggs samples were different from the other groups, and it also revealed the distinct structure among the eggs, nymph and adult of Periplaneta americana (Fig. 5).
Relative Abundance and Core Gut Microbiome of the eggs, nymph and adult of Periplaneta americana The top 10 representative phyla and genera according to relative abundance of the gut bacteria in eggs, nymph and adult stages that were displayed in Fig

Discussion
With the development of next-generation sequencing (NGS) technology, bacteria gene sequencing has become more convenient (Macas et al 2011;Aloisio et al 2016). In this study, we used 16S rRNA gene Illumina HiSeq sequencing to study the gut microbiome in eggs, nymph and adult life stages of Periplaneta americana. The results from Alpha-diversity analysis showed that the richness and diversity of gut microbiome in eggs stage was significant lower than that in nymph and adult stages. However, the numbers of the OTUs shared by eggs, nymph and adult stages were high, and the predominant bacteria in the three life stages were similar. The results suggested that the gut microbiome of Periplaneta americana might be inherited to the offspring from their parents.
Microbial communities are particularly prominent in the digestive tract of most insects and animals (Rowland et al 2017). Like other animals, the insect gut microbiome is an important factor which may be related to many physiological functions of their host such as food digestion, tolerance, development and immunity, etc.

Conclusions
Periplaneta americana is a widely distributed pest of public health importance found in the tropics and subtropics.
It is a serious urban pest due to its ability to act as a carrier and transfer many disease-causing organisms. This     Unifrac method with OTUs. The closer of the distance between points the samples in the graph, the higher their similarity. Stress lower than 0.2 indicates that the NMDS analysis is reliable.   While the LDA score greater than the estimated value, the species with significant difference. The length of the histogram represents the LDA score which indicates the degree of influence of species with significant difference between different life stages. The default score is 4.0.