Overview of sequencing analysis
A total of 2541888 reads were obtained from 14 samples, with mean reads of 181563 per sample. A total of 1221 OTU's were obtained in the study. Good's coverage of >99%, indicated the sufficient sequencing depth to represent all the bacterial communities. Alpha-diversity indices (Ace, Chao 1, and Shannon) of different developmental stages of Indian white shrimp were calculated. Ace and Chao 1 are richness indicators, which measures different kinds of organisms present. The richness index Chao1 varied from 579.86±22.25 to 841.22±16.82 and ACE varied from 564.56±11.72 to 828.70±14.17. Our observations suggested that the richness of microbes steadily increased from egg till mysis and gradually stabalised in post larval stages though there was an increase in PL12 stage. However, Shannon is a diversity indicator, which takes into account the richness of the microbes and how evenly they are distributed. The diversity index Shannon showed that the mysis stage was more diverse and PL12 was less diverse inspite of having increased richness (Table 1).
Bacterial diversity and composition
P. indicus developmental stages microbiome contained a diverse community composition characterized into 21, 34, 29, 29, 27, 30 and 33 phyla, which were further classified to 243, 586, 584, 542, 457, 491 and 566 genera for egg, nauplii, zoea, mysis, PL1, PL6 and PL12 respectively (Table S1). The major phyla associated with the early developmental stages of P. indicus were Proteobacteria, Bacteroidetes, Actinobacterota, Firmicutes, Planctomycetes and Verrucomicrobiota. Proteobacteria and Bacteroidetes were the top two dominant bacterial phyla in all the developmental stages of the shrimp and the rest varied in the abundance and prevalence (Fig. 2). The Proteobacteria was the most dominating phylum in all the developmental stages studied. Nearly half of the phyla belong to Proteobacteria till larvae reach mysis stage, which further increased to more than 60% during the PL stages. Bacteroidetes was the second most dominating between 22-35% in the early stages, which further reduced to about 10% during the late post larval stages (Table 2).
Bacterial community associated with different developmental stages
The most abundant genera from the most abundant phyla (Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes) were compared in all the developmental stages (Fig. 3). Alteromonas was the most dominant genera in egg (19.67%) and nauplii (12.55%). Winogradskyella (12.64%), Mesoflavibacter (10.97%), Haloferula (5.23 %) and Rhodobacteraceae_unclassified (4.61%) were dominant in egg; while Nautella (9.02%), Tenacibaculum (5.94%), Vibrio (5.64%) and Pseudoalteromonas (5.42%) were observed in nauplii. The top five bacterial genera in zoea stage were Roseibacillus (12.85%), Saprospiraceae_unclassified (6.92%), Rubinisphaeraceae_unclassified (6.73%), Rhodobacteraceae_unclassified (6.44%) and uncultured (5.29%); while uncultured (8.17%), NS3a_marine_group (6.59%), Pseudoalteromonas (6.40%), Rhodobacteraceae_unclassified (5.90%), Kordiimonas (5.24%) were dominant in mysis. Rhodobacteraceae_unclassified was found to be common dominant genera between zoea and mysis. In PL1, Vibrionaceae_unclassified (14.77%), Kordia (11.01%), Lactobacillus (3.60%), Vibrio (7.35%), Pseudomonas (6%) were present. Pseudomonas started increasing in postlarvae stages (PL1 (6%), PL6 (13.51%) and PL12 (13.05%); while Ascidiaceihabitans was common in PL6 (19.95%) and PL12 (19.05%), Gammaproteobacteria_unclassified (PL6: 14.01%, PL12: 11.20%) and Pseudarthrobacter (PL6: 4.19%, PL12: 6.01%) were also observed to be common between PL6 and PL12 (Fig. 4, Table 2).
Comparison of the bacterial community structures
Principal coordinate analysis (PCoA) based on the Bray-Curtis distance was used at the feature level to compare the composition of bacteria associated with P. indicus at early developmental stages (Fig. 5). The bacterial communities associated with shrimp were different across all developmental stages. However, the bacterial profiles in PL6 and PL12 were not significantly different as observed in β diversity analysis. The results indicate that the bacterial compositions became stable once they enter post larval stage. PERMANOVA analysis, confirm that bacterial profiles were strongly associated with shrimp developmental stages (F-value: 2.1962; R-squared: 0.65308; p-value < 0.005).
Shared bacterial community associated with shrimp at early life stages
Venn diagram showed 70 shared genera that were associated with all the developmental stages (Fig. 6). The shared genera were classified to phyla Proteobacteria, Bacteroidetes, Planctomycetes, Actinobacteria, Firmicutes, Cyanobacteria, Chloroflexi and Bdellovibrionota mainly dominated by Proteobacteria. Further, unique genera specific to egg (6), nauplii (65), zoea (44), mysis (30), PL1 (17) and PL12 (57) were observed in the study. Linear discriminant analysis effect size (LEfSe) was performed to identify the specific taxa significantly varied in abundance in early developmental stages. In total, 34 genera, varying significantly were identified with LDA scores >4 (Fig. S1).