Microbial community composition associated with early developmental stages of the Indian white shrimp, Penaeus indicus

Gut microbiota is known to influence the physiology, health, nutrient absorption, reproduction, and other metabolic activities of aquatic organisms. Microbial composition can influence intestinal immunity and are considered as health indicators. Information on gut microbial composition provides potential application possibilities to improve shrimp health and production. In the absence of such information for Penaeus indicus, the present study reports the microbial community structure associated with its early developmental stages. Bacterial community associated with the early developmental stages (egg, nauplii, zoea, mysis, PL1, PL6 and PL12) from two hatchery cycles were analysed employing 16S rRNA high throughput sequencing. Proteobacteria and Bacteroidetes, were the two dominant phyla in P. indicus development stages. Sequential sampling revealed the constant change in the bacterial composition at genus level. Alteromonas was dominant in egg and nauplii stage, whilst Ascidiaceihabitans (formerly Roseobacter) was the dominant genera in both PL6 and PL12. The bacterial composition was highly dynamic in early stages and our study suggests that the mysis stage is the critical phase in transforming the microbial composition and it gets stabilised by early post larval stages. This is the first report on the composition of microbiota in early developmental stages of P. indicus. Based on these results the formation of microbial composition seems to be influenced by feeding at early stages. The study provides valuable information to device intervention strategies for healthy seed production.


Introduction
Complex symbiotic microbiota inhabits the intestine and is known to play significant role in the host physiology, health, nutrient absorption, reproduction, and other metabolic activities in terrestrial and aquatic animals (Holt et al. 2020;Rajeev et al. 2021;Butt and Volkoff 2019;Kho and Lal 2018;Li et al. 2018;Nelson et al. 2013). Diversity of gut bacterial communities shift significantly during early growth stages and stabilizes later (Stephens et al. 2016), and the abundance of certain group of bacteria can be considered as "health or disease indicators" (Zheng et al. 2017). Studies characterizing the colonization of gut microbiota in aquatic animals (Stephens et al. 2016;Angthong et al. 2020;Liu et al. 2020), and the influence of a bacterial community or an individual bacterial species, on intestinal immunity (Holt et al. 2020;Rajeev et al. 2021;Zheng et al. 2020) have been reported recently. However, very few have attempted to describe the dynamics of microbes in the early developmental stages including egg, nauplii, zoea, mysis and early post larvae of shrimp when they are vulnerable to diseases.
Information on microbiota provides potential application possibilities to improve the production of food animals. Recent studies have reported the formation of microbiota, which get stabilized by post larval stages (PL5-PL15), indicating the role of early feeding, food and feeding habits, feed additives in shaping the gut microbiome in penaeid shrimp P. monodon (Angthong et al. 2020) and P. vannamei (Wang et al. 2020). Recently, we have reported the gut microbial composition in adult P. indicus obtained from wild and farmed environments (Patil et al. 2021 information on the formation of microbiota in the early developmental stages of Indian white shrimp, P. indicus is not available. Shrimp farming is a major aquaculture activity in several south-American and south-Asian nations including India (FAO 2020;Naylor et al. 2021). P. indicus is endemic to Indo-West Pacific and Middle Eastern regions (Sajeela et al. 2019) and suggested as an indigenous complimentary species (Vijayan 2019) alongside P. vannamei, a globally dominant aquaculture species. Infections due to bacterial pathogens is considered as a major challenge in P. indicus seed production in hatcheries requiring scientific intervention for economical sustainability. Understanding the dynamics of establishment of species-specific microbiota will help in devising intervention strategies to produce healthy shrimp seeds. The present study reports the investigation of gut microbial communities from early developmental stages (egg to post larva) of P. indicus using 16S rRNA high throughput sequencing.

Shrimp seed production and sampling
Indian white shrimp (Penaeus indicus) larvae were produced in two full-sib batches at Muttukadu Experimental Station of ICAR-Central Institute of Brackishwater Aquaculture, Chennai, India. Healthy brooders were shifted to spawning room and maintained in UV sterilized seawater (35 ppt) in 250 L tanks. After spawning, the eggs were disinfected (Povidoneiodine 100 ppm) and allowed to hatch in 500 L tanks. After hatching, the healthy phototactic nauplius was collected and stocked in larval rearing tanks (100 no/L). The first feeding was started from zoea-1 stage with live algae, Thalassiosira sp. (40-80,000 cells/mL) till post larvae. The post-larval stages were fed with brine shrimp nauplius (Artemia) at 4-5 no/PL. Ten percent water exchange was carried out daily from mysis stage and no antibiotic, probiotic was added throughout the study. Samples from both the batches were collected at egg, nauplii, zoea (Z2), mysis (M2), PL1, PL6 and PL12 stages (Fig. 1) PL12: 25) were homogenised using sterile pestle for DNA extraction. Genomic DNA was extracted using the QIAamp DNA stool mini kit (Qiagen, Germany) following the manufacturer's protocol. The DNA concentration and purity were determined using NanoDrop ND-1000 spectrophotometer (Thermo Scientific, USA) and stored at −80 °C until used.
The amplicon libraries were prepared using the Nextera XT index kit (Illumina Inc. USA) as per metagenomic sequencing library preparation protocol. Amplification of bacterial 16S rRNA gene was carried out using the primers F GCC TAC GGGNGGC WGC AG and R ACTACHVGGG TAT CTA ATC C. The amplicon libraries were purified by AMPPure XP beads and quantified using Qubit Fluorometer. Library QC was checked on Agilent 4200 Tape station system and sequenced using MiSeq platform.

Amplicon sequence processing and microbiome analysis
The raw sequences were processed using MOTHUR pipeline (v. 1.42) (Schloss et al. 2009) to filter reads, create contigs; reduce noise as per standard MiSeq procedure. Sequences were aligned, clustered, and identified taxonomically with SILVA database (http:// arb-silva. de) release 138. Chimera. vsearch option was used to identify and remove chimeras. Sequences with 97% identity threshold were classified into operational taxonomic units (OTUs) at genetic distances of 0.03. Alpha diversity indexes were calculated from rarefied samples using calculators for richness and diversity indices of the bacterial community (i.e., Chao1, ACE, and Shannon). Statistically significant correlation with the coordinates was tested for the PCoA plot (p < 0.05). Venn diagram was prepared using a web-based tool, interactiVenn (Heberle et al. 2015). Non-parametric t-test was carried out using linear discriminant analysis (LEfSe) to determine the significantly differing OTU's between the groups. The data output was subjected to detailed statistical and meta-analysis using MicrobiomeAnalyst (Dhariwal et al. 2017) and Microsoft Excel for visualisation.

Data availability
The datasets of 16S rRNA amplicon sequences obtained in this study were submitted to the NCBI BioProject ID (PRJNA718452).

Overview of sequencing analysis
A total of 2,541,888 reads were obtained from 14 samples, with mean reads of 181,563 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, Chao1, and Shannon) of different developmental stages of Indian white shrimp were calculated. Ace and Chao1 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 stabilised 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 bacterial diversity was high at mysis stage and PL12 was having higher richness but less diversity (Table 1).
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 belonged to Proteobacteria till the larvae reached mysis stage, which further increased to more than 60% during the PL stages. Bacteroidetes was the second most dominating between 22 and 35% in the early stages, which further reduced to about 10% during the late post larval stages (Table 2).

Comparison of the bacterial community structures
Principal coordinate analysis (PCoA) based on the Bray-Curtis distance was used at the OTUs level to compare the composition of bacteria associated with P. indicus at early developmental stages (Fig. 5). The bacterial communities associated with the shrimp varied across all developmental stages and the pattern in post larval stage was more similar compared to egg, nauplii, zoea and mysis. The bacterial profiles in PL6 and PL12 were not significantly different as observed in β diversity analysis. The results indicate that the developmental stages have substantial influence on the bacterial compositions, which 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 2 : 0.65308; p-value < 0.005).

Discussion
It is increasingly evident that host-microbiota interaction plays a major role in growth and developmental stages of both terrestrial and aquatic animals. Co-evolution of host and microbes has always been overlooked during the evolution of aquatic organisms ). Further, it was always considered that the embryo/egg is sterile and has no microbiota. However, with the advancement of technology, the presence of bacteria in the eggs of Cod and Halibut was reported previously (Hansen and Olafsen 1989) and it is well understood that the microbiota is transferred vertically and horizontally. Recent studies in P. monodon (Angthong et al. 2020), P. vannamei (Zheng et al. 2017;Wang et al. 2020), Macrobrachium rosenbergii ; Procambarus clarkii (Zhang et al. 2020), Panulirus ornatus (Ooi et al. 2017) have shown the presence of microbiota in the eggs and early developmental stages of these aquatic crustaceans. Understanding these host-microbial community relationship will help in devising intervention strategies for healthy shrimp production. Here, we report the microbiota associated with the early developmental stages of P. indicus for the first time employing high throughput sequence analysis.  Analysis of 16S rRNA remains the standard culture-independent approach to investigate microbial diversity (Klindworth et al. 2013). Alpha diversity is a measure of compositional complexity within an ecosystem. Ace and Chao1 are richness indicators, which measures different kinds of organisms present. Shannon is diversity indicator, which takes into account the richness of the microbes and how evenly they are distributed. Our observations suggested that the richness of microbes steadily increased from egg till mysis and gradually stabilised in post larval stages though there was an increase in PL12 stage. The diversity index Shannon showed that the mysis stage was more diverse and PL12 was less diverse, though the species richness was high in PL12. The observed richness in PL12 might be due to the presence of some rare microbes which are not evenly distributed. The zoea and mysis stages were highly diverse, which might be due to the initiation of feeding during these stages. Several studies tracking gut microbial composition during developmental stages have implicated the feeding to be the cause of bacterial community changes (Holt et al. 2020;Angthong et al. 2020;Wang et al. 2020). PCoA analysis showed that the bacterial communities of PL6 and PL12 from full sib families were similar compared to earlier stages. Shrimps undergo metamorphosis through various developmental stages and the observed shift in the microbial composition can be attributed to the physiological development and the   (1) Egg (2) Nauplii (1) Nauplii (2) Zoea (2) Zoea (1) Mysis (2) Mysis (1) PL1 (2) PL1 (1) PL12 (2) PL6 (2) PL12 (1) PL6 (1) Sample type Egg Nauplii Zoea Mysis PL1 PL6 PL12 diet requirement of each stage (Angthong et al. 2020). In the present study, the eggs hatched into nauplii, which survive utilizing yolk reserves and in zoea and mysis stage, they were fed with live algae (Thalassiosira sp.). A transient period of development from egg to mysis with changing physiology and diet might the reason for observed variations in the bacterial community structures. During postlarvae (PL1, PL6, PL12) they were fed with brine shrimp nauplius (Artemia). The comparatively longer duration of post larval stage with little physiological change and the uniform diet might be the reason for observed stable microbiota during these stages. Hence, the morphological and physiological changes, along with diet modifications might impact the shift in bacterial diversity. The bacterial profile at each developmental stage varied slightly between the families as the growth progress, which is reported to be due to the possible genetic variation in different shrimp families (Angthong et al. 2020). Further studies with large number of full-sib batches and multiple replicates could explain the influence of family and individual on bacterial profile. The present study identified 70 shared genera, classified to phyla Proteobacteria, Bacteroidetes, Planctomycetes, Actinobacteria, Firmicutes, Cyanobacteria, Chloroflexi and Bdellovibrionota, during the developmental stages of P. indicus. Proteobacteria and Bacteroidetes were the top two dominant bacterial phyla associated with all the developmental stages, while Actinobacterota, Firmicutes, Planctomycetes and Verrucomicrobiota varied in prevalence and abundance. Proteobacteria and Bacteroidetes are considered as resident microbiota of several aquatic organisms such as Neocaridina denticulate (Cheung et al. 2015), P. chinensis (Liu et al. 2011), P. vannamei (Wang et al. 2020), P. monodon (Rungrassamee et al. 2016(Rungrassamee et al. , 2013(Rungrassamee et al. , 2014. Angthong et al. (Angthong et al. 2020 and Wang et al. (Wang et al. 2020) reported that Proteobacteria and Bacteroidetes are the dominant phyla in the early developmental stages in in P. monodon, M. rosenbergii and P. vannamei, respectively in agreement with the present study.
Each developmental stages showed the dominance of different microflora indicating the role of host factors in shaping the microbiota of an organism. Alteromonas (19.67% and 12.55%) were the major bacterial genera in egg and nauplii, which diminished in later stages. The symbiotic association of Alteromonas sp with the embryo of marine crustacean Palaemon macrodactylus, protecting from fungal infection by producing antifungal metabolite was reported (Turnes et al. 1989). The dominance of genus Alteromonas in the egg and nauplii of P. indicus suggest similar role during the developmental stage. Further, Vibrio was observed in all the stages, but was abundant in PL1 stage. Studies have shown that Vibrio is commonly associated with several aquatic organisms and our observations agree with the observations of Angthong et al. (2020) in the developmental stages of P. monodon. Initiation of feeding during the zoea and mysis stages may be the reason for the observed colonisation by several group of microbes without being dominated by any specific group. Ascidiaceihabitans (formerly Roseobacter) was the dominant genera in both PL6 and PL12. Roseobacter are reported to be associated with the gut of several aquatic organisms and have essential metabolic capabilities to utilize proteins (Luo and Moran 2014;Curiel et al. 2018), which help in growth of an organism. Recently, several species of Roseobacter are reported to have probiotic activity (Yao et al. 2018).
The dominant association of genera Winogradskyella and Mesoflavibacter under phylum Bacteroidetes with egg stage was observed, however their role in the development of larvae is not known. Tenacibaculum is normally associated with the marine algae (Crenn et al. 2018) and the presence of these genera in various stages of shrimp development may be associated with the planktonic feed provided in the hatchery. Kordia was dominant during PL1 stage, and it is known to utilize polysaccharides originated by marine phytoplankton or algae (Lim et al. 2020) and it is reported to increase in the presence of algal bloom and utilize the organic matter. Further, the genus under Actinobacterota, Firmicutes, Planctomycetes and Verrucomicrobiota were also reported. In a similar study in M. rosenbergii, strong association of Proteobacteria, Bacteroidetes and Actinobacterota was observed , while Planctomycetes and Verrucomicrobiota were present in relation to feeding and environment. Similarly, the shift in the microbial dynamics at different life stages has been reported from other invertebrates like butterfly, silkworm Hammer et al. 2014).
Our study suggests that the mysis stage is the critical phase in transforming the microbial composition and gets stabilised by post larval stages. During the early stage, shrimp nauplii develop using yolk. The possible entry of microbial community associated with early feeding in addition to other metabolic activities in the gut might be the reason for the observed changes in the bacterial community. Similar observation in P. monodon was suggested to be due to the influence of host physiology and diets (Angthong et al. 2020). Further, several studies have reported that the wild shrimp share similar microbial communities with the domesticated shrimps though they inhabit different environments having different bacterioplankton communities (Patil et al. 2021;Rungrassamee et al. 2014). The literature suggest that the microbial communities of host differ from the environment as host intestinal environment is expected to exert strong selective pressure on establishment of particular gut microbial community (Angthong et al. 2020;Liu et al. 2020;Rungrassamee et al. 2014;Johnson et al. 2008;Meziti et al. 2012;Schmidt et al. 2015). Hence, gut microbial structure seems to be strongly determined by the host than the environment. Further studies including a greater number of replicates are required for enhanced resolution of the bacterial diversity specific to each developmental stage of the species.
To our knowledge this is the first report on the characterization of microbiota associated with early developmental stages of P. indicus. The bacterial composition was highly dynamic, and stage specific dominance of genera was observed. Modulation of gut microbial composition through supplementation of beneficial microbes at the mysis stage might improve survival and help in healthy and disease-free shrimp seed production. Further studies including multiple families is required to confirm the influence of external feeding on the microbial diversity.