**1. Introduction**

Sponges (Porifera) constitute an important component of marine and freshwater ecosystems because of their species' richness, abundance, and key functional roles [1–4]. Sponges are inhabited by a wide variety of microorganisms, including archaea, heterotrophic bacteria, cyanobacteria, microscopic algae (green, red, cryptophytic, diatoms), dinoflagellates, and fungi; these microorganisms account for up to 50% of their biomass [5–7]. Sponge bacterial communities tend to be dominated by Gamma-, Alphaproteo-bacteria, Actinobacteria, Cyanobacteria, Chloroflexi and Poribacteria [8–10].

Sponge symbiotic communities are based on complex functional relationships that were formed during the adaptation of the entire community to environmental conditions [11,12]. Microorganisms can be alternative sources of energy and carbon for the sponge, take part in the nitrogen cycle, protect against oxidative stress, and produce various bioactive metabolites [9,12,13]. In aquatic ecology, metagenomic approaches make it possible to investigate complex microbial communities and their interactions with the host and with the environment.

**Citation:** Itskovich, V.; Kaluzhnaya, O.; Glyzina, O.; Prathiviraj, R.; Seghal Kiran, G.; Selvin, J. Microbiome Changes of Endemic Lake Baikal Sponges during Bleaching Syndrome Development. *Diversity* **2021**, *13*, 653. https://doi.org/10.3390/d13120653

Academic Editor: Michael Wink

Received: 16 November 2021 Accepted: 2 December 2021 Published: 8 December 2021

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Studies on the effects of increased temperature and ocean acidification on marine Porifera revealed that microbial symbiotic communities play an important role in maintaining sponge health and survival [11,14]. Under stress, sponge species undergo compositional and functional shifts in the microbiome [10,15–19]. Most research has been conducted on the microbial associations of marine sponges. Symbiotic associations of freshwater sponges have been insufficiently studied, even though these sponges also have diverse microbiomes including Actinobacteria, alpha-, beta-proteobacteria, Verrucomicrobia, and Flavobacterium [20–26].

Lake Baikal is the world's oldest and deepest lake, estimated to be 35 million years old with a maximum depth of 1647 m. The Lake Baikal endemic sponge family Lubomirskiidae constitutes the bulk of the benthic biomass and includes 14 described species and two subspecies [27–29]. Sponges form the main part of the benthos biomass and, as sedentary biofiltrators, play an important role in the lake's ecology. *Lubomirskia baikalensis Pallas, 1776* is a benthic littoral and sublittoral species that inhabits depths from 3–120 m. *Lubomirskia baikalensis* is a branched sponge that can grow to over 1 m and forms underwater forests at depths of 8–15 m. *Baikalospongia intermedia* Dybowsky,1880 is an overgrowing sponge that occurs at all depths, including the deep-water zone. *B. intermedia* and *L. baikalensis* are the most widespread species [29,30].

An ecological crisis has been observed in the littoral area of the lake since 2011 [31]. An important sign of the ecological crisis is the mass death of the sponges, and in some regions of Lake Baikal, 100% of the individuals are affected [31–34]. Studying Baikal sponge microbial communities could help reveal if there is a connection between bacterial symbionts and a mass sponge bleaching event that was recently detected.

Climate warming, coastal eutrophication, accumulation of toxic industrial contaminants, or infection with pathogens are indicated as possible reasons for the mass death of Baikal sponges [31–36]. The revealed narrow temperature optimum of *L. baikalensis* indicates the probable effect of increased temperature in the lake on disease development [33]. These findings correlate with the findings of numerous studies of sea sponges and corals, which also showed the influence of climate warming on the development of several diseases of these organisms [11,37–41]. However, metagenomic studies of the effect of disease on the composition of the symbiotic community of Baikal sponges are rare.

The diversity of 16S rRNA genes in the microbial community of diseased *L. baikalensis* revealed the predominance of Cyanobacteria and low abundance of Bacteroidetes and Betaproteobacteria [32]. It was also found that the mucous films on the surface of diseased sponges were formed by cyanobacteria of the order Oscillatoriales, which included representatives of the genera *Tychonema*, *Phormidium*, and *Leptolyngbya* [42]. Other results of 16S rRNA gene sequencing revealed that, in diseased sponges, the most represented OTUs belong to the families Oscillatoriaceae, Cytophagaceae, Flavobacteriaceae, Chitinophagaceae, Sphingobacteriaceae, Burkholderiaceae, Rhodobacteraceae, Comamonadaceae, Oxalobacteraceae and Xanthomonadaceae [35]. A comparison of healthy and diseased sponge microbiomes showed an increase in the number of Bacteroidetes and Proteobacteria, and the absence of a specific pathogen in diseased samples [36]. Bacteroidetes and Proteobacteria (families Flavobacteriaceae, Burkholderiaceae and Moraxellaceae) are abundant in diseased sponges [43]. It was confirmed that mat-forming cyanobacteria *Tychonema* plays a special role in the disease and death of Baikal sponges [44]. Moreover, studies of diseased sponge microbiomes were mostly carried out for *L. baikalensis*, even though other species are also susceptible to disease. Thus, the currently available data on the Baikal sponge microbial communities are limited and contradictory.

In this work, we carried out a comparative metagenomic analysis of the diseased and healthy sponges of two Lubomirskiidae species, *L. baikalensis* and *B. intermedia*, which were collected at two different time points.
