**4. Discussion**

A phylum-based taxonomic classification showed that the microbiome genera that were most abundant across all samples belonged to Acidobacteria, Bacteroidetes, Actinobacteria and Cyanobacteria. Chlorophyta is also abundant in microbiomes. Moreover, the composition of the microbiome is nonspecifically altered in diseased *L. baikalensis*. These phyla were found in moderate abundance in the samples of both healthy and diseased individuals. Our samples of healthy and diseased *L. baikalensis* were collected at two different collection points and at different times (2006 and 2015). Therefore, fluctuations in the microbial community composition among healthy sponges can be caused by different environmental conditions in Lake Baikal during a given time period. In marine sponges, the microbial composition has substantial intraspecific and interspecific variability, and varies by depth and season [15]. Thus, when comparing healthy and diseased *L. baikalensis* samples, we did not reveal specific changes in diseased sponge bacterial composition. We have shown that, among healthy *L. baikalensis*, there are significant differences in the content of Chlorophyta, Bacteroidetes, Cyanobacteria and other phyla. These results are consistent with some previously obtained results. When studying the 16SrRNA gene diversity of *L. baikalensis* microbial communities in discolored tissue areas, Cyanobacteria were dominant and there was a low abundance of Bacteroidetes and Betaproteobacteria compared to healthy samples [32]. Other 16S rRNA gene sequencing results revealed that, in diseased sponges, Cyanobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria showed more than five-fold increases in abundance compared to healthy sponges [35]. Simultaneously, a comparison of the microbiomes of healthy and diseased sponges collected in 2011 and 2015 showed an increase in the number of Bacteroidetes and Proteobacteria [36]. There was no clear difference between the microbiomes of diseased and healthy sponges; for example, a high content of Verrucomicrobia (class Methylacidiphilae) was characteristic of both diseased and healthy sponges [36].

Cyanobacteria probably play an important role in the development of the disease of the Baikal sponges [42,44]. Proliferation of benthic cyanobacteria on Lake Baikal can be observed during all seasons [31,44]. The influence of the season on the microbiome and diseases of the Baikal sponges has not been studied previously. Although other studies analyzed samples collected in different seasons, there was no comparative analysis between them. With our limited number of samples, we demonstrate a difference in the microbiome of healthy *L. baikalensis* collected in different seasons, but more samples need to be analyzed.

Most previous studies of diseased sponge microbiomes were only carried out for *L. baikalensis*, even though other species are also susceptible to disease. For the first time, we performed a comparative analysis of diseased and healthy *B. intermedia*; we identified a decrease in Chlorophyta and an increase in Bacteroidetes and Cyanobacteria in diseased specimen. Until now, very few studies have been conducted on the microbiome in *B. intermedia* [21,24,26]. Proteobacteria, Actinobacteria, Planctomycetes, Cloroflexi, Verrucomicrobia, Acidobacteria, Chlorobi and Nitrospirae were identified within the *B. intermedia* microbiome [21]. It was noted that deep-sea habitat conditions affected the taxonomic diversity of microbiome bacteria and the presence of functionally significant microorganisms in communities. In microbial associations of *Baikalospongia* sp., the bacterial phyla Bacteroidetes, Proteobacteria, and Actinobacteria were predominantly identified [24]. Seo et al. [26] found differences in the bacterial species composition and diversity among *B. intermedia, L. baikalensis*, and *S. papyracea*. The bacterial communities in *B. intermedia* and *L. baikalensis* were highly similar because both species were collected from shallow zones, and Cyanobacteria and Proteobacteria accounted for the highest proportion [26]. Our data showed that Chlorophyta, Acidobacteria, Bacteroidetes, Proteobacteria and Cyanobacteria were predominant in healthy *B. intermedia*. Thus, different Baikal sponge species and different samples of the same species have differences in microbial composition; however, at present, we cannot say whether they are host- or habitat-specific.

At the genus level, the Opitutus (Verrucomicrobia) content increased or appeared in all diseased sponges. Verrucomicrobia, which are predominantly heterotrophic microorganisms that decompose hydrocarbon substrates, are widespread in marine, freshwater, soil, and hot spring ecosystems. Verrucomicrobia are characteristic of Baikal sponge communities [32]. It has been shown that Verrucomicrobia abundance is positively correlated with an increased nutrient content, phosphate availability, and seasonal algal blooms, and can change depending on the season [24,32,42]. The number of Planctomyces also increased in diseased *L. baikalensis* samples. Planctomycetes in freshwater ecosystems are generally considered minor phyla. Representatives of this phylum participate in the an-aerobic oxidation of ammonium and have the ability to degrade hydrocarbons produced by phytoplankton [53]. The genus *Nitrospira* also appeared or increased in abundance in diseased *L. baikalensis*, which reflected a general trend toward an increase in cyanobacteria in diseased sponges. Additionally, in diseased sponges, tendencies toward a decrease in the number or disappearance of some proteobacteria and planctomycetes were noted. *Streptomyces* and *Acidovorax* disappeared from diseased sponges. The number of Isosphaera, which are acidophilic planktomycetes capable of degrading numerous heteropolysaccharides, decreased in two diseased *L. baikalensis* samples.

In marine sponges, elevated temperature can disrupt the functionally important microbial symbionts [39,54,55]. Microbial community changes upon exposure to elevated temperature can manifest as a loss of specific bacterial and archaeal taxa, and increases in opportunistic microorganisms [11,39,55]. Global warming has led to several sponge mortality events [37,40,41,56,57]; in Lake Baikal, a probable cause of sponge disease is also increased water temperature. The occurrence of diseased sponges throughout Lake Baikal, including in ecologically less disturbed areas [34], also supports this idea. Previously, a decrease in the heat shock protein (HSP70) content of diseased sponges with various types

of lesions was shown, which indicates a suppression of their physiological and energetic states [33]. This suppression may occur even before the development of visible signs such as lesions, which are the result of the weakening of the spongy immunity. For example, in corals, the microbiome can shift prior to bleaching [58,59]. The composition of the sponge bacterial community is probably also influenced by the degree of disease involvement. Because the extent of disease involvement is difficult to visually determine when collecting sponges, samples identified as healthy may already be at the stage of early destruction of the microbiome. This may explain the lack of a clear picture of microbial composition changes in diseased sponges. In addition, it is possible that studies of changes in the eukaryotic community composition will also elucidate the reasons for the destruction of diseased sponge microbiomes.
