*2.6. KEGG Enrichment of DEGs*

Functional enrichment analysis of DEGs in different groups was determined using KOBAS (https://kobas.cbi.pku.edu.cn/home.do, accessed on 6 June 2019) leverage of the KEGG database. Genes were classified according to the pathways they participate in or the functions they perform, and the biological processes most relevant to biological phenomena were identified. The Benjamini and Hochberg method was used for multiple test correction, with *p* ≤ 0.05 indicating that there was significant enrichment in the GO enrichment function or KEGG pathway.

#### **3. Results and Analysis**

#### *3.1. Comparison of Survival Rate and Survival Time of D. tibetana from Different Origins*

The survival rate and survival time of *D. tibetana* that originated in each area are shown in Figures 2 and 3. The survival rate of *D. tibetana* from the three areas dropped rapidly after the fifth instar (Figure 2). In general, the survival rate of the NMKC strain was higher than those from the other two areas. However, the NMKC strain had the shortest survival time and failed to complete the seventh instar. This was followed by the PC strain and finally the ZTGC strain, which had the longest survival time and for which there were still survivors after the ninth instar. The longest survival time of each strain was 26 d for NMKC, 35 d for PC35, and 53 d for ZGTC (Figure 3).

**Figure 2.** Survival rates of *Daphniopsis tibetana* from different origins.\*: Have difference; \*\*: Have a significant difference.

**Figure 3.** Life span of *Daphniopsis tibetana* from different origins. Means with different lowercase letters are significantly different (*p* < 0.05). a: no significant difference.

*3.2. Comparison of Growth and Reproduction of D. tibetana from Different Origins*

Growth rates of the NMKC and PC strains were significantly higher than that of the ZGTC strain (*p* < 0.05; Figure 4). The body lengths of the NMKC, PC, and ZGTC strains increased by 1.21 ± 0.91 mm (growth rate, 153.6 ± 12.1%), 1.17 ± 0.13 mm (growth rate, 136.4 ± 16.1%), and 0.70 ± 0.07 mm (growth rate, 86.2 ± 7.6%).

**Figure 4.** Growth rate of body length of *Daphniopsis tibetana* from different origins. Means with different lowercase letters are significantly different (*p* < 0.05).

Under the conditions of laboratory seawater acclimation (15 ± 1 ◦C), the NMKC strain had the shortest prenatal development period (19.6 ± 0.25 d), followed by the PC strain (20.25 ± 0.25 d), and the ZGTC strain had the longest prenatal development period (24.5 ± 0.5 d) (Figure 5). The prenatal development period of the ZGTC strain was significantly different from those of the other two strains (*p* < 0.05).

**Figure 5.** Prenatal development time of *Daphniopsis tibetana* from different origins. Means with different lowercase letters are significantly different (*p* < 0.05). a: no significant difference; b: significant difference.

The average number of offspring per litter for the NMKC and PC strains was higher (*p* < 0.05) than that of the ZGTC strain (*p* < 0.05) (Figure 6). Among them, the NMKC strain had the most offspring per litter (11.5 ± 0.65), followed by the PC strain (9.5 ± 0.57), and then the ZGTC strain (3.0 ± 1.0).

**Figure 6.** Average number of offspring per litter of *Daphniopsis tibetana* from different origins. Means with different lowercase letters are significantly different (*p* < 0.05). a: no significant difference; b: significant difference.

#### *3.3. Comparison of Population Growth Parameters of D. tibetana from Different Origins*

The intrinsic growth rate and net reproductive capacity of the NMKC and PC strains were significantly higher than those of the ZGTC strain (*p* < 0.05), and the generation cycle of ZGTC was significantly longer than those of the other two strains (*p* < 0.05) (Table 1). However, the weekly growth rates of the three strains were not significantly different (*p* > 0.05).



Note: a: no significant difference; b: significant difference.

#### *3.4. Embryo Development*

The development time and cumulative development time of each instar of each *D. tibetana* strain are shown in Figures 7 and 8. In general, the three *D. tibetana* strains developed to the sixth instar. Among them, the NMKC strain had the shortest survival time; this strain survived after the sixth instar, but soon died and failed to complete the seventh instar. The PC strain developed to the eighth instar. ZGTC had the longest development time but failed to complete the 10th instar. The cumulative development time of the ZGTC strain was longer than those of the other two *D. tibetana* strains.

**Figure 7.** Instar development time of *Daphniopsis tibetana* from different origins.

**Figure 8.** Cumulative development time of *Daphniopsis tibetana* from different origins.

#### *3.5. Transcriptomic Analysis of D. tibetana*

Compared with Y, 7252 differentially expressed genes were generated after acclimation with seawater, among which 4146 were up-regulated and 3106 were down-regulated. The results indicated that *D. tibetana* had significant gene expression difference after acclimation in seawater (Figure 9).

Results of GO enrichment analysis performed on the DEGs of wild-type and domesticated *D. tibetana* are shown in Figure 10. The DEGs between wild-type and domesticated *D. tibetana* were mainly enriched in four biological process terms (establishment of localization, transport, single-organism operation, and single-organism localization), one cellular component term (extracellular region), and two molecular function terms (substrate-specific transporter activity and transporter activity).

**Figure 9.** Gene expression differences between wild-type and domesticated *Daphniopsis tibetana*.

**Figure 10.** GO enrichment analysis of DEGs between wild-type and domesticated Daphniopsis.

Compared with wild-type *D. tibetana*, domesticated *D. tibetana* had higher enrichment of RNA transport, protein digestion and absorption, and protein processing in endoplasmic reticulum pathways (Figure 11).

**Figure 11.** Scatterplot of DEGs between wild-type and domesticated *Daphniopsis tibetana* enriched in the KEGG pathways.

#### **4. Discussion**

ZGTC salinity is greater and the water body larger than that of PC, although the ecological environments are similar, and *D. tibetana* is less dense than in NMKC. The salinity of NMKC is between 15–25 ppt, and the composition of salt ions is different from that of other lakes because of its unique geographical location and water environment characteristics; this could be why the *D. tibetana* biomass is greater in NMKC.

A previous study on the salt lakes in northern Tibet revealed that the fish biomass in these salt lakes is low, and *D. tibetana* has become the main food for some water birds in Tibet [9]. Because most of the salt lakes are not connected, the inhabiting activities of birds may be the main reason that *D. tibetana* can be distributed in each salt lake even though the water ecosystem of each salt lake is different and there is a certain amount of geographical isolation. This may be the main reason why *D. tibetana* formed different strains.

Different geographical populations of the same *Daphnia* species must adapt to the specific ecological environment of their habitat; therefore, certain interspecies differences occur. In May and July 2001, Zhao [10] investigated the biological and ecological characteristics of 22 lakes in northern Tibet; the lake salinities ranged between 1 and 390 ppt, and 95 taxa phytoplankton and 42 zooplankton taxa were recorded. Moreover, Na+ and Mg2+ were the main cations in lake water; however, CO3 <sup>2</sup>−was the dominant anion under low salinity, whereas Cl− was the dominant anion with increasing salinity. This is consistent with the results of our laboratory's investigation in a few of Tibet's salt lakes in September 2018. Therefore, this experiment used the optimum temperature (15 ◦C) and salinity (15 ppt) for *D. tibetana* survival and growth to further explore the dynamic changes of *D. tibetana* seasonal populations in three different areas [9].

Under certain environmental conditions, a change in the intrinsic growth rate of a population can reflect small changes in the environment and is an important indicator of the reproductive ability of a species [11,12].This study found that there was no significant difference in the *D. tibetana* intrinsic growth rate, weekly growth rate, and generation cycle between the NMKC and PC strains, but the net reproductive capacity of NMKC was significantly less than that of PC. This is because individuals of the NMKC strain gave birth only once during their entire life cycle, whereas individuals of the PC strain gave birth more than once. However, the ANOVA results for the experimental data of these two strains showed that the average prenatal development period, average reproductive volume per litter, and growth rate of body length were not significant (*p* > 0.05). This finding shows that the *D. tibetana* of NMKC and PC may be the same geographic population. However, on average, the prenatal development period of the NMKC strain was shorter, and the average reproductive capacity per individual was the largest. This may be because the water used in this experiment was closer to the salinity of NMKC and had less impact on this strain. Compared with the other two groups, ZGTC had obvious differences in intrinsic growth rate and net reproductive capacity; this may result from the geographical isolation and salinity changes having important impacts on *D. tibetana* biology.

From the perspective of salinity, the three lakes are all inland salt lakes; however, the populations of these cladocerans in different areas have very different adaptability to salinity domestication [13].The salinity of NMKC and PC are both 16 ppt, whereas that of ZGTC is 21 ppt. Under the experimental conditions, the salinity used was closer to that of NMKC and PC; therefore, compared with the ZGTC strain, the NMKC and PC strains had the characteristics of shorter prenatal development period and larger average reproductive volume per individual. However, because of the dry climate in Tibet, slow changes in salinity during the evaporation and concentration of water also play a natural role in domesticating aquatic organisms.

There is little difference between the pH values of NMKC, PC, and ZGTC (9.54, 9.86, and 10.06, respectively). Moreover, the temperatures of NMKC, PC, and ZGT Care 13 ◦C, 16.5 ◦C, and 11.5 ◦C, respectively, and the control temperature in this experiment (15 ± 0.5 ◦C) was closer to NMKC and PC. Zhao [14] noted that geographical isolation and salinity changes have important impacts on the genetic diversity of *D. tibetana* from different water bodies. Additionally, Wang [7] found that there were obvious interspecies differences caused by geographical isolation. This study compared the distribution of *D. tibetana* in Tibet with some biological observations of indoor domesticated strains and further confirmed that there are differences in genetic diversity among different geographic populations of *D. tibetana*. However, this difference cannot be attributed simply to geographical isolation. It may be that in Tibet, *D. tibetana* has genetic diversity differences that result from long-term adaptation to different ecological factors. This difference is based mainly on what factors directly or indirectly affect the organisms and can be used to identify differences among different geographic populations.

In addition, 7252 DEGs were identified based on the third-generation transcriptome sequencing data of wild-type and domesticated *D. tibetana* that were analyzed in the laboratory. After *D. tibetana* was moved from the wild to the laboratory, numerous DEGs were generated. Significant enrichment of GO terms revealed that the DEGs are mainly involved in molecular functions, such as substrate-specific transporter activity and transporter activity, and they are mainly located in the cellular components of the extracellular region. Moreover, the majority of DEGs were associated with biological processes and were enriched in the establishment of localization, transport, single-organism operation, and single-organism localization. In the KEGG pathway enrichment analysis of DEGs, the RNA transport pathway, protein digestion and absorption pathway, and protein processing in endoplasmic reticulum pathway were highly enriched. Through these annotations, a large amount of wild-type and domesticated *D. tibetana* transcriptome information, which can more effectively help us understand the genetic characteristics of *D. tibetana* at the molecular level, was obtained. This is of great significance for further exploration of gene function in the future and provides basic data for exploring the functional genes related to *D. tibetana* resistance to environmental stress and studying related physiological functions.

#### **5. Conclusions**

Under laboratory domestication at a temperature of 15 ± 0.5 ◦C and a salinity of 15–16 ppt, the ZGTC strain had the longest life span, but the NMKC and PC strains had significantly higher growth rates of body length than the ZGTC strain (*p* < 0.05).The prenatal development period of the NMKC strain was the shortest (19.6 ± 0.25 d), but the average number of offspring per litter was the largest (11.5 ± 0.65). The intrinsic growth rate and net reproductive capacity of the NMKC and PC strains were significantly higher than those of the ZGTC population (p < 0.05). Three generations of transcriptome sequencing of wild-type D. tibetana after it was moved from the wild to the laboratory were performed in the laboratory, and correlation analysis was performed on the determined DEGs. In total, 7252 DEGs were generated in the comparison between wild-type and domesticated D. tibetana after seawater domestication, of which 4146 were up-regulated and 3106 were down-regulated. After seawater domestication, a series of biological processes and related genes in D. tibetana cells were affected. In GO enrichment analysis, the DEGs were mainly enriched in four biological process terms (establishment of localization, transport, single organism operation, and single organism localization), one cellular component term (extracellular region), and two molecular function terms (substrate-specific transporter activity and transporter activity. In KEGG pathway enrichment analysis, the DEGs were highly enriched in the RNA transport pathway, protein digestion and absorption pathway, and protein processing in the endoplasmic reticulum pathway.

**Author Contributions:** Conceptualization, W.Z. (Wan Zhang) and J.Z.; methodology, W.Z. (Wen Zhao); software, W.Z. (Wan Zhang); validation, J.W., S.W. and D.Y.; formal analysis, J.Z.; investigation, W.Z. (Wen Zhao); resources, W.Z. (Wen Zhao); data curation, W.Z. (Wen Zhao); writing—original draft preparation, W.Z. (Wan Zhang); writing—review and editing, J.Z.; visualization, W.Z. (Wan Zhang); supervision, W.Z. (Wen Zhao); project administration, J.W.; funding acquisition, W.Z. (Wen Zhao). All authors have read and agreed to the published version of the manuscript.

**Funding:** The paper was supported by National Key R&D Plan Blue Granary Science and Technology Innovation Project (grant No.2020YFD0900200).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Because we still have a lot of follow-up studies going on, the data is not available.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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