Sars, 1903 belongs to Daphniidae and is a rare saltwater cladoceran [1
] that is mainly distributed in high-altitude areas such as Tibet, Qinghai, and Xinjiang in China. D. tibetana
preferentially live in low temperatures. Moreover, they are well-suited for living at high altitudes in cold and nutrient-poor saline water bodies. D. tibetana
also plays an important role in the study of cladoceran biology [2
With the rapid development of mariculture, the demand for live farmed animals is increasing. China and other countries have conducted several series of studies on the cultivation and domestication of cladocerans. Among them, a euryhaline species of cold water, D. tibetana
is suitable for the water temperature conditions used during the nursery period of northern marine fish and shrimp. Its low temperature tolerance makes D. tibetana
preferable to warm-adapted species that need to be raised for cultivation in high-temperature environments. D. tibetana
has a longer developmental period and lower fecundity than Moinidae and Daphnia
but higher fecundity than marine copepods and marine zooplankton [3
]. Additionally, the amino acid composition of D. tibetana
can fully meet the essential amino acid needs of most marine and freshwater fish and shrimp, and the contents of certain unsaturated fatty acids in the body are even higher than those of many common species, such as Moina mongolica
spp., Tigiopus japonica
, Brachionus plicatilis
, and Artemia.
To date, there have been reports on the influence of D. tibetana
morphology and structure [4
], living habits [5
], ecological distribution [3
], and classification and evolution [6
], and on how environmental factors influence D. tibetana
population growth and physiological metabolism [7
]. However, there has been no research on the biology of D. tibetana
from three lakes in Tibet (Lake Namukacuo, NMKC; Lake Pengcuo, PC; and Lake Zigetangcuo, ZGTC) or on the genetic difference between wild-type and seawater-domesticated D. tibetana
. This article reports on and compares some biological observations of D. tibetana
from these three locations that were domesticated indoors to enrich the biological data on D. tibetana.
This information can be used for in-depth study of indoor seawater domestication and large-scale cultivation of D. tibetana
2. Materials and Methods
2.1. Source and Domestication of Test Animals
is an inland saline cladoceran that is widely distributed in saltwater lakes in Tibet and Qinghai, China, including NMKC (31°83′ N, 89°79′ E), PC (31°89′ N, 90°95′ E), and ZTGC (32°00′ N, 90°90′ E) (Figure 1
The D. tibetana used in the experiment were collected from NMKC, PC, and ZGTC in Tibet in October 2018. Daphniopsis tibetana were brought back to the laboratory and domesticated in diluted seawater with a salinity of 15–16 ppt at 15 ± 0.5 °C and fed with Chlorella pyrenoidosa. To avoid the negative impact of individual differences on the experiment, observation of life history started with the larval-stage of D. tibetana of the same maternal line. We isolated one gravid mother prior to the experiment and only used its offspring. For the process of domestication, pure water was added to sterilized seawater to adjust the salinity, and the seawater was diluted to 15~16 as culture water. The room temperature was controlled by air conditioning, and the temperature was adjusted to 15 ± 0.5 °C. The Chlorella pyrenoidesa was used as feed with one daily feeding. The culture medium was not replaced during the observation period.
2.2. Experimental Design
Some 30 larval D. tibetana cultured under seawater domestication conditions during the same period were selected for experimentation. One larval D. tibetana each was placed in a 16-mL test tube. During the experiment, its death, molting, time of first birth, interval between births, and number of births were observed and recorded. The experiment was conducted until the D. tibetana died.
2.3. Data Analysis
The population growth parameter was calculated by the following formula:
In the formula, x is the age period (d), lx is the survival rate at stage x (%), mx is the birth rate at stage x, rm is the intrinsic growth rate (d−1), R0 is the net reproductive volume (individual), T is the generation period (d), and λ is the weekly growth rate (d−1).
Microsoft Excel 2010 was used to process test data, and IBM SPSS Statistics 23 (IBM Corporation, Armonk, NY, USA) was used for one-way ANOVA and Duncan’s multiple comparison test to test for significance and variance homogeneity. The arithmetic mean of replicate groups was taken and expressed as mean ± standard deviation; p < 0.05 indicated significant difference; p < 0.01 indicated extremely significant difference.
The growth rate of body length was calculated as follows:
In the formula, LGR is the growth rate body length (%), L0 is the initial body length of the D. tibetana used in the experiment, and L1 is the body length at the time of death in the experiment.
2.4. Transcriptome Sequencing
Approximately 400 D. tibetana were randomly selected from each treatment group as a biological replicate. Total RNA was extracted from D. tibetana using TRIzol (Invitrogen, Carlsbad, CA, USA), and DNase I (TaKara, Dalian, China) was used to remove gene DNA. Using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA), the concentration and purity of the extracted RNA were detected by ND-2000 microspectrophotometer (Thermo Scientific, Wilmington, DE, USA) to ensure the integrity of all RNA samples (OD260/OD280 = 1.8–2.2, OD260/OD230 ≥ 2.0, RIN ≥ 8.5, 28S/18S ≥ 1.0) and perform transcriptome sequencing. The mRNA sequencing was conducted using the HiSeq platform, and library construction was performed using the Illumina TruSeq™ RNA sample prep kit method as follows: total RNA was extracted (>1 μg), and mRNA was then enriched, fragmented, and inverted into cDNA; then, adapter ligation and illumina sequencing were performed.
2.5. Differentially Expressed Genes (DEGs)
To explore the differential gene expression of wild-type and domesticated D. tibetana
, the expression levels of protein-coding genes were calculated by the FPKM method. DEGs were screened, and differential gene expression volcano plots were drawn. Quantitative analysis of gene expression levels was conducted using RSEM (https://deweylab.biostat.wisc.edu/resm/
accessed on 6 June 2019); after obtaining the number of read counts of gene transcripts, DEGseq (http://bioconductor.org/packages/stats/bioc/DESeq/
accessed on 6 June 2019) software was used to analyze gene expression differences between samples. The significance of differential expression was measured by FPKM (fragments per kilobases per million reads) using false discovery rate (FDR) and fold change (FC) as criteria. When a gene exhibited both FDR < 0.05 and |log2FC| > 1, it was considered differentially expressed.
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.
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+
were the main cations in lake water; however, CO32−
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
].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
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.
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.