Biodiversity and distribution of microzooplankton in Spirulina (Arthrospira) platensis mass cultures throughout China

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Biodiversity and distribution of microzooplankton in Spirulina (Arthrospira) platensis mass cultures throughout China

T

Danni Yuana,b,c,1, Xueling Zhana,b,1, Mengyun Wanga,b,c, Xianhui Wanga,b, Weisong Fengd, ⁎ ⁎ Yingchun Gonga,b, , Qiang Hua,b,e, a

Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Key Laboratory for Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China c University of Chinese Academy of Sciences, Beijing 100049, China d Center for Freshwater Ecology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China e SDIC Microalgae Biotechnology Center, China Electronics Engineering Design Institute, Beijing 100142, China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Biodiversity Microzooplankton Spirulina cultivation Harmful species Contamination

Spirulina (Arthrospira) platensis is the most commonly produced microalgae for commercial applications, such as nutraceuticals and feed. While crop productivity of commercial Spirulina farms is often compromised by grazers and contaminating microzooplanktons, the biodiversity and identity of the most harmful microzooplanktons in Spirulina farms have not been extensively studied. As China is the number one producer of Spirulina in the world, comprehensive information on the biodiversity and identity of microzooplanktons in Spirulina farms is essential for the long-term commercial viability of these farms. Therefore, we determined the biodiversity and identity of the major microzooplanktons that are present in eight commercial Spirulina cultivation sites throughout China. Furthermore, we identified the major grazers that appear to directly affect the productivity of Spirulina cultures. Among twenty-three species that include 2 flagellates, 2 amoebae, 15 ciliates, and 4 rotifers, Brachionus plicatilis, Frontonia sp. and one unknown Heterolobosean amoeba appeared to be the most harmful to Spirulina due to their high density and ability to graze Spirulina. The similarity of the biodiversity and abundance of the microzooplankton was > 80% among two out of eight mass cultivation sites (C and D), while the remaining cultivation sites exhibited their own unique microzooplankton biodiversity characteristics. Redundancy analysis (RDA) showed that there was a positive relationship between harmful species of Brachionus plicatilis and salinity, while the other two harmful species of Heterolobosean amoeba and Frontonia sp. had a positive relationship with oxidation-reduction potential (ORP). As this is the first report to identify the major harmful microzooplankton species in commercial Spirulina farms, our study not only provides a theoretical basis for the relationship between environmental factors and biodiversity of harmful grazers but also lays a scientific foundation for developing effective monitoring and management strategies for commercial Spirulina farms.

1. Introduction Among the various microalgal species that are currently used for commercial applications, Spirulina is produced in the greatest quantity for the production of nutraceuticals, aquaculture feed, food, and cosmetics [1–3]. China annually produces over 10,000 tons of Spirulina biomass, which represents > 70% of the global supply. Spirulina has been successfully cultivated for many decades at commercial scale throughout the world, as it can be cultivated in extreme conditions, such as high alkalinity, salinity, and temperature. This robustness allows Spirulina to outcompete other contaminating microalgae or grazers

⁎

1

[4,5]. Additionally, its large and spiral morphology helps Spirulina to resist grazing by various microzooplanktons. However, Spirulina cultures often crash based on personal communication with algal producers, and the productivity of Spirulina is mainly affected by the invasion of various grazers [6]. Algivore zooplankton are most harmful to microalgae cultivation because they can rapidly reduce algal biomass through direct grazing, causing serious economic losses [7]. The grazers and unwanted algae present major challenges in mass cultivation of microalgae and complicate the management of industrial algal farms [6]. Wang et al. found that the algivore rotifer Brachionus plicatilis was the main contaminating

Corresponding authors at: Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. E-mail addresses: [email protected] (Y. Gong), [email protected] (Q. Hu). Danni Yuan and Xueling Zhan equally contributed to the work.

https://doi.org/10.1016/j.algal.2017.12.009 Received 12 September 2017; Received in revised form 29 November 2017; Accepted 21 December 2017 2211-9264/ © 2017 Published by Elsevier B.V.

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Table 1 Geographical positions and descriptions of Spirulina cultivation systems from different companies in China. Sites

Geographical position

Size of ponds

Medium depth

Sampling time

A B C D E F G H

N19°59′6.52″ E110°27′48.92″ N25°43′46.54″ E119°26′17.20″ N25°03′44.57″ E102°39′57.78″ N25°03′33.18″ E102°39′39.69″ N27°15′29.72″ E114°42′20.48″ N33°14′38.80″ E120°44′3.29″ N37°29′19.72″ E118°37′47.09″ N39°05′58.42″ E107°59′18.87″

550–560 m2 1200 m2 800 m2 800 m2 900–1800 m2 710–776 m2 300–640 m2 300–640 m2

15–20 cm 32–33 cm 18–20 cm 18–20 cm 30 cm 17–25 cm 30 cm 15–20 cm

Sept. 21 Sept. 20 Sept. 26 Sept. 26 Jul. 30 Aug. 3 Aug. 6 Aug. 19a

a

The samples of H site were collected from Spirulina cultures in 2014, while the rest of the samples were collected in 2015.

covered (Fig. 2). Sampling was conducted from July 30th to September 16th, 2015 for sites A-G and on August 19th, 2014 for site H (Table 1). Six ponds were randomly selected in each sampling site, and samples consisted of mixtures from three points in each pond. Samples of the original culture including algae and contaminating organisms were collected in two 50 mL centrifuge tubes. One tube with 50 mL culture was fixed with Lugol's solution to a final concentration of 1.0% for quantitative analysis [13]. The remainder was used for morphological analysis and molecular identification of microzooplanktons. Additional samples (250 mL) were collected and subjected to various analyses of abiotic factors.

species and cause of major losses of Spirulina biomass productivity [8]. Microalgal contaminants, protozoa and fungi negatively affect the overall productivity of open outdoor cultures of Spirulina platensis [5,9]. However, there are few publications on the comprehensive biodiversity of major grazers or on how to manage the grazing of microalgal ‘crops’ by predators [10]. As there is limited information on microzooplankton species in mass cultivation ponds of Spirulina [9,11], the aim of this research was to determine the biodiversity of microzooplankton and to identify the major harmful grazers in commercial Spirulina farms throughout China. We further attempted to establish relationships between various environmental factors and the occurrence of harmful grazers and the biodiversity of microzooplanktons in these large-scale Spirulina culture ponds.

2.2. Analyses of pond abiotic factors 2. Materials and methods

During this study, water temperature (WT), salinity, pH, conductivity, dissolved oxygen (DO) and Oxidation-Reduction Potential (ORP) were measured on site with a YSI Professional Plus meter (Yellow Springs, USA). Samples (250 mL) were collected under the water surface for nutrient analysis. The samples were filtered through 0.45-μm millipore filters immediately after collection and stored at − 20 °C for future analyses. The total nitrogen (TN) and phosphorus (TP) were determined by standard methods [14], and dissolved organic carbon (DOC) was quantitated with a TOC-L analyzer (Shimadzu Corporation).

2.1. Cultivation of Spirulina platensis and sample collection The microalga Spirulina platensis was semi-continuously cultivated with modified Zarrouk medium [12] by eight companies (Table 1; Fig. 1) located in seven provinces that represent the main regions of Spirulina mass cultivation throughout China. All culture systems were open raceway ponds of various sizes that ranged from 300 to 1800 m2 of water surface, and the ponds from four sites (C, D, F, and H) were

Fig. 1. Spirulina culture sampling sites throughout China. A, in Hainan province; B, in Fujian province; C and D, in Yunnan province; E, in Jiangxi province; F, in Jiangsu province; G, in Shandong province; H, in Inner Mongolia.

H G

E

C, D

B

A

Sampling site 39

F

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Fig. 2. The corresponding photos of ponds at each sampling sites in Fig. 1.

SeaView [23]. Maximum likelihood (ML) and Bayesian inference (BI) were used to build trees. ML analysis was conducted online with PhyML 3.0 (http://atgc.lirmm.fr/phyml/; [24]), and BI was performed using MrBayes 3.0b [25]. In addition to the 19 sequences of contaminated microzooplankton in the algal cultures that were found in our study, another 37 sequences (including 36 sequences of the most closely related sequences to ours and 1 sequence of Candida albicans as the outgroup) were retrieved from the NCBI database. The tree topologies were inferred using the model selected as the best-fit model of nucleotide substitution by AIC in Modeltest 3.7 [26] as implemented in PAUP 4.0b10. For maximum likelihood analysis, the distance data were bootstrap resampled 1000 times, and for Bayesian analysis, the chain length for our analysis was 1,000,000 generations with trees sampled every 100 generations, and the first 4000 generations were discarded as burn-in.

2.3. Identification of microzooplanktons The initial identification of protozoa and rotifers was conducted based on morphological characteristics according to Shen [15], Patterson [16] and Wang [17]. In addition, small subunit ribosomal DNA (SSU rDNA) rRNA gene sequences were used to confirm and improve the initial morphology-based identification of species. 2.3.1. Light microscopy Observations and photomicrography of specimens were performed with the differential interference contrast method using an Olympus microscope BX53 with an attached Olympus digital camera DP80 (Olympus, Japan). To calculate the abundance of microzooplankton, a 100 μL phytoplankton counting chamber was used to count the abundance of microzooplankton with the upright microscope (CX-31, Olympus, Japan). Each sample was counted twice [18], and the following formula [19] was used to determine final microzooplankton abundance: N = Vs ∗ (N1 + N2) / (2 ∗ Vj ∗ V), where N is the sample of microzooplankton abundance, Vs represents the concentrated volume, Vj represents the counting volume, N1 and N2 are counting numbers, and V is the sampling volume.

2.4. Data analysis To shed light on the dynamics of microzooplankton among different sampling locations, all the microzooplankton abundance data was used to construct the dendrogram with the software Origin 8.5. To evaluate the effects of environmental factors on microzooplankton abundance and distributions, the similarity of microzooplankton abundances and environmental factors among 8 sites were analyzed with the Bray-Curtis method using the software PRIME, and all abiotic and biotic data were log10 (X + 1) transformed. Moreover, redundancy analysis (RDA) was carried out using the Canoco program for canonical community ordination, and both environmental variables and species data were transformed using the Hellinger distance prior to analysis [27]. In the RDA plots, explanatory variables (environmental factors) are depicted as red arrows and response variables (microzooplankton species) as blue arrows that are either positively corresponding to the environmental factor (same quadrant), negatively corresponding (opposite quadrant) or unrelated (90 degree angle quadrants) [28].

2.3.2. DNA amplification, sequencing and phylogenetic analysis For each species, approximately one hundred cells were isolated from Spirulina cultures using fine glass pipettes. DNA extraction was performed with a DNeasy Blood & Tissue Kit (Qiagen, Germany) following the manufacturer's instructions. SSU rDNA was amplified by polymerase chain reaction (PCR) with universal eukaryotic primers (F: 5′-AACCTGGTTGATCCTGCCAGT-3′; R: 5′-TGATCCTTCTGCAGGTTCACCTAC-3′) [20]. The PCR products were approximately 1800 bp. For the species Gastrostyla steinii and Meseres sp., the primer LSU-R (5′-GTTAGTTTCTTTTCCTCCGC-3′) [21] was used as the reverse primer for amplification which will obtain PCR products of 2200 bp. The samples were amplified in a 20 μL volume, containing 10 μL 2 × GoTaq Green Master Mix (Promega, USA), 0.5 μL of each primer (10 μM) and 2 μL template DNA. The PCR program was 94 °C 10 min, followed by 5 cycles of 94 °C for 1 min, 48 °C for 1.5 min, and 72 °C for 2.5 min, and then followed by 30 cycles of 94 °C for 1 min, 55 °C for 1.5 min, 72 °C for 2.5 min, and a final extension at 72 °C for 10 min. PCR products were checked on an agarose gel and purified using a gel extraction kit (Omega, USA). Products were ligated to pGEM-T vectors (Promega, USA) and transformed into E. coli. Finally, 3–5 positive colonies were chosen for sequencing (Tianyi Huiyuan, Wuhan). For phylogenetic analysis, multiple alignments of 56 sequences were performed using ClustalX 1.81 [22] and were manually modified using

3. Results 3.1. Environmental factors in Spirulina mass cultures All companies used a modified Zarrouk's medium [12] to cultivate Spirulina. The physical and chemical conditions of the different mass culture systems are described in Table 2. The water temperature and DO were high at site E (36.17 °C and 15.32 mg L− 1, respectively), and low at sites H (27.55 °C) and F (8.82 mg L− 1), respectively. The conductivity (15,621 μs cm− 1), salinity (8.62‰), pH (10.17) and DOC (154.48 mg L− 1) were high at site H, while low concentrations of 40

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Table 2 Abiotic factors (mean values) of Spirulina cultivation systems which located in eight different sampling sites (A–H) as given in Table 1.

WT (°C) DO (mg L− 1) C (μs cm− 1) Salinity (‰) pH ORP (mV) TN(mg L− 1) TP(mg L− 1) DOC (mg L− 1)

A

B

C

D

E

F

G

H

30.37 12.34 4520 2.16 9.48 32.20 15.13 9.63 34.89

34.83 15.16 6996 3.14 9.71 11.96 99.44 19.14 26.92

32.32 10.64 12,695 6.25 9.96 166.94 47.62 15.35 104.69

31.58 10.98 13,394 6.76 10.08 150.24 23.97 9.26 119.88

36.17 15.32 6789 2.97 9.70 − 19.24 85.55 22.83 25.17

35.94 8.82 10,017 4.53 9.39 87.91 51.70 42.84 24.29

28.43 14.29 11,297 6.57 9.61 − 68.06 23.54 42.21 53.28

27.55 9.27 15,621 8.62 10.17 −25.90 56.46 30.92 154.48

WT: water temperature; DO: dissolved oxygen; C: conductivity; ORP: oxidation-reduction potential; TN: total nitrogen; TP: total phosphorus; DOC: dissolved organic carbon dioxide.

conductivity (4520 μs cm− 1) and salinity (2.16‰) were detected at site A, and low concentrations of pH (9.39) and DOC (24.29 mg L− 1) were detected at site F, respectively. During the investigation, average contents of ORP ranged from −68.06 mV at site G to 166.94 mV at site C, while average concentrations of TN and TP were highest at sites B (99.44 mg L− 1) and F (42.84 mg L− 1). The cluster analysis showed > 80% similarity of environmental factors including oxygen concentration, water temperature, pH, salinity and ORP, TN, TP and DOC concentrations in Spirulina cultures at eight sites (Fig. S1).

radiating mostly from the poles; the longest filopodia can reach 2 times the body diameter. Pseudopodia are sometimes branched. One nucleus is obvious (Fig. 3D). Food vacuoles and contractile vacuoles are easily observed. The cells can form cysts. They were observed to ingest Spirulina well (Fig. 3C–D) but were present at low concentrations (6 inds. mL− 1, Table 4). 3.2.3. Ciliates (1) Cyclidium glaucoma (Fig. 3H), KY886366 Body is slightly long-ovoid and small, while somewhat dorsalventrally is compressed (18–26 μm in length and 8–15 μm in width). Paroral membranes look like a translucent pocket. No cilia are present on the anterior truncation; a single caudal cilium longer than somatic cilia and one contractile vacuole are located at the posterior end. The adoral zone is approximately 1/2 of body length. They are bactivores and were not observed ingesting Spirulina cells. (2) Cyclidium marinum (Fig. 3I), KY886367 Body is 20–30 μm in length and 15–20 μm in width. Their morphological characteristics are very similar to Cyclidium glaucoma except that the body is slightly round-ovoid and the adoral zone of Cyclidium marinum is approximately 1/3 of the body. Cyclidium spp. were very common in Spirulina cultures, as they were found at seven places with the highest abundance of 323 inds. mL− 1 in our work. (3) Monodinium sp. (Fig. 3J), KY887579 Body is barrel-shaped, approximately 40 μm long and 20 μm wide. Many granules spread in the endoplasm. A short conical proboscis with expansible cytostome at the tip is at the central section of the anterior end. Somatic cilia are degenerate. Body is surrounded by one anterior girdles of pectinelles. They are omnivores and were not observed to graze Spirulina cells, but they did feed on other ciliates. They rarely occurred in the Spirulina cultures, as they were found at only sites F (Jiangsu province) and H (Inner Mongolia), with the highest abundance of 4 inds. mL− 1 (Site F). (4) Dysteria sp. (no photo), KY922819 Unfortunately, we did not observe this species under the microscope, but obtained its SSU rDNA gene sequence from environmental DNA samples, which showed that it was most related to Dysteria cristata (KC753488) with a similarity of 99% after a database search and sequence comparison. (5) Euplotes encysticus (Fig. 3L), KY922820 Body is asymmetrically oval and is dorso-ventrally flattened; the posterior end is slightly wider than the anterior. It is 80–90 μm in length and 50–65 μm in width; many light-reflecting granules and few food vacuoles often fill the colorless cytoplasm. The adoral zone of membranelles is approximately 3/4 of the body length with a narrow peristome at the anterior. One single contractile vacuole is located to the right of transverse cirri, and the cell usually moves by crawling. They were observed to graze Spirulina,

3.2. Identification of microzooplankton Combining morphological observations (Figs. 3–4) with molecular analysis (Table 3; Fig. 5), the 23 identified taxa included 2 flagellates, 2 amoebae, 15 ciliates, and 4 rotifers. Ciliates mainly belonged to Oligohymenophorea, Spirotrichea, Litostomatea and Phyllopharyngea (Fig. 5). 3.2.1. Flagellates (1) Cafeteria roenbergensis (no photo), KY886365 Unfortunately, we did not observe this species under the microscope but did obtain its SSU rDNA gene sequence from environmental DNA samples, which showed that it was most related to Cafeteria roenbergensis (AF174364) with 99% similarity after blasting. (2) Ochromonas sp. (Fig. 3A, B), KY887582 Elliptical or spherical body is about 10 μm long, but the cell becomes larger when it feeds. It has one or two golden chloroplasts. Two unequal length flagella are observed: a short flagellum that bends backwards, and a slightly curved long flagellum that extends in front of the cell. Small size compared to Spirulina; they were observed effectively ingesting Spirulina (Fig. 3A–B). 3.2.2. Amoebae (1) Heterolobosean amoeba (Fig. 3E–F), MF490458 Body is elongated or spherical (60–100 μm in length). It is usually limax and monopodial or polypoidal and can form eruptive pseudopodia; sometimes it can form fine uroidal filaments and bulbous uroid when moving. A round nucleus with a central nucleolus is clearly seen in the body; large food vacuoles with algae and rounded inclusions are filled in the cytoplasm. They were observed ingesting Spirulina (Fig. 3E) regularly and were found at four places (Table 4) with the highest abundance of 109 inds. mL− 1 at a commercial site in Hainan province (Site A). (2) Nuclearia sp. (Fig. 3C, D), KY887581 Body is at least 10 μm in diameter. The cell usually has two forms: a spherical floating form and an elongate flattened amoeboid form. The spherical form has thin filopodia radiating from all areas of the cell surface and the flattened form has thin hyaline pseudopodia 41

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Fig. 3. Morphological observations of protozoans isolated from mass cultivation of Spirulina. A, B: Ochromonas sp.; C, D: Nuclearia sp.; E, F: Heterolobosean amoeba; G: unknown Suctorida; H: Cyclidium glaucoma; I: Cyclidium marinum; J: Monodinium sp.; K: Meseres sp.; L: Euplotes encysticus; M: Euplotes vannus; N: Spathidium stammeri; O: Vorticella microstoma; P: Schmidingerothrix sp. Scale bars: 10 μm for A–B, and H–J; 20 μm for C–G, and K–P.

of undulating membrane is located on the right edge while three similarly structured peniculi are located on the left edge. Somatic cilia evenly spread throughout the body. Lots of trichocysts are located under the ectoplasm. One single contractile vacuole is sometimes with radiating collecting canals. This species is very common in Spirulina culture, and reached a concentration of 71 inds. mL− 1 in Yunnan province (Site D). They were observed to graze Spirulina cells very actively, and some incompletely digested algal fragments were found inside the cell body (Fig. 4A–B). (8) Gastrostyla steinii (Fig. 4C), KY922822 Body is elliptical and not easy to bend, approximately 110–180 μm long and 50–80 μm wide. The dorso-ventral region of the body is flat while the mid-dorso has ridges. It usually swims by rotating at a fast speed in the water. No particles are in the pellicle, but oil balls, inclusions and crystal lines can be seen in the body. The adoral zone is 40% of the body length, with one large contractile

but not vigorously. (6) Euplotes vannus (Fig. 3M), KY922821 Inflexible body is elliptical and D-shaped. It is 75–200 μm long and 65–90 μm wide. The adoral zone is approximately 2/3 of the body length. It has only one contractile vacuole. The cell usually moves by slow gliding. They were observed to graze Spirulina, but not vigorously. Euplotes spp. were very rare in Spirulina cultures, as they were only found from Shandong province with very low concentrations (Site G, Table 4). (7) Frontonia sp. (Fig. 4A–B), KY887580 Body is ellipsoid and slightly flattened, approximately 100–160 μm in length and 50–100 μm in width. The anterior end is more broadly rounded than the posterior. Oral area locates on 1/3 ventral surface of the anterior. It has a preoral suture and a postoral suture terminating at the posterior end of the cell. A piece 42

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Fig. 4. Morphological observations of representative ciliates and rotifers isolated from mass cultivation of Spirulina. A, B: Frontonia sp.; C: Gastrostyla steinii; D: Hemiurosomoida longa; E, F: Brachionus calyciflorus; G, H: Brachionus plicatilis; I: Cephalodella sp.; J: Philodina sp. Scale bars: 20 μm.

vacuole with a diameter of 20 μm in the left middle of the body. They occurred in the algal cultures rarely (Table 3) and were observed ingesting Spirulina cells. (9) Hemiurosomoida longa (Fig. 4D), KY912033 Body is elliptical, 50–100 μm long and 18–40 μm wide. The adoral zone occupies 25–35% of the body length. The left and right marginal cirri rows are not confluent at the posterior end. They rarely occurred in the algal cultures (Table 3) and were sporadically observed ingesting Spirulina cells. (10) Meseres sp. (Fig. 3K), KY922823 Body is broad-ellipsoid; the widest area of the body is below the anterior adoral membranelles and the posterior is rarely slightly

tapering. The anterior end of the cell is surrounded by a number of adoral membranelles. The cytoplasm is usually filled with many greasy shining globules, irregularly shaped crystals and some food vacuoles containing green algae. The species moves perpetually by gyrating and jumping. The species occurred in the algal cultures occasionally, as they were found in five places (Table 4) with a maximum abundance of 25 inds. mL− 1 in Inner Mongolia (Site H). They are bactivores and were not observed to graze Spirulina cells. (11) Schmidingerothrix sp. (Fig. 3P), KY922824 Body is long and highly flexible and sometimes presents as Sshaped or pisciform with a slight right marginal bulge just posterior to the mid-body and a short but distinct tail. Some lipid 43

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Body is elongated and flexible. The posterior end is usually tapered and narrow. The number of macronuclei ranges from 2 to 4, and a single contractile vacuole is usually located in the left-mid body. Unfortunately, we did not take a high-quality picture for this species under the microscope, but obtained its SSU rDNA gene sequence, which showed it was most closely related to Urosoma salmastra (KF951419) with a similarity of 98%. The species occurred in the algal cultures occasionally and was only found at two places (Table 4), with the highest abundance of 5 inds. mL− 1 in Jiangsu province (Site F). They were bactivores and were not observed to graze Spirulina cells. (15) Unknown Suctorida (Fig. 3G) Two growth stages were present: the young without sucking tentacles has cilia and can swim freely; the adult without cilia is spherical and radiates numerous sucking capitate tentacles of various lengths over its entire surface. One macronucleus and a contractile vacuole can be observed. Reproduction occurs by budding. The species rarely occurred in the algal cultures and was only found at two places (Table 4), with the highest abundance of 69 inds. mL− 1 in Inner Mongolia (Site H). They were omnivores and were not observed to graze Spirulina cells, but did consume some other ciliates. Because we did not have enough samples, no SSU rDNA gene sequences were obtained for this species.

Table 3 List of microzooplankton taxa observed in Spirulina cultures. The frequency, functional group taxa and accession ID (sequenced in this study) are also provided. Group

Species

Frequencya

Functional groupsb

Accession ID

Flagellates

Cafeteria roenbergensis Ochromonas sp. Nuclearia sp. Heterolobosean amoeba Cyclidium glaucoma Cyclidium marinum Monodinium sp. Dysteria sp. Euplotes encysticus Euplotes vannus Frontonia sp. Gastrostyla steinii Hemiurosomoida longa Meseres sp. Schmidingerothrix sp. Spathidium stammeri Urosoma sp. Vorticella microstoma Unknown Suctorida Brachionus calyciflorus Brachionus plicatilis Cephalodella sp. Philodina sp. 23

r r r o

B A, B A, B A, B

KY886365 KY887582 KY887581 MF490458

f f r r r r f r r o r o r o r o

B B R B A, B A, B A,B A,B A B B R B B, A R O

KY886366 KY886367 KY887579 KY922819 KY922820 KY922821 KY887580 KY922822 KY912033 KY922823 KY922824 KY922825 KY922826 KY886364

f r r

O O O

KY886363 -

Amoebae

Ciliates

Rotifers

Total

3.2.4. Rotifers (1) Brachionus calyciflorus (Fig. 4E, F), KY886364 Body is 200–400 μm in length and 180–195 μm in width; it has a broad, thin and smooth lorica on which four spines are located on the anterior and two on the side near the posterior. The lengths of the four anterior spines are nearly equal, but sometimes the two middle spines are longer than the others. Ciliated corona and malleate-shaped trophi can be easily observed. The foot is un-segmented and has dense annular grooves; foot aperture is sub-terminal, and a pair of tong-shaped toes occurs at the end of the foot. The species occurred in the Spirulina cultures occasionally and was found at three sites (Table 4), with the highest abundance of 33 inds. mL− 1 in Yunnan province (Site D). They were heterotrophic and were observed to graze Spirulina cells, bacteria, molds, protozoa, and organic particles. (2) Brachionus plicatilis (Fig. 4G, H), KY886363 Very similar to B. calyciflorus. Body length is approximately 125–315 μm, and has a ciliated corona. The trunk is surrounded by a broad lorica with a smooth or dotted surface. Three pairs of asymmetric spines are on the anterior dorsal margin of the lorica while two pairs of rounded lobules are on anterior ventral margin; the former six spines with wide bases and relatively sharp triangular apices are roughly similar in length; the trophi is “Malleate” and symmetrical. The un-segmented foot can contact and swing freely; the foot aperture is sub-terminal and on the ventral plate. One pair of tong-shaped toes is located at the posterior of the foot. Oval resting eggs are attached to the posterior part of the lorica when carried. The size and shape of the lorica and the shape of spines vary greatly within species. The species predominantly occurred in the Spirulina cultures, and could be observed in almost all sampling ponds (Table 4). The concentration reached as high as 70 inds. mL− 1 in Shandong province (Site G). They were observed to actively graze Spirulina cells, while bacteria, molds, protozoa, and organic particles can also be consumed. (3) Cephalodella sp. (Fig. 4I) Body is roughly cylindrical and approximately 120–220 μm long. The head is separated from the trunk unlike the foot. The corona, with long marginal cilia and two lateral tufts of swimming cilia, is usually oblique and convex. It has a virgate trophi, and the foot

-: We didn't get the sequences for these species. a r, rare; o, occasional; f, frequent. b A, algivores; B, bactivores-detrivores; O, omnivores; R: raptors; N: do not have the ability to prey on plankton; according to Shen [15], Wang [17] and our observation to classify.

droplets and food vacuoles can be observed in the colorless cytoplasm. It can move by gliding. The species rarely occurred in the algal cultures (Table 3). They are bactivores and were not observed to graze Spirulina cells. (12) Spathidium stammeri (Fig. 3N), KY922825 Body is bottle shaped and 30–400 μm in length. The anterior end is inclined and flattened with a broadly spatulate cytostome, while the posterior end is rounded. The cytostome lacks well-developed feeding cilia but contains some trichocysts and extrusomes. The body is evenly ciliated with longitudinal rows; one single contractile vacuole is located at the posterior end. The species occurred in the algal culture at three sites (A, F and H) with very low abundance (1–6 inds. mL− 1) (Table 4). They are omnivores and were not observed to graze Spirulina cells but rather other ciliates and flagellates. (13) Vorticella microstoma (Fig. 3O) Body is nearly a spherosome, approximately 35–83 μm long and 22–50 μm wide. The adoral zone of membranelles usually expands outward forming a narrow peristomal lip, approximately 12–25 μm wide. It has a contractile stalk supported by a smooth spasmoneme. The stalk is 20–250 μm long and 1.5–4 μm wide. One single and large contractile vacuole is under the peristomal lip and in the left oral vestibule. Thin transverse bands can be observed on the body surface. It can reproduce by direct fission. The species occurred in the algal cultures occasionally and was found at four sites (Table 4), with a very low abundance of 4 inds. mL− 1 in Hainan province (Site A). They are algivores and bactivores, mainly feeding on bacteria, while unicellular algae can also sometimes serve as food. Because we could not obtain enough samples, no SSU rDNA gene sequences were obtained for this species. (14) Urosoma sp. (no photo), KY922826 44

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Fig. 5. Small subunit ribosomal DNA (SSU rDNA) phylogeny of 19 contaminant species (in bold) in Spirulina cultures. The fungi Candida albicans was used to root the tree. This is the maximumlikelihood tree obtained by PhyML analyses of 56 sequences using 2194 aligned characters. Bootstrap support values after 1000 replicates and Bayesian posterior probabilities are indicated at nodes when they are above 50% and 0.70, respectively. The black circles represent support values at or above 90%/0.95. The red box represents branch lengths that are compressed to 1/3 of the original. A scale bar value of 0.1 indicates a sequence divergence of 10%. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Because we could not recover enough samples, no SSU rDNA gene sequences were obtained for this species.

bears one pair of toes, which are generally long and thin. The species rarely occurred in the Spirulina cultures and was found at only one cultivation site (Jiangsu province; Site F) at a very low abundance of 2 inds. mL− 1 (Table 4). They were heterotrophic and were observed to graze Spirulina cells as well as bacteria, molds, protozoa, and organic particles. No SSU rDNA gene sequences were obtained for this species. (4) Philodina sp. (Fig. 4J) Body is 320–460 μm long. One pair of eyespots is located in the back brain of the back tentacles; the distance between the two big and conspicuous eyespots is wide. It has ramate trophi. The ciliated corona has two symmetrical parts. Four toes, of which only two can usually be observed, are at the end of foot. It can form resting eggs when the environment is not suitable. The species rarely occurred in the Spirulina cultures and was only found from one location (Jiangxi province; Site E) with a very low abundance of 2 inds. mL− 1 (Table 4). They were heterotrophic and were observed to graze Spirulina cells as well as bacteria, molds, protozoa, and organic particles.

3.3. Distribution and abundance of microzooplankton As shown in Table 4, 16 species or species mixtures that were observed under the microscope were used for abundance analysis. Brachionus plicatilis (Fig. 4G–H), Frontonia sp. (Fig. 4A–B) and Cyclidium sp. (Fig. 3H–I) were the most frequent species and were commonly found in all sampling sites with comparatively high densities. Because both B. plicatilis and Frontonia sp. were found to vigorously graze Spirulina, they were regarded as the most harmful to Spirulina culture. However, bactivore species of Cyclidium sp. did not appear to have a direct relationship with the decrease of Spirulina. One Heterolobosean amoeba, three ciliates (Meseres sp., Spathidium stammeri, and Vorticella microstoma) and one rotifer (Brachionus calyciflorus) occasionally occurred in the cultures. In our study, Heterolobosean amoeba was often observed with food vacuoles containing a large number of Spirulina (Fig. 3E), indicating that it might be 45

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Table 4 The highest density (inds. mL− 1) of microzooplankton detected in Spirulina cultures from eight different sampling sites (A–H) as given in Table 1.

Amoebae Ciliates

Rotifers

Total

Heterolobosean amoeba Nuclearia sp. Cyclidium sp.a Monodinium sp. Euplotes sp.b Frontonia sp. Hemiurosomoida longa Meseres sp. Spathidium stammeri Vorticella microstoma Urosoma sp. Unknown Suctorida Cysts Brachionus calyciflorus Brachionus plicatilis Cephalodella sp. Philodina sp. Eggs 16c

A

B

C

D

E

F

G

H

109 0 64 0 0 51 0 3 6 4 0 0 36 5 43 0 0 0 9

1 0 323 0 0 23 0 0 0 1 0 0 7 0 17 0 0 0 6

0 0 87 0 0 38 0 0 0 1 0 0 17 19 19 0 0 0 6

1 0 59 0 0 71 0 0 0 0 0 0 34 33 27 0 0 0 6

0 0 0 0 0 1 0 1 0 0 2 0 0 0 18 0 2 3 6

9 3 67 4 0 2 0 6 2 3 5 5 0 0 18 2 0 1 13

0 0 46 0 1 0 2 17 0 0 0 0 0 0 70 0 0 20 6

0 0 204 3 0 4 0 25 1 0 0 69 0 0 42 0 0 8 8

a

Mixtures of Cyclidium glaucoma and Cyclidium marinum. Mixtures of Euplotes encysticus and Euplotes vannus, which were detected by molecular methods but were difficult to differentiate under the microscope. c Excluding cyst and egg. b

one of the major predators in Spirulina cultivation. Other species, including one Nuclearia sp. amoeba, five ciliates (Monodinium sp., Euplotes sp., Hemiurosomoida longa, Urosoma sp., and an unknown Suctorida), and two rotifers (Cephalodella sp., Philodina sp.) only occurred in one or two sampling sites and were regarded as rare species. Among these, although their concentrations were very low in general, both Nuclearia sp. (Fig. 3C–D) and Ochromonas sp. (Fig. 3A–B) showed significant grazing ability on Spirulina. With regard to feeding characterization, among 23 reported species, 1 species only fed on algae, 8 species can feed both algae and bacteria but prefer algae, 7 species only feed bacteria, 3 species mainly feed other ciliates, 4 species can feed bacteria, algae, and other ciliate or debris (Table 3). In 48 ponds from 6 sampling sites, it was found that the algivore and bactivore-detrivore species were the dominant groups in the culture systems (Fig. 6A), the proportion that the sum of algivore and bactivore-detrivore species occupied the total abundances ranged from 64% to 100%, and the abundances of algivore and bactivoredetrivore groups could reach as high as 167 inds. mL− 1 and 323 inds. mL− 1 respectively. With regard to taxon, ciliates were the most dominant group (15 species in total) with the highest abundance of 331 inds. mL− 1, followed by rotifers with the highest abundance of 255 inds. mL− 1 (Fig. 6B). Combining the data from Table 4, Cyclidium sp. and Frontonia sp. contributed the main ciliate group, while Brachionus plicatilis and B. calyciflorus contributed the main rotifer group. Amoebae only occurred occasionally, however, their concentration could be as high as 109 inds. mL− 1 in some sites (Table 4, Fig. 6B).

Fig. 6. Microzooplankton abundance in pond cultures from different sampling locations. A: functional group (A': algivores; B': bactivores-detrivores; O: omnivores; N: do not have the ability to prey on plankton); B: micro-zooplankton taxa.

Fig. 7. Dendrogram of cluster analysis based on microzooplankton diversity and abundance (mean value of six ponds for each point). A–H signifies pond site location as given in Table 1.

environmental factors (Fig. 8). One group included the lower latitude sites A, B, C and D, and four species were found with a relatively higher abundance in this group (Brachionus calyciflorus, Heterolobosean amoeba, Frontonia sp., Spathidium stammeri and cyst). The other group included the other four higher latitude sampling sites, and three species occurred in this group with a relatively higher abundance (Brachionus plicatilis, Meseres sp., unknown Suctorida and eggs). Based on the dimension of the angles formed by the red arrows (environmental factors) and blue arrows (microzooplankton species), the RDA plots (Fig. 8) showed that the majority of the contaminants have positive relationship with salinity (such as Monodinium sp., Meseres sp., Suctoria sp., and Brachionus plicatilis), and ORP (such as Heterolobosean amoeba, Frontonia sp., Spathidium stammeri, Brachionus calyciflorus, and cyst). The main species of Brachionus plicatilis, Frontonia sp. and Cyclidium sp. exhibited a positive relationship with salinity, ORP and DOC (Fig. 8; Table S2), respectively. The Heterolobosean amoeba was a harmful but occasional species, and its abundance was positively correlated with ORP content in our study.

3.4. Correlations of microzooplankton with environmental variables In addition to two sites that have high similarity (sites C, D, 88%), the other sampling sites only exhibited similarities ranging from 30% to 80% in terms of microzooplankton composition and abundance through clustering analysis (Fig. 7). It was also found that sites A, B, C, and D with lower latitude clustered one group, while sites F, G, and H with higher latitude clustered another group. For redundancy analysis (RDA), the data from 12 species (abundances > 4 inds. mL− 1) and 8 environmental variables (except conductivity) from eight sites 48 ponds (6 ponds for each sampling site) were analyzed in total. The RDA ordination showed that all samples could be separated into two groups with different latitudinal ranges by 46

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medium water quality; some species are omnivores that feed on other ciliates, but they may play other functional roles in the population dynamics of Spirulina ponds. For the amoebae, only Heterolobosean amoeba and Nuclearia sp. were found. The harmful species of Heterolobosean amoeba regularly grazed Spirulina (Fig. 3E). However, many articles have described the living environment of Heterolobosean amoeba but do not describe the feeding behavior of the species [34,35]. Although Nuclearia sp. can also graze Spirulina, their frequency of occurrence was not as high as Heterolobosean amoeba. For the flagellates, only Cafeteria roenbergensis and Ochromonas sp. were detected. The heterotrophic nanoflagellate Cafeteria roenbergensis is a predator of marine bacteria, but it is also a prey for heterotrophic dinoflagellates and ciliates. Therefore, it plays an important role in transferring bacteria to predators in microbial loops [36]. Burkert et al. reported that the flagellate Ochromonas had a strong impact on the Microcystis population, since it was able to feed on cells of unicellular Microcystis [37]. We also found that some Ochromonas sp. can graze on Spirulina (Fig. 3A–B). Our results on the biodiversity of microzooplankton population indicate that Brachionus plicatilis, Frontonia sp. and Cyclidium sp. are the dominant species in the commercial cultivation sites of Spirulina in China. The feeding habits of Brachionus plicatilis and Frontonia sp. suggest that they are the most harmful species to Spirulina among the species that were identified in our study, as they were observed to vigorously ingest/digest Spirulina. Although Heterolobosean amoeba was present at a high density at one site and were not present throughout China, they cannot be neglected in mass culture of Spirulina as the species has a high grazing ability. Our results provide new insights into the biodiversity of microzooplanktons in large mass cultures of Spirulina. Our study presents new information on the microzooplankton species that may need to be monitored for their impact on the overall productivity of Spirulina culture.

Fig. 8. Redundancy analysis (RDA) of relationship between microzooplankton and environmental factors for all pond sites (A–H) as given in Table 1. Significant environmental variables are indicated by red arrows and microzooplankton by blue arrows. Samples are represented with symbols that stand for the corresponding companies. Species data were log-transformed. (Hete, Heterolobosean amoeba; Cycl, Cyclidium sp.; Mono, Monodinium sp.; Eupl, Euplotes sp.; Fron, Frontonia sp.; Uros, Urosoma sp.; Mese, Meseres sp.; Spat, Spathidium stammeri; Vort, Vorticella microstoma; Suct, Suctoria sp.; BraP, Brachionus plicatilis; BraC, Brachionus calyciflorus. WT: Water temperature; DO: Dissolved Oxygen; ORP: Oxidation-Reduction Potential; TN: Total nitrogen; TP: Total phosphorus; DOC: Dissolved organic carbondioxide.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4. Discussion 4.1. Harmful microzooplankton composition in Spirulina mass culture

4.2. Relationship between microzooplankton and environmental factors Although it was reported that nutrient limitation as well as inhibitory pH can be effectively used to manage contaminating species in microalgal cultures [29], the existence of predatory grazers in algal ponds is a major problem for microalgae cultivation management. Therefore, we systematically analyzed the composition of microzooplanktons and determined the major predators in Spirulina ponds by combining morphological observations and molecular tools. Our results present the biodiversity of 23 species that consist of 2 flagellates, 2 amoebae, 15 ciliates and 4 rotifers. Among the four rotifers, the frequent species Brachionus plicatilis was the most harmful to Spirulina culture, while the occasional species Brachionus calyciflorus and rare species Cephalodella sp. and Philodina sp. did not appear to threaten Spirulina cultivation, even though they are omnivores. Brachionus plicatilis is a filter feeding rotifer and often feeds on algae, and it has been considered as a cosmopolitan and euryoecious species [30]. According to Yin [31], B. plicatilis can feed on several kinds of microalgae and has high consumption rates on Arthrospira fusiformis [32]. Therefore, Brachionus plicatilis can be considered one of the most detrimental species to Spirulina culture. Among the 15 species of ciliates that were found in Spirulina cultures, only Frontonia sp. showed high grazing ability. Species of Frontonia sp. are commonly seen in marine and freshwater habitats [33]. These large ciliates have high grazing ability on phytoplankton, nearly twice that of rotifers [32]. Although Euplotes sp., Gastrostyla steinii and Vorticella microstoma are algivores and bactivores, they appear to not be harmful for Spirulina culture. This may be because of their low abundance or their feeding habits on bacteria that were observed in this study. Though some species, such as Cyclidium, are bactivores that grazes on heterotrophic bacteria rather than algae as their main food source [32], they may play other roles in adjusting the

Very limited information is available in the literature on the ecology and biodiversity of microzooplanktons, especially with regards to harmful grazers in an artificial pond such as Spirulina culture beds and their interactions with various environmental parameters. Therefore, we investigated the possible relationships between various abiotic factors and the biodiversity of microzooplanktons that might affect Spirulina culture. As knowledge of the ecological interactions of aquatic insects proved to be useful for contaminant control [38], our results also lay a foundation to devise approaches for the early detection and control of microzooplanktons that are harmful to Spirulina. Based on our results, we can further develop risk mitigation and remediation strategies for large-scale Spirulina cultivation against harmful predators [39]. The main species (Brachionus plicatilis, Frontonia sp. and Cyclidium sp.) had a wide range of environmental adaptability as they occurred in nearly all sampling sites (Table 4). The abundance of Brachionus plicatilis was highly correlated with the salinity of cultures (Fig. 8; Table S2). The rotifer Brachionus plicatilis tolerates salinities ranging from 1 to 97% [30]. Salinity values ranged from 2.16‰ to 8.62‰ in these eight sampling sites. Salinity changes to higher and lower values resulted in a reduced percentage of mobile rotifers (Brachionus plicatilis), but only a small fraction of rotifers became immobilized after transferring to higher salinity [40]. It was reported that B. plicatilis can grow and reproduce better under conditions of 5–10 ppt at 18 °C and 10–15 ppt at 24 °C or 30 °C than at lower salinity when using microalgae as food source [31]. The ciliate Frontonia sp. was found at seven sampling sites with a relatively high abundance and had a positive and significant correlation with ORP (Fig. 8; Table S2). The Oxidation Reduction Potential (ORP) value represents an activity level of electrons, and it is applicable to the 47

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et al. [47] reported that selection of markers for DNA barcoding required careful consideration and evaluation since the highly conserved gene 18S rRNA may also have some level of intraspecific variation. Some specimens were morphologically determined to the genus level as a close sequence match was not found in GenBank, so a taxonomic species name could not be confirmed [49], such as Heterolobosean amoeba and Unknown Suctorida. Vorticella microstoma could not be detected by molecular methods as it is difficult to collect for DNA extraction. We wish to highlight that molecular analyses cannot replace classification based on morphological features. Combining morphology with molecular analysis is an effective approach to obtain the best taxonomic information of a particular species [49].

determination of the degree of oxidation or reduction. ORP contents ranged from −68.06 mV to 166.94 mV in this study. A low ORP indicates the presence of organic matters and other reducing materials and reflects active oxidation-reduction processes that decrease the volume of oxygen [41]. Harmful species of Heterolobosean amoeba abundance also correlated positively with ORP (Fig. 8; Table S2). This is consistent with the results in Frontonia sp., and Heterolobosean amoebas frequently graze Spirulina algae rather than organic materials. The abundance of bactivore Cyclidium sp. showed a positive relationship with DOC (Fig. 8; Table S2). Dissolved organic carbon (DOC) represents the proximal substrate of heterotrophic planktonic bacteria production, and the sum of DOC inputs restricts how much C is available for bacterial respiration [42]. The contents of DOC at eight sampling sites ranged from 24.29 to 154.48 mg L− 1, with significant differences. Difference can be caused by the semi-continuous mode of cultivation used at different times at the eight sampling sites, which could frequently cause the occurrence of bacteria. The recycling operation can easily result in the accumulation of organic matter due to decomposition and death of algae [38]. Bactivore Cyclidium sp. abundances increased with accumulated DOC contents and cannot impact Spirulina biomass loss directly. In addition to physical and chemical factors, latitude may affect microzooplankton biodiversity and distribution in these eight sampling sites. The species distributed differently at different latitude. Higher abundance of Brachionus plicatilis, Meseres sp. and unknown Suctorida were found at four higher latitude sampling sites (E, F, G and H), while Brachionus calyciflorus was only found at the lower latitude sites (sites A, B, C and D). A possible explanation is that the different sampling sites have different occurrence rates of species because of the differences in water alkalinity at various geographical locations [43]. The interaction of climatic and geological factors can determine the alkalinity of lakes, which controls the type and number of ions transported from the drainage basin [44]. Shurin [45] also found that latitude can affect protist diversity in 36 different lakes along with geochemical factors, with lower diversity in high-latitude lakes.

5. Conclusions Our study is the first report to identify the major harmful microzooplankton species in commercial Spirulina farms. Totally, twentythree species of microzooplankton were identified, among which, Brachionus plicatilis, Frontonia sp. and one unknown Heterolobosean amoeba appeared to be the most harmful to Spirulina due to their high density and ability to graze Spirulina. Redundancy analysis (RDA) showed that the abundance of harmful species had a positive relationship with the salinity and oxidation-reduction potential (ORP) in the Spirulina cultures. Our study not only provides a theoretical basis for the relationship between environmental factors and biodiversity of harmful grazers but also lays a scientific foundation for developing effective monitoring and management strategies for commercial Spirulina farms. Declaration of authors' contributions Danni Yuan and Xueling Zhan contributed equally to the work. Yingchun Gong and Qiang Hu conceived of the idea and designed the experiments. Danni Yuan performed experiments and data collection, and Xueling Zhan drafted the species description and produced the phylogenetic tree. Mengyun Wang contributed to the isolation, morphological observation and molecular identification. Xianhui Wang did molecular identification. Weisong Feng made morphological observation and identification for some flagellates and rotifers. Yingchun Gong and Qiang Hu obtained the funding. Danni Yuan and Xueling Zhan drafted the article. Yingchun Gong ([email protected]) and Qiang Hu ([email protected]) revised it critically for important intellectual content, and made final approval of the version to be submitted.

4.3. Contaminant identification methods For species identification, DNA sequence data has been employed for > 20 years to clarify taxonomic boundaries in groups where morphology-based approaches are difficult [46]. Current taxonomy based on morphological characters as detected by light-microscopy may be insufficient to discriminate possible species complexes [47]. We detected 16 species of microzooplankton through morphological observation, while 19 species were found when molecular tools were used (Table 3). Molecular methods have clear advantages over morphological approaches when identifying very small or low density microzooplanktons. For example, some flagellates (Cafeteria roenbergensis and Ochromonas sp.) and low-abundance ciliates (Gastrostyla steinii and Schmidingerothrix sp.) were difficult to identify by microscopy. Knowlton [48] also posited that there was a barrier to accurate assessment of biodiversity on “cryptic” species in marine ecology when taxa lack readily observable morphological characters useful for distinguishing species. Moreover, molecular methods have clear advantages over morphological approaches when identifying exceedingly small interstitial taxa [46,49]. In this study, the ultrastructure-based characters were not measured when identifying species based on traditional characters. With respect to Cyclidium sp. (Cyclidium glaucoma and Cyclidium marinum) and Euplotes sp. (Euplotes encysticus and Euplotes vannus), two species were respectively detected through molecular methods in this study, but using the morphological method, it was difficult to identify them down to the species level due to their similar morphology. However, some species of Suctoria sp., cyst, Philodina sp., Cephalodella sp. and rotifer eggs were poorly detected with the eukaryotic primer set or were restricted by the limited numbers of reliable published sequences for species identification in GenBank. Bhadury

Acknowledgments This work was funded by the China Electronics Engineering Design Institute, State Development & Investment Corporation (No. Y34115-1Z01-0007), and Chinese Academy of Sciences (No. ZDRW-ZS-2017-2-2). The authors thank Mimi Yao and Chaojun Wei for their help on microzooplankton sampling, Prof. Minsung Park from Institute of Hydrobiology, Chinese Academy of Sciences for his constructive advice and revising on the manuscript, and also the operators of the Spirulina farms for their help on conducting the present study. Informed consent, human/animal rights No conflicts, informed consent, human or animal rights applicable. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.algal.2017.12.009. 48

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