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Life cycle of the suctorian Ephelota plana attached to the Euphausia pacifica

Yoshinari Endoa,*, Daiki Fujiia,1, Goh Nishitania, Peter H. Wiebeb

aLaboratory of Biological Oceanography, Graduate School of Agricultural Science, Tohoku

University, Sendai 981-8555, Japan,

bWoods Hole Oceanographic Institution, Woods Hole, MA 02543, U.S.A.

*Corresponding author at: Laboratory of Biological Oceanography, Graduate School of Agricultural

Science, Tohoku University, Sendai 981-8555, Japan

E-mail address: [email protected]

1Present address: Marine Biological Research Institute of Japan Co. Ltd., Tokyo, 142-0042, Japan

Running title: Life cycle of Ephelota plana

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ABSTRACT

The hypothesis that life cycle of an epibiotic suctorian ciliate Ephelota plana is adapted to the molt cycle of the krill Euphausia pacifica collected in Saanich Inlet, Canada was evaluated. The infestation prevalence of E. plana and the number of individuals attached increased from postmolt stage to premolt stage of E. pacifica, and concurrently cell growth of E. plana was observed. Budding individuals of E. plana first appeared at early premolt stage and increased to 21% at late premolt stage.

Thus the life cycle of E. plana seems to be adapted to the molt cycle of E. pacifica.

KEYWORDS: suctorians, Ephelota plana, life cycle, Euphausia pacifica, molt cycle

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1. Introduction

Various are known to infest euphausiids, an important marine group in marine ecosystems, ranging from highly lethal ones such as Pseudocollinia spp., which kill the euphausiid host within 40 h of infestation (Gómez-Gutiérrez et al. 2003, 2012; Lynn et al. 2014) to seemingly harmless ones such as Ephelota spp. (Nicol 1983; Rakusa-Suszczewski and Nemoto 1989).

Euphausiids molt regularly throughout their life, and this behavior helps them in ridding ectoparasites

(Tarling and Cuzin-Roudy 2008). Some ciliates, however, are adapted to the molt cycle of euphausiids. For example, Landers et al. (2006) reported that an apostome ciliate Gymnodinioides pacifica is an epibiotic exuviotroph that encysts on the setae of the appendages, telson, and antennae of Euphausia pacifica. This species excysts and enters the exoskeleton of the host after molting, where it feeds on exuvial fluid.

Suctorians belonging to the genus Ephelota are found attached to various euphausiid species:

Meganyctiphanes norvegica (Nicol 1983; Tarling and Cuzin-Roudy 2008), Euphausia superba

(Rakusa-Suszczewski and Nemoto 1989; Stankovic et al. 2002), Euphausia pacifica (Endo et al.

1985; Fernandez-Leborans 2011), E. crystallorophias (Stawiszynska-Janas and Kittel 1983), and

Thysanoessa macrura (Stankovic et al. 2002). They are characterized by the absence of a lorica and by the presence of two types of tentacles (suctorian and prehensile tentacles), as well as a stalk and a branched macronucleus (Kahl 1934; Guilcher 1951; Jankowski 1967). About a dozen species of

Ephelota have been described (Sawyer et al. 1976). The life cycle of Ephelota, however, is poorly known. Kobayashi et al. (2011) was the first to describe the growth of Ephelota gigantea that settled on plastic nets placed on wakame long-line culture along northeastern coast of Japan. They found that the stalk elongated first, followed by enlargement of body length and body width, with body width increasing linearly with time. Over 5-6 days, E. gigantea increased 4-5-fold by external budding.

Tazioli and Di Camillo (2013) reported on the life cycle of Ephelota gemmipara that settled on the 4 hydroid Eudendrium racemosum in the Adriatic Sea throughout the year. This Ephelota species produces multiple buds that detach from the parental cells to settle on the hydroid colony from May to

September when the abundance of the host and water temperature are high. Sexual reproduction and the encystment occur when the temperature and the abundance of the hydroid decrease. No such studies have been done, however, on the life cycle of Ephelota that attaches to .

It is known that Ephelota sp. attaches to the euphausiid Euphausia pacifica, the dominant euphausiid in the North Pacific (Endo et al. 1985; Fernandez-Leborans 2011). Until recently,

Ephelota species that attached to krill have not been identified to species, because the taxonomy of this genus is not well established.

This study tested the hypothesis that the life cycle of Ephelota sp. is adapted to the molt cycle of

Euphausia pacifica. In order to evaluate that the infestation rate, individual number attached, attachment site, cell size, and reproductive state of Ephelota sp. were examined for each molt stage of the euphausiid Euphausia pacifica collected from Canadian waters.

2. Materials and methods

2.1. Sampling and biological measurements

Krill samples were collected with MOCNESS (Wiebe et al. 1985) on 23 June, 2011 (from 17:00 to

18:20 hours) in the upper 160 m of Saanich Inlet, Canada (48°39’N, 123°30’W) and preserved with

90% ethanol. The North Pacific krill Euphausia pacifica Hansen, 1911were sorted under a dissecting microscope (OLYMPUS SZX12). Sex was determined based on the presence/absence of a petasma.

Finally, the molt stage of each individual was determined by examining either antennal scale or uropod under a light microscope (OLYMPUS BX51). When individual of Ephelota suctorians were found, they were detached from the host and photographs were taken with a digital camera

(OLYMPUS U-CMAD3). The image was incorporated into Image J (https://imagej.nih.gov/ij/) and 5 the body length and width of the Ephelota suctorians were measured. At the same time, they were examined for budding individuals.

Molt stages were determined according to Buchholz (1982, 1991) as follows:

A: Early postmolt stage, body soft, tissue granular or few structures.

BC: Late postmolt stage, body hard, stripe pattern present in tissue, no epidermal pockets.

D1 : Early premolt stage, body hard, stripe pattern and invaginations present in tissue, no second

cuticle.

D2: Late premolt stage, body hard, second cuticle present.

D3: Immediate premolt stage, body soft, all cuticle structures doubled.

The system is based on macroscopic integumental changes and the development of setae originally proposed by Drach (1939). Just after the actual molt, the new chitinous cuticle has no additional layers (early postmolt), after which the cuticle hardens and several layers are added. The integument is then completed and the epidermis is no longer active (late postmolt). In premolt, the epidermis again becomes active and secretes a new cuticle. As preserved samples were used, the D0 stage, the earliest premolt stage, was included in the BC stage according to Tarling et al. (1999).

2.2. DNA extraction and analysis

Genomic DNA was extracted from Ephelota sp. that were attached to Euphausia pacifica. Details of DNA extraction and analysis are given in Sato et al. (2015). In short the procedure is as follows.

Cells were washed by several transfers in filtered (pore size 0.2 µm) seawater and distilled water to remove as much extraneous matter as possible. PCR tubes (0.2 mL) each containing 50 µL of 10%

Chelex® suspension (Bio-Rad Laboratories Inc., Richmond, CA) and single cells of Ephelota sp. were heated at 95°C for 20 min to extract genomic DNA according to Richlen and Barber (2005). 6

Extracted DNAs were used as templates to amplify the target regions. All PCRs were performed on a thermal cycler in a reaction mixture (25.0 µL) containing 1.0 µL of template DNA, 0.2 mM of each deoxynucleoside triphosphate (dNTP), 1×PCR buffer, 2.0 mM MgSO4, 0.4 U of KOD-Plus-ver. 2

DNA polymerase (Toyobo, Osaka, Japan; with intensive 3’→5’ exonuclease activity), and 0.2 µM of each primer. For amplifying the nuclear 18S rRNA gene of the Ephelota plana, three primer pairs

(18S-1F1/18S-1R632, 18S-2F576/18S-2R1209, and 18S-3F1129/18S-R1772) were used (Nishitani et al. 2012). The PCR products were sequenced directly with an automated DNA sequencer (ABI

3100; Applied Biosystems). The forward and reverse complementary sequences were combined, and the three nucleotide sequences obtained with the three primer pairs were aligned using GENETYX software (Genetyx Corp., Tokyo, Japan). Partial sequences of the nuclear 18S rRNA gene were aligned with the sequences of other related species obtained from GenBank by using the ClustalW algorithm (Thompson et al. 1994).

2.3. Statistical analysis

The Kruskal-Wallis test was used for the difference in the mean number of Ehelota sp. per krill among molt stages and Wilcoxon rank sum test was used for the difference in the median body length and width of E. sp. between krill molt stages using MATLAB R2015b (The MathWorks Inc. 2015).

3. Results

A total of 147 individuals of Euphausia pacifica were examined. These individuals consisted of 68 males and 79 females. A precise length measurement of the individuals was not made because there was no relationship between krill body length and infestation prevalence or attached number of

Ephelota sp. for Euphausia pacifica collected off Iwate Prefecture, Japan (Y. Endo unpublished data).

Similar results were reported for Euphausia superba (Rakusa-Suszczewski and Nemoto 1989). All 7

147 individuals were identified as adult since secondary sexual characteristics were apparent and the body lengths were larger than 10 mm (Endo 1981). Molt stage composition was as follows: stage A;

14 indiv., BC; 53 indiv., D1; 20 indiv., D2; 58 indiv., and D3; 2 indiv.

A total of 227 individuals of Ephelota suctorians were detached and measured. The suctorians that attached to E. pacifica were identified as Ephelota plana, since they had a truncated cone body shape, and stalk was widest at its junction with the body, gradually narrowing toward distal end. These characteristics are very similar to those reported for this species that were attached to E. pacifica collected from Iwate Prefecture, Japan (Fernandez-Leborans 2011) (Fig. 1). The body length was

22.06-174.92 µm ( =73.72 µm, n=227), and body width was 31.54-236.29 µm ( =94.45 µm). The mean body length of Ephelota plana is 1.9 times larger and the mean body width is 1.4 times larger on the Saanich Inlet E. pacifica than the Ephelota plana that were attached to Euphasia pacifica collected from northeastern coast of Japan (Fernandez-Leborans 2011).

Analysis of rDNA of 5 individuals of Ephelota sp. that attached to Euphausia pacifica collected from Canadian waters and 8 individuals of Ephelota sp. that attached to Euphausia pacifica collected from Kamaishi off Iwate Prefecture, northeastern Japan showed that rDNA sequence is the same. The rDNA sequence was registered in GenBank (DDBJ Accession no. AB979871). The size of 8 individual Ephelota sp. collected from Kamaishi and analyzed for rDNA was smaller than those from

Canada, but looked very similar to those reported by Fernandez-Leborans (2011) with a body length of 42.1-89.5 µm and a body width of 19.6-105.3 µm (Sato et al. 2015). These results suggest that they are the same species, but the size of Ephelota plana is larger in Canadian waters compared with

Japanese coastal waters for some unknown reasons.

Infestation prevalence of Ephelota plana increased gradually with molt stage (Fig. 2A) from 14.3%

(2/14) at stage A to 51.7% (30/58) at stage D2. There were only 2 krill individuals at stage D3 and no infestation was observed at this stage. Mean number of Ephelota plana individuals per krill also 8 increased with molt stage, from 1.0 individuals at stage A to more than 4.0 individuals at stages D1 and D2 (Fig. 2B). Kruskal-Wallis test showed the mean number differed significantly among molt stages (p=0.0178).

Sixty-four percent (145/227) of Ephelota plana attached to the ventral side of abdomen and the inner part of the tergites of abdomen of Euphausia pacifica, followed by pleopods (20%, 46/227), eyestalks (7%, 16/227), and gills (3%, 7/227) (Fig. 3). Therefore, most of them (83.7%) were attached to the “abdominal parts” (abdomen + pleopod). When attachment site was examined for each molt stage, more Ephelota plana were attached to the “abdominal parts” at first (91.2-100% at stages A and

BC), but expanded into various part of the body as molt stage progressed (Fig. 4). At stage D2, 81.7 %

(116/142) of Ephelota plana were attached to the the “abdominal parts” and the remaining 18.3% were attached to other parts of the body. Of the “abdominal parts”, the importance of the pleopods as an attachment site increased gradually from 9.7% (3/28) at stage BC to 29.3% (34/116) at stage D2, while the percentage of the abdomen decreased from 90.3% at stage BC to 70.7% at stage D2. This decreasing trend in the percentage of the abdomen and increasing trend in the pleopods for the attachment site were similar for male and female Euphausia pacifica.

Median body length of Ephelota plana increased from 47.9 µm at stage A to 74.3 µm at stage D1 and was a similar value at stage D2 (Fig. 5A). There was no significant difference, however, in median body length of Ephelota plana between any pair of molt stages of Euphausia pacifica

(Wilcoxon rank sum test). Median body width of Ephelota plana increased steadily from 53.1 µm at stage A to 97.1 µm at stage D2 (Fig. 5B). There were significant differences in median body width between molt stages A and D2 (Wilcoxon rank sum test, p=0.0467), BC and D1 (p=0.0351), and BC and D2 (p=0.00069).

Budding individuals first appeared at stage D1 (6.1%, 3/49) and increased to 21.4% (30/140) at stage D2 (Fig. 2C). 9

4. Discussion

Tarling and Cuzin-Roudy (2008) reported that Ephelota sp. infestation depended on the molt frequency and age in Antarctic krill, namely there was more infestation for krill with a longer molt interval and older age. They suggested that the control of external parasitism was also a major advantage for krill molting at high frequency. Therefore, they considered Ephelota sp. infestation from the viewpoint of krill health. In contrast, Ephelota sp. infestation on krill was considered from the viewpoint of life cycle of Ephelota plana, especially adaptation of Ephelota plana to molt cycle of

Euphausia pacifica.

Ephelota that attached to krill will be shed with the exuviae when molting. Therefore, as part of its life cycle, the infestation prevalence and the number of Ephelota individuals increase with molt stage of E. pacifica. Thus just after molting, very few Ephelota plana are attached to E. pacifica individuals and then the number increases as the molt stage progresses. This same pattern was reported for

Euphausia superba (Tarling and Cuzin-Roudy, 2008).

The attachment site of Ephelota plana was mainly on the abdomen (64%), followed by pleopods

(20%), eyestalks (7%) and gills (3%). The attachment site was similar to the North Atlantic krill,

Meganyctiphanes norvegica (Nicol, 1983), and Euphausia superba (Rakusa-Suszczewski and

Nemoto 1989). When the attachment site was examined for E. pacifica molt stages, at late postmolt stage (BC), as many as 82.4% of the Ephelota plana were attached to the abdomen. The percentage of attachment to the abdomen, however, gradually decreased to 69.4% at D1 to 57.7% at D2 as the molt stage advanced. Therefore, attachment might have begun from abdomen and spread onto other parts of the body. New settlement of Ephelota swarmers from other krill individuals or settlement of swarmers from the same krill individuals may be possible since budding Ephelota were observed from D1 stage onwards. In order for swarmers to move from one krill individual to another, krill have 10 to be in close contact like when copulating because swarmers have to make use of boundary layers to stay in close contact with the krill. Conversely, Rakusa-Suszczewski and Nemoto (1989) hypothesized that Ephelota sp. mostly attached between second and fourth pleopods because that part of Euphausia superba is washed by the strongest water current while swimming and enables the attachment of Ephelota swarmers. Once a swarmer is outside of the boundary layer, it will be difficult for it to attach to a krill individual since in the case of Ephelota gigantea (Noble 1929; Kobayashi et al.

2011) swarmers do not swim but crawl on the substrate.

Epizoic diatoms are found on specific body part of host copepods, which is related to their copulatory and noncopulatory clasping behavior. Therefore, these diatoms are thought to be transferred to other animals during these activities (Russel and Norris 1971; Gibson 1979; Hiromi et al. 1985). While there is no report on the mating behavior of Euphausia pacifica, the mating behavior of Euphausia superba was observed in the field (Naito et al. 1986; Kawaguchi et al. 2011).

Kawaguchi et al. (2011) reported that the behavior consists of 5 processes, “chase”, “probe”,

“embrace”, “flex”, and “push.” Intimate contact between male and female takes place in “flex” position when the male wraps his abdomen around the female’s abdomen. This is a very good opportunity for Ephelota swarmers to move to the abdominal parts of other krill individual.

To our knowledge, this is the first report that the cell size of Ephelota sp. increased with the molt stage of krill. Body width of Ephelota gigantea is suggested to be a good indicator of its growth

(Kobayashi et al. 2011; Sato et al. 2015). Assuming an intermolt period of Euphausia pacifica, we can estimate the growth rate of Ephelota plana. The ambient water temperatures at the sample collection was 8.0-9.5°C in the water column, so the intermolt period would be 6 days according to

Lasker (1966). The difference between the smallest body width of Ephelota plana that were attached to stage A and largest body width of Ephelota plana that were attached to stage D2 was 191.9 µm.

Therefore, the daily increase was estimated to be 32.0 µm (191.9/6), which is comparable to that 11 reported for Ephelota gigantea, 29.7 µm per day (Kobayashi et al. 2011).

Budding individuals of Ephelota plana appeared at molt stages D1 and D2, with the highest budding rate of 21.4% at molt stage D2. Therefore, more than 1/5 of Ephelota plana are ready to liberate buds, or swarmers, which then can move to other krill individuals before molting and avoid sinking with the exuviae. Thus Ephelota plana appears adapted to the molt cycle of the krill

Euphausia pacifica. Based on an observation of the development time of Ephelota gigantea that were attached to the plastic net, Kobayashi et al. (2011) reported that budding occurred 5-7 days after attachment. Therefore, the E. gigantea budding has very similar timing to the budding process for

Ephelota plana that attached to the krill Euphausia pacifica. These two Ephelota species showed similar timing of budding probably because they inhabit sea areas with similar water temperatures.

There is no information on reproduction timing of Ephelota sp. that attaches to Euphausia superba, but budding is expected to take place later in the colder Antarctic Ocean.

5. Conclusions

Infestation prevalence of Ephelota plana on E. pacifica increased from 14.3% at postmolt stage (A) to 51.7% at premolt stage (D2) and concurrently cell growth of E. plana was observed. A steady increase of body width of E. plana was observed with the molt stage development of E. pacifica. The attachment site of E. plana was mainly on the abdominal parts followed by eyestalks and gills. When the attachment site was examined for molt stages, the attachment appears to have begun from abdomen and spread to other part of the krill body. Budding individuals first appeared at early premolt stage (D1) and increased to 21% at late premolt stage (D2). Therefore, the life cycle of E. plana seems to be adapted to the molt cycle of E. pacifica. The growth rate of E. plana was estimated to be about 32 µm d-1, which is similar to that of Ephelota gigantea that attached to wakame seaweed

(Kobayashi et al. 2011). This is the first report to show the growth rate of an Ephelota suctorian that attaches to crustaceans. 12

Acknowledgements

Plankton sampling was conducted by P.H.W. as part of the summer 2011 Marine Bioacoustics course taught at the University of Washington’s Friday Harbor Laboratories. Ship time and other logistics associated with the course were supported by a grant from the Office of Naval Research’s Marine

Mammals and Biology Program. We thank captain of the R/V Centennial and the students for their kind help in collecting krill samples.

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Figure legends

Fig. 1. Ephelota plana. General aspect of the body. BL: body length, BW: body width.

Fig. 2. Infestation prevalence (A, %), number of Ephelota plana attached per krill (B), and the percentage of budding individuals of Ephelota plana (C) for each molt stage of Euphausia pacifica.

Vertical bars represent 1 SD.

Fig. 3. Percentage of attachment sites of Ephelota plana on the body of Euphausia pacifica compiled for all krill individuals examined in this study.

Fig. 4. Percentage of attachment sites of Ephelota plana for each molt stage of Euphausia pacifica. A: n = 2, BC: n = 34, D1: n = 49, D2: n = 142.

Fig. 5. Box plots showing the median (the line in the middle of the box) and the 25th and 75th percentiles (the lower and upper lines of the box, respectively) for body length (A) and body width

(B) of Ephelota plana that attached to each molt stage of E. pacifica. The whiskers show 5th and 95th percentiles.