Green Fluorescence of Cytaeis Hydroids Living in Association with Nassarius Gastropods in the Red Sea

Green Fluorescent Proteins (GFPs) have been reported from a wide diversity of medusae, but only a few observations of green fluorescence have been reported for hydroid colonies. In this study, we report on fluorescence displayed by hydroid polyps of the genus Cytaeis Eschscholtz, 1829 (Hydrozoa: Anthoathecata: Filifera) found at night time in the southern Red Sea (Saudi Arabia) living on shells of the gastropod Nassarius margaritifer (Dunker, 1847) (Neogastropoda: Buccinoidea: Nassariidae). We examined the fluorescence of these polyps and compare with previously reported data. Intensive green fluorescence with a spectral peak at 518 nm was detected in the hypostome of the Cytaeis polyps, unlike in previous reports that reported fluorescence either in the basal parts of polyps or in other locations on hydroid colonies. These results suggest that fluorescence may be widespread not only in medusae, but also in polyps, and also suggests that the patterns of fluorescence localization can vary in closely related species. The fluorescence of polyps may be potentially useful for field identification of cryptic species and study of geographical distributions of such hydroids and their hosts.


Introduction
Fluorescent proteins (FPs) related to the Green Fluorescent Protein (GFP) family have been reported in many taxa of marine organisms, including Cnidaria (Hydrozoa and Anthozoa), Ctenophora, Arthropoda, and Cephalochordata [1,2]. Artificially engineered FPs have a broad range of excitation and emission spectra and are used for in vivo fluorescence labeling in a variety of different cellular systems and transgenic organisms [2,3]. Progress in FP technologies has arisen from the discovery of novel natural FPs with unusual properties.
However, fluorescence has only been reported from the hydroids of six species, the majority within the family Campanulariidae (Clytia hemisphaerica, Obelia geniculata, O. longissima, O. dichotoma, and O. bidentata). Campanulariid hydroids have fluorescence in their photocytes [5]. Photocytes with GF have been detected in the upright shoots of O. geniculata (Linnaeus, 1758), and GF is concentrated in the tip of the pedicel of O. bidentata Clark, 1875 [9]. Recently, a presumably species-specific fluorescence pattern (dispersed spots of fluorescence) was noted in the hypostome of O. dichotoma (Linnaeus, 1758) [20]. Additionally, oval-shaped spots of GF have been detected in the middle part of the hydrocaulus of Clytia [14].
The only other hydroid in which fluorescence has been detected is Cytaeis uchidae Rees, 1962 within the family Cytaeididae. In C. uchidae from Japan, GF was found in the epithelium below the tentacles [17]. Additionally, the medusae of C. uchidae have fluorescence in the epithelium of the exumbrella and subumbrella as well as in the gonads [17].
During a recent biodiversity survey in the southern Red Sea, several Cytaeis-like hydroid colonies displaying fluorescence were collected. The hydroids were epibiotic on small gastropod molluscs. In this study, we examined the fluorescence patterns of these hydroid specimens and compared the observed patterns of distribution and fluorescence with past literature in order to ascertain if such fluorescence patterns are species-specific. Species identification of hydroid colonies remains problematic, and species-specific fluorescence patterns may be a useful diagnostic tool in field identification and taxonomy.

Ethics statement
This research was carried out under the general auspices of King Abdullah University of Science and Technology's (KAUST) arrangements for marine research with the Saudi Arabian Coast Guard and the Saudi Arabian Presidency of Meteorology and Environment. These are the relevant Saudi Arabian authorities governing all sea-going research actions in the Saudi marine environment. KAUST has negotiated a general and broad permission for marine research in Saudi Arabian Red Sea waters with these two agencies and thus there is no permit number to provide. The animal use protocol was performed in accordance with Bioethics Committee of Lomonosov Moscow State University and KAUST's Institutional Biosafety and BioEthics Committee (at present, the committees do not provide a specific approval numbers for non-vertebrate animal research).  (Fig 1). The gastropods were on the surface of the sandy bottom, actively burying into the sand in response to the blue light of an underwater flashlight.

Specimen collection, morphological examination
The colonies of hydroid polyps were attached to the outer shell surfaces of actively moving N. margaritifer and were observed underwater and aboard the research vessel immediately after collection in petri dishes. 47 polyps were additionally observed under a Leica MZ6 stereomicroscope. Photographs were taken with a Canon 550D camera and MP-E macro lens.
The fluorescence of polyps was observed and photographed at nighttime underwater (in situ) and aboard the research vessel by means of a UV torch NIGHTSEA FL-1 dive light with a yellow filter. Collected specimens of N. margaritifer with hydroids attached were then preserved in 96% ethanol (to avoid fast degradation of fluorescence signal and DNA stored in -20°C) or 10% salt water formalin; 2 specimens of N. margaritifer with attached hydroids preserved in 96% ethanol are deposited in the Zoological Museum of Lomonosov Moscow State University (catalog number Ea-169).
Fluorescence images of 52 polyps were also taken using a SZX12 (Olympus, Japan) stereomicroscope with GFP filter set (excitation at 460-490 nm, emission at 520-550 nm). Emission spectra of specimens were measured directly from a fluorescent portion of the hydroid polyp using the same stereomicroscope equipped with a SMS 2 VIS (Pannhoff Optische Messtechnik, Germany) spectrophotometer, which can record spectra from a small area within the field of view (shown in Fig 2B).

Molecular identification
DNA was extracted from a total of two ethanol-preserved hydroid specimens using a Diatom DNA Prep 100 Kit (Isogene, Moscow, Russia). Fragments of nuclear (18S ribosomal DNA, internal transcribed spacer region (ITS1+5.8S+ITS2), 28S ribosomal DNA) and mitochondrial (cytochrome oxidase I (COI), 16S ribosomal DNA) were amplified using an Encyclo Plus PCR kit (Evrogen, Moscow, Russia) and specific primers [21][22][23][24][25]. The following PCR conditions were used: 3 min at 95°C, 37 cycles of 94°C for 20s, followed by annealing at respective temperatures for 30s, 72°C for 1 min 30s, and then a final elongation at 72°C for 5 min. PCR products were purified with preparative electrophoresis in 1% agarose gel and DNA bands of appropriate length were excised from the gels and extracted using a GelPrep spin-column kit (Cytokine, Saint Petersburg, Russia). Extracted DNA was sequenced on an ABI 3730 capillary sequencer in both directions.
The sequences were assembled and edited using MEGA 6.0 [26]. Sequences obtained in this study were deposited in GenBank (Accession Numbers KR233812-KR233813, KT326191) and compared for similarity with previous hydroid sequences using the National Center for Biotechnology Information's Basic Local Alignment Search Tool (NCBI BLAST). Sequences were compared only by similarity (i.e., DNA barcoding [27]) to identify specimens; no further phylogenetic analyses were performed.
Alive and preserved specimens have same patterns and color in situ and lab, and bright green fluorescence was observed in the hypostome of all polyps (Figs 2A & 3C). The emission spectrum measured directly from the hypostome of seven polyps The spectra have been collected at the same time and on different shells had a peak at 518 nm (Fig 2A and 2B). Variability or anatomical differences in expression of green fluorescence was not found. All studied specimens of hydroids of different sizes and from different shells expressed strong signal of the same fluorescence, which was always in the hypostome as shown.

Genetic analyses
All but one of 1730 base pairs of 18S rDNA sequences from hydroid specimens were identical to previously reported sequences of Cytaeis uchidae and Podocorynoides minima, both from Japan (GenBank Accession Numbers JQ407405 and EU883546, respectively). The acquired 28S rRNA sequence was 1050 bp in length and 99% (1041/1050 bp) identical to a previously reported sequence from P. minima (EU883552). The acquired 28S rRNA sequence was identical to a previously reported 253 bp 28S rDNA fragment of C. uchidae (JQ410764). The 100% similarity of ribosomal RNA sequences between C. uchidae (Cytaedidae) and P. minima (Rathkeidae), species belonging to different families, was unexpected and suggests either a mislabeling of data or misidentification of specimens. The numbers of available hydrozoan COI sequences in GenBank are limited, and there are no sequences designated as 'Cytaeis' or 'Podocorynoides'. Our obtained COI sequence was most similar (86%) to sequences reported from Nemopsis bachei (KC440111, KC440112, KC440115). However, three 16S mitochondrial rDNA sequences of P. minima and one of Cytaeis capitata are present in GenBank, and the novel 16S rRNA sequences from the Red Sea specimens were 92% similar to a sequence of P. minima from Japan (EU883541) and 91% similar to a sequence of Cytaeis capitata from Indonesia (KP776769), but only 77% similar to two other 16S rDNA sequences identified as P. minima from New Zealand and the Mediterranean (AM183125, AM411420, respectively). In the cladistic tree (Fig 4), 16S sequences of P. minima from New Zealand and Mediterranean fell within a well-supported clade including Lizzia (GenBank Accession Number AM411423) and Rathkea 1-3 (GenBank Accession Numbers AM411416, EU3055483, AM411416, respectively), while the third sequence of P. minima from Japan grouped with C. capitata and our Red Sea specimen. The grouping of P. minima with Lizzia and Rathkea, members of the Rathkeidae family, is consistent with current taxonomy of filiferan hydrozoans [28]. Therefore, we consider that the 16S rRNA sequence EU883541 from a hydrozoan from Japan has been misidentified, as the nuclear rRNA sequences from the same hydrozoan (EU883546, EU883552) [29] are 100% similar with sequences of C. uchidae (JQ407405, JQ410764) [30]. Therefore, although initially confusing, our molecular data confirm the morphological identification of the Red Sea hydrozoan as a representative of the genus Cytaeis.

Discussion
The identification of species of genus Cytaeis is based on features (size of polyps, numbers of tentacles, and presence of peridermal cap) that are often fragmentarily present, or present only depending on development stage or physiological state of the colony (S1 Table). With the lack of reliable morphological characters to distinguish hydroids of Cytaeis, our findings of intense fluorescence in the hypostome of specimens in this study that are different from other congeners suggests this fluorescence characteristic may be useful feature for species related diagnoses.
Two species of Cytaeis (C. nassa (Millard, 1959)) and C. tetrastyla (Eschscholtz, 1829) have been reported from the Red Sea [31]. The medusa of C. tetrastyla (hydroid form still unknown) has been reported circumglobally in the tropics and subtropics [28,[32][33] and may represent a complex of species [34][35]. The hydroid Cytaeis nassa with known medusae buds but an unknown medusa has been found in the Red Sea on shells of the gastropods Nassarius arcularia (Linnaeus, 1758) and N. fenistratus (Marrat, 1877). The species was also reported on shells of N. fenistratus and N. coronatus (Bruguière, 1789) from South Africa (Inhaca Island) and on N. arcularia and N. albescens (Dunker, 1846) from Madagascar [36]. The polyps of Cytaeis in this study and those observed before at the Red Sea, as well as the medusa of C. tetrastyla, may represent records of either the same species or a complex of cryptic species. This taxonomic uncertainty combined with the large number of records of Cytaeis associated with Nassarius from different localities (S2 Table) together with our molecular results lead us to identify these Red Sea specimens as Cytaeis sp.
Hydrozoan species typically have FPs with maximum emission at 495-510 nm [6,9,10,37,38]. In addition, cyan, yellow, and orange FPs and non-fluorescent purple chromoprotein have been isolated from Obelia and Phialidium (Clytia) species and an unidentified jellyfish (Anthoathecata) [6,10,15,16,38]. Here, we found that the FP in Cytaeis sp. from the Red Sea had a maximum emission at 518 nm. Similar green FPs with slightly red-shifted spectra (emission peak at 515-525 nm) have been characterized in anthozoan species, such as Acropora eurostoma, Echinophyllia echinata, and Favites abdita [39]. Thus, our finding of slightly redshifted emission spectra in Cytaeis sp. can be considered unusual but not unprecedented. While it is not outside the range of expected emission, it may be a species-specific character within the genus Cytaeis.
Green fluorescent proteins (GFPs) have often been noted in the medusa stage of hydroids but there have also been several reports of GFPs in early development stages (eggs or planulae) as well as in hydroid colonies. Fluorescence in Obelia colonies is localized in the upright parts: in the hydrocaulus, the tip of pedicels, or in polyps [9,20]. In Clytia hemisphaerica, fluorescence is localized to the endoderm of the stolon, the hydranth, and polyp tentacles [18]. From the transcriptome of C. hemisphaerica, four GFPs with distinct stage-and tissue-specific expression profiles were identified [18]. Fluorescence of the manubrium and gonad tissue showed a fluorescence emission peak at 508-510 nm, whereas in the eggs and tentacle bulbs the emission peaked at 500-502 nm. In other Phialidium (Clytia) species fluorescence was not detected in the planula stage but green fluorescent cells appeared after metamorphosis in the peduncle at the base of the stalk [7].
Fluorescence has also been seen in anthoathecata hydroids in the congeneric hydroid Cytaeis uchidae from Japan [17]. The fluorescence of C. uchidae is localized in different parts of polyps when compared to our results from Cytaeis sp. from the Red Sea. Thus, based on past literature and the results of this study, it appears that even within congeners, fluorescence can be localized within different areas of hydroids [9,17,20]. Whether this character is species-specific or varies within the natural ranges of species remains to be seen, but as accurate identification of hydroid polyps is currently very difficult due to lack of morphology characters, the utility of fluorescence localization needs to be examined in more detail.
Associations between hydroids and molluscs have long been recognized (reviewed in [40]), although few studies have investigated the ecological significance of these symbioses (e.g., [41]). The ecological implications of the symbioses between Nassarius snails and Cytaeis hydroids have been studied [42] but the ecological role of the fluorescence in Cytaeis remains poorly understood. Fluorescence in the hypostome of Cytaeis sp. has probable ecological significance as prey are likely to be attracted to the tentacles and mouth of the polyps [43]. If this is the case, the fluorescence of polyps could be utilized in sampling and study of nocturnal activity as well as in elucidating the distribution of the hydroids and their hosts. Fluorescence utilized to attract prey to hydroids living on nocturnal hosts needs moonlight for stimulation of fluorescence. The sunlight during sunset or sunrise can be another source of light. However, many aspects of this association remain understudied, including; diurnal/nocturnal activity of the Nassarius snails and Cytaeis hydroids, host specificity, and intra-and interspecies variation of fluorescence of hydroids at different developmental stages. Finally, the physiological state of Cytaeis hydroids and environmental conditions during which fluorescence is observed also require additional study.
Supporting Information S1 Fig. (a) cnidocysts of Cytaeis sp. a. undischarged cnidocysts in a tentacle of a polyp; (b) discharged eurytele from a tentacle of a polyp; (c) discharged desmoneme from a tentacle of a polyp. Scale bar 20 μm (applicable to all images). (TIF) S1