Self/Non-Self Recognition Affects Cnida Discharge and Tentacle Contraction in the Sea Anemone Haliplanella luciae.
The distinction between self and non-self is fundamental to a variety of basic biological phenomena, including predatorprey interactions, reproduction, spatial competition, immunity, and hosting symbionts. This distinction can result from the recognition of the presence or absence of non-self molecules or the recognition of the presence or absence of self molecules (Rinkevich, 2012). Certain species of sea anemone are clonal, sessile animals that live tightly packed among other anemones on hard substrates (Lubbock, 1980; Augustin and Bosch, 2010; Rosengarten and Nicotra, 2011; Rinkevich, 2012). Crowding of anemones favors self/non-self encounters with other anemones. For example, an encounter might involve a stimulus or combination of stimuli being presented to a stationary, resident anemone by an encroaching anemone. Stimulation of one or more receptor systems in the resident and/or the encroaching anemone might result in an activation of a wide variety of effector systems, including cnida discharge, mesenterial filament extrusion, contraction of tentacles and/or the body column, bending of the body column away from the opposing anemone, bleaching, and development and deployment of catch tentacles or acrorhagi (Purcell and Kitting, 1982; Chornesky, 1983; Kaplan, 1983; Watson and Mariscal, 1983; Buss et al., 1984, 2012; Hidaka, 1985; Sebens and Miles, 1988; Ayre and Grosberg, 1995; Rinkevich, 2012).
The initial encounters between nearby opposing anemones involve chemical and/or mechanical stimuli based on mucus and tissue contact between the anemones. As mentioned above, these stimuli result in the activation of multiple effectors, including cnida discharge from feeding tentacles. Cnidae are membrane-enclosed organelles derived from the Golgi apparatus, each consisting of a capsule and an eversible tubule (Watson and Wood, 1988; Ostman, 2000). In acontiate sea anemones, feeding tentacles contain two different types of cnidae: spirocysts and nematocysts. Spirocysts are deployed to adhere to the surface of target organisms so as to aid in prey capture (Mariscal et al., 1976; Purcell, 1977; Krayesky et al., 2010; Thorington and Hessinger, 1990). Discharging nematocysts function by injecting venom into targets, entangling appendages, or adhering to the surface of target organisms (Ostman, 2000; Fautin, 2009). The most common type of nematocyst in feeding tentacles of acontiate sea anemones, including the sea anemone Haliplanella luciae (Verrill, 1898), is the microbasic p-mastigophore (Watson and Mariscal, 1983). Discharge of these nematocysts is regulated by a variety of receptor systems responding to specific chemical and mechanical stimuli, including prey-derived compounds, presence of nearby vibrations at specific frequencies and amplitudes, and force of contact (Watson and Hessinger, 1994; Watson and Hudson, 1994; Watson et al., 1998, 2000; Krayesky et al., 2010; Todaro and Watson, 2012).
Sea anemones employ their innervated muscular system for mouth opening, prey capture and delivery of captured prey to the mouth, retraction of tentacles for defense, locomotion including pedal disk creeping, burrowing, peristalsis for digestion and egestion of waste materials, and contraction or extension of the body column or tentacles (Leclere and Rottinger, 2017). The multifunctional muscle cell in anthozoans is called an epitheliomuscular cell. It is located in the ectodermal layer and also in the endodermal layer of the body wall. Circumferential and longitudinal muscle fibers are oriented perpendicular to each other to allow lengthening or shortening of the column or tentacles, respectively. The nerve net innervates the endodermal and ectodermal muscle layers (Marlow et al., 2009). Neurons integrate sensory input so as to regulate muscular contractions. In anemones, neurons are bipolar and multipolar. The diffuse spatial organization of the nerve net within the epitheliomuscular system allows propagation of neural signals in multiple directions from stimuli delivered to a particular part of the animal.
In this paper, we used Haliplanella luciae to test combinations of self/non-self stimuli for their possible effects on each of two effector systems: cnida discharge and tentacle contraction. Although chemical cues evidently are derived both from mucus and tissue, the most important chemical cues are derived from cell surface components. Cnida discharge and tentacle contraction were significantly greater when anemones were tested with the combination of interspecific anemone mucus and interspecific anemone tissue. These effector responses were suppressed when anemones were tested in the combined presence of conspecific anemone mucus and conspecific anemone tissue. Thus, several receptor systems integrate cues to generate graded effector responses during the initial interactions of anemones during competition for space.
Materials and Methods
Specimens of Haliplanella luciae (Verrill, 1898) were collected near Florida State University Marine Laboratory, Turkey Point, Florida. Longtime H. luciae stock cultures were maintained in shallow glass dishes with natural seawater at 32%c and 14 [degrees]C, with a 12 h : 12h light: dark photoperiod. The anemones were fed to repletion twice weekly with freshly hatched Artemia nauplii, followed by a change in seawater.
Mucus collection and preparation
Mucus rings were collected from the columns of healthy conspecific and interspecific anemones H. luciae and Nematostella vectensis Stephenson, 1935, respectively, using fine forceps two or three days after feeding. Collected mucus was homogenized on ice for 10 min using a Dounce (Kimble/Kontes, Vineland, NJ) homogenizer. Protein concentration of the homogenized mucus solutions was quantified using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Solutions of homogenized mucus were stored at -20 [degrees]C.
Assay for testing nematocyst discharge
Specimens of Haliplanella were transferred to 35-mm-diameter plastic petri dishes filled with natural seawater and were allowed 2 h to recover from handling. Seawater in the conspecific and interspecific dishes was replaced with homogenized mucus in seawater to a final concentration of 0.5 [micro]g m[L.sup.-1]; control dishes were replaced with new seawater. After 10 min, tentacles were touched with nonvibrating test probes. Test probes were composed of 2-cm segments of nylon fishing line coated in 25% (w/v) gelatin. After contacting tentacles, test probes were fixed in 2.5% glutaraldehyde in seawater for 30 min at room temperature (22 [degrees]C). Test probes were prepared as wet mounts and viewed using phase contrast optics. Discharged microbasic p-mastigophores were quantified in a single field of view at 400 x magnification. Each data point represents a mean number of nematocysts counted per field of view for two replicate experiments, each consisting of eight test probes. Each anemone was touched only once (total n = 16 [+ or -] SEM). Statistical analyses were completed using JMP Statistical Discovery Software version 13.0 (SAS Institute, Cary, NC). Data were analyzed using an ANOVA followed by a least significant difference (LSD) post hoc test. In every case, significance is reported as P [less than or equal to] 0.05 (SEM based on n = 16) (Russell and Watson, 1995).
Assay for testing spirocyst discharge
Specimens of Haliplanella were transferred from the mass culture to 35-mm diameter plastic petri dishes filled with natural seawater and were tested for spirocyst discharge by using a modification of methods previously described (Krayesky et al., 2010). Seawater in experimental treatments was replaced with homogenized mucus in seawater to a final concentration of 0.05, 0.15, 0.5, 1.5, and 5.0 [micro]g m[L.sup.-1] for both conspecific and interspecific mucus; seawater in control dishes was replaced with fresh seawater. Tentacles were touched with test probes after 10 min. In this study, test probes were constructed using cut segments of uncoated glass coverslips, each measuring 2 mm x 22 mm. Coverslips were cleaned, scored by a diamond scribe, and then carefully cracked along the score line. This modification from the previous method of imaging discharged spirocysts overcomes the issue of having to count cnidae while focusing through multiple focal planes that accompanied the use of the cylindrical nylon test probes constructed from fishing line. The use of flat segments of cover-slips touched to tentacles to trigger discharge resulted in an improved microscopic image and therefore allowed a more accurate quantification of spirocysts discharged onto the probe. After test probes contacted tentacles, they were fixed in 4% paraformaldehyde in Millonig's phosphate buffer at 22 [degrees]C for 30 min (Watson et al., 2009). After fixation, probes were rinsed in phosphate-buffered saline (PBS), and then the probes were transferred to a 1/30,000 dilution of goat anti-rabbit immunoglobulin G (IgG) secondary antibody conjugated to alkaline phosphatase in PBS for 60 min. After rinsing in PBS, probes were transferred to solution containing the substrate BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate in conjunction with nitro blue tetrazolium) for 10 min (Sigma-Fast BCIP/NBT, Sigma-Aldrich, St. Louis, MO). Probes were then rinsed in PBS and viewed as wet mounts, being sure to have the test surface of the glass probe facedown on the glass slide. Wet mounts were viewed at 200 x (total magnification) with bright-field optics. Images were obtained using a STL-11000 SBIG (Santa Barbara, CA) cooled CCD camera controlled by Maxim-DL software (Diffraction Limited, Ontario, Canada). Images were edited in ImageJ (National Institutes of Health [NIH], Bethesda, MD) to enhance contrast and reduce background noise. Then a visual threshold function was used to determine the area occupied by the highly contrasted discharged spirocyst tubules. The mean area of a single discharged spirocyst tubule (n = 10) was used to calculate total number of spirocysts discharged onto the surface of test probes. Interestingly, a comparable variation in the area of a single undischarged spirocyst capsule and a single discharged spirocyst tubule lends confidence to the idea that this method of quantifying discharged spirocysts is accurate. Final data points consisted of the mean and standard error (n = 8), with a total of 8 probes per treatment. Data were analyzed using an ANOVA with an LSD post hoc test employed to make pairwise comparisons between experimental data points and controls.
Behavioral observations of anemones touched with self- and non-self tissue
A fully crossed factorial experiment was performed to evaluate the influence of two variables on anemone behavior. The first variable was mucus obtained from different sources, including none (no mucus control), conspecific, and interspecific. The second variable was the source of tissues used to contact the anemone, including fine forceps alone (no tissue control), conspecific tissue held in fine forceps, and interspecific tissue held in fine forceps.
Specimens of Haliplanella were sized and selected for having oral disks measuring about 5 mm in diameter. Anemones were transferred to 35-mm plastic petri dishes filled with sea-water and were allowed to rest for 2 h to recover from handling. Mucus had been previously collected and processed as described above. Test tissues were collected on the same day as the day of experimentation. Each trial was video-recorded. To obtain test tissue from H. luciae, specimens were anesthetized for 1 h in full-strength potassium seawater (KSW) consisting of millimolar concentrations of the following reagents: NaCl 323, MgS[O.sub.4] 26, Mg[Cl.sub.2] 24, KCl 100, Ca[Cl.sub.2] 12, and NaHC[O.sub.3 ]2. From anesthetized anemones, a piece of the column and foot measuring 2 mm x 2 mm was dissected and temporarily stored in anesthetic. Likewise, specimens of Nematostella vectensis were anesthetized for 1 h in half-strength KSW composed of millimolar concentrations of the following reagents: NaCl 161, MgS[O.sub.4] 13, Mg[Cl.sub.2] 12, KCl 50, Ca[Cl.sub.2] 6, and NaHC[O.sub.3] I (Watson et al., 2009). In this case, the test tissue from Nematostella consisted of a piece of the lower body column measuring 2 mm x 2 mm that was dissected and stored in anesthetic.
Seawater in experimental treatments was replaced with the appropriate homogenized mucus in seawater diluted from frozen stocks to a final concentration of 0.5 [micro]g m[L.sup.-1] mucus for conspecific and interspecific treatments. Seawater in control dishes was replaced with new seawater. After a 5-min immersion in the mucus-seawater solution, a normally extended tentacle was touched with fine forceps alone, conspecific tissue held in fine forceps, or interspecific tissue held in fine forceps. Tissue pieces were dragged through seawater (about 20 mm) en route to contacting the tentacle. After contact, the test tissue was immediately pulled directly away, to minimize the possibility of secondary contact. Each anemone was tested once. The behavioral responses of Haliplanella to contact were captured in video recordings, using an Imaging Source (Charlotte, NC) DMK 21AU04.AS camera with a Helios 44m-4 lens (Krasnogorsk, Russia) and using IC Capture 2.0 software (Imaging Source) to control the camera and to process images. Video recordings were composed of image streams obtained at 7.5 fps and were recorded for a duration of 2 s before, during, and after contact.
The video recordings were analyzed frame by frame using a manual tracking plug-in (MTrackJ) within ImageJ software (NIH). Analyses were restricted to a total of 15 frames, with 7 frames recorded just before the frame in which contact occurred and 7 frames recorded just after the contact frame. The movements of the tentacle tip touched with the test tissue or just forceps (control) and the movements of the tip of the furthest tentacle from the point of contact located on the opposite side of the anemone were tracked through all 15 frames. The total distance (mm) and maximum velocity (mm [s.sup.-1]) of movements were calculated for both of these tentacles for each replicate experiment (n = 6). Data points shown in graphs are based on the mean values [+ or -] standard error, n = 6. Data were analyzed using a two-way ANOVA to test main effects (mucus and tissue) and their interaction effect on distance and velocity of tentacle movement, with significance (P[less than or equal to] 0.05) tested using LSD post hoc tests.
In seawater alone and in the absence of vibrations, test probes touched to tentacles had a mean ([+ or -]SEM) number of discharged nematocysts of 18 [+ or -]2 per field of view (controls) (Fig. 1). In the presence of conspecific mucus, the mean number of nematocysts discharged into test probes was 21 [+ or -] 1, which was not significantly different from the controls (P = 0.2). In the presence of interspecific mucus, the level of mean discharge of nematocysts into probes was 26 [+ or -] 2, which is significantly different from controls and conspecific treatments (P = 0.0003 and 0.035, respectively). In summary, exposure to interspecific mucus significantly enhanced discharge of nematocysts by approximately 44% as compared to seawater alone.
The previously published assay for estimating discharged spirocysts employed short segments of uncoated fishing line touched to anemone tentacles (Krayesky et al., 2010). Test probes were then stained with the secondary antibody detection protocol detailed above in Methods. The cylindrical shape of the nylon probe and, consequently, the multiple focal planes necessary to quantify the number of spirocysts made this method cumbersome, especially for large patches (Fig. 2). We here modified the assay by incorporating cut segments of uncoated glass coverslips to overcome the difficulty caused by having multiple focal planes. The coverslip method produced high-contrast images of the patches of darkly stained spirocyst tubules in a single focal plane (Fig. 3). Based on images of intact spirocysts prepared from wet mounts, a single undischarged spirocyst had a mean ([+ or -]SD, to illustrate the spread and variability of data) area of 38.43 [+ or -] 6.61 [micro][m.sup.2] (n = 8) (Table 1). When using the coverslip method to collect discharged spirocysts, a single discharged spirocyst had a mean area of 261.79 [+ or -] 61.30 [micro][m.sup.2] (n = 8). We compared the coefficient of variation in the total area measured for undischarged (17%) and discharged (23%) spirocysts. Only 6% of the variation observed was unexplained by the natural variation in spirocyst size. This low unaccounted-for variation indicated that the coverslip method combined with the ImageJ analysis can reliably estimate spirocyst discharge.
In seawater alone and in the absence of vibrations, test probes had a mean of 135 [+ or -] 31 discharged spirocysts adhering to them (Fig. 4). This response was set as the control for comparison to other treatments. In the presence of conspecific mucus at each concentration, discharge of spirocysts onto test probes was insignificantly different from the control, ranging from 56 [+ or -] 12 to 123 [+ or -] 38 per field of view. In the presence of interspecific mucus, discharge of spirocysts onto test probes was significantly different from controls for mucus concentrations of 0.5 [micro]g m[L.sup.-1] (231 [+ or -]28), 1.5 [micro]g [ml.sup.-1] (246 [+ or -]37), and 5.0 [micro]g m[L.sup.-1] (257 [+ or -] 30). Pairwise comparisons between treatments of the same concentration are highly significant at mucus concentrations of 0.5, 1.5, and 5 [micro]g m[L.sup.-1] (P = 0.0014, 0.0002, and 0.0001, respectively). In summary, exposure to interspecific mucus significantly enhanced discharge of spirocysts by approximately 90% as compared to seawater alone.
The analysis of video recordings of intact anemones touched with test substrates in the presence or absence of mucus indicated that much of the movement of tentacles directly contacted by the test substrate was likely caused by the substrate physically moving that tentacle (here referred to as the "contact tentacle") (Video 1, available online). Therefore, the results reported here focus primarily on the movement of the tentacle located on the opposite side of the oral disk (here referred to as the "furthest tentacle"). When anemone tentacles were contacted with fine forceps, there was a consistent response of the furthest tentacle to contract. The following data refer to the furthest tentacle for each treatment. With the use of forceps and absence of mucus, the furthest tentacle moved an average distance of 0.84 mm (Fig. 5). The presence of conspecific mucus reduced the average distance to 0.75 mm, and the presence of interspecific mucus increased the distance to 1.07 mm. With the use of conspecific tissue and absence of mucus, the furthest tentacle moved an average distance of 0.53 mm. The presence of conspecific mucus reduced the average distance to 0.15 mm, and the presence of interspecific mucus increased the distance to 0.59 mm. With the use of interspecific tissue and absence of mucus, the furthest tentacle moved an average distance of 1.92 mm. The presence of conspecific mucus decreased the distance to 1.46 mm, and the presence of interspecific mucus increased the distance to 3.02. Exposure to conspecific tissue and mucus resulted in the minimum response (Video 1, available online), while exposure to interspecific tissue and mucus resulted in the maximum response (Video 2, available online). In every case, conspecific mucus dampened the mean response relative to the absence of mucus. Also, in every case, interspecific mucus enhanced the mean response relative to the absence of mucus. This same overall pattern was found in the velocity data: a dampening in the presence of conspecific stimuli and an enhancement in the presence of interspecific stimuli (Fig. 6). The main effects of tissue and mucus are significant in distance (P = 0.001 and 0.0039, respectively) and velocity (P = 0.0001 and 0.0174, respectively) responses. There is no evidence that the effect of mucus depends on tissue or that the effect of tissue depends on mucus. There was no significant interaction between these two factors in distance or velocity (P = 0.1261 and 0.3302, respectively) responses.
Previously published studies show that cnida discharge is regulated in part by prey-derived compounds, nearby vibrations at specific frequencies and amplitudes, and force of contact (Watson and Hessinger, 1994; Watson and Hudson, 1994; Watson et al., 1998, 2000; Krayesky et al., 2010; Todaro and Watson, 2012). We believe that data presented in this study show for the first time that cnida discharge also is regulated by anemone-derived mucus and by the source of anemone tissues used to contact anemones. Cnida discharge is enhanced compared to control levels in the presence of interspecific mucus, both for nematocysts and for spirocysts. Thus, specific chemoreceptors are likely to be activated by one or more components in mucus. This stimulatory effect of interspecific mucus exhibits dose dependency. Once interspecific mucus concentration increases to a threshold, spirocyst discharge is significantly greater than control levels. Discharged microbasic p-mastigophore nematocysts potentially damage the neighboring anemone, because they are penetrant-type nematocysts and contain venom. Spirocysts may be defensive, possibly deployed as a protective net by trapping incoming tubules of discharging nematocysts from neighboring anemones. In addition to affecting cnida discharge, the contact of tentacles with anemone tissues (or forceps as controls) differentially stimulated the contraction of tentacles located on the opposite side of the body relative to the tentacle that was contacted. This response was influenced by the source of anemone mucus included in the bath containing the experimental anemones. Thus, mucus and tissue type affect tentacle contraction on the opposite side of the anemone relative to the point of contact.
In these experiments, interspecific stimuli most enhanced the effector responses. Mucus provides the anemone an advanced warning that a neighboring anemone is nearby and is potentially approaching to compete for space and access to prey. When we combine pre-exposure of mucus with the tissue contact stimulus, we see that both cues are integrated into one muscular contraction across the anemone, with tissue type having the greatest effect. Interspecific stimuli enhance tentacle contraction. Moreover, conspecific stimuli appear to inhibit tentacle contraction as compared to contact with a foreign object (forceps). The tentacle contraction responses reported here are not restricted to the point of contact. Contraction in the furthest tentacle is enhanced with interspecific stimuli and inhibited with conspecific stimuli when compared to the forceps control. Taken together, these findings raise questions about which molecules in the mucus and tissue are responsible for the stimulation of the receptor-effector system. In addition, one must wonder specifically why stimuli from conspecific anemones produce weak responses on the opposite side of the anemone as compared to stimuli from interspecific anemones. One would assume that in both cases, the same circuitry is involved. Neurons integrate sensory input into muscular contractions. The nerve net system in sea anemones allows neural signals to rapidly propagate across the whole body. Perhaps body-wide nervous communication is somehow dampened by stimuli accompanying con-specific contact. This seems to be the first paper to investigate the short-term effector responses, on the scale of seconds to minutes, to self/non-self stimuli in sea anemones.
Mucus is temporally the first cue of an approaching anemone and would activate chemoreceptors only. Tissue contact may involve activation of both chemoreceptors and mechanoreceptors. Unique cell surface molecules, along with the residual mucus, on the tissue presented to the anemone may be the ligands responsible for the inhibitory responses seen in conspecific interactions and possibly a different set of related molecules in the excitatory responses seen in interspecific interactions. The forceps control would interact with only mechanoreceptors. These chemoreceptors and mechanoreceptors must activate a pathway that regulates effector responses. The nitric oxide pathway is one pathway in particular that has known involvement in both cnida discharge and muscle contraction effector systems in sea anemones (Salleo et al., 1996; Morrall et al., 2000; Kass-Simon and Pierobon, 2007; Cristino et al., 2008; Anctil, 2009; Colasanti et al., 2010). Perhaps the presence or absence of self or non-self molecules is linked to the circuitry that stimulates or inhibits the nitric oxide pathway in sea anemones. Future experiments will explore this possibility.
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KATRINA A. GUNDLACH (*) AND GLEN M. WATSON
Department of Biology, University of Louisiana, Lafayette, Louisiana 70504-3602
Received 25 January 2018: Accepted 29 June 2018; Published online 3 October 2018.
(*) To whom correspondence should be addressed. E-mail: email@example.com.
Abbreviations: LSD, least significant difference: PBS, phosphate-buffered saline.
Online enhancements: videos.
Table 1 Comparison of the variation in area of undischarged and discharged spirocysts Spirocyst state Mean area ([micro][m.sup.2]) SD CV (%) Undischarged 38.43 6.61 17 Discharged 261.79 61.30 23 SD, standard deviation; CV, coefficient of variation.
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|Author:||Gundlach, Katrina A.; Watson, Glen M.|
|Publication:||The Biological Bulletin|
|Date:||Oct 1, 2018|
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