Morphological Variation of Champia Parvula (C. Agardh) Harvey in Syntheic Medium with Special Attention to the Influence of Bromide

While under investigation as a toxicity-testing organism, female gametophytes of the small marine red alga, Champia parvula (C. Agardh) Harvey exhibited a loss of diaphragm formation in indeterminately growing branch tips. Further study revealed that this change of morphology occurred when bromide was omitted from nutrient enriched, synthetic seawater (Thursby and Harlin 1984). The goals of this project were to verify and further document the loss of .diaphragm formation in C. parvula under bromide minus and other culture conditions and to observe developmental events associated with the loss of diaphragm formation. The working hypothesis was that diaphragm formation is dependent on bromide. To test this hypothesis, diaphragm formation in C. parvula branch tips was morphometricly measured by monitoring first segment length (FSL ), the region from the apex to the first observable diaphragm. Overall growth of C. parvula branch tips was monitored by measuring tip length (TL), the distance from the apex to the base of the tip. FSL was selected as an appropriate measure of diaphragm formation by comparing FSL to the increase in segment number in branch tips over time. Tips in synthetic media lacking bromide showed an increase in FSL, but only a small increase in segment number indicating that formation of new diaphragms and, therefore, new segments had stopped. In contrast, branch tips in control treatments showed an increase in segment number but little change in FSLs over time indicating continuous, periodic formation of new diaphragms. The sensitivity of diaphragm formation to bromide availability was observed in both tetrasporophytes and female gametophytes in two versions of synthetic media. However, the two versions of synthetic media with bromide (used as controls)

The goals of this project were to verify and further document the loss of . diaphragm formation in C. parvula under bromide minus and other culture conditions and to observe developmental events associated with the loss of diaphragm formation. The working hypothesis was that diaphragm formation is dependent on bromide.
To test this hypothesis, diaphragm formation in C. parvula branch tips was morphometricly measured by monitoring first segment length (FSL ), the region from the apex to the first observable diaphragm. Overall growth of C. parvula branch tips was monitored by measuring tip length (TL), the distance from the apex to the base of the tip.
FSL was selected as an appropriate measure of diaphragm formation by comparing FSL to the increase in segment number in branch tips over time. Tips in synthetic media lacking bromide showed an increase in FSL, but only a small increase in segment number indicating that formation of new diaphragms and, therefore, new segments had stopped.
In contrast, branch tips in control treatments showed an increase in segment number but little change in FSLs over time indicating continuous, periodic formation of new diaphragms. The sensitivity of diaphragm formation to bromide availability was observed in both tetrasporophytes and female gametophytes in two versions of synthetic media. However, the two versions of synthetic media with bromide (used as controls) 11 proved unsuccessful in maintaining diaphragm formation indefinitely. Toward the end of experiments, FSL was inconsistent among branch tips cultured in synthetic media, and by two weeks, branch tips from synthetic media were more similar in length to tips from the bromide free media than to tips from NSW. This finding undermined the hypothesis that loss of diaphragm formation depends on the availability of bromide.
Lack of bromide may induce stress in C. parvula and loss of diaphragm formation may be a generalized stress response. This possibility is supported in that C. parvula showed a reduced reproductive capability in synthetic media as compared to natural seawater, and that altering nitrogen sources led to loss of diaphragm formation. Features of C. parvula that were not significantly affected in synthetic media with and without bromide were growth (as measured by TL), branch formation, and the development of hyphal filaments and gland cells.
The effort to test whether bromide is required for diaphragm formation in C.
parvula was not conclusive. However, the investigation did support previous research performed with C. parvula --developmental work by  and culturing studies by . A staining technique was tried in the investigation that, if pursued, may provide the histological information that is needed to determine the role of bromide in diaphragm formation in C. parvula. Suggestions for pursuing this research in the future are provided.  Table 1 Notation for media used in the investigation Table 2 Data from three nonreplicate tests on cystocarp and carpospore production by female tissue cultured in NSW and Sm Sections of branch tips treated in NSW, Sm and Sm-Br

INTRODUCTION
Champia parvula (C. Agardh) Harvey is a small, marine red alga with a cosmopolitan, subtidal distribution , Abbott and Hollenberg 1976. The morphology of this alga may be described as a branched, segmented tube. A single layer of cortical cells form the walls of the tube whereas single layers of cells, called diaphragms or septa, intersect the tube at regular intervals ).
When C. parvula is switched from nutrient-enriched natural seawater to nutrient enriched synthetic seawater lacking bromide, diaphragm formation at branch tips eventually ceases . The result is an apparent lengthening of the first segment (the region from the apex to the first observable diaphragm) as the alga continues apical growth. Over time, aseptate branch tips become flattened and misshapen .
Although much literature exists on the presence of halogenated secondary metabolites in algae, the role of halides (i.e., chloride, bromide, and iodide) in the metabolic processes is not well understood (i.e., Fenical 1975, Butler and. Proposed metabolic roles for halides include the maintenance of osmotic potential , incorporation into secondary metabolites that inhibit bacterial growth, epiphytism or herbivory ; and synthesis ofhaloperoxidases or halogenated phenolics that may be involved in cross-linking of polymeric substances . A handful of studies have documented morphological variation in macroalgae relative to the availability or concentration of halides . With the exception of a study of stalk formation in a diatom , no studies have investigated the relationship of the development of a morphological feature in an alga relative to the availability of a halide.  studied the morphology of C. parvula collected from the wild and built upon the understanding of the structure of this alga by comparing his findings to the work of previous investigators. Through careful examination of sectioned and whole mount C. parvula, Bigelow observed that diaphragms are formed about 30 um from the apex and proposed that diaphragm cells arise from hyphal filament cells which themselves form from the divisions of apical cells. As the apical cells repeatedly divide first anticlinally (perpendicular to the branch axis) and then periclinally (parallel to the branch axis), the cells toward the exterior of the branch become cortical tissue whereas cells toward the inside become the first hyphal cells. These initial cells are elongated compared to cortical cells and compose uniseriate filaments that span the length of branch tips. Early on in their development, near the apex, hyphal filament cells, located at intervals around the interior of the branch apex, either· bud a small round cell (the gland cell) or another "outgrowth", a cell that will give rise to a diaphragm. These cells, coming from each hyphal filament cell, meet in the center of the thallus forming the diaphragm .
Building on the work of  and , the goals in this investigation were to verify and document the loss of diaphragm formation in C.
parvu/a under bromide-free and other culture conditions and to observe developmental events associated with the loss of diaphragm formation. The working hypothesis was that diaphragm formation is dependent on bromide. This phenomenon is easily studied in C.
2 parvula. The alga exhibits quick, indeterminate growth permitting its continuous clonal propagation in laboratory culture .  ). The tetrasporophyte never resumed tetraspore production during these experiments, and the possibility exists that this isolate underwent a spontaneous mutation. It has been reported that C. parvula exhibits spontaneous mutation of genes for pigmentation and morphology Thursby 1980, Thursby and. For example, on fertilized, female yellow mutants (alleles designated asyell-1,yell-2, andyell-3), carposporophytes failed to develop normally and to produce viable carpospores ).

General information about
The triphasic life cycle of C. parvula involves isomorphic and free-living female and male gametophytes and a tetrasporophyte. The third phase, the carposporophyte, is reduced and essentially parasitic on the female gametophyte and develops subsequent to 3 fertilization   Pinter 1980, Dworetzky 1983).
Glassware was washed with RBS-35 (Pierce Chemical Co., Rockford, IL) and then rinsed thoroughly in hot tap water followed by distilled water. Residue from the detergent was removed by soaking glassware in a 1 :5 solution of concentrated HCl: distilled water. After acid treatment, glassware was rinsed thoroughly in distilled water.
Prior to use, all glassware was autoclaved for 16 min. at 121° C, 18 p.s.i. .
Cultures were kept at 22° C ± 2° C in Percival Growth Chambers on a 16 h:8 h light:dark cycle. Lighting from above was 14 -17 umol m· 2 s· 1 from cool-white fluorescent lights. Measurements of lighting were made with a Quantum/Radiometer/Photometer, Model Ll-189 (Li-Cor, Lincoln, NB).

Experimental culturing conditions
Experimental cultures were treated in the same manner as stock cultures  with a few exceptions. Culture medium was aerated continuously and changed every 5 or 7 days. During experiments, flasks were randomly repositioned in the culture chambers on a daily basis. Lighting from above and below using cool-white fluorescent lights was either 30 -40 umol m· 2 s-1 or 14 -22 umol m-2 s-1 • The latter light level was achieved with shade cloth.     Additionally, branching patterns and the appearance of plants were documented. First segment length on female and tetrasporophyte branch tips averaged 0.105 ± 0.04 mm (n = 60) and ranged from 0.047 mm to 0.283 mm.

Statistical analysis
Values for each set of five tips were pooled with values from replicate flasks within a single experiment or from replicate runs of an experiment. Additionally, the tetrasporophyte and female isolates, cultured separately during an experiment, were 13 treated as replicates. Data was pooled once interaction effects were determined to be minimal or not significant by plotting the data and performing two-factor ANOVA using tissue type or experiment as one factor compared to either sampling day or treatment.
Following this initial analysis for tissue type and replicate experiments, sampling day and treatment were used as factors in two-factor ANOV A for all experiments. In most cases, data was log (base e) transformed prior to ANOV A to both normalize and confer equal variance among treatments. However, in many cases, the data did not pass tests for normality and equal variance even after transformation, but treatment effects were often highly significant at p:::::;; 0.01. ANOVA is described as "robust" meaning it is resistant to Type 1 errors and yields accurate results for data sets with equal sample sizes (as was the case in these experiments) that are not normally distributed and that lack equal variance among means for treatments . The Student-Newman-Keuls test was used for multiple comparisons when the data was found to show significant differences (p = 0.05). Statistics were performed using SigmaStat statistical software (Jandel Scientific Corp., San Rafael, CA).

Histology
During experiments, tissue was collected on certain sample days and fixed in FAA (formalin-alcohol-acetic acid, see Appendix A) for 24 -48 hours. This coagulative fixative prevented the delicate C. parvula tissue from collapsing during processing. Due to the fact that FAA does not preserve cytoplasm, cell outlines were easily distinguished in thick sections. Following fixation, tissue was dehydrated using the following, finely graded ethanol series: 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 95 %, and two exchanges of 100%. The tissue was kept in labeled tubes to which the ethanol solutions were added. The tubes were continuously mixed on a rotary shaker at room temperature and each dehydration step lasted 30 minutes.
Tissue was infiltrated with JB-4 Plus embedding media (JB-4 embedding kit, Polysciences, Inc.) according to the manufacturer's instructions. Sectioning of blocks was performed on a Sorvall JB-4 microtome using glass knives. In some instances, pieces of the blocks were sawed off and reoriented to get cross-sections of tips; cyanoacrylate adhesive (i.e., super glue) was used to reattach block pieces. Thick sections (2 -5 um) were transferred to filtered water droplets on glass slides, cleaned with 70% ethanol. Sections were dried down on the slides for at least a day using a warming plate.
The following stains were applied to sections, but were not used routinely due to the lack of contrast and lack of differential staining of classes of molecules: Saffranin/Fast Green; 1 % Acid Fuchshin in distilled water; 0.1 % Fast Green, pH 2; Alcian Blue/Alcian Yellow ; 0.3% Alcian Blue w/v in 3% acetic acid, pH 2.5 (Apple 1994); Heath's Toluidine Blue, Methylene Blue and Neutral Red . The strongest staining reaction, with the most contrast, occurred with 1 % Toluidine Blue with 1 % borax. Sections were stained by flooding slides for 1-3 min. on a wanning plate. Slides were rinsed with distilled water and air-dried. A coverslip and mounting medium were not used to view sections; however, for photomicrography, sections were mounted in immersion oil that has the same refractive index as JB-4 Plus methacrylate.
Jn an attempt to trace cell lineage in branch tips, whole mounts of C. parvula were stained with DAPI fluorescent nuclear stain  and with aceto-iron-haematoxylin-chloral hydrate stain (Wittman 1965, Coomans and. Prior to staining with DAPI, tissue was fixed in 3: 1 95 % ethanol: glacial acetic acid for a day or more, transferred to 70% chloral hydrate for 1 -2 hours, and finally squashed on a glass slide (technique~ modified from . The squashed tissue was soaked in a small amount of 0.5 ug/ml DAPI in natural sea water plus GP2 enrichment solution (2 ml I 100 ml sea water) (see Appendix A) and microwaved for 10 sec. Afterward, the tissue was remounted in water and observed using brightfield and fluorescence microscopy. Tissue was also fixed in 3:1 95% ethanol: glacial acetic acid prior to staining with the acetoiron-haematoxylin-chloral hydrate method. Following fixation, tissue was mounted in a glass slide and heated in a microwave oven for 10 sec. After excess fixative was removed, a drop of stain was added to the tissue on the slide. The slide was briefly heated over an ethanol burner until the edges of the stain turned mauve. The stain is made up of 5 ml of stock staining solution plus 2 g of chloral hydrate. This solution was filtered and then allowed to age for 24 hours prior to use. The stock staining solution consisted of 4 g ofhaematoxylin, 1 g iron alum (FeNJ4 (S04)2-12H20; a.k.a. ferric aluminum sulfate) . Interestingly, new branches had formed in these tips, yet these too lacked diaphragms (personal observation).
In each case, tips were brought into focus so that the outline of the cuticle margin was clear (arrow). Diaphragms, seen as darkened lines perpendicular to the branch axis  all tissue was transferred to NSW. Each data point represents a mean± 2S.E. (n = 10).
Significant differences described in the text are based on p < 0.01. shown). TLs within sampling days were not significantly different, but TLs for a given sampling day was significantly larger than TLs for the previous sampling day.

32
Jn order to exclude changes in salinity and pH as causes for the variability in FSL for tips cultured in synthetic media, these parameters were examined for the recovery experiment (Figure 6a and 6b ), the experiment using St and lower light levels (Figure 7 Reproduction in NSW and Sm was investigated in three non-replicate tests (Table   2). Both cystocarps and spores were produced in NSW and Sm (tests #1 and #2).
However, cystocarps were generally smaller on plants cultured in Sm than in NSW.
Additionally, based on observations of settled spores on the bottoms of culture flasks, the number of spores produced in NSW seemed greater than the number of spores produced in Sm. When spores were allowed to continue to develop in the absence of adult plants and in the medium in which they originated, 1. 7 g and 4 mg of germling material was collected in NSW, and 1.0 g and 3.5 mg of germling material was collected in Sm for tests #1 and #2, respectively. Tetraspores were not present on germlings collected from NSW or Sm; however, while germlings from NSW had diaphragms, germlings from Sm lacked diaphragms.
At the end oftest #3, plants grown in NSW had vase-shaped cystocarps and had produced many spores (as was evident on the bottom of the flask). Although, cystocarps were evident in Sm, settled spores on the bottom of the culture flask were not visible.
Additionally, it was noted the first segment length (FSL) in this tissue was relatively long compared to FSL for tissue grown in NSW. Cystocarps and spores were not produced by tissue cultured in Sm-Br.
Growth oftetrasporophyte and female tissue in NSW, Sm, and Sm-Br was investigated (data not shown). Tissue was examined for each treatment on the day the media was changed and at the end of the culturing period. In each run of the experiment (n = 9), diaphragm formation was continuous in NSW; microscopic observation revealed " " " " Smnone None " " " " Br that first segment lengths (FSLs) were relatively small (no measurements were made) and that diaphragms were regularly spaced on branch tips. However, in Sm, diaphragms were not as closely spaced as they were in the tissue cultured in NSW. In Sm-Br, it was noted during media changes that diaphragm formation in branch tips had ceased and the apical regions of the tips were malformed. By the end of each run (ranging from Day 10 to Day 20), diaphragm formation had ceased in both Sm and Sm-Br. The misshapen appearance of branch tips of tissue cultured in Sm-Br, was less frequently seen in Sm. Significant differences (p = 0.05) in the mean weight of tissue(± 2S.E.) collected occurred between the female tissue cultured in NSW ( 4.30 ± 1.0 g) and tetrasporophyte tissue cultured in Sm-Br (1.25 ± 0.6 g). When the data for female and tetrasporophyte tissue were combined, significant differences in the mean mass of tissue collected occurred between tissue cultured in NSW (3.5 ± 1.2 g) and Sm-Br (1.44 ± 0.46 g).
In an extended investigation of growth of C. parvula, data for mean cell perimeter per segment from the early experiment (see first paragraph in Results) was plotted. For all sampling days in this experiment (1, 2, 3, 5, 7 and 14), mean cell perimeters for NSW and Sm were similar to each other for corresponding segments (Figure 1 O; data not shown for Days 2, 3, 5 and 7). In both treatments, mean cell perimeters increased with the segment number. In other words, cells expanded with age as their place along the branch axis increased in distance from the apex due to new apical growth and new diaphragm, and therefore, segment formation. By comparing the data points for all treatments for Day 1 (filled in symbols) to the data points for Sm-TM, it is evident that  55 56 fall above (12-2) and below the diaphragm through which the cell was passing ( Figure   11).
The histology on cultured C. parvula was supported in the literature and through observation of C. parvula collected from the wild (Figure 13a and 13b ).   The culturing experiments of  and the hypothesis that bromide is required for diaphragm f01mation were supported by the results from the early stages of my investigation. In two nutrient enriched synthetic seawater media (Sm and St), for both tetrasporophyte and female isolates, diaphragm formation did stop in C.
parvula when bromide was unavailable. In nutrient enriched, synthetic media lacking bromide, first segment lengths (FSLs) are significantly large after 3 or 5 days of culture The initial observation that bromide was correlated with diaphragm formation was made by  during their work to formulate a synthetic medium for culture of C. parvu/a. In this study, the loss of diaphragm formation in C. parvula tissue was stated to have occurred in synthetic media that lacked bromide. Additionally, in this investigation, loss of diaphragm formation was seen both in St and St-Br in which nitrate was replaced with nitrite. In contrast,  subjected female c. parvula to numerous stressful conditions in their investigation where loss of diaphragm formation might have occurred but did not. These conditions were as follows: (1) c. parvula was exposed to ten organic toxins resulting in inhibited reproduction between males and females, (2) under crowded conditions in culture, tissue turned yellow, (3) lack of vitamins inhibited growth, but growth was stimulated when EDTA was added in the absence of vitamins, (4) iron deficiency resulted in limited growth and pink tissue, (5) phosphorous deficiency resulted in white precipitate forming on tissue; this was considered to be CaC03, and ( 6) in the absence of nitrate, tissue died, and under nitrate deficiency, tissue turned yellow. Excess branching in C. p arvula may be related to changes in nutrient conditions during culturing (a possible source for stress) (Steele and Thursby 1980). For example, when tetrasporophytes switched from ammonia to nitrate as a nitrogen source, excess branching occurred. However, during this present investigation, excess branching was not seen in any of the treatments for female gametophytes or tetrasporophytes.
There are two accounts of lack of diaphragm formation in C. parvula that are unrelated to bromide availability. In an early report, Steele and Thursby (1980) stated that under their culture conditions, four female mutants resulted that exhibited a lack of diaphragm formation (Steele and Thursby 1980). Steele and Thursby (1980) speculated that diaphragm formation had either failed to form at all or completely during development. The mutant females were similar in size to normal females, and, most interestingly, fertilization occurred resulting in the appearance of normal looking cystocarps on these females. Lack of diaphragm formation has also been noted for C.
parvula that has been exposed to antibiotics (Alexander 0. Frost, personal communication). Antibiotics work by blocking bacterial metabolism; red algal metabolism may be similarly susceptible to the effects of antibiotics. It is important to note that a close relative of C. parvula, Lomentaria baileyana (also in the Rhodymeniales, Rhodophyta) also has hollow branch tips spanned longitudinally by byphal filaments, however, the filaments are highly branched perhaps to compensate structurally for the lack of diaphragms in this alga.
The work initiated with DAPI and the aceto-iron-haemotoxylin-chloral hydrate stains provides the most promise for determining the developmental events that occur in synthetic media with and without bromide. These stains on tissue squashes reveal information on how cells are arranged and associated in a thallus. Tissues like diaphragms and hyphal filaments fall into the category of secondary tissues in macroalgae. There is a need for more research on secondary, non-reproductive tissue in macroalgae. Much of the recent work on algal development using modem techniques has been done on primary tissues, especially the growth of shoots and rhizoids (Coomans and Hommersand 1990, Appendix C).
Future investigations into the role of bromide in C. parvula should perhaps rely on the use ofDAPI stain with tissue squashes. The finding that the cuticle of tissue 'cultured in synthetic media minus bromide is not maintained and seems susceptible to microbial attack, may point to discovering a role of bromide in C. parvula. The discovery using DAPI that cells of C. parvula eventually become multinucleate is supported by the extensive red algal work performed by Coleman ( 1986, 1987), and provides a new line of research with this alga.
That bromide has been identified as a possible player in maintaining the morphology of a red alga is interesting given the volume of work that has been done and is being done on halogens in red algae (Butler and Walter 1993;Appendix D). referred to as modified 'f medium or GP2.  developed the medium for C. parvula based on  and Spotte et al. (1984).  most recently used the medium for culturing C. parvula.
A. Add the following compounds and solutions in order to 1 L ofR-0 DiH20: Bring volume to 1000 ml with R-0 DiH20. Cobalt: Add 0.0476 g C0Cl2-6H20 to a boiling solution of 2 ml 0.2 M EDTA and 100 ml R-0 Di-H20. Follow procedure as above.
Copper: Add 0.01 g CuS04-SH20 to a boiling solution of 0.4 ml 0.2 M EDTA and 100 ml R-0 Di-H20. Follow the procedure as above. For a sulfate-free solution, substitute CuCl2).
Molybdenum: Add 0.24 g Na2Mo04-2H20 to a boiling solution of 10 ml 0.2 M EDTA and 100 ml R-0 Di-H20. Follow the procedure as above.
Iron: Add 1.34 g FeC6H507-SH20 to a boiling solution of 40 ml 0.2 M EDTA and 100 ml R-0 Di-H20. Follow procedure as above. Preparation of 400 ml working stock solution of S-3 vitamins: Add 20 g of i-Inositol to 300 ml R-0 Di-H20 in a 500 ml beaker on a stirring hot plate. Acidify to near pH 4.5 with concentrated HCl. Add other vitamins, except Capanthothenate in the order listed above. The solution should be gently heated and stirred during these additions. Adjust the pH to 4.5 if necessary and autoclave the solution.
Dissolve 0.5 g of Ca-panthothenate in 25 ml Di-H20 at room temperature.
After autoclaved solution is cool, add 20 ml of the Ca-panthothenate solution via sterile filcration. Bring this solution up to 400 ml with sterile R-0 Di-H20 and add to sterile storage bottles. Store at -70 C.
Preparation of working stock solution of S-3 vitamins: Thaw S-3 stock bottle and remove 1 ml under sterile conditions. Add the 1 ml of S-3 via sterile filtration to 100 ml of sterile R-0 Di-H20. Asepticly dispense 2 ml aliquots in to sterile 10 ml screw top test tubes. Store aliquots at -20 C.
H. Special conditions: * Sulfate-free media: Omit MgS04 and replace volume with MgCl2 (adding a total volume ofMgCl2 of20 ml). Additionally, replace STM-1 with STM-1 minus -S04.    In discussing the development of red algae,  lists events that typically must occur during development. Cell number increases, cell size and shape changes, and cells differentiate. As a function of the apical regions of branch tissue, continuous development multicellular diaphragms in C. parvula would require repeated cell division, variation in the plane of cell division, cell expansion, and designation of cell function. Additionally, the timing of these developmental events would be important.
There is good documentation of the patterns of development for red macroalgae , Dixon 1971.
However, investigations of the factors which induce or affect the fundamental developmental processes such as cell division, expansion, and differentiation are few in number (Dixon 1971). The environmental morphogenic factors that influence algal macrophytes include levels and types of irradiation, nutrition, gravity, herbivory, and surface energy . Regarding the red algal literature, irradiation is perhaps the most investigated of these factors . For example, photoperiod ) and light intensity Comer 1962, Edwards 1977) can affect the rate of cell division.
And, photoperiod , Sylvester 1987xx) and unidirectional light (L'Hardy-Halos 1971) have been implicated in the regulation of cell expansion. Other factors examined in red algal morphogenesis include the age of a cell Dixon 1975, Garbary 1979), cell position in the thallus (Duffield et al. 1972, Murray and, and cell tissue type, either rhizoidal or of the shoot . The differentiation of primary tissue such as rhizo!dal and shoot tissue in algal macrophytes is relatively well studied ). Red algae are particularly useful subjects for studies on cell differentiation because cellular events occur within the confines of simple construction and because vegetative regeneration and high growth rates facilitate their culture (Dixon 1971). For example, excised tissue from many red algae will regenerate by developing shoot cells from the end of the filament or cell that was nearest the apex and rhizoidal cells from the end that was nearest the base of the thallus (Dixon 1973, W aaland and. Similar phenomena have not been explored for the development of secondary tissue (i.e. , the hyphal and diaphragm tissue in C. parvula) in red algae . Physical factors that affect cell differentiation in primary red algal tissue include cell position in the thallus (L' Hardy-Halos 1971) and proximity to a site of wounding . To note, recent work in the area ofred algal molecular biology has determined that red algae do not have true cell differentiation as defined by that seen in higher plants and animals .
In light of what is known about the activity of hormones in higher plants, there is a reasonable amount of speculation on hormones in algal macrophytes ). Produced at the cellular level, hormones, also known as growth substances, are considered to be endogenous morphogenic factors . To date, the only identified and characterized endogenous growth substance for macroalgae is rhodomorphin ). Rhodomorphin, found only in red algae, is produced by repair cells which elongate in a rhizoidal manner to bridge a gap of dead tissue created by wounding . As a wound response substance, rhodomorphin appears to induce cell division and differentiation, and cell-cell attraction during repair events . The activity of isolated rhodomorphins is species-specific . In a more recent study, a rhodomorphin-like substance was localized in a red alga using fluorescently-labeled lectins; like rhodomorphin, the compound induces cell attraction and polar growth during repair and is a glycoprotein . Aside from the work ofWaaland ) and , research on isolated compounds or the effects of known growth hormones (i.e., auxin) has not yielded clear evidence for the presence of endogenous morphogenic chemicals in any algal macrophyte (Waaland 1984, Lobban and.

APPENDIX D: Halogens in Algae
An attempt was made in this study to detect peroxidase activity in C. parvula.
The rationale for this search was based on the following literature review.
Halides and halogenated compounds in macroalgae uM) for I and IO , respectively .
Macroalgae (and other marine organisms) have easy access to halogens in the marine environment and, not surprisingly, halogenated compounds are commonly found in many marine organisms . Among the red algae, representatives from nine families have been found to contain halogenated secondary metabolites , Wolle 1968. Bromine in the form of bromide appears to be the most commonly incorporated . There is some speculation on the metabolic roles for sequestered halides and halogenated compounds.
The putative role of halides and halogenated compounds in macroalgae is that they act as protective compounds against bacteria, epiphytes or herbivores , Butler and Wallcer 1993, Laturnus et al. 1996. Haloperoxidases, required for sequestering halogens and synthesizing halogenated compounds, may mediate adhesion in algae by cross-linking polymeric or polyphenolic substances ).
Studies that have addressed the requirement of halides for development and growth in macroalgae are few. At least one study found that the refractile inclusions in a certain red algal genera (family Bonnemaisoniaceae) seemed to be a storage site for bromide ). When bromide was excluded from a synthetic culture medium, the refractile vesicles failed to form ).

discovered that
Polysiphonia has an absolute demand for iodide and that optimal growth requires both iodide and bromide. The zoospores of the brown alga, Petalonia, were found to follow different developmental pathways according to the availability of iodine .
McLachlan (1977) found that normal growth of Fucus embryos required at least 50 uM bromine. Interestingly, bromine was implicated as a requirement in Fucus after the finding that omission of a trace metal solution containing bromine greatly inhibited growth . Most recently, stalk formation and attachment in a marine diatom were found to be bromide-dependent . However, high concentrations of iodide inhibited both stalk formation and diatom attachment to a substratum, even in the presence of bromide . However, stalk formation was also inhibited despite bromide availability when the diatoms were grown "in sulfate-free media .
Haloperoxidases in macroalgae  proposed that bromide acts as a substrate for an endogenous peroxidase. By crosslinking the extracellular polysaccharides secreted by the marine diatom, such a bromide-dependent peroxidase could render the compounds insoluble and appropriate for stalk formation . These findings are supported by the work of yreeland et al. (1998). Vanadate bromoperoxidase has been identified in Fucus embryos by .
The activity of vanadium-and bromide-dependent peroxidase  and the attachment of Fucus zygotes ) requires sulfate. Attachment is thought to be mediated, at least in part, by the peroxidase which indirectly activates secreted polyphenols to "crosslink wall polymers to form a glue which binds to both hydrophobic and hydrophilic surfaces" . More recently,  has proposed a "fiber-phenolic-catalyst mechanism of alga adhesion" within the cell wall matrix. Polyphenolics are activated to bond nonspecifically to acidic carbohydrate fibers and to a substratum via the catalytic activity of vanadium bromoperoxidase. The complete details of the mechanism are not fully understood due to the difficulties in working algal peroxidases explained in detail in  and discussed in personal communication with Dr. V. Vreeland (1996).
The history of identifying haloperoxidases in macroalgae are as follows. Murphy and 6 hEocha ( 1973) provided the first respected evidence for the presence of peroxidase activity in red algae; research from 30 years before had identified peroxidase but the conclusions were tainted by claims that the results were based on artifacts. The interest in isolating peroxidase from a red alga was inspired by the possibility that peroxidase mediates the biosynthesis of halogenated compounds .
Upon isolating peroxidase,  found that their compound was unable to oxidize iodide, tested because it has the lowest redox potential for 2X-----> X2 where X represents a halide. The researchers concluded that the isolate was not involved in the synthesis of halogenated compounds; they did not test to see if the peroxidase reacted with bromide (Murphy and 6 hEocha 1973). However,  later characterized a brominating peroxidase from this alga.  found that 55 out of 72 marine algae tested had bromoperoxidase activity, and that the majority of the 55 were representatives ofRhodophyta. A vanadium-dependent bromoperoxidase has been recently found in Corallina officinalis, a red alga ). Vreeland et al.(1996) andWaite et. al. (1997) localized vanadium-dependent bromoperoxidases in attached Ectocarpus spores and in the mucilage of Fucus embryos (both brown algae) and in the frustule of the diatom, Achnanthes.

Conclusion
Haloperoxidases may be involved in cell wall activity in macroalgae or simply in producing unique secondary metabolites. The peroxidase work with C. parvula was not conclusive and this may be in part due to not having had enough tissue from this hollow, delicate alga to detect peroxidase activity.  offers some consolation: "The inability to detect either phenolic compounds or haloperoxidases  in algal extracts is not necessarily an indication that these compounds are not present (e.g. see Mehrtens 1994). Some cultured Antarctic brown algae showed no halogenating activity in crude extracts, although they produced large amounts of polyhalomethanes, which can result only from a halogenation reaction (Laturnus et al. 1996)."  explain further that detection of haloperoxidases and involved polyphenols is very difficult due to molecular interactions and reactions with algal carbohydrates.
The role of halogenated compounds in algal systems is not well understood (Laturnus et al. 1996). A push toward understanding the role within macroalgae has not occurred among scientists for four possible reasons: (1) interest in halogenated compounds has leaned toward uncovering their pharmacological uses particularly since the compounds seem to work as antimicrobial and antiviral agents , (2) the concern that ozone depletion particularly over Antarctica is due to volatile halogenated compounds from macroalgae (Laturnus et al. 1996), (3) the difficulty in working with algal haloperoxidases which are required for synthesizing halogenated compounds , and (4) the seemingly underdeveloped field macroalgal developmental biology outside of the investigations on Fucus embryos. The latter reason may in part be explained with the fact that present day understanding of animal and higher plant developmental biology is relatively much greater than the present day understanding of macroalgal developmental biology. The imbalance in 'understanding in this narrow field is comparatively true for many other topics in biology when one does a cross comparison of scientific effort by kingdom. 101