Gamete Morphology of Six Species of Sea Urchins in Saipan, CNMI. Julia Fuller Mentor: Dr. Roger Goodwill Summer 2014 Bio 494

Transcription

1 Gamete Morphology of Six Species of Sea Urchins in Saipan, CNMI Julia Fuller Mentor: Dr. Roger Goodwill Summer 2014 Bio 494

2 Abstract Using scanning electron microscopy, gamete morphology was examined of six sea urchin species collected from Saipan, Commonwealth of the Northern Mariana Islands, Echinometra mathaei, Parasalenia poehlii, Eucidaris metularia, Colobocentrotus atratus, Mespilia globulus and Nudechinus scotiopremnus. Regular sea urchin spermatozoa is characterized by a conical head, circular mitochondrion, and flagellate tail. Observation showed no variation from this however sperm head width : length ratio was found to vary between species with the most variation in C. atratus. General egg morphology is known to consist of large, round eggs with a vitelline membrane. Results showed variation of egg size with the largest for M. globulus was almost three-times that of E. metularia. Surface structures present on the vitelline membrane were also found to differ for different species. These results were compared with an earlier study of gamete morphology in four species of bathyl echinothuriid echinoids collected in the Bahama Islands along with a study of four shallow-water species collected off the coast of Japan. The results of these comparisons were an observed sperm head length measuring shorter than those of Japan and the Bahamas, excluding C. atratus which measured similar to those in Japan. Key words: sea urchins, sperm, egg, morphology, membrane, Echinometra mathaei, Parasalenia poehlii, Eucidaris metularia, Colobocentrotus atratus, Mespilia globulus, Nudechinus scotiopremnus Introduction Within Echinoidea, gross sperm morphology has been examined for more than 100 species with results showing general gamete structure as highly conserved (Chia et al. 1975, Eckelbarger et al. 1989b, Marks et al. 2008). The general shape of mature echinoid sperm is characterized by a conical head (Chia et al. 1975) composed largely of the cone-shaped nucleus topped with a small acrosome, a midpiece with a single, and circular mitochondrion (Drozdov and Vinnikova 2010), followed with a flagellate tail (Marks et al. 2008). Echinoid eggs are round and covered by vitelline and jelly-like membranes (Drozdov and Vinnikova 2010). Current theory on sperm morphology focuses on morphological differences in sperm of species from different oceans. Studies have shown that sperm collected from the Pacific showed major morphological differences in sperm shape, size, and ultrastructure from specimens collected from the Atlantic. Western Pacific populations sperm head length was twice that of central Pacific populations (Marks et al. 2008, Landry et al. 2003). Sperm head 1

3 length was also found to be elongated in species collected from the deep sea environment, at depths ranging from 600 to 900 m (Eckelbarger et al. 1989b). Sperm size can be polymorphic among populations within a single species, however, variation in length more than twofold represents differences among species (Chia et al. 1975, Amy 1983, Landry et al. 2003).The divergence in sperm morphology within a species has been viewed as the result of recent speciation (Landry et al. 2003). In the few studies related to sea urchin gamete morphology from a single area, results have shown that echinoid sperm are conservative in morphology exhibiting little interspecific structural variation (Eckelbarger et al. 1989a). Spermatozoa were found to differ significantly by the structure of their acrosome complex (Drozdov and Vinnikova 2010) and unique staining in the acrosome vesicle (Eckelbarger et al. 1989a). The presence of lipid droplets has been observed in certain species within a particular locality (Mita et al. 2002). Intraspecific variation of egg size and egg-jelly thickness has been found (Marks et al. 2008) along with significant differences in the carriage of chromatophores (Drozdov and Vinnikova 2010). This study describes morphological features of both male and female gametes of Echinometra mathaei, Parasalenia poehlii, Eucidaris metularia, Colobocentrotus atratus, Mespilia globulus and male gametes of Nudechinus scotiopremnus collected in Saipan, Commonwealth of the Northern Mariana Islands. Material and Methods Six Echinometra mathaei, two Parasalenia poehlii, two Eucidaris metularia, four Mespilia globulus, four Colobocentrotus atratus, and one Nudechinus scotiopremnus were collected in June 2014 using snorkel gear and scuba diving gear from subtidal habitats in Saipan, Commonwealth of the Northern Mariana Islands (15.18 N E) and transferred to a marine lab. Spawning was induced by injection of 0.55 M KCl into the coelomic cavity; gametes were 2

4 collected in 1 ml syringe as they were released from the gonoduct in sea water. Samples were smeared on glass slides to be stained later or immediately fixed for scanning electron microscopy by adding 4% glutaraldehyde in 0.1 M sodium cacodylate with 0.35 M sucrose, ph of 7.4. Within three weeks samples were transported to the University of Hawaii at Manoa and prepared for scanning electron microscopy. Eggs were prepared by washing in 0.1 M cacodylate buffer with 0.35 M sucrose for two 15 minute intervals. Each sample was postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for one hour. Dehydration of the sample was performed through a graded ethanol series (30%, 50%, 70%, 85%, 95%) with one change at 30% and 50% and two changes of 70%, 85%, and 95% every three to five minutes. At 85% samples were transferred to 78 μm pore size microporous specimen capsules to complete the dehydration. A 100% ethanol dehydration was performed with three changes at ten minute intervals, the last change in the critical point dryer. Samples were dried in a Tousimis Samdri- 795 critical point dryer according to standard protocol. Sperm samples were prepared using 13 mm Millipore Isopore 0.2 μm polycarbonate filters held in Millipore Swinnex filter holders. Samples were washed in 0.1 M cacodylate buffer with 0.35 M sucrose twice for five minutes each then postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 30 minutes. Dehydration was performed using a graded ethanol series (10%, 20%, 30%, 50%, 70%, 85%, 95%) with two changes of each dilution, the first for five minutes and the second for 15 minutes followed by three changes at ten minute intervals in 100% ethanol, the last change in the critical point dryer. Samples were then dried in a Tousimis Samdri-795 critical point dryer according to standard protocol. Specimens were mounted on aluminum stubs with double-stick carbon tape, coated with gold/palladium in a Hummer 6.2 sputter coater and examined and measured on a Hitachi S-4800 Field Emission Scanning Electron Microscope at an accelerating voltage of 5 kv. Measurements were analyzed using one-way ANOVA and Tukey pairwise comparison methods through Minitab 17. 3

5 Results The mature spermatozoa of E. mathaei, P. poehlii, E. metularis, M. globulus, and N. scotiopremnus were found to be conservative in morphology with similar sperm head shape, mitochondria, and flagellate tail. Spermatozoa of C. atratus, however, differed significantly in head shape showing a narrower and longer head along with a less specified mitochondrion (Figure 1). Spermatozoa were found to differ within 0.58 μm in length and 0.25 μm in width. Colobocentrotus atratus had a significant difference in head shape. Sperm head length of C. atratus was significantly different from the other species (P<0.001). Nudechinus scotiopremnus sperm length was significantly different from all other species (P<0.05) except P. poehlii and M. globulus (P>0.5). The mean sperm head length (excluding mitochondrion) of the six species ranged from the longest, C. atratus (7.14 μm) to the shortest, N. scotiopremnus (2.18 μm). Excluding C. atratus the remaining five species had a mean head length between 2 μm and 3 μm (Figure 2). In measuring sperm head width four groupings were calculated that were not significantly different from each other (P>0.5). However significant differences were found in comparing individual species measurements. The sperm head width of E. metularia was significantly different from E. mathaei, C. atratus, M. globulus and N. scotiopremnus (P<0.5). Parasalenia poehlii was significantly different from E. mathaei, C. atratus, and M. globulus (P<0.5). Nudechinus scotiopremnus was signigicantly different from P. poehlii, E. metularia, and C. atratus (P<0.5). Echinometra mathaei was significantly different from P. poehlii, E. metularia, C. atratus, and N. scotiopremnus (P<0.5). Mespilia globulus was different from P. poehlii, E. metularia, and C. atratus (P<0.5). Colobocentrotus atratus was significantly different from all other species (P<0.5). Measurements were generally similar with the smallest width at 1.2 μm (C. atratus) and the largest at 1.57 μm (E. metularia) (Figure 3). Sperm tail diameter measurements of N. scotiopremnus were significantly different from E. mathaei and C. atratus (P<0.001) and P. poehlii and E. metularia (P<0.05) (Figure 4). 4

6 Echinometra mathaei, Parasalenia poehlii, Eucidaris metularia, Colobocentrotus atratus, and Mespilia globulus were all found to have spherical eggs but vary in the appearance of the vitelline membrane. Surface structures were found on each species membrane but varied in size, shape, and number. Eucidaris metularia had the most distinct membrane appearance. The proteins were longer and grew away from the egg surface giving the membrane a somewhat furry appearance (Figure 5). Significant differences were observed in egg diameter among some species (P<0.001). Colobocentrotus atratus and Parasalenia poehlii egg diameter was not significantly different (P=0.777). Egg diameter ranged from the largest, M. globulus (88.46 μm), to the smallest, E. metularia (30.22 μm) (Figure 6). Egg samples were not collected from Nudechinus scotiopremnus. 5

7 Figure 1. The scanning electron microscopy images of the head regions of spermatozoa for each species. (a): Echinometra mathaei, (b): Parasalenia poehlii, (c): Eucidaris metularia, (d): Colobocentrotus atratus, (e): Mespilia globulus, (f): Nudechinus scotiopremnus. 6

8 Figure 2. The calculated mean plus or minus the standard deviation of observed measurements of spermatozoa head lengths measured using scanning electron microscopy image measurements. Figure 3. The calculated mean plus or minus the standard deviation of observed measurements of spermatozoa head widths measured at the widest point using scanning electron microscopy image measurements. 7

9 Figure 4. The calculated mean plus or minus the standard deviation of observed measurements of spermatozoa tail diameters measured using scanning electron microscopy image measurements. 8

10 Figure 5. The scanning electron microscopy images showing general morphology of eggs along with a close-up image of egg outer membrane surface for each species. (a): Echinometra mathaei, (b): close up view of Echinometra mathaei, (c): Parasalenia poehlii, (d): close up view of Parasalenia poehlii, (e): Eucidaris metularia, (f): close up view of Eucidaris metularia, (g): Colobocentrotus atratus, (h): close up view of Colobocentrotus atratus, (i): Mespilia globulus, (j): close up view of Mespilia globulus. 9

11 Figure 6. The calculated mean plus or minus the standard deviation of observed measurements of eggs measured using scanning microscopy image measurements. Diameter was recorded as the longer of the two diameters measured on each egg. Discussion Recent studies have shown differences in general size and morphology of spermatozoa between shallow and deep-water sea urchin species (Eckelbarger et al. 1989a). This research describes additional modifications of gametes from various shallow-water sea urchin species. The spermatozoa in this study showed limited variation between species. General head shape was found to consist of a nucleus and mitochondrion with a flagellate tail. Other studies found that variation in morphology exists between species but the general structure was the same. Eggs also showed limited variation between species. Body size varied widely in comparison to spermatozoa size. Vitelline membrane structures varied which may be due to the selective permeability of specific urchin species spermatozoa. All samples were collected within a three week period in June which may have contributed to the variance in gametes. Some sea urchins may not have been sexually mature 10

12 enough or ready to spawn at that time. Estimation by the software package could also contribute to minor differences in measurements. Gamete ultrastructure of four deep sea echinothuriids Phormosoma placenta, Sperosoma antillense, Araseosoma fenestratum and Araeosoma belli (Eckelbarger et al. 1989a) and sperm ultrastructure in three shallow-water echinothuriids Asthenosoma ijimai, Araeosoma owstoni, and Hapalosoma gemmiferum (Amemiya et al. 1980) found similar structures. One notable difference was sperm head length. In shallow-water species head length was 7 μm A. ijimai, 6 μm A. owstoni, and 4 μm in H. gemmiferum. Deep sea species head length measured at 12 μm Phormosoma placenta, 8.5 μm A. fenestratum, and 9 μm in A. belli and Sperosoma antillense. The reasons for this variance has not been addressed. Little is known about the vitelline membrane of the egg or the structures present on the membrane. Clearly the next phase of this research must address the reasons for the variance within and between species. Literature Cited Amemiya, S., T. Suyemitsu, and I. Uemura Morphological observations on the spermatozoa of echinothurid sea urchins. Development, Growth, and Differentiation 22: Amy, R. L Gamete sizes and developmental time tables of five tropical sea urchins. Bulletin of Marine Science 33: Chia, F., D. Atwood, and B. Crawford Comparative morphology of echinoderm sperm and possible phylogenetic implications. American Zoological 15: Drozdov A. L., and V. V. Vinnikova Morphology of gametes in sea urchins from Peter the Great Bay, Sea of Japan. Russian Journal of Developmental Biology 41 (1): Eckelbarger, K. J., C. M. Young, and J.L. Cameron. 1989a. Modified sperm ultrastructure in four species of soft-bodied echinoids (Echinodermata: Echinothuriidae) from the bathyal zone of the deep sea. Biological Bulletin 177:

13 Eckelbarger, K. J., C. M. Young, and J. L. Cameron. 1989b. Modified sperm in echinoderms from the bathyal and abyssal zones of the deep sea. In Reproduction, Genetics and Distributions of Marine Organisms. J. S. Ryland and P. A. Tyler, eds. Olsen & Olsen, Fredensbog, Denmark. pp Landry, C., L. B. Geyer, Y. Arakaki, T. Uehara, and S. R. Palumbi Recent speciation in the Indo-West Pacific: rapid evolution of gamete recognition and sperm morphology in cryptic species of sea urchin. Proceedings of the Royal Society of London. Series B: Biological Sciences (London) 270: Marks, J. A., C. H. Biermann, W. F. Eanes, and H. Kryvi Sperm polymorphism within the sea urchin Strongylocentrotus droebachiensis: divergence between Pacific and Atlantic Oceans. Biological Bulletin 215: Mita, M., T. Uehara, and M. Nakamura Comparative studies on the energy metabolism in sperm of four closely related species of sea urchins (genus Echinometra) in Okinawa. Zoological Science 19: