The role of epiphytic periphyton and macroinvertebrate grazers in the trophic flux of seagrass communities around Santiago Island, Bolinao, Philippines

Studies of epiphytic periphyton and grazing epifauna were carried out at up to 9 sites in the seagrass beds around Santiago Island, Bolinao, Luzon, Philippines between February and May 1991. Sites were chosen to represent a range of seagrass habitats within different sediment regimes and with differing exposure to wind, currents and wave action.Dry weight (DW) and ash-free dry weight (AFDW) was determined for periphyton removed from randomly selected shoots (n=20) of Cymodocea serrulata and Enhalus acoroides collected from Pislatan and Malilnep (shore). Biomass of periphyton per unit area of seagrass bed was calculated from periphyton biomass per plant and mean plant density based on unpublished data. Additional periphyton samples (n=5), collected from Lucero (reef flat), Malilnep (shore) and Pislatan were analysed for total organic carbon and total nitrogen (CHN Analyser). Samples were also examined under a compound microscope and the percentage cover of constituents was estimated by the point intersect method. Primary productivity estimates using respirometry (light-dark bottle) were obtained. Four artificial seagrass units (ASU), comprised of thin plastic strips (ten, 25 mm × 500 mm) suspended from a frame were placed among seagrasses at Pislatan and Malilnep (shore) for 4 weeks to form a cover of periphyton. Seven bottles containing ambient seawater were arranged on the seagrass bed and into each of five bottles was placed a periphyton-coated strip (25 mm x 80 mm). Two bottles without periphyton were controls. Bottles were sealed and incubated for 20 min, either under ambient sunlight, or in complete darkness. Incubations were repeated three times for light exposure and twice for dark, and made under cloudless conditions at both Malilnep and Pislatan in early May between 10:00 and 14:00 h. After each incubation, the bottles were transferred to a boat where the ASU strips were removed. Oxygen concentration in each bottle was measured with a Yellowsprings Scientific: Instruments (YSI) polagraphic O2 probe. Photosynthetic pigments were extracted from the periphyton-coated strips. Absorbance of extracts was read at 666 and 730 nm (Varian spectrophotometer), and the concentration of chlorophyll a was calculated. Four samples of surface (1 cm) sediment were collected from the Malilnep (shore), Lucero (reef flat) and Pislatan seagrass beds. These were dried to constant weight and subsampled (n=3) for determination of AFDW to DW ratio, silt, clay, sand and gravel contents, and N and total organic C.The number and type of grazing invertebrates occurring on randomly selected specimens of Enhalus acoroides, Cymodocea serrulata and Thalassia hemprichii (n = 50) during daylight in April 1991 was estimated for all nine sites. At Malilnep (shore) and Pislatan, the number of grazers present the upper and lower half of each plant was recorded. The variation in abundance and distribution of different grazing invertebrates occurring on Enhalus acoroides and Cymodocea serrulata over one 24-hour cycle was assessed at Pislatan. Dominant grazers at Malilnep (shore) and Pislatan were collected randomly (n = 60-70 per species) from plants during daylight for measurement of shell length. The occurrence of grazers on sediments within each of the nine sites was determined by collecting and counting animals within randomly placed 300 mm × 300 mm (in 100 mm x 100 mm partitions) quadrats (n = 10). Sediment was examined to a depth of about 5 mm. A preliminary estimate was made of the abundance of smaller epifauna on Thallasia hemprichii, Enhalus acoroides and Cymodocea serrulata from four entire plants of each species, collected during daylight at Malilnep (shore) and preserved in separate bags. After l hour, the contents were washed through a 0.5 mm sieve and the epifauna preserved for later examination. The feeding behaviour of the gastropods Strombus mutabilis and Cerithium tenellum was monitored at the Bolinao Marine Laboratory. Ingestion rate (I) of individuals of these two species was estimated by two independent techniques. In the faeces quantification technique, five groups of freshly collected snails (ten per group) were placed in separate containers with aerated seawater and equal quantities of periphyton-covered fronds of Cymodocea serrulata and Enhalus acoroides collected from the snails' habitat. Over 3.5 days, the faeces produced in each container after successive day and night periods were collected for separate DW and AFDW determinations. Subsamples of periphyton scraped from seagrass fronds were similarly analysed for AFDW and organic carbon. In the grazer manipulation technique, a series of experiments was conducted at Malilnep (shore) using short-term caging of undisturbed plants of Cymodocea serrulata as the feeding substratum. In each experiment, ten plants were enclosed within cages made of Perspex tubing, which was sealed to macrograzers, but provided with mesh covered 3 cm holes to permit water exchange. Half of the plant-chamber replicates were free of snails and other large macrograzers, and two snails (of one species) were introduced to the remaining replicates. The chambers were sealed at the base by a split rubber bung, which enclosed the stem of the plant and then attached to a pole driven into the sediment. After 24 hours grazed and ungrazed plants were harvested for estimation of biomass. The experiment was repeated once for each snail species. Periphyton biomass on each plant was determined as described above.To investigate the degree of selection of food types by snails, a separate enclosure experiment was conducted at Malilnep (shore) using ten separate plants caged as above, three with Strombus mutabilis, three with Cerithium tenellum and four as controls. Plants were harvested after 24 h and periphyton on each plant preserved for microscopic analysis of composition. Stomach contents of ten specimens of the dominant grazers, Strombus mutabilis and Cerithium tenellum, were examined, and the major components of the diet quantified using the method for microscopic analysis of periphyton composition. This study was initiated to:1. quantify the availability of epiphytic periphyton food resources in a tropical seagrass community2. quantify the use of epiphytic periphyton by epifaunal grazers Voucher specimens of identified algae are kept at the University of the Philippines Marine Science Institute (UPMSI).

1991年2月至5月间，研究团队于菲律宾吕宋岛博利瑙地区圣地亚哥岛周边的海草床中选取至多9个采样点，开展了附生周丛生物（epiphytic periphyton）与植食性底上动物（grazing epifauna）的相关研究。采样点的选取覆盖了不同沉积环境、受风、流及波浪作用强度各异的多种海草生境类型。

研究人员从皮斯拉坦（Pislatan）与马里内普（Malilnep，岸线站点）采集的齿叶丝粉藻（Cymodocea serrulata）和海菖蒲（Enhalus acoroides）的随机选取的20株植株上刮取周丛生物，测定其干重（dry weight, DW）与灰分干重（ash-free dry weight, AFDW）。基于单位植株周丛生物生物量与未公开的植株平均密度数据，计算得到海草床单位面积内的周丛生物生物量。

额外从卢塞罗（Lucero，礁坪站点）、马里内普（岸线站点）与皮斯拉坦采集了5份周丛生物样本，采用碳氮分析仪（CHN Analyser）测定其总有机碳与总氮含量。通过复合显微镜对样本进行观测，并采用点截法（point intersect method）估算各组分的盖度百分比。

采用呼吸计量法（light-dark bottle，亮暗瓶法）开展初级生产力估算。在皮斯拉坦与马里内普（岸线站点）的海草床中放置4组人工海草单元（artificial seagrass unit, ASU），该单元由悬挂于支架上的10条25 mm × 500 mm的薄塑料条构成，放置时长为4周，以使其表面形成周丛生物覆盖层。

在海草床中布设7瓶原位海水，其中5瓶分别放入1片25 mm × 80 mm的周丛生物覆盖塑料条，剩余2瓶未放置塑料条作为对照组。将瓶体密封后，分别在原位自然光下与完全黑暗环境中培养20分钟。光照组实验重复3次，黑暗组重复2次，实验均于5月初晴朗天气下在马里内普与皮斯拉坦站点的10:00至14:00时段开展。每次培养结束后，将瓶体转移至船上，取出塑料条。采用Yellow Springs Scientific Instruments（YSI）极谱式溶氧探头测定瓶内溶氧浓度。

从周丛生物覆盖的塑料条中提取光合色素，使用瓦里安分光光度计（Varian spectrophotometer）在666 nm与730 nm波长处读取吸光度，并据此计算叶绿素a浓度。

从马里内普（岸线站点）、卢塞罗（礁坪站点）与皮斯拉坦的海草床中采集4份表层（1 cm深度）沉积物样本，将其烘干至恒重后，分取3份子样本，用以测定灰分干重与干重比值、粉砂、黏土、砂与砾石含量，以及总氮与总有机碳含量。

1991年4月日间，对全部9个采样点上随机选取的50株海菖蒲、齿叶丝粉藻与泰来藻（Thalassia hemprichii）样本上的植食性无脊椎动物的数量与类群进行了统计。在马里内普与皮斯拉坦站点，研究人员记录了每株植株上下半部分的植食动物数量。在皮斯拉坦站点，研究人员评估了海菖蒲与齿叶丝粉藻上不同植食性无脊椎动物的丰度与分布在24小时周期内的变化情况。

从马里内普与皮斯拉坦站点的植株上随机采集优势植食动物（每个物种60~70只），测量其壳长。通过在每个采样点随机放置10个300 mm × 300 mm（分为100 mm × 100 mm分区）的样方（quadrats），收集并计数样方内沉积物中的动物，以此确定9个站点沉积物中植食动物的出现情况。沉积物采样深度约为5 mm。

对马里内普（岸线站点）日间采集的各4株完整泰来藻、海菖蒲与齿叶丝粉藻样本上的小型底上动物丰度进行初步估算，将样本分别装入密封袋中保存。1小时后，将样本经0.5 mm孔径筛网冲洗，留存底上动物样本以供后续镜检。

在博利瑙海洋实验室监测了腹足类（gastropods）动物锈唇凤凰螺（Strombus mutabilis）与秀丽蟹守螺（Cerithium tenellum）的摄食行为。通过两种独立方法估算这两个物种的个体摄食率（ingestion rate, I）。

第一种方法为粪便定量法：将新鲜采集的蜗牛分为5组，每组10只，分别置于装有充氧海水的容器中，加入等量从其栖息地采集的覆盖有周丛生物的齿叶丝粉藻与海菖蒲叶状体。在3.5天内，分别收集昼夜时段内各容器产生的粪便，用以测定干重与灰分干重。从海草叶状体上刮取的周丛生物子样本同样进行灰分干重与有机碳含量分析。

第二种方法为植食动物操控实验法：在马里内普（岸线站点）开展系列短期实验，以未受干扰的齿叶丝粉藻植株作为摄食基质，使用有机玻璃管制作的笼箱进行围隔。每个笼箱包含10株植株，笼箱可阻挡大型植食动物，但设有覆盖网纱的3 cm孔径孔洞以保证水体交换。一半笼箱不放置螺类与其他大型植食动物作为对照组，另一半笼箱则放入2只同种螺类。笼箱底部采用开缝橡胶塞密封，包裹植株茎部后固定于插入沉积物的立杆上。24小时后，收获被摄食与未被摄食的植株以估算生物量。该实验针对两种螺类各重复一次。每株植株上的周丛生物生物量测定方法如前所述。

为探究螺类对食物类型的选择偏好，在马里内普（岸线站点）开展了另一组围隔实验：设置10株围隔植株，其中3株放置锈唇凤凰螺，3株放置秀丽蟹守螺，剩余4株作为对照组。24小时后收获植株，将植株上的周丛生物保存以供镜检分析其组成。

对10只优势植食动物（锈唇凤凰螺与秀丽蟹守螺）的胃容物进行镜检，采用周丛生物组成的显微分析方法量化其日粮的主要组分。

本研究的立项目标如下：
1. 量化热带海草群落中附生周丛生物食物资源的可获得性
2. 量化底上植食动物对附生周丛生物的利用情况

经鉴定的藻类凭证标本（voucher specimens）保存于菲律宾大学海洋科学研究所（University of the Philippines Marine Science Institute, UPMSI）。