Filter feeding bivalves are key players in dealing with current environmental processes due to effects of climate change. With increasing carbon dioxide levels, algal blooms algal blooms are becoming more abundant and coastal acidification is becoming more prevalent amongst estuaries around the world. This study examines the bivalve, Plebidonaxdeltoids, and sees if this species has a preferred algal preference. The design of this experiment included 5 different treatments with 3 different algal species (Isochrysis galbana, Thalassiosira weissflogii, Nannochloropsis spp.). Two other treatments were also used,one with all algae and one without any algae. The results yielded significant data amongst the Nannochloropsis spp. treatment and significance between Nannochloropsis spp. and all other treatments. This study brings up the question, if species such as pipis are willing to selectively decide which type of food they want, can they be used to remediate algal blooms and solve other corresponding events? The findings in this study can serve as a pilot study for others in the field of marine bivalve behavior.

Eutrophication due to anthropogenic input of nutrients is a major issue in parts around the world both in estuaries and coastal waters (Brodie et al. 2010, Wallace et al. 2014). River runoff from agricultural fields and other nutrient rich lands lead to addition of nutrients such as inorganic nitrogen and phosphorous (Packett et al. 2009 & Bell 1992). With this increase in nutrients phytoplankton blooms become more prevalent leading to higher rates of microbial degradation (respiration) of the algae (Brodie etal. 2010, Wallace et al. 2014). Microbial degradation of this algal biomass leads to large decreases in dissolved oxygen levels causing large hypoxic zones killing fish and other marine life (Brodie et al. 2010, Wallace et al. 2014). In addition to hypoxia, microbial respiration releases metabolic CO₂ that reacts with the carbonate chemistry in the water leading to decreases in pH and increases in acidification (Wallace et al. 2014). Algal blooms therefore are major threats to marine ecosystems and should be controlled or prevented in order to preserve marine life.

Phytoplankton are photosynthetic single celled organisms that play a large role in oceanographic processes (“A database”2015).Some species produce toxins that affect shellfish and others form blooms that can cause hypoxia and poor water quality as mentioned (“A database” 2015, Wallace et al. 2014). These photosynthesizers are the base of the marine trophic pyramid and affect secondary production and consumers. Some main groups of phytoplankton that are present within the Queensland waters are diatoms and dinoflagellates (“Coastal Algal” 2010). Diatoms have an outer skeleton (frustule) made of silica and are present in both inestuaries as well as along the coasts (“Coastal Algal” 2010). Dinoflagellates are mostly unicellular, are the most common bloom forming phytoplankton, and are responsible for some major forms of shellfish poisoning (“Coastal Algal” 2010, “A database,” 2015). Two of the species, Thalassiosira weissflogii and Isochrysis galbana,used during this experiment belong to diatom and dinoflagellate phytoplankton groups. The last algal species, Nannochloropsis spp., belongs to another phylum of phytoplankton that is also an important primary producer.

The target species, (Plebidonax deltoids), belongs to the phylum Mollusca, class Bivalvia,and order Venerida. They are laterally compressed with a shell made of two parts that are hinged together by an elastic ligament and closed by adductor muscles from the mantle (Brusca et al., 2016). They are filter feeders that use their siphons to feed upon an array of algal species (Moy, 2014). Bivalves can be in either marine or freshwater and are very diverse spanning 9,200 species that are displayed in all depths of the ocean (Brusca et al., 2016). P. deltoids, being prevalent along the sandy beaches of Australia, uses its muscular foot to burrow in the sand and filter feed the surf diatoms that wash up with the waves (Moy, 2014). P. deltoids isn’t usually a species used for marine remediation purposes, but being a filter feeder it has the potential to be one. In addition to being a filter feeder, P. deltoids is also an important food source for sea birds and humans alike (Common Pipi, 2016). Humans can use them as bait when fishing and even in pasta and other dishes for dinner (Common Pipi, 2016).

During this experiment, P. deltoids was exposed to 3 different algal species to observe someform of feeding selectivity. By understanding this preference, if found, can support conservation efforts dealing with eutrophication and algal blooms in estuaries across Australia. It was hypothesized that P.deltoids would have a preference towards diatom species as it is the most common phytoplankton group encountered in its natural habitat. 

This study was carried out using pipis (Plebidonax deltoids) taken from Tugen Beach along the Gold Coast in Queensland, Australia. Pipis were taken out ofthe sand during low tide approximately a quarter mile along the beach. All pipis were between 4-6 centimeters and were transferred to the marine aquarium at the University of Queensland. A total of 30 pipis were used during the experiment with 6 allocated per treatment.

There were 5 different treatments in the experiment,one without any algae that served as a control and the other 4 with algae. The first treatment used Nannochloropsis spp.(1-2 μm), the second used Isochrysis galbana (5-6 μm), the third used Thalassiosira weissflogii (7-20 μm), and the last treatment was a combination of all three species (“Thalassiosira weissfloggi” 2010, Paes 2016, Tan 2017, “Typical Nutritional” 2005).All treatments began at a constant biovolume (65 x 10⁷ μm³/mL) to ensure that the amount of biomass per mL was the same due to the differing sizes of the algae. The average biovolume of each cell type was used to calculate the biomass that was needed in each replicate per treatment (“Chapter 3 Development”2009).

Circular containers were used for each replicate with one pipi per container. The algae solutions were then made by diluting volumes of Instant Algae® of each species in the 200 mL container of seawater. The solutions were mixed thoroughly and initial absorbance readings were taken. Before placing the pipis in each individual container, they were placed in a separate container with seawater to destress and open up for approximately 45 minutes. After each pipi was placed in its container, samples were taken every half hour for 2 and a half hours.

The Beckman Coulter DU® 720 spectrophotometer was used to obtain absorbance readings throughout the experiment. The samples were read at OD 550 nm instead of OD 660 nm to capture the greatest particulate signal as every species of algae had varying amounts of chlorophyll a per cell (“TypicalNutritional” 2005).

Statistical Analyses:

Statistical testing was performed using R version 3.3.1. All treatments were graphed with absorbance versus time to obtain the slope per replicate using Excel. The data was log transformed to correct for violations of normality and equal variance before analyzing significant differences amongst treatments. A one-way ANOVA was performed to recognize differences amongst treatments with reference to their slopes of absorbance versus time. Tukey’s Multiple Comparison Test was performed after the one-way ANOVA to recognize differences between treatments.

Algae concentrations did decrease in all treatments but not all replicates. The average slope of algae concentrations per species did decrease across treatments with Nannochloropsisspp. having the largest change in concentration and the containers with no algae had the smallest change. As in figure 4, although each treatment had different starting absorbance readings they decreased in a somewhat linear fashion.

It was seen that most treatments had poor coefficients of determination with the highest being Nannochloropsis spp. at 0.75 although they all had negative relationships with initial absorbance values. A one-way ANOVA was performed for the slopes of each treatment. It was found that the results were significant across treatments (p < 0.05).  The Tukey test found significant differences between the Nannochloropsis spp. treatment and all other treatments(p < 0.05).

Nannochloropsis spp. was found to be significant amongst all other treatments. It was shown in the analysis that Nannochloropsis spp. had the largest change from beginning absorbance to end absorbance along with the largest slope. Nutritional content of Nannochloropsis spp. may have played a part in the selective feeding of the pipis. Compared to both Isochrysis and T. weissflogii, Nannochloropsis spp. has the highest protein, lipid, and calories per dry weight (“Typical Nutritional” 2005). With this difference the pipis may have had an incentive to feed upon Nannochloropsis spp. rather than the less nutritious Isochrysis galbana and T. weissflogii. Although the diatom Asterionella spp. is a main source of food for pipis which also happens to be a diatom, it was displayed in the results that they filtered more Nannochloropsis spp. (Ferguson 2013).

In addition to nutrition content, Nannochloropsis spp. is also the smallest algae used in this experiment.Being the smallest may have given them highest preference amongst other algae as it could have been easier for the pipis to filter. The size of diatoms such as T. weissflogii are almost triple the size of Nannochloropsis spp. which may lead to the pipis being less likely to eat them due to energetic restraints(“Typical Nutritional” 2005).

Clams and other bivalves have been proven to be intertwined with nutrient cycling as well as water quality along coasts and estuarine environments (Nakamura et al. 2000, Nasci et al. 1999, Nelson et al. 2004). Some bivalves such as oysters and mussels have proven that they can be used to partially filter out nutrient polluted water by decreasing the amount of total suspended solids and chlorophyll a concentrations (Nelson et al. 2004). Based on the findings from this study, a following experiment can be developed using pipis or another species of clam as options for bioremediation in nutrient polluted water. In addition to filtering the water, some species of clams can also be used as biomarkers toward assessing water quality and nutrient cycling in certain marine environments (Nakamura et al. 2000, Nasci et al. 1999).  By understanding these relationships, pipis and other related species may be used to further research in relationships of bivalves and the health of marine ecosystems.

Although Nannochloropsis spp. was found to be significant it may have been due to some errors in experimental design or machine accuracy. As displayed in figure 4, the absorbance readings of Nannochloropsis spp. were much higher due to a higher amount of cells in the solution in order to match the same biovolume amongst the other treatments. Since the Nannochloropsis spp. cells were so small, a large amount of the concentrated algae was added into the seawater and with more algae a larger absorbance reading was read. The higher absorbance readings that were seen could have been due to more particulates in the water compared to the much lower readings of the other treatments. Also, since the other treatments had such low values of absorbance the spectrophotometer may have had some error when detecting such a low concentration. Retrieving each sample may have been skewed as well since the containers were not mixed before retrieval in fear that the pipis may stop feeding. Unfortunately, the weight of each pipi was unable to be taken due to time constraints. The weight and tissue to shell ratio may have been additional factors as well in reference to each individual pipis filtration rate.

Although significant data did arise from this experiment,it is important to follow up this pilot study with others in order to come to a conclusion on how pipis along with other filter feeding bivalves can be used to their highest potential with reference to eutrophication, coastal acidification, and other environmental issues. This study also takes a step forward into learning more about the ecological relationship between pipis and nutrient poor areas such as sandy beaches versus estuaries. Follow up studies that can strengthen or refute the findings of this experiment can involve a larger sample size, using species of algae native to Queensland waters, using abundance of cells versus biovolume for treatments, or a comparison to other bivalve species. Especially as global environmental changes continue to occur,research among filter feeders and species dealing with calcification are becoming increasingly important.   

Many thanks to our course coordinator, Bernard Degnan,professor Sandie Degnan, and tutors on providing help and advice for this project. Also, thanks to Thomas Ulrich and Carl Chew for enduring with me to find every pipi needed for this experiment.

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