Physiology Group Project

Study Outline

Effect of Food Concentration on the Respiration Rate of the

Asian Green Mussel, Perna viridis

Introduction

The Asian green mussel or Perna viridis is a species of bivalve under the family
Mytilidae. They are commonly found in the estuarine areas and intertidal zones of the Indo-
Pacific region and Southeast Asia. They are classified as filter feeders and considers
phytoplankton as their main diet component (Iqbal et al., 2017) The changes in growth rate
abundance as well as distribution of suspension-feeding bivalves such as Perna viridis may have
adverse effects on benthic and pelagic ecosystems. Filter feeders such as Perna viridis serve as
bioindicators by evaluating the organic and inorganic contaminants that accumulate in the
bivalve’s tissues (Mamon, 2016). Because of their important ecological functions they are
considered as key species in estuarine and coastal habitats (Huhn et al., 2017). Aside from its
ecological importance, bivalves such as Perna viridis are also considered as an important food
source and provide economic benefits in countries throughout Asia. Perna viridis provides an
inexpensive source of protein as well as polyunsaturated fatty acids. It also contributes to the
intake of essential minerals, trace metals as well vitamins with high pharmaceutical values
(Chakraborty et al., 2011)

P. viridis can grow up to 80-100mm in length, with the smooth, elongated shell and the
external surface characterized by concentric growth rings. The periostracum color ranges from
dark brownish- green (anterior) to olive and light green (posterior) and the inner shell surface is
white, smooth and iridescent with a bluish-green hue (Menzel, 2018). Juveniles are bright green
in color but as the organism matures, the color dulls into dark geen to brown (Rajagopal et al.
2005).
 In the first quarter of 2018, the mussel production in the Philippines reached 9.2 thousand
metric tons which is 54.18% higher than the 2017 volume. The volume of mussel production in
Western Visayas, however, decreased by 4.52% compared to last year. This decrease is
associated with the effect of siltation on wild spat growth in Sapian Bay, Capiz (Philippine
Statistics Authority, 2018), which is one of the major mussel producing areas in the country.

In most animals, digestion and respiration may seem to be separate systems but in reality,
both of them work together in providing energy to the cells of the body. The oxygen provided by
respiration is utilized in burning food molecules to be an energy source for the body
(Hendrickson, 2017). In most bivalves, the gills are also used for feeding aside from respiration.
They have enlarged and folded gills which serve as filtering apparatus. As water passes through
the mantle cavity, particles are sieved from the water by a film of mucus in conjunction with
special cilia which then leads to a ciliated groove after which to the mouth (Giribet, 2008).

Objectives

This study generally aims to study the relationship of different food concentrations to the
rate of Perna viridis respiration. Specifically, the study aims to determine the effect of the
different concentrations of Isochrysis spp. to the respiration rate of Perna viridis.

Significance

Data collected in this study may benefit Perna viridis farmers by providing data on the
mussel’s response to excessive or insufficient amounts of food available to the mussels in
relation with their respiration rate. It may also provide an overview of the respiratory responses
of Perna viridis when exposed to varying food concentrations in the natural setting which may
help future conservation efforts in case such phenomenon happens.

Review of Related Articles

Green mussels such as Perna viridis are considered as invasive species due to its wide
range of tolerance to various environmental conditions. They may tolerate temperatures ranging
from 15 °C up to 32.5 °C without difficulties. They also have great adaptive capabilities in terms
of salinity where they can survive salinity levels as low as 20 %. Oxygen consumption in
mussels is directly related to its filtration rate. Optimum filtration rates occur at 30 °C and 30 -
35% salinity. Filtration rates are also affected by mussel size and the light-dark cycle aside from
variations in temperature and salinity (Rajagopal et al., 2006).

A study done on Perna viridis for tolerance to hypoxia by Huhn and his colleagues last
2016 displayed the acclimatization process on Perna viridis for their experiment. After sample
collection, the salinity on the sampling site was maintained on the acclimatization tanks and the
temperature was a degree lower than that of the sampling site. Before experimentation, mussels
were initially cleaned of epibionts and are acclimatized for 5 (sample type 1) and 14 days
(sample type 2). All other variables (amount and type of food, water exchange rates, aeration and
mussel to water ratios) were same for both sample types. During acclimatization, mussels were
kept in tanks with 80 L of aerated seawater at a density of 1 mussel per liter. They were fed with
0.01 ml of the concentrated filter feeder food Sera Marin Coralliquid (SMC) which corresponds
to an individual every day. Half of the water in the tanks were also changed daily (Huhn et al.,
2016)

A study done on 2000 about the influence of algal concentration, salinity and body size
on the ingestion rates of cultivable Indian bivalves showed another means of means of bivalve
acclimatization. Initially they collected bivalve sample of Perna viridis (64-67 mm and 100-105
mm), Crassostrea madrasensis (65-70 mm and 100-105 mm) and Paphia malabrica (30-32 mm
and 45- 47 mm) from their bivalve farm site. Samples were then acclimatize for two weeks at
approximately at a temperature of 30°C and a salinity of 32 ppt in 10 liter plastic basins with
continuous oxygen supply. The water in the basins were replaced every second day and the
animals were fed on axenic cultures of microalga namely Isochrysis galbana (Rajesh et al.,
2000).

According to www.fondriest.com, a site which provides information in monitoring the

environment, there are three methods available for measuring dissolved oxygen concentrations.
These are the dissolved oxygen meter and sensor method, colorimetric method and titrimetric
method. The dissolved oxygen sensor method utilizes various sensors which could be optical (by
the use of dyes) or electrochemical (polarographic or galvanic). Colorimetric method on the
other hand utilizes color change by the use of reactants such as indigo carmine and rhodazine D.
Lastly, the titrimetic method or usually called the traditional method utilizes a starch indicator
for titration. In this method, the volume of oxygen is considered to be equivalent to the volume
of sample titrated before the starch indicator changes its color.

A study done to determine the respiration rates of a fan mussel Pinna nobilis
demonstrated a method in determining differences in respiration rates. On the last day of
maintenance period, mussels were starved to minimize diet interferences on respiration. The
specimens were placed vertically into the closed respiration chambers with oxygen-saturated
water 1 h before starting the experiments. Pump aeration was used to mix the water before
introducing the specimens. Oxygen measurements by using a probe were done on different
depths to prevent biases. A chamber without the presence of animals was always used to record
oxygen loss without the presence of mussels. The respiration rates were recorded when the
mussels valves are open using a specific oxymeter galvanic probe. At the start of the experiment
the electrode used was calibrated to 100% oxygen solubility. Measurements were done every
hour for 5 hours, to reduce the risk of O2 partial pressure fall and excessive concentration of
mussel excretions. The process was repeated three times for each subject with breaks of 72 hours
between individual experiments in every treatment (Trigos et al., 2014).
Materials and Methods

Test organism

Samples of green mussels (Perna viridis) will be gathered from Ivisan, Capiz and will be
transported to the UPV Multi-Species Hatchery. The samples attached to the bamboo poles will
be covered with cloth soaked with seawater to preserve moisture and put into storage boxes.
Each mussel is detached carefully by cutting their byssal threads (Wang et al., 2011), and the
epibionts are removed and its shell height will be measured (Trigos et al., 2014).

Selection and Stocking

The samples will be transferred into 50 x 75 cm tanks prior to the acclimatization. The tanks will
have proper aeration system and the seawater will be obtained from the coast near the hatchery.

Acclimatization of the Test Organism

The acclimatization of the samples will be done for seven days prior the experiment proper. The
samples will be put into tanks with normal room temperature. The salinity will be maintained at
30-35 ppt. Physicochemical parameters of the water will be monitored daily. It will be fed with a
standard amount of Isochrysis sp. everyday day after stocking to prevent stress.

Experimental Design

This study will follow Completely Randomized Design (CRD) (Figure 1) using 300 samples of
juvenile mussels which will be distributed into three treatments with three replicates. Treatment
1 – Control, Isochrysis sp desired feeding value; Treatment 2 – twice the amount of Isochrysis sp
desired feeding value; Treatment 3 – half of the Isochrysis sp desired feeding value; and
Treatment 4 – Unfiltered Seawater + Isochrysis sp desired feeding value. All treatments except
Treatment 4 have filtered seawater. Each treatment has three replicates. Each tank will have 10
mussels. Water change will be done every day in the morning.
 T1R3 T4R1 T3R2 T2R3

T1R1 T4R3 T3R3 T2R2

T2R1 T4R2 T1R2 T2R1

Figure 1. Experimental layout using CRD in three replicates

Experiment proper

One week after the acclimatization, the experiment will begin. The samples will be
subjected to different food concentrations. This will serve as the basis in determining differences
in the respiration rate. The dissolved oxygen will be recorded by the use of an oxygen recording
device and will be specifically measured using a parameter.

Statistical analysis

One-way Analysis of Variance (ANOVA) will be used to determine the significant differences
on the means of water parameters, size, food intake, and respiration rate of the organism. If
found significant, Duncan’s Multiple Range Test (DMRT) will be used to determine the pair of
means that differ. Pearson correlation coefficients will be computed to know the relation of the
size of the samples and the respiration rate. The data will then be analysed using SPSS.
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