上海海洋大学

Shanghai Ocean University China

College of Fisheries and Life Science, SHOU

上海海洋大学 水 产 与 生 命 学 院

Study Plan for Doctoral Degree Program

INFLUENCE OF OCEAN ACIDIFICATION ON

PHYSIOLOGY, BEHAVIOR, SURVIVAL, GROWTH, AND
BIODIVERSITY OF DIFFERENT MARINE ANIMALS

By

WAQAS

Research Supervisor

Dr. YOUJI WANG

Department of Aquatic Physiology

School/Department Applying for College of Fisheries and Life

Science
APPLYING FOR AQUACULTURE
Abstract

Fossil fuels burning, cement production, deforestation, and many other activities lead to an
increase in the CO2 level in the environment. The ultimate consequences result in acidification of
the ocean and severely affect marine animals. The current research study is aimed to investigate
objectives;(1) the major factors that contribute to ocean acidification. (2) the impact of ocean
acidification on the physiology and behavior of marine animals more specifically the reproductive
physiology and behavior will be studied. And (3) the chronic Impact of ocean acidification on
marine biodiversity. (4) impact of ocean acidification on survival and growth of different marine
fishes (5) search for control strategies that minimize ocean acidification. This study is
economically and environmentally having far significance is the ocean acidification is mainly
caused due to increased Carbon dioxide in the environment. Which leads to corrosive effects of
the physiology of different marine organisms

Key Words; Ocean acidification, Aquatic environment, marine animals, Carbon dioxide, pH.
1. Introduction and background
Due to the burning of fossil fuels, deforestation, cement production, and other human activities,
CO2 level increases which lead to ocean acidification, also referred to as the "other CO2 problem
(Dony et al., 2009) is a direct consequence of increasing atmospheric carbon dioxide (CO2)
concentrations. This process has important societal benefits: the ocean has significantly reduced
climate change by consuming about a quarter of the overall human output of CO2. However, it
also has less beneficial effects, as dissolved CO2 affects the chemistry of seawater, with a
succession of potentially harmful impacts on marine ecology, ecosystem resources, and human
society. The rise in seawater acidity, arising from the release of hydrogen ions (H+), is the
starting point for such improvements. On the logarithmic pH scale, acidity is measured, with H+
concentrations at pH 7.0 being ten times greater than at pH 8.0. The mean pH in the surface
ocean has fallen by 0.1 units since pre-industrial times, a linear acidity rises of~26 percent.
Unless CO2 emissions are quickly produced, with a shortened, average surface pH is predicted
Elevated degree of certainty-to drop by another ~0.3 Units by the year 2100(Sabine et al., 2004;
Gattuso, 2011). This reflects a rise in acidity of about 170 percent relative to pre-industrial levels.
The actual shift would depend on potential CO2 emissions, with ocean response variations, both
regional and local. In the last decade, several scientific studies have unambiguously shown that
a wide variety of marine species are susceptible to pH changes of such magnitude, affecting their
physiology, health, and survival, often (but not always) in a negative way (Gattuso, 2011;
Kroeker et al., 2013). However, even less certain are the effects of ocean acidification for marine
food chains, fisheries, biogeochemistry, and the human use of marine resources. Ocean
acidification is not the only environmental disruption that will be faced by species in the future,
since it will occur in conjunction with other stressors (e.g. elevated temperature and
deoxygenation)(Bijma et al., 2013). It is not possible to assume that the biological effects of
multiple stressors occurring together are additive; instead, their combined impacts may be
amplified (through synergism) or decreased due to interactions (antagonism). It is not possible
to assume that the biological effects of multiple stressors occurring together are additive; instead,
their combined impacts may be amplified (through synergism) or decreased due to interactions
(antagonism) Gattuso, 2011; Kroeker et al.,m 2013).
1.1. Chemical Basis of Ocean acidification
Ocean acidification can be defined in relatively narrow terms, limiting its meaning to a long-
term decrease in seawater pH on a global scale, which is currently mainly due to an increase in
atmospheric CO2 driven by humans, which will almost certainly intensify. At many locations,
with the longest atmospheric CO2 time series from the Mauna Loa observatory, the CO2-pH
relationship has now been observed. The ocean acidification concept above focuses on the
reaction of dissolved anthropogenic CO2 with water to form carbonic acid (H2CO3), which
dissociates to form bicarbonate ions (HCO3-) and hydrogen ions (H+, pH-scale quantified).
There is also an additional carbonate ion reaction (CO32-; naturally occurring in seawater),
lowering their concentration (Gattuso, 2011; Kroeker et al., 2013). As a consequence,
modifications to the carbonate system of seawater will more commonly be viewed as the
mechanism of ocean acidification. Although pH values are of great interest, calculating them
with high precision is not straightforward. Instead, other measured parameters, such as dissolved
carbon dioxide(pCO2), total dissolved inorganic carbon (DIC), and total alkalinity (TA) are also
determined. (TA; the combined proton-acceptor abundance, i.e. negatively charged ions
interacting with a heavy acid). Noteworthy is one more chemical reaction. A drop in the
concentration of carbonate ions in seawater affects the stability of solid-form calcium carbonate
(CaCO3), which may be present as bedrock (such as chalk or calcareous), dead shells, or as an
external or structural part of living organisms, such as mollusks (such as mollusks, oysters and
sea-snails), echinoderms (such as sea urchin Crustaceans, warm and cold-water corals and
calcifying algae (e.g., crabs and lobsters) (Bijma et al., 2013).
2. Objective of the study
the current study will focus on (1) investigating the major factors that contribute to ocean
acidification. (2) to evaluate the impact of ocean acidification on the physiology and behavior of
marine animals more specifically the reproductive physiology and behavior will be studied. And
(3) the chronic Impact of ocean acidification on marine biodiversity. (4) impacts of ocean
acidification on survival and growth of different marine fishes (5) to search for control strategies
that minimize ocean acidification. This study is economically and environmentally having far
significance is the ocean acidification is mainly caused by increased Carbon dioxide in the
environment. Which leads to corrosive effects of the physiology of different marine organisms
(Bijma et al., 2013). While the acidification of the ocean is increased every single day as CO2
level is increasing in the environment due to rapid development Gattuso, 2011; Kroeker et al.,
2013).
3. Research Questions
1. What are the main factors contributing to ocean acidification?
2. Does ocean acidification having any effects on the behavior and physiology of marine animals?
3. Is there any impact of ocean acidification on marine biodiversity?
4. Is there any effects of ocean acidification on the growth and survival of different fishes?
5. What are the main strategies to control ocean acidification?
4. Research hypothesis
H01: Ocean acidification having no significant effects on the behavior and physiology of marine
animals.
H1: Ocean acidification having significant effects on the behavior and physiology of marine
animals.
H02:There is an insignificant impact of ocean acidification on marine biodiversity.
H2: There significant impact of ocean acidification on marine biodiversity.
H03: There insignificant effects of ocean acidification on the growth and survival of different
fishes.
3
H : There significant effects of ocean acidification on the growth and survival of different fishes.
5. Methodology of the study
5.1. Experimental Animals and Acclimation
Since larvae are generally more susceptible to environmental changes and the recruitment of fish
population can be significantly affected by their nutrition condition (Ishimatsuet al., 2008
Constable et al., 2014), larval of different marine animals will be investigated in the present study
Larvae of regular size (length of 4.71±0.45 cm, weight at 2.59±0.71 g) from one spawning event
using multiple parents will be collected from the Shanghai local ocean. The hatchery and rearing
of these larvae will be conducted in the open sea net cages with seawater at the ambient pH
(∼8.10).
Larvae sea breams will be immediately transferred to the laboratory, Department of Aquatic
Physiology College of Fisheries and Life Science, Shanghai Ocean University, and will be
acclimated for a week in a 500 L tank filled with 350 L of aerated, flowing seawater(the
temperature at 23.71±0.08◦C, pH at 8.10±0.01, and salinity at 20.74±0.01) before the
experiment. During the acclimation period, larvae will be fed with commercial pellet
feed(diameter of 1.5 mm) twice daily After the acclimation, healthy individuals without physical
injury will be used for the experiments.
5.2. Ocean Acidification Treatment and Seawater Chemistry Monitoring
According to the near-future OA scenarios predicted by the IPCC, pH levels of 8.1, 7.8, and 7.4
will be employed to simulate the pH levels at present and in the years 2100 and 2250,
respectively. According to the method of Zhao et al. (2017), the stimulation of the acidified
scenario will be achieved by bubbling dry air or a mixture of carbon dioxide and dry air with
different but constant percentages. Once the pH of each experimental tank reached equilibrium
at the corresponding desired pH through aeration, 90 larvae will be randomly selected from the
acclimation tank and will be equally assigned into 9 (3 treatments×3 replicates)experimental
tanks (total volume of 50 L) each containing 30 L of still seawater, pre-adjusted to the
corresponding experimental pH values. The exposure will be conducted in an air-conditioned
indoor laboratory (temperature 24◦C) with an exposure time of 15 days and the sea larvae will
be fed with commercial pellet food at a satiation rate twice daily. An hour after feeding, seawater
in each experimental tank will be replaced with seawater pre-prepared at the corresponding
experimental pH values. During the experiment, the seawater of each tank will be continuously
aerated with the corresponding dry air or CO2-air mixture to maintain the stability of seawater
carbonate chemistry. To ensure that the chemical parameters of seawater in each tank will
consistent throughout the entire experiment, pH, salinity, and temperature were monitored daily
and total alkalinity (TA) will be determined once a week. Hep HNBS of each trial will be
measured by a pH meter (PB-10, Sartorius) and calibrated with NBS standard buffers. Salinity
will be measured with a conductivity meter (Multi 3410, WTW) and a mercury thermometer
gaged temperature. TA will be determined using potentiometric titration with the constants
supplied by Mehrbach et al., (1939) and refitted by Dickson and Millero (1987) and the KSO4
dissociation constant from Dixson et al. (2010).
5.3. Foraging Behavior Experiments and Video Analysis
Foraging behavior experiments will be performed following published methods with
modifications (Ferrari et al., 2012; Dodd et al., 2015; Pistevos et al., 2015). After exposure to
corresponding pCO2 levels for 15 days and then food deprivation for 24 h, 5 larvae individuals
will be randomly selected from each experimental tank and will be transferred to one end of a
white plastic tray (105 × 80 × 30 cm) containing 100 L of sand-filtered still seawater. Since it
has been suggested that the behavioral effects of OA will last in fish individuals for 1 or 2 days
and the pH of testing seawater will not affect the behavioral responses observed (Munday et al.,
2010, 2016), seawater at ambient pH 8.1 will be used for the behavioral experiment in the present
study. The 5 sea bream individuals will be tested simultaneously as one replicate in the analysis.
Individuals will be allowed to acclimate for 30 s before the introduction of commercial food
pellets (50.10 ± 0.81 g), placed approximately 100 cm away from the acclimation area, at the
other end of the tray. The food pellets will be held in a glass petri dish with an opaque white
cover with holes on the top, a design that allows the dispersal of olfactory cues but prevents
visual detection of the food. The plastic baffle plate, which will be used to restrain the fish in the
acclimation area before the assay, will be removed and video data of the foraging behavior will
be collected with an HD digital video camera (T90, Aigo®, China) 3 min after the food was
introduced. Three replicates will be performed for each experimental group and fish tested will
be discarded to ensure that each individual will be only tested once. The curvilinear swimming
velocity (VCL) used to approach the food source, the linearity (LIN) and wobble (WOB) of the
fish swimming path, and the time taken to leave the acclimation area and reach the food will be
determined using the open-source software ImageJ (National Institutes of Health, Bethesda,
Maryland, United States) following the method described by Wilsonleedy and Ingermann (2007)
and Shi et al., (2017).
5.4. Content Estimation of Neurotransmitters
After 15-day exposure to corresponding pCO2 levels, 6 individuals will be randomly selected
from each experimental treatment tank and dissected on ice. Brain tissue of each individual will
be carefully removed and used for the determination of in vivo GABA and ACh contents using
commercial ELISA kits (MLBIO Biotechnology Co., Ltd., Shanghai, China) following the
manufacturer’s instructions. After weighing, samples will be homogenized in ice-cold PBS (0.01
M, pH 7.4) followed by centrifugation at 2000 rpm for 20 min at 4°C. Twenty microliters of the
supernatant will be mixed with an 80 μL working reagent containing a chromogenic reagent.
After incubation at room temperature for 20 min, the absorption values at 450 nm will be
measured with a microplate reader (Thermo Multiskan Go, United States). The contents of
GABA and ACh will be subsequently determined using the corresponding standard curves.
5.5. Statistical analysis
All the numerical data will be analyzed using SPSS and Graphpad Prism. Values will be represented
as Mean ± SEM. One-way, two-way ANOVA, paired Student’s t-test and two-tailed tests will be
used for analysis. A probability value P < 0.05, P < 0.01 and P < 0.0001 will be considered
statistically significant.
5.6. A 3 Years Ph.D. Study Schedule

2021- 2022 2022-2023 2023-2024

activities Quarter1 Quarter2 Quarter3 Quarter1 Quarter2 Quarter3 Quarter1 Quarter2 Quarter3

Course
study

litrature
survey

Collection
of Sample

experimental
desingning

data
Collection

Research
Papers
writing

Thesis
writing

Research
thesis
defense

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