Republic of the Philippines

ZAMBOANGA STATE COLLEGE OF MARINE SCIENCES AND TECHNOLOGY

Fort Pilar, Zamboanga City
Tel No. 992-3092/Tel No: (062) 991-0643 Telefax: (062) 991-0777
website:http:www.zscmstedu.ph

Instructional Materials and Development Office (IMDO)

Office of the Vice President for Academic Affairs

MODULE FOR

FISH 6
PHYSIOLOGY OF AQUATIC
ORGANISMS

NERISA G. BONGO
Assistant Professor IV
College of Fisheries and Marine Sciences
August 20
 Fish 6 Physiology of Aquatic Organisms

COURSE CREDITS: 3 Units

CONTACT HOURS/WEEK: 3 hours

PRE-REQUISITE: None

COURSE DESCRIPTION

The course emphasizes on the physiological mechanisms conserved across taxa and those that
are unique to a particular aquatic animal group. Some case studies on how particular groups of animals
cope physiologically with extreme environments and with contrasting environments at different parts of
their life cycle (e.g. anadromy in salmon, catadromy in eels) are also discussed.

PROGRAM LEARNING OUTCOMES

The graduates have the ability to:
a. Articulate and discuss the latest developments in the specific field of practice (PQF level 6 descriptor)
b. Effectively communicate orally and in writing
c. Work effectively and independently in multi-disciplinary and multi-cultural teams (PQF level 6
descriptor)
d. Act in recognition of professional, social, and ethical responsibility
e. Preserve and promote “Filipino historical and cultural heritage” (based on RA 7722)
f. Formulate plans and programs in the conservation, protection, development and sustainability of
resources, and in the marketing of products
g. Engage in activities related to education and/or research-development–extension continuum
h. Develop, operate and manage aquaculture production systems
i. Utilize fisheries resources using innovative fishing methods which are responsible and sustainable
j. Apply post-harvest practices that are compliant to international standards for food safety and quality
k. Manage and protect the integrity and quality of aquatic ecosystems and resources

COURSE OUTCOMES

At the end of this series of 8 modules, the students should be able to:
1. Discuss the physiological challenges that the aquatic organisms encountered;
2. Discuss the trophic modes, food acquisition and their adaptations;
3. Explain the fish nutrition, energy budgets and fish metabolism; and
4. Explain the organismic biology of fish.

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 Vision, Mission and Core Values

i. Vision: A world-class institution for higher learning, research, development

and innovation in fisheries, marine sciences, maritime education, and
technology by 2024.

ii. Mission: Provide quality education and relevant research and extension
to produce globally competitive human capital for fisheries and marine-
based industries.

iii. Core Values: CARE- C-Commitment A-Attitude R-Relationship E-

Excellence

Copyright:
This module is a property of © Zamboanga State College of Marine Sciences and Technology
All rights reserved. No part of this module may be reproduced or distributed in any form or by any means,
or stored in a database or retrieval system, without prior written permission of the Zamboanga State
College of Marine Sciences and Technology.

Author:

Nerisa G. Bongo
Assistant Professor IV
College of Fisheries and Marine Sciences
BS Fisheries Program

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 List of Modules
Module
Time Frame Module Content
No.
CHARACTERISTICS OF AQUATIC ENVIRONMENTS AND THE
1 Week 1-2
PHYSIOLOGICAL CHALLENGES OF AQUATIC ORGANISMS
2 Week 3-4 TROPHIC MODES AND FOOD ACQUISITION

3 Week 5-6 FISH NUTRITION, BIOENERGETICS AND METABOLISM

4 Week 7-8 OSMOREGULATION

5 Week 9-10 GAS EXCHANGE AND CIRCULATION

Week 11-12 REPRODUCTION: DIVERSITY; SEX DETERMINATIONAND

6
DIFFERENTIATION

Week 13-14 FERTILAZATION; CHEMICAL SIGNALING AMONG AND BETWEEN

7
SPECIES –SEMIOCHEMICALS AND PHEROMONES

Week 15-16 REPRODUCTIVE DYSFUNCTION OF FISH ASSOCIATED WITH

8
CONTAMINANTS

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 How to Use this Module

Before you begin with the module, read the description of the different module sections which are
represented by the icons below.

Learning Objectives. Describe what you are expected to learn by the end of the
module.

Introduction. This section includes a general idea about the topic which provides a
background of the module.

Gives the time frame to accomplish a particular module and the due date of the
submission of learning activities.

Lecture Proper. Contains the detailed lessons of the topics in the module

Videos to watch to get a better understanding of the topics. Video links are provided in
the module and are available in the learning packet

Activities. Activities which take place within this module, either as an individual task or
a group activity with fellow students on your course.

Self-Assessment. This section contains review questions or problem sets to test your
understanding to the lesson.

Summary. Brief and concise points of every lesson are provided at the end of every
module.

References. List of books, articles, websites and links used in the module

This icon means that you are ready to take the assessment.

This module is accompanied by a learning packet which contains learning materials such as e-books,
manuals, journal articles, videos, and other materials.

To get help, contact your course professor:

0905-168-7266 Nerisa Bongo

nbongo@zscmst.com.ph Nerisa Bongo

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 Welcome to Fish 6 – Physiology of Aquatic Fauna. This is a 3-unit course which covers three hours
lecture. Modules 1-4 comprise the midterm and consist of the concepts on the physiology of aquatic animals
with emphasis on challenges of the aquatic environment to the animals, trophic modes, bioenergetics and
osmoregulation. The finals on the other hand has Modules 5-8 which tackles on gas exchange and
circulation, reproduction, fertilization, reproductive dysfunctions.

Our objective here is to provide students with enough information on concepts of physiology and
competency as to the aspects of energy partitioning, feeding characteristics, reproductive performance of the
aquatic animals for them to be equipped with sufficient expertise in that particular field as they will be on their
job in fisheries in the near future.

Timing and Organizing Your Study

You are given 6 hours, spread over two weeks (3 hours/week), to accomplish a module. It is advised
to finish one unit (one chapter) per week to comply all the assignments and assessments given in each
module. Follow the organization of this module and avoid jumping to the subsequent sections without
accomplishing the exercises in the previous one. Watch the videos given intently and read and understand
the reading material before answering the exercises given.

Study Skills

Writing, reading and listening are of importance in this course. It is recommended that you spend
significant time on each module and avoid multi-tasking to maintain focus. Make sure to read and understand
the covered lessons per week to cope up with the activities and exercises given. Videos will be posted online
or copied in a flash drive for your reference. Follow the outline presented in the table of contents above for
your guide.

Assessments/Activities

In this module, you will be asked to respond to the self-tests questions. Make sure to accomplish all
of them before submitting the module. Submission of two assignments or assessments from each module is
expected. Assessments/activities given will be submitted either online or offline. For online submission, the
student may send their assignments through Google Classroom. For offline submission, student may copy
their assignments/assessments in a flash drive and submit it to the teacher in charge together with the module

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 Module 8: Reproductive Dysfunction Of Fish Associated With Contaminants

Learning Objectives
After studying this module, you will be able to:
1) Identify the different fish contaminants;
2) Describe reproductive dysfunction of fishes; and
3) Explain the role of hypothalamic-pituitary glands in reproductive dysfunction of fishes

Introduction

Reproduction and early development of organisms are one of the most sensitive targets with
respect to biological effects from chemical compounds and environmental stress in general. Reproductive
disorders are important effects of environmental stress since they could have effects at the population
level.
During the past decades, vast quantities of different contaminants have been released into terrestrial
and marine environments due to land erosion, industrial, agricultural, urban, technological applications and
other anthropogenic activities. Most of the substances known as xenobiotic include metals, pesticides,
herbicides, anti-fouling compounds, nanoparticles, and plastics. However, environment is threatened not
only by chemical insults but also by climate changes, which includes global warming and ocean acidification.
Either chemical pollution or climatic modifications are creating a raising alarm in the scientific community and
governments due to the potential toxic, genotoxic and carcinogenic impact on living organisms and humans.
A large body of evidence indicates that chemical pollution interferes with hormone function giving rise to the
so‐called endocrine disruption. Due to the vulnerability of hormone‐receptor systems, it appears that some
endocrine disruptors (EDs) affect the normal reproductive functions and embryo development. Perturbation
of wildlife hormonal pathways generate reproductive failures and abnormalities in the reproductive organs of
fish, birds, reptiles, and mammals.

Lecture Proper

Contaminants
Metals are the most common pollutants, and although some of them are essential for physiological
functions, they become toxic at high concentration and together with nonessential metals exert adverse
effects on either animal or human reproductive functions. The use of engineered nanomaterials in industry,
consumer products, and biomedicine is rapidly expanding giving rise to socio-economic advantages but
posing at risk environment and its biota for the high toxicity related to nanoparticles release and persistence
in water, air, and soil.

By using animal models, a large body of studies are evaluating the effects of several nanoparticles
on human health and the reproductive systems of both male and female. These are clearly demonstrating
different impacts of nanoparticles on reproductive organs and systems structure, function, and physiology
and the generation of adverse effects on germ cells and in turn on fertility potential. In our experience, two
nanoparticles widely used in manufacturing products as nickel and copper oxide exert spermiotoxic impact
in two different marine invertebrates.

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In particular, we demonstrated an adverse effect on some parameters underlying sperm function as
concentration, motility, morphology, mitochondrial potential, and the generation of oxidative stress which in
turn affected the membrane and genome integrity. This sperm quality impairment resulted in an impairment
of the fertilizing ability in both the species supporting the current concern for fertility reduction of marine
species and the potential implications on reproductive fitness and survival of the aquatic biota.

Plastics are considered environmental contaminants of emerging concerns. They are constituents of
several manufactures as bottles, shopping bags, paints and cosmetics, furthermore they are used for
biomedical applications in drug delivery. The increase of plastic production over the last 60 years has led to
the estimation that they will persist in in many habitats (Plastics Europe, 2015) from 10 to 100 years, therefore
plastic and their derivative are defined as persistent organic pollutants. In the environment, plastic is known
to deteriorate and fragment occurring in different sizes, according to which it is classified The increase of
plastic production over the last 60 years has led to the estimation that they will persist in many habitats
(Plastics Europe, 2015) from 10 to 100 years, therefore plastic and their derivate are defined as persistent
organic pollutants. In the environment, plastic is known to deteriorate and fragment occurring in different
sizes, according to which it is classified in macro‐ (≥1 c m , m e s o ‐ (1–10 mm), micro‐ (1–1000 μm) and
nano plastics

Fig. 1. Effects of environmental stressors on reproduction and development. A series of chemo-physical

stressors as metal, nanoparticles, plastics, antifoul ants, global warming, and ocean acidification impair
gamete morphology and embryo/larvae development. (Source: Google Image)

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Reproductive Dysfunction

In fishes, as in other vertebrates, reproduction is primarily controlled by the hypothalamic pituitary gonadal
axis. The hypothalamic neuroendocrine system regulates synthesis and release of the pituitary
gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh). Both gonadotropins are
essential in the endocrine control of reproduction by regulating steroidogenesis and germ cell development
through interactions with their respective receptors in gonadal tissues.

Fig. 2. Schematic representation of the reproductive axis in fish, its major components and phases, and its
environmental and endocrine control. (Source: Google Image)

Reproductive Dysfunction

In fishes, as in other vertebrates, reproduction is primarily controlled by the hypothalamic pituitary

gonadal axis. The hypothalamic neuroendocrine system regulates synthesis and release of the pituitary
gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh). Both gonadotropins are
essential in the endocrine control of reproduction by regulating steroidogenesis and germ cell development
through interactions with their respective receptors in gonadal tissues.
Under natural conditions the endocrine–reproductive axis, linked to appropriate environmental cues,
ensures that maturation and reproduction occur at a time and under conditions that maximize reproductive
success. In captivity, these processes are frequently disrupted, resulting in sexual dysfunction. Sexual
dysfunction can manifest itself in different ways, from partial inhibition of some reproductive processes,
resulting in a reduction in the quantity or quality of gametes, to a complete failure to spawn. Female fishes
seem to be more susceptible to sexual dysfunction than males. Sexual dysfunction in cultivated females
takes three forms. The rarest is the failure of vitellogenesis, the second is probably the most common: the
oocytes complete vitellogenesis, but do not undergo oocyte maturation and so ovulation and spawning fail.
The third problem, seen in salmonids, is that ovulation occurs but spawning does not proceed naturally.
These problems are probably a result of the stress imposed by the culture conditions allied to the failure of

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those conditions to provide the appropriate physical and social environment required for successful
spawning.

1. Exposure to mercury

Disruption of male reproductive function The cycle of spermatogenesis in which stem cells divide
into mature spermatozoa is controlled by LH and FSH. Throughout this cycle, the testes increase in size,
which can be measured by an increase in the gonadosomatic index (GSI; gonad wt/body wt · 100) and usually
is maximal in the breeding season. Disruption of testes function can be classified as either cytotoxicological
or endocrine in origin. Cytotoxic effects are characterized by damage to cellular integrity or alterations in cell
function, whereas endocrine effects involve the disruption of specific cells as a result of alterations in pituitary
secretion or gonadal steroidogenesis.

Testicular morphology and spermatogenesis

Compared to control fish, testicular growth and spermatogenesis in murrel were arrested following
exposure to inorganic mercury (10 _g/L) or a mercurial fungicide (200 _g/L) for six months]. Necrotic areas
were visible within the testes of fish exposed to the mercurial fungicide, suggesting direct cytotoxic effects;
however, no obvious signs of degeneration were observed after inorganic mercury exposure.
Spermatogenesis also was inhibited and GSI decreased in male walking catfish after 180 d of exposure to
organic and inorganic mercury at 30 to 50 _g/L. Similarly, guppies (Poecilia reticulata) exposed to 5.6 _g/L
of methyl mercury for three months and medaka (Oryzias latipes) exposed to 40 _g/L of methyl mercury for
8 d exhibited signs of testicular atrophy and arrested spermiation. In a study they concluded that necrosis of
the sperm was occurring during the process of spermatogenesis, and those authors proposed a mechanism
involving the disruption of the mitotic spindle. Mercury has been shown to result in depolymerization of
microtubules that form the mitotic spindle, which is a possible mechanism underlying mercury induced
genotoxicity in mammalian cells. Disruption of testicular function also has been observed in exposures to
environmentally relevant levels of methyl mercury. Dietary exposure for six months to methyl mercury
concentrations (0.1–1.0 _g/g) similar to those encountered by piscivorous fish in North America resulted in a
significant decrease of GSI in juvenile male walleye with mean mercury body burdens of 0.25 to 2.37 _g/g
wet weight. The normal morphology of the testes was significantly altered, with the appearance of atrophy.
Decreased spermatogenesis and atrophied seminiferous tubules were observed in male Nile tilapia
(Oreochromis niloticus) after a seven month exposure to methyl mercury. Intraperitoneal implants in the
male tilapia released methyl mercury gradually, with final muscle concentrations ranging from 0.08 to 0.54
_g/g wet weight (assuming 80% water content), which are similar to levels reported for largemouth bass in
the northeastern United States. From these experiments, it can be concluded that both inorganic and organic
mercury exposure via a variety of exposure routes can arrest gonadal development in male fish. This effect
may be cytotoxic in nature, because effects at the level of the cell include structural alterations in Leydig cells
and necrotic cell death. The inhibition of spermatogenesis in the murrel was correlated with small and inactive
gonadotrophs of the pituitary. The data are inconclusive regarding the impacts of mercury exposure on
testicular growth. In addition to the studies mentioned previously, in several studies no change in GSI
occurred.

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In wild-caught northern pike with muscle mercury content ranging from 0.12 to 0.62 _g/g wet weight, no
significant correlations were found between mercury content and GSI or gonadal sex steroid concentration;
however, the small sample size (n _ 7) and high variability may have masked any subtle effects. Dietary
methyl mercury decreased sex steroid levels but had no effect on gonadal development in male fathead
minnows with mean total mercury carcass concentrations of 0.86 to 3.84 _g/g wet weight after 250 d of
exposure. Similarly, GSI was not affected in male tilapia exposed to methyl mercury, despite changes in
testicular morphology and spermatogenic arrest. This indicates that histology may provide a more sensitive
indicator than GSI for assessing the effects of mercury on gonadal development. Mechanisms underlying
testicular atrophy resulting from mercury exposure are unknown, but they may involve alterations in mitotic
activity. Alternatively, mercury exposure can reduce both the size and number of gonadotrophs. In goldfish,
human chorionic gonadotropin, which mimics endogenous gonadotropins, acts as a cell survival factor during
the late stage of spermatogenesis. Therefore, methylmercury-induced testicular atrophy may be caused by
the induction of apoptosis through alterations in pituitary derived survival factors. Atrophy in developing testes
could directly impact individual reproductive potential.

Testicular hormone production

Gonadotropic regulation of spermatogenesis and spermiogenesis in fish is mediate androgen

secreted by the interstitial cells. Murrel testes undergoing spermiation contain active interstitial cells
characterized by large, rounded nuclei with prominent nucleoli. In contrast, inactive and atrophied interstitial
cells were observed in murrels exposed to inorganic mercury (10 _g/L) or a mercurial fungicide (200_g/L) for
six months.

Exposure to methyl mercury resulted in inflammation and fibrosis of the interstitium in male guppies.
These cytotoxic effects imply potential adverse effects on the steroidogenic potential of the interstitial cells.
The biosynthesis of androgens and estrogen requires 3_-hydroxy-_5-steroid dehydrogenase (3_HSD); a
decrease in 3_HSD activity has been used to indicate decreased steroidogenic activity. Exposure male
catfish to organic and inorganic mercury at 30 to 40 _g/L for six months and was then measured 3_HSD
activity ex vivo in sections of testes. The activity of 3_HSD was inhibited completely in testes from fish
exposed to methyl mercury and, to a lesser degree, in those from fish exposed to inorganic mercury. The
loss of enzyme activity may be a direct action of mercury on the testes or an indirect one at the hypothalamic–
pituitary level through inhibition of gonadotropin secretion, because 3_HSD is influenced by gonadotropin.
Furthermore, impaired steroidogenesis may have led to the inhibition of gonadal growth and
spermatogenesis, as reported in a similar experiment. In male fish, androgens control gonadal development,
secondary sexual characteristics, and sexual behavior . Testosterone and/or 11ketotestosterone secretion is
high during gonadal recrudescence and declines before spermiation.

Plasma testosterone levels significantly decreased in male fathead minnows fed diets containing
0.87 to 3.93 _g/g of methyl mercury for 250. Decreases in plasma 11-ketotestosterone also were observed
in male Nile tilapia (O. niloticus) after a six-month exposure to methyl mercury and in largemouth bass after
an eight-week exposure to 3.12 to 6.25_g/g of dietary methyl mercury; no concurrent decrease in 17_-
estradiol was observed in either exposure.

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These studies provide further evidence that mercury alters steroidogenesis in male fish. Further studies,
however, are required to determine whether the effect is direct, at the gonad level, and/or indirect, via
disruption of the hypothalamus or pituitary. In wild fish, the effects of mercury accumulation on androgens,
particularly 11-ketotestosterone, are unclear. Similar to the results of laboratory studies, both plasma
testosterone and 11-ketotestosterone were negatively and significantly correlated with muscle mercury
content (mean value, 0.17 _g/g wet. wt) in sexually immature white sturgeon (Acipenser transmonatus).

Increased steroidogenic enzyme activity and a concurrent increase in plasma steroid levels were
observed in female fish exposed to inorganic mercury. Positive correlations also were observed between 11-
ketotestosterone and muscle mercury concentrations in female smallmouth bass (0.21–0.51 _g/g wet wt.)
and common carp (0.06–0.33 _g/g wet wt.). Field surveys are complicated by the presence of multiple
contaminants in addition to methyl mercury, which may impact steroidogenesis. Both studies suggest that
mercury exposure resulted in a decreased mobilization of lipids from the liver to gonads. Impaired lipid
metabolism may be responsible, in part, for the inhibition of spermatogenic and steroidogenic activities in
mercury-treated fish.

Sperm morphology and motility

The morphology and motility of sperm are useful in assessing the total impact of mercurials on the
male reproductive system, because they are critical factors for fertilization success in teleosts. Instantaneous
(5-s) and short-term (_24-h) exposure of sperm are representative of the effect of mercury on sperm released
into a polluted aquatic environment. Short-term (21- to 80s) exposure of milt to 1.0 _g/L of inorganic mercury
decreased the motility of African catfish (Clarius gariepinus) sperm. A short-term (2- to 5-min) aqueous
exposure of mummichog sperm to 50 _g/L of methyl mercury resulted in decreased motility without any
apparent morphological effects. Instantaneous exposure of goldfish sperm to inorganic mercury reduced
curvilinear velocity at 1 _g/L, percentage of motile sperm at 10 _g/L, and flagella length at 100 _g/L. The
decreased motility of sperm and morphological changes in the flagellum reported in some species may be a
result of mercury binding to protein sulfhydryl groups located on the membranes of the nucleus, mid piece,
and tail. Mercury can react with sulfhydryl groups on microtubules and inhibit microtubule assembly in vitro,
which may affect the microtubule sliding mechanism required to generate waves along the flagellum. Mercury
also may interfere with the function of sperm mitochondria, resulting in a decrease in energy production. The
success of fertilization events likely are reduced or inhibited completely by alterations in sperm motility and
morphology. It is now known, however, that fertilization success is dependent on the ratio of sperm to egg
used in each test, because high ratios will mask toxic effects by providing sufficient spermatozoa to overcome
the spermiotoxic effects. Sperm also can be exposed to mercury in seminal fluids (i.e., in vivo) of
contaminated fish. A 24-h exposure in an extender buffer has been designed to mimic this type of exposure
in vitro. Goldfish sperm, which exhibited no effects after instantaneous exposure, had decreased flagella
length and motility after preincubation in an extender buffer with 100_g/L of inorganic mercury. Attempts have
been made to measure the effects of in vivo sperm exposure on fertilization success. Thus far, the results
have been inconclusive because of the inherent difficulty in assessing the effects on sperm in isolation from
other reproductive impairments.

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Disruption of female reproductive function

Mercury exposure also can affect reproductive endpoints in female fish. Mercury has been shown to
affect ovarian morphology, delay oocyte development, and inhibit steroid hormone synthesis. As with the
testes, it is difficult to determine whether mercury affects the ovaries directly or indirectly, via alterations at
the level of hypothalamus or pituitary, and whether the effects are the cause or the result of changes in
steroidogenesis. Unlike in males, female gamete production involves the synthesis and incorporation of a
yolk protein, which may transfer some of the maternal mercury burden to the embryo. The effects of mercury
on the female reproductive system discussed in the next section are separated into change at the level of
ovarian morphology and egg development, steroid hormone and vitellogenin synthesis, fecundity and
spawning, and ultimately, egg and embryo survival.

Ovarian morphology and oogenesis

Oogenesis in teleosts involves the maturation from immature or previtellogenic oocytes through the
accumulation of yolk vesicles during vitellogenesis and, finally, the formation of mature oocytes. This cycle
of oogenesis was significantly repressed in the freshwater murrel after exposure to inorganic mercury or
mercurial fungicide (10–20 _g/L), with a concurrent decrease in GSI. After 180 d of exposure, only immature
oocytes completely devoid of vitellogenin were present, whereas the control ovaries were fully developed,
with a large number of mature and maturing oocytes. Degenerative changes—namely, nucleolar necrosis
and atresia (follicular degeneration)—also were observed in some of the fungicide and inorganic mercury
treatment groups.

Ovarian recrudescence was arrested with some atretic changes and a decrease in GSI of walking
catfish exposed over six months to inorganic and organic mercury (30–40 _g/L). Atretic follicles also were
more common in female Nile tilapia (O. niloticus) after exposure to methyl mercury implants; however, no
change in GSI was observed. Common spiny loach (Lepidocephalichthys thermalis) exposed to inorganic
mercury for 10 d at 100 _g/L had extensive vacuolation in the oocortex, necrosis of the oolemma, and
hypertrophy of follicular cells, leading to atresia of the oocytes after 20 d. In sexually mature largemouth bass,
the frequency of atretic oocytes was positively correlated with sediment mercury concentrations in a
Tennessee, USA, reservoir system. These degenerative changes are suggestive of cytotoxic actions on the
ovary. Apoptosis is the main trigger for follicle atresia in mammals and is involved in normal teleost ovarian
development and regression.

A significant increase in the number of apoptotic cells was measured in ovaries of fathead minnows
exposed to dietary methyl mercury as juveniles through to sexual maturity (_250 d), with resultant mean total
mercury carcass concentrations of 0.92 to 3.84 _g/g wet weight. Thus, the induction of apoptosis in
steroidogenic gonadal cells is one possible mechanism by which sex steroid levels are reduced in fish
exposed to methylmercury.

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Steroidogenesis

In female fish, 17_-estradiol is the major reproductive hormone and is produced primarily in the
follicle of the developing oocyte. Luteinizing hormone stimulates the production of testosterone, which is then
converted into 17_- estradiol via the aromatase enzyme. One of the primary roles of 17_-estradiol is to
stimulate liver production of the yolk protein, vitellogenin, which is then incorporated into the maturing oocyte
to later serve as nourishment for the developing fish larva. Similar to testosterone, gonadal 17_-estradiol
secretion and subsequent vitellogenin production both change with maturation of the oocytes; thus, plasma
levels can be used to monitor the reproductive stage of fish. In female Nile tilapia (O. niloticus), 17_-estradiol
levels decreased over the summer months and remained low until November, when the hormone level surged
to prepare for the new reproductive season. The synthesis of steroid hormones involves a series of enzymatic
reactions that may be sensitive to mercury exposure.

Vitellogenesis

Estradiol stimulates the liver to synthesize the phospholipo glycoprotein yolk precursor vitellogenin in
oviparous vertebrates. Altered expression of vitellogenin mRNA and its protein is one of the most commonly
used biomarkers of aquatic contaminant exposure. In males, vitellogenin normally is not expressed, but it
can be induced by exposure to estrogenic chemicals. In control female catfish, serum vitellogenin levels
doubled after 17_-estradiol injection. After injection of inorganic mercury, however, serum vitellogenin levels
decreased and did not respond to subsequent 17_-estradiol injection. Vitellogenin synthesis also was
inhibited in catfish after a 90- to 180-d exposure to inorganic mercury (50_g/L) and organic mercury (40
_g/L), as indicated by a decrease in 32P-labeled phosphoprotein content in the liver and ovaries. This
inhibition of vitellogenin synthesis on exposure to inorganic or organic mercury may be a result of impaired
steroidogenesis or alterations in estrogen signaling pathways. Recent studies have demonstrated alterations
in vitellogenin mRNA levels in livers from fish exposed to dietary methyl mercury in laboratory-based and in
situ exposures.

Lipids

During the gonadal cycle, a reciprocal relationship exists between hepatic and gonadal lipids, the
balance of which may be altered by pollutants, such as mercury. In the peak of reproductive activity, hepatic
lipid levels decrease, with a concomitant increase in gonadal levels.
Phospholipids are an important component in the structure of proliferating and developing germ cells
as reserve food in the form of yolk or as energy sources. Cholesterol, on the other hand, is the precursor for
steroid hormones. Lipid content increased in the liver (and muscle) with a concurrent decrease in the ovaries
of bronze featherback (Notopterus notopterus) exposed for 30 d to inorganic mercury (44–88 _g/L). Lipid
levels (total lipids, phospholipids, and cholesterol) also increased in the liver of walking catfish, with a
simultaneous decrease in all forms of lipids in the ovaries after a 180-d exposure to inorganic mercury (50
_g/L) and methyl mercury (40 _g/L). Murrel exposed to 200 _g/L of inorganic mercury, however, had a
significant reduction of lipid and cholesterol in the liver and ovary . In summary, these results suggest a
mercury induced change in lipid retention and/or inhibition of seasonal mobilization of lipids from liver to ovary

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during ovarian recrudescence. Disturbance of the lipid balance by mercury may result in other disruptions,
including impairment of vitellogenesis (implicated in arrest of oogenesis) and steroidogenesis.

Effects on ovulation and spawning

Mercury exposure can alter fecundity (number of eggs produced) and spawning of female fish. Zebrafish
produced fewer eggs following exposure (19–25 d) to 1.0 _g/L of a mercurial fungicide. A multigenerational
study with brook trout (Salvelinus fontinalis) exposed to aqueous methyl mercury observed no spawning in
second-generation trout exposed to 0.93_g/L or more methyl mercury for 108 weeks. Similarly, no spawning
occurred in fathead minnows (whole-body concentrations, 4.47 _g Hg/g wet wt) after 41 weeks of inorganic
mercury exposure at and above 1.02 _g/L [106]. Fertilization success was reduced in offspring (whole-body
mercury concentrations, 1.1–1.2 _g/g wet wt) of mummichog fed methyl mercury- contaminated diets.
Recently, two studies were performed with fathead minnows exposed to dietary methyl mercury as juvenile
fish through sexual maturation to determine the effects on reproduction. In these studies, an overall decrease
in spawning success was observed as a consequence of reduced gonadal growth correlated to a decrease
in 17_-estradiol levels, decreased fecundity, and increased number of days to spawning in fish, with carcass
mercury concentrations ranging from 0.57 to 3.68 _g/g wet weight. Days to spawning appears to be
influenced by the female, because contaminated females that mated with control males took 3 d longer to
spawn, on average, than control females that mated with contaminated males. In male fathead minnows with
carcass mercury concentrations of 0.71 to 4.23 _g/g wet weight, spawning behavior and spawning success
were reduced after dietary methyl mercury exposure as juveniles through to sexual maturity. Because
androgens are critical in regulating reproductive behavior, methyl mercury- induced impairment of
reproduction may be caused by alterations in sex steroids that, in turn, result in altered behavior. A female-
specific delay in spawning can be especially detrimental if sperm release is not similarly delayed. Delayed
spawning can have significant effects on the offspring, because it may disrupt the timing of feeding relative
to seasonal food resources.

If young fish are unable to find food as an indirect effect of mercury exposure, their growth may be affected,
leading to increased susceptibility to predation and reduced survival.

Eggs and embryos

Mercury in eggs originates primarily from maternal transfer and is predominantly (_90%) in the
methylated form. Originally, it was believed that maternal transfer occurred by mobilization and transfer of
stored mercury to the gonads during oogenesis. The actual amount transferred, however, is only 0.2 to 2.3%
of the maternal burden. In contrast, organochlorines are transferred to the eggs at 5.5 to 25.5% of the whole-
body burden. The diet of the maternal adult during oogenesis, rather than the body burden, is now accepted
as the principal source of methyl mercury in eggs. Therefore, the exposure of embryos to maternally
transferred methyl mercury depends on the levels of mercury in the prey of the adult during oogenesis, which
can vary intra- and inter-annually.

Mercury exposure may diminish the reproductive potential of maternal fish by reducing the hatching
success of embryos. Hatching success of zebra fish embryos in unpolluted water was decreased after

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maternal exposure (19–25 d) to 0.2_g/L of a mercurial fungicide. Maternal exposure also may alter the
sensitivity of eggs to mercury in the environment. Mummichog eggs from a polluted site and a control site
were exposed to methyl mercury (0.5–5.0 mg/L) for 20 min before fertilization with untreated sperm. Eggs
from the polluted creek demonstrated a higher tolerance to methyl mercury.

Scanning-electron microscopy revealed that the control eggs exposed to 1.0 mg/L of methyl mercury
for 20 min had an increased incidence of spontaneous micropyle blockage, resulting in reduced fertilization
success. These studies indicate that pre exposure of eggs to mercury in maternal fish can decrease hatching
success in the laboratory, whereas multigenerational exposure in wild populations may convey increased
resistance to mercury pollution. In addition to maternal transfer, mercury bioaccumulation in eggs and
embryos can occur via bio concentration of mercury from the water. On spawning, eggs are released into the
aquatic environment, where they may be exposed to additional methyl mercury.

2. Exposure to Pesticides

The freshwater bodies are particularly at the risk of pesticide pollution due to the penetration of
pesticide into them via run off, drifting, leaching, and drainage. The excessive use of noxious pesticides, for
example, carbamates and organophosphates, has degraded the environment and threatened the existence
of non-target life forms including fish fauna via affecting their cholinergic systems. Pesticides deteriorate the
health of fish by impairing their metabolic process that occasionally causes death of fish.

Fish are particularly sensitive to a wide variety of pesticide chemicals, and toxic conditions may arise,
not only from the spillage or deliberate discharge of these chemicals into rivers and lakes, but also from
approved agricultural practices if their use is excessive. Many applications outside agriculture, such as
sylviculture, horticulture or public health, can also lead to a detrimental influence on fish populations. The
nature of the effects on fish can be very variable. Apart from causing death, either directly, or due to
starvation, by destruction of food organisms, many pesticides have been shown to affect growth rate,
reproduction and behavior, with evidence of tissue damage.

Pollution of water resources with agricultural water drainage has a great risk on fish reproduction.
Organophosphorus pesticides such as Malathion and dimethaote are frequently used due to their highly
effectiveness for controlling agriculture pests. These pesticides were found that have endocrine disrupting
effect on fish reproduction through lowering sex steroid hormones (estradiol and testosterone). Endocrine
disrupting pesticides also have been implicated in the impairment of fish fecundity, semen quality, hatchability
and survivability of fishes. Sex steroid hormones, vitellogenin, organosomatic indices and histopathology are
considered as biomarker tools used for assessing disrupting effect of pesticides on fish.

3. Exposure to nanomaterials

Nanoparticles like those of copper, gold, palladium and silver have drawn much attention of the
scientific community due to a combination of their shape and size dependent- unique chemical and physical
properties (Garai et a. 2018). These in turn have found increasing applications in pharmacology bio-
diagnostics, medicine, drug-delivery, catalysis, solar cells, purification of water, etc.NPs are finally found in

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aquatic and terrestrial environments, where they are ingested by living organisms in which they accumulate,
before being eliminated by the immune system or other pathways. In organisms, NPs represent foreign
elements with their own physicochemical properties, so they may interfere with the normal physiological
mechanisms of the embryos, growing animals, and adults.

Due to the increasing use of nanomaterials in human daily life and probable release of these substances into
the environment, several studies had been done to investigate the effect of these like the presence of
colloidal silver nanoparticles (cAgNPs) (Johari, 2014) in the aquatic environment on the reproduction of
rainbow trout as a model aquatic organism and copper nanoparticles (CuNPs and CuONPs) and gold
nanoparticles using zebrafish as the test animal and tilapia for zinc-oxide nanoparticles to name a few.

A significant decrease was observed in that the end of yolk-sac absorption in rainbow trout and while
the hatching rates of all cAgNPs exposed treatments was significantly lower than the control (Johari, 2014).
Also significant and dose-dependent decrease in duration of sperm motility was observed after in vitro sperm
exposure to cAgNPs (0>0.001>0.01>0.1 mg/l). According to these results, presence of 0.1 mg/l cAgNPs in
the water environment can affect the fertilization and reproduction success of rainbow trout. Thus, the
presence of silver nanoparticles in the aquatic environment can interfere with fish reproduction and thereby
fish populations will be at risk.

The physiochemical characteristics of surrounding water mediums, such as in aquaria, ponds, lakes,
and seas, also affect the Cu dissolution, speciation, and toxicity, in addition to the dosage, physiological
concentrations, and internal structure of aquatic organisms (Malhorta, et al. 2020).

4. Effects of plastics/microplastics

The minuscule fibers, which are made of polyester, polypropylene and other types of plastics, are shed or
washed off of synthetic textiles used in clothing and other consumer and industrial products. Once shed, they
enter wastewater and accumulate in oceans, rivers and lakes worldwide, accounting for more than 90% of
micro plastic pollution in some areas.

Plastic ingestion can happen both intentionally and accidentally depending on the foraging strategy
of the animal: some predatory fish might mistake plastic for food and filter-feeders might ingest them
unintentionally while feeding. However, sometimes the pathway of plastic ingestion remains unclear if the
fish is able to use both filter-feeding and directional feeding. Ingestion is observed in both predatory fish, such
as bigeye tuna (Thunnus obesus) in the central North Atlantic, and filter-feeders, such as herring (Clupea
harengus) and horse mackerel (Trachurus trachurus) in the North Sea and English Channel.

The effects of microplastics (MP) on aquatic organisms are currently the subject of intense research.
Microplastic fibers are linked to respiratory and reproductive changes in fish. In a surprising study, not only
did fish exposed to microplastics reproduce less but their offspring, who weren’t directly exposed to plastic
particles, also had fewer young, suggesting the effects can linger into subsequent generations.

In addition, exposure to microplastics causes cellular changes in fish. Female fish exposed to fibers
containing polypropylene produced more eggs over time, suggesting that chemicals that may be leaching
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from the microfibers are acting as endocrine disruptors. The further ingestion of plastics are shown in Fig. 3
and Fig. 4 below.

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 To have more understanding, click the link:
https://www.youtube.com/watch?v=BWI60aRyG4
https://www.youtube.com./watch?v=cUjjtCMdkUnI
https://www.youtube.com./watch?v=x9_8pvPKq2Y
https://www.youtube.com./watch?v=AXx16j15DVY
https://www.youtube.com./watch?v=h6nhOChpMck

Learning Activity

Read carefully the procedure/questions in each activity. A rubric will be used to rate your work.
Exercise 8.1- Comic Strip Procedure: Create your own comic strips on the topic: Reproductive dysfunction
of fishes following the usual protocol on production of comic strips (e.g. at least 6 frame, characters preferably
fishes and other aquatic organisms, moral lesson).

Self-Assessment

Briefly answer the following questions. Rubric will be used to evaluate your work.
1. Explain effects of environmental stressors like ocean acidification to reproduction of fishes.
2. Nanoparticles are widely used in manufacturing products. Describe how these NPs could possibly be
harmful to fishes.
3. Differentiate the effect of mercury exposure of male catfish from female catfish.

Metals are the most common pollutants, and although some of them are essential for physiological functions,
they become toxic at high concentration and together with nonessential metals exert adverse effects on either
animal and human reproductive functions. Other contaminants are nanoparticles, plastics and
organophosphate pesticide.
Exposure to mercury.The cycle of spermatogenesis in which stem cells divide into mature
spermatozoa is controlled by LH and FSH. Throughout this cycle, the testes increase in size, which can be
measured by an increase in the gonadosomatic index (GSI; gonad wt/body wt · 100) and usually is maximal
in the breeding season. Disruption of testes function can be classified as either cytotoxicological or endocrine
in origin. Cytotoxic effects are characterized by damage to cellular integrity or alterations in cell function,
whereas endocrine effects involve the disruption of specific cells as a result of alterations in pituitary secretion
or gonadal steroidogenesis.
Mercury exposure also can affect reproductive endpoints in female fish. Mercury has been shown to
affect ovarian morphology, delay oocyte development, and inhibit steroid hormone synthesis. As with the
testes, it is difficult to determine whether mercury affects the ovaries directly or indirectly, via alterations at
the level of hypothalamus or pituitary, and whether the effects are the cause or the result of changes in
steroidogenesis. Unlike in males, female gamete production involves the synthesis and incorporation of a

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yolk protein, which may transfer some of the maternal mercury burden to the embryo. The effects of mercury
on the female reproductive system discussed in the next section are separated into change at the level of
ovarian morphology and egg development, steroid hormone and vitellogenin synthesis, fecundity and
spawning, and ultimately, egg and embryo survival.
Exposure to pesticide. Many applications outside agriculture, such as sylviculture, horticulture or
public health, can also lead to a detrimental influence on fish populations. The nature of the effects on fish
can be very variable. Apart from causing death, either directly, or due to starvation, by destruction of food
organisms, many pesticides have been shown to affect growth rate, reproduction and behaviour, with
evidence of tissue damage.
Exposure to nanomaterials. NPs are finally found in aquatic and terrestrial environments, where they
are ingested by living organisms in which they accumulate, before being eliminated by the immune system
or other pathways. In organisms, NPs represent foreign elements with their own physicochemical properties,
so they may interfere with the normal physiological mechanisms of the embryos, growing animals, and adults.
Effects of microplastics. Microplastic fibers are linked to respiratory and reproductive changes in
fish. In a surprising study, not only did fish exposed to microplastics reproduce less but their offspring, who
weren’t directly exposed to plastic particles, also had fewer young, suggesting the effects can linger into
subsequent generations.

References

1. Abd El-Gawad, E.A., Abass, A.A., Shaheen, A.A. 2012. Risk induced by pesticides on fish
production. The Global Journal of Fisheries and Aqua. Res. - Vol. No. 5, Proc. of the 5th
Global Fisheries & Aqua. Research Conf., Egypt, pp. 329 – 338.
2. Akhtar, M.S., Cijji, A., Sarma, D. Rajesh, M., Kamalam, B.S., Sharma, P. and Singh,A.K. 2017.
Reproductive dysfunction in females of endangered golden mahseer (Tor putitora) in
captivity.Anim. Reprod. Sci. 182: 95-103. .
3. Alkaladi, A., Afifi, M., Ali, H., Saddick, S. 2020. Hormonal and molecular alterations induced by
sub-lethal toxicity of zinc oxide nanoparticles on Oreochromis niloticus. Saudi Journal of
Biological Sciences 27: 1296-1301.
4. Ansari, B.A. and Kumar, K.. 1987. Malathion toxicity: effect on the ovary of the zebra fish
Brachydanio rerio (Cyprinidae). Int. Rev. Ges. Hydrobiol. 72, 517-528.
5. Arora, N. and Kulshrestha, S. K.. 1984. Comparison of the toxic effects of two pesticides on
the testes of a freshwater teleost Chana striatus B1. Acta Hydrochim.Hydrobiol. 12, 435-441.
6. Barboza, L.G.A., Lopes, C., Oliveira, P., Besa, F., Otero, V., Henriques, B., Raimundo, J.
Caetano, M., Vale, C., Guilhermino, L. 2020. Microplastics in wild fish from North East Atlantic
Ocean and its potential for causing neurotoxic effects, lipid oxidative damage and human
health risks associated with ingestion exposure. Science of the Total Environment, 717:1-14.
7. Billard, R. and Roubaub, P. 1985. The effect of metals and cyanide on fertilization in rainbow
trout (Salmo gairdneri). Water Res 19, 209-244.
8. Dayal, N., Singh, D., Patil, P., Thakur, M., Vanage, G.,Joshi, D.S. 2017. Effect of
bioaccumulation of gold nanoparticles on ovarian morphology of female zebrafish (Danio rerio).
World Journal of Pathology, 9: 17p.
9. DeSa, L.C., Oliveria, M., Ribiero, F., Rocha, T.L., Futter, M.N. 2018. Studies of the effects of
microplastics on aquatic organisms: what do we know and where should we focus in the
future? Science of the Total Environment 645:1029-1039.

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 10. Doering, J., G. Ankley, B. Blackwell, J. Cavallin, A. Cole, K. Dean, K. Fay, D. Feifarek, K.
Jensen, M. Kahl, ,A. Kittelson, C. Lalone, S. Poole, E. Randolph, C. Tilton, and D. Villeneuve.
2019 A cross-species model for predicting reproductive dysfunction in fishes exposed to
aromatase inhibiting chemicals. Twin Ports Early-Career Researchers Symposium, Duluth,
MN.
11. Exbarayat, J-M., Moudilou, E.N., Lapied, E. 2015. Harmful effects of nanoparticles on animals.
Journal of Nanotechnology, 10 p. ID 861092.
12. Guzman, J.M., Luckenbach,J.A.,Goetz, F.W., Fairgrieve, W.T., Middleton, M.A., Swanson, P.
2015. Reproductive dysfunction in cultured sablefish (Anoplopoma fimbria). Bull. Fish. Res.
Agen. No. 40: 111-119.
13. Johari, S.A. 2014. Toxicity effect of colloidal silver nanoparticles on fertilization capacity and
reproduction success of rainbow trout (Oncorhynchy mykiss). Journal of Nanomedicine
Research, 1(1):1-4.
14. Kerekes, F. Koll’ar, T., Gazsi, G. K’asal, E., Urb’anyi, B., Csenki-Bakos, Z. and Horv’ath, A.
2020. Investigation of Fertilizing Capacity of Zebrafish (Danio rerio) Sperm Exposed to Heavy
Metals An International Journal. pp.1-10. DOI: 10.1177/1559325820919597 1
15. Kime, D.E. 1995. The effects of pollution on reproduction in fish. Reviews in Biology and
Fisheries 5 : 52–95.
16. Lal, B. 2007. Pesticide—induced reproductive dysfunction in Indian fishes. Fish Physiol
Biochem 33, 455–462. https://doi.org/10.1007/s10695-007-9171-4
17. Malhorta, N., Ger, T.R., Uapipatanakul, J.C., Huang, K, Chan, H.-C., Hsiao, C-D. 2020.
Review of copper and copper nanoparticles toxicity in fish. 2020. Nanomaterials, 10,1126:28 p.
18. Svensson, O., Gräns, J., Celander, M C., Havenhand, J., Leder, E H. et al. 2017. Immigrant
reproductive dysfunction facilitates ecological speciation. Evolution, 71(10): 2510-252
19. Wooton, R.J., and Smith, C. 2014. Reproductive Biology of Fishes. John Wiley and Sons,
Ltd. 496p.
20. Wu, R. 2009. Effects of hypoxia on Fish Reproduction and Development. Fish Physiology
27: 79- 141.
21. Yousefian, M. and Mousav, S.E. 2011. The mechanism of reproduction and hormonal function
in finfish species: A review. Scientific Research and Essays 6(17):3561-3570.
22. Links https://www.youtube.com./watch?v=dn-kzrsLg8I
https://www.youtube.com./watch?v=G0GSi-Em5I8
https://www.youtube.com./watch?v=IZCADC65Q4Q
https://www.youtube.com./watch?v=N6-qom43h94

Congratulations! You may now take the assessment!

Contact your course professor for the assessment schedule.

The due date of submission is the last day of the second week. The submission may be
done via e-mail, messenger, or via designated drop boxes.

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