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Effects of ammonia on shrimp physiology and immunity:
a review
Mingming Zhao
1,2
, Defu Yao
1,2
, Shengkang Li
1,2
, Yueling Zhang
1,2
and Jude Juventus Aweya
1,2
1 Institute of Marine Sciences and Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, China
2 STU-UMT Joint Shellfish Research Laboratory, Shantou University, Shantoua, China
Correspondence
Jude Juventus Aweya and Yueling Zhang,
Institute of Marine Sciences, College of
Science, Shantou University, Shantou,
Guangdong 515063, China. Emails:
jjaweya@stu.edu.cn (JJA); zhangyl@stu.edu.cn
(YZ)
Received 17 December 2019; In Revised form
7 March 2020; accepted 16 March 2020.
Abstract
The rapid expansion in shrimp farming comes with problems such as inadequate
culture technology, disease outbreak, water pollution and other environmental
degradation-related problems. Prominent among these is the level of ammonia,
which has been rising steadily in the last couple of years, mainly due to farming
practices and other anthropogenic activities, and therefore has had consequent
effects on marine life, especially shrimp. While optimal ammonia levels are a good
source of nitrogen for marine phytoplankton that increases the level of dissolved
oxygen in water as well serves as food for shrimp, high levels of ammonia are
harmful to shrimp. When shrimp pond ammonia levels go beyond the tolerance
limits, it inhibits chitinase expression, moulting, growth, phenoloxidase and hae-
molymph antimicrobial activity, thereby attenuating shrimp innate immune
response. In this review, we bring together recent information on the effects of
ammonia stress on the growth, physiology, biochemistry, ammonia-metabolizing
enzymes and immunity of shrimp. We also propose areas of research that can be
explored in the breeding of new ammonia-tolerant and disease-resistant shrimp
with robust immune and/or physiological systems that could withstand environ-
mental stress and pathogens.
Key words: ammonia, aquaculture, immunity, shrimp culture, water pollution.
Introduction
Aquaculture provides food as well as huge economic bene-
fits to farmers and the economy.In the last two decades,
the total volume of international exports of fishery com-
modities rose steadily from 45 million tons in 1997 to
64 million tons in 2017, while proceeds from these exports
increased from $53.4 billion in 1997 to $156.4 billion in
2017 (FAO, 2019). Total global shrimp (mainly Litopenaeus
vannamai and Penaeus monodon) output from aquaculture
production is among the top 20 aquaculture products.
Thus, the total export volume of shrimp and shrimp prod-
ucts has seen a steady increase from 1.69 million tons in
1997 (3.77% of the world trade) to 4.36 million tons
(6.72% of the world trade) in 2017 (FAO, 2019). Unfortu-
nately, the rapid growth and expansion in shrimp farming
have also resulted in degradation and pollution of the aqua-
culture fields and surrounding environment. For instance,
a growing number of studies have shown aquaculture fields
and ponds to have undergone drastic changes in salinity,
pH, temperature, ammonia, etc., which impact on aquatic
organisms and their immune response (Xu et al., 2018;
Han et al., 2018; Qiu et al., 2018; Lou et al., 2019). The
most noxious among these parameters is ammonia, which
is reported to affect numerous marine species (see
Table 1).
In shrimp aquaculture, ammonia is also one of the main
water pollutants and in fact the main limiting factor that
causes rapid increase in shrimp mortality and therefore
brings about huge economic losses to farmers (Cobo et al.,
2014; Sun et al., 2018). There is a high tendency for non-
lethal levels of ammonia to be exceeded in aquaculture due
to the nature of this farming practice; hence, various
shrimp species including L. vannamei,Fenneropenaeus chi-
nensis and P. monodon could easily be impacted (Cobo
et al., 2014; Chen et al., 2016; Cui et al., 2017; Wang et al.,
2017). Chemically, ammonia (NH
3
) is nitrogen (N) in the
form of free ammonia or ionic ammonia in water, the con-
centration of which depends mainly on the pH, tempera-
ture and salinity of the environment. At pH <8.75,
©2020 John Wiley & Sons Australia, Ltd 1
Reviews in Aquaculture, 1–18 doi: 10.1111/raq.12429
Table 1 Effects of ammonia stress on marine species
Organism Species Size/life stage Total ammonia
nitrogen
Increased factor Decreased factor Tissue Reference
Crustacea Portunus trituberculatus 104.8 !9.6 g 1.0–20.0 mg L
"1
–Phagocytic and
antibacterial activity,
THC, a
2
-M
Haemolymph Yue et al. (2010b)
Scylla serrata Juvenile 10–140 mg L
"1
pH Na
+
, Ca
2+
Haemolymph Romano and Zeng.
(2007a)
Molluscs Chlamys farreri 14.90 !1.36 g 20.0 mg L
"1
IDH, HSP70, HSP90,
GSase
CEA Haemolymph, serum Wang et al. (2012)
Corbicula fluminea 1.5 !0.2 cm 10 and 25 mg L
"1
NF-jB, MAPK TLR4 Digestive gland Zhang et al. (2019)
Ruditapes philippinarum 3.5 !0.3 cm 0.1–0.5 mg L
"1
Apoptosis ratio MTP, Ca
2+
-ATPase,
H
+
-ATPase, K
+
-ATPase
Gill, haemocytes Cong et al. (2019)
Fish Ctenopharyngodon idellus Juvenile 0.5–18.0 mg L
"1
Antioxidative enzymes,
Antioxidants
CAT Liver, gills, muscle Yao et al. (2019)
Hippoglossus hippoglossus Juvenile 0.06–0.17 mg L
"1
pH, HCO
3
"
, PCO
2
Blood Paust et al. (2011)
Hypophthalmythys nobilis Larvae 0.06–0.264 mg L
"1
SGR, weight, GSH SOD Whole-body
homogenates
Sun et al. (2012)
Megalobrama amblycephala 13.8 !0.04 g 10 mg L
"1
ACH50, NO, SOD,
CAT, HSP70, HSP90
Cortisol, glucose, MDA Plasma, liver Zhang et al. (2015a)
Monopterus albus 150 –250 g 50 mmol L
"1
- NKCC1 Brain Ip et al. (2013)
Monopterus cuchia 150-170 g 50 mMMDA, H
2
O
2
, HSP70, HSP90, - Plasma Hangzo et al. (2017)
Oreochromis Niloticus Juvenile 5–10 mg L
"1
TBARS, PCO, XO, AO, CAT,
c-GT, c-GCS
- Liver, white muscle Hegazi et al. (2010)
Pelteobagrus fulvidraco Juvenile 5.70 mg L
"1
Glutamine, TBARS, SOD, GPX, GR, brain Li et al. (2016)
Scophthalmus maximus Juvenile 20–40 mg L
"1
CRH, ACTH, SOD, CAT,
HSP70, HSP90, MDA
GH, LZM, C3, C4,
IgM, GSH, IGF-1
Plasma, liver Rui et al. (2016)
Sebastes schlegelii 38.36 !3.45 g 0.1–1.0 mg L
"1
Glucose, GOT, GPT RBC, WBC, total protein Serum Shin et al. (2016)
Sebastes schlegelii 38.36 !3.45 g 0.1–1.0 mg L
"1
SOD, CAT, GST, Cortisol,
HSP70
GSH, phagocytosis,
lysozyme activity
Liver, plasma Kim et al. (2015)
Takifugu obscurus 25.5 !1.8 g 1.43–7.14 mMROS, BAFF, TNF-a, HSP90,
HSP70, CAT, P53
–Liver, blood Chang et al. (2015)
ACH50, alternative complement pathway activities; ACTH, adrenocorticotropic hormone; AO, aldehyde oxidase; BAFF, B-cell activating factor; C3, complement 3; C4, complement 4; CAD, catalase
activities declined; CAT, catalase; CEA, cellular energy allocation; CRH, corticotropin-releasing hormone; GH, growth hormone; Gln, glutamine; GOT, glutamic oxalate transaminase; GPT, glutamic
pyruvate transaminase; GPX, glutathione peroxidase; GR, glutathione reductase activities; GSase, glutamine synthetase; GSH, glutathion; GSH, glutathione; GST, glutathione s-transferase; HCO3
"
,
blood bicarbonate; HSP70, heat shock protein 70; HSP90, heat shock protein 90; IDH, isocitrate dehydrogenase; IGF-1, insulin-like growth factor-1; IgM, immunoglobulin M; LZM, lysozyme; MDA,
malondialdehyde; MTP, mitochondrial transmembrane potential; NKCC1, Na
+
:K
+
:2Cl
-
cotransporter 1; NO, nitrogen monoxide; P53, a tumour suppressor gene; PCO, protein carbonyl group; PCO
2
,
partial pressure of CO
2
; RBC, red blood cell; ROS, reactive oxygen species; SGR, specific growth rates; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; THC, total haemocyte
count; TLR4, toll-like receptor 4; TNF-a, pleiotropic pro-inflammatory cytokine; WBC, white blood cell; XO, xanthine oxidase; c-GCS, c-glutamyl cysteinyl synthetase; c-GT, c-glutamyl transpeptidase.
Reviews in Aquaculture, 1–18
©2020 John Wiley & Sons Australia, Ltd
2
M. Zhao et al.
ammonia mainly exists in the form of NH
4+
, but when the
pH is higher than 9.75, ammonia mainly exists in the form
of NH
3
(Molins-Legua et al., 2006). There is therefore a
close relationship between the concentration of ammonia
and marine life, as this directly affects their normal activi-
ties.
Ammonia is not only the main limiting factor in crus-
tacean aquaculture (Chen et al., 2012b; Cobo et al., 2014;
Xiao et al., 2019), but also a major environmental pollu-
tant, and therefore one of the most important water quality
parameters used for monitoring environmental pollution
(Yue et al., 2010a). The use of ammonia as a water quality
parameter is due to its impact on important physiological
and pathophysiological processes such as growth, meta-
morphosis, osmotic regulation, immunity, reproduction,
metabolism, survival and excretion in shrimp and other
crustaceans (Hong et al., 2007; Rodriguez-Ramos et al.,
2008; Bouwman et al., 2011; Romano & Zeng, 2013; Chang
et al., 2015; Cui et al., 2017). High ammonia levels could
suppress immune parameters such as total haemocyte
count (THCs), phagocytic activity, phenoloxidase (PO)
that mediates melanin synthesis (Cerenius & Soderhall,
2004), and antibacterial activity through the second mes-
senger including cAMP (cyclic adenosine monophos-
phate)-, CaM (calmodulin)- and cGMP (cyclic guanosine
monophosphate)-dependent pathways (Zhang et al.,
2018a). Ammonia stress does not only affect shrimp growth
and survival (Cobo et al., 2014; Qiu et al., 2018), but also
increase shrimp sensitivity to pathogens (Cui et al., 2017).
From the foregoing reports, it is evident that high ammonia
stress is an important factor that impacts on shrimp sur-
vival and disease tolerance. However, few studies have so
far explored the mechanisms of immune regulation in
shrimp under conditions of excess ammonia exposure. This
review therefore brings together circumstantial evidence
from various studies that connect and relate ammonia
stress to immunomodulation in shrimp.
Effect of ammonia levels on shrimp growth and
survival
Sources of ammonia
Ammonia, nitrite and nitrate are the most common forms
of dissolved inorganic nitrogen ions, which are important
in aquatic life, although ammonia could also be the most
toxic form (Romano & Zeng, 2013). As a nutrient salt,
ammonia is widely found in seawater and is one of the
important indexes used for monitoring environmental pol-
lution (O’Connor Sraj et al., 2018). Seawater ammonia is
an important part of the upper ocean nitrogen cycle, and
one of the essential nutrients for marine phytoplankton
(Olowe & Kumarasamy, 2017). Therefore, the consump-
tion of ammonium ions by phytoplankton in surface waters
of natural seawater results in nanomolar levels of ammonia
in seawater (Hashihama et al., 2015).
Ammonia is the main product of protein catabolism,
unconsumed feed and faeces of aquatic animals in marine
environment (Chang et al., 2015). Thus, high ammonia in
the environment results in its accumulation in aquaculture
animals (Hong et al., 2007). The excretion of ammonia by
aquatic organisms therefore depends on its levels in the
aquaculture environment. In shrimp, ammonia is mainly
eliminated from the body or haemolymph (usually higher)
into the surrounding environment due to concentration
gradient (Habaki et al., 2011).
In aquaculture, ammonia could be generated from many
sources including nitrogen-containing exogenous sub-
stances, production of ammonia by organic matter used as
feed, faeces, dead aquatic animals, culture density, algae,
exchange of ammonia between aquatic organisms and the
surrounding water, as well as from ammonia metabolism.
Given that shrimp culture systems differ (i.e. intensive,
semi-intensive and super-intensive), ammonia levels, its
change during the culture circle and its impact on shrimp
vary (see Table 2). Ammonia is therefore the main metabo-
lite of nitrogen-containing compounds and an important
inorganic pollutant that accumulate in water bodies
(Alonso & Camargo, 2004; Prenter et al., 2004). While
ammonia is an important index used to monitor or evalu-
ate water quality (Yue et al., 2010b), it is also an important
environmental factor that reflects the survival status of
aquaculture animals (Bouwman et al., 2011).
Toxic effects of ammonia
It has been shown that when ammonia levels in water
exceed the tolerance limit of shrimp, it could cause direct
damage to tissues such as gill and hepatopancreas, as well
as affect respiration, metabolism, immunity, osmotic regu-
lation, excretion, moulting and growth (Liang et al., 2016;
Zhou et al., 2017; Qiu et al., 2018). High ammonia could
therefore attenuate resistance to pathogenic infections and/
or cause mortality of shrimp (Cobo et al., 2014; Lu et al.,
2016; Cui et al., 2017). In addition to its direct damaging
effects on many tissues and organs of aquatic animals (Li
et al., 2015), ammonia toxicity or stress also results in
oxidative damage and increase in the levels of free reactive
oxygen species (ROS) in shrimp (Ching et al., 2009; Zhang
et al., 2015b). Changes in environmental factors, such as
salinity, pH and temperature, give rise to fluctuations in
ammonia concentration in water, which decreases the feed-
ing rate, growth rate and immune function of aquatic
organisms as well as predispose them to diseases (Cui et al.,
2017; Han et al., 2018; Qiu et al., 2018; Lou et al., 2019).
For instance, when ammonia levels in aquaculture water
reach a certain mass concentration, non-ionic ammonia
Reviews in Aquaculture, 1–18
©2020 John Wiley & Sons Australia, Ltd 3
Ammonia stress and shrimp immunity
Table 2 Total ammonia nitrogen (TAN) levels during shrimp culture
Species Size/life stage Culture perod Density
(shrimp
m
"2
)
Culture system Salinity TAN Food
conversion
ratio
Survival
(%)
Growth rate References
Litopenaeus vannamei 6.0 g 10 weeks 132 Super-intensive 21.6–38.8 ppt 0.39–1.68 ppm 1.28 95.19 1.03 g
per week
Browdy and Moss.
(2005)
Litopenaeus vannamei Postlarvae 120 days 20 Semi-intensive 42.0 !4.6 g L
"1
0.06–0.10 mg L
"1
2.16 65.5 Porchascornejo
et al. (2015)
Litopenaeus vannamei Postlarvae 165 days 35 –37.07 g L
"1
0.115–0.533 mg L
"1
1.43 84.84 0.18 g day
"1
Patil et al. (2016)
166 days 56 High-intensive 36.8 g L
"1
0.121–0.802 mg L
"1
1.92 61.53 0.19 g day
"1
Litopenaeus vannamei 7.4 !0.1 g 30 days 28 Intensive 32 g L
"1
0.08–0.2 mg L
"1
–94 1.17% Wu et al. (2013)
Litopenaeus vannamei 0.87 !0.03 g July
to September
656 Intensive 16.79 g L
"1
0.04 "11.9 mg L
"1
–75.48 Chen et al. (2018)
Penaeus monodon 0.05 g 122 1.5 Extensive –0.160–163.88 µgL
"1
3.2 45 5.06% Ramanathan
et al. (2013).
123 5 Semi-intensive –0.148 –171.38 µgL
"1
3.4 44 4.95%
Penaeus monodon Postlarvae 17 weeks 25 Intensive 21 ppt 0.015–0.32 ppm –97.7 –Corre et al. (2016)
Penaeus monodon Postlarvae April
to September
6–24.6 ppt 0.32–0.98 mg L
"1
––– Fernandes
et al. (2010)
Penaeus monodon Juveniles 90 days 25 Intensive 20 ppt 0.2768 mg L
"1
1.83 62 0.11 g day
"1
Thakur and Lin.
(2003)
50 Intensive 20 ppt 0.5191 mg L
"1
1.52 78 0.14 g day
"1
Penaeus penicillatus Postlarvae 141 days 286 Super-intensive 15.33 ~21.00 ppt 0.022–46.110 mg L
"1
–44.3 –Chen et al. (2007)
Reviews in Aquaculture, 1–18
©2020 John Wiley & Sons Australia, Ltd
4
M. Zhao et al.
could easily enter shrimp body via cell membranes, result-
ing in physiological imbalance and inhibition of growth
(Naqvi et al., 2007; Xiao et al., 2019). Moulting is strongly
impacted by high ammonia levels, as the epidermis-se-
creted chitinase that degrades the inner layers of old
exoskeleton to synthesize a new one during moulting (ecd-
ysis) (Zou & Bonvillain, 2004; Salma et al., 2012) has been
shown to be significantly decreased in shrimp upon ammo-
nia stress treatment (Lu et al., 2016; Zhou et al., 2017; Lu
et al., 2018; Li et al., 2018b) (see Table 4). In terms of
immune response, elevated ammonia levels could induce
an immune response in shrimp (Spriggs et al., 2010; Lu
et al., 2016), as high levels of ammonia decrease shrimp
immunity and increase their susceptibility to pathogens
(Qiu et al., 2018). Similarly, increased levels of ammonia
reduce the oxygen-carrying capacity of haemocyanin,
which is believed to be one of the main mechanisms by
which ammonia causes toxicity in shrimp (Chand & Sahoo,
2006). Recent transcriptional analysis of shrimp hep-
atopancreas samples has also revealed a decrease in the
expression of genes associated with prophenoloxidase in
response to ammonia stress (Sun et al., 2018). Toxic effects
of ammonia have been reported in shrimp and other crus-
taceans, especially effects on osmotic regulation (Cui et al.,
2017) and metabolism (Liang et al., 2016). Exposure to
high levels of ammonia could perturb amino acid metabo-
lism, by increasing the expression of some genes related to
ammonia detoxification and excretion (Xiao et al., 2019).
For instance, in L. vannamei, NH
4+
affects the activity of
ammonia-metabolizing enzymes and ammonia excretion
(Romano & Zeng, 2010; Pinto et al., 2016).
The main target organs affected by ammonia
Shrimp gills are in direct contact with the external environ-
ment, for which reason they are directly affected by envi-
ronmental factors such as ammonia stress. While most
studies have shown that ammonia can directly damage gill
tissues of crustacean, resulting in anoxia, ammonia also
decreases immunity, as well as causes metabolic
dysfunction and increases their sensitivity to pathogens
(Spencer et al., 2008; Sung et al., 2011; Zhou et al., 2017).
For instance, in L. vannamei, high levels of ammonia are
reported to cause structural and pathological gill damage,
resulting in oedema, inflammation (haemolymph and
haemocytic infiltration), melanization and necrosis (Fre-
goso-L!
opez et al., 2017), especially in the labyrinth of the
antennal gland (Fregoso-L!
opez et al., 2018). Similarly,
shrimp intestine is involved in nutrient digestion and
absorption, and hence, ammonia stress changes intestinal
metabolism and therefore affects its essential role in the
immune response (Duan et al., 2017). When L. vannamei
were exposed to high ammonia stress, it caused significant
damage to the intestine mucosal, stripping off most of the
epithelial cells on the basement membrane accompanied by
necrosis (Duan et al., 2018a). As the main digestive or
metabolic organ in shrimp, the hepatopancreas plays a
major role in ammonia stress response (Qiu et al., 2018). It
is therefore not surprising that excess ammonia exposure
could induce oxidative stress and apoptosis in the hep-
atopancreas of L. vannamei (Liang et al., 2016) (see
Table 3). During high ammonia exposure, an increased use
of carbohydrates has been observed, while eyestalk ablation
dramatically decreases glucose and lactate levels, which sug-
gest that eyestalk hormone might be involved in glucose
metabolism to meet the high-energy requirements under
ammonia stress conditions (Cui et al., 2017). In addition,
high levels of non-ionic ammonia in water bodies do not
only increase oxygen consumption by crustaceans, but also
decrease the concentration of oxy-haemocyanin, as well as
destroy the excretory system and osmotic balance in these
organisms (Qiu et al., 2018).
Effects of ammonia stress on shrimp physiology
and biochemistry
Effects of ammonia stress on homeostasis
Crustaceans have strong ability to maintain lower haemo-
lymph ammonia levels relative to their surrounding (Mar-
tin et al., 2011). Thus, ammonia accumulation in shrimp is
Table 3 Effects of ammonia stress on some shrimp tissues
Tissue Size/life stage Ammonia
level (mg L
"1
)
Pathological features*References
Gill Postlarvae 13.9 Oedema, inflammation (haemolymph,
haemocytic infiltration), melanization, necrosis
Fregoso-L!
opez et al. (2017)
Antennal gland Postlarvae 13.9 Inflammation, haemocytic melanized nodules
and pyknotic nuclei
Fregoso-L!
opez et al. (2018)
Intestine mucosa Juvenile 20 Stripping of epithelial cells from basement
membrane and necrosis
Duan et al. (2018a)
Hepatopancreas 6.5 cm 20 Apoptosis of hepatopancreas cells Liang et al. (2016)
*Pathological features of tissue sections observed under light microscope.
Reviews in Aquaculture, 1–18
©2020 John Wiley & Sons Australia, Ltd 5
Ammonia stress and shrimp immunity
influenced by factors such as branchial permeability, speci-
ation of nitrogenous waste, mechanisms to reduce passive
diffusion, ability to excrete through a concentration gradi-
ent, internal detoxification processes and environmental
factors (Romano & Zeng, 2013). Ammonia (NH
3
) diffuses
through the lipid bilayers of gills and is protonated to
NH
4+
, before being transported to the gill apical membrane
by Na
+
/K
+
-ATPase, where it is excreted into the environ-
ment through Na
+
/NH
4+
transport (Valencia-Castaneda
et al., 2018) (see Fig. 1). Because NH
4+
and K
+
have similar
hydrated radius, NH
4+
competes with K
+
for K
+
-transport
proteins (Weihrauch & O’Donnell, 2015). When ammonia
levels are high, both NH
4+
excretion and gill Na
+
/K
+
-
ATPase activity increase in crustaceans (Romano & Zeng,
2011). The Na
+
/K
+
-ATPase pump, which is regulated by
the crustacean hyperglycaemic hormone (CHH) through
cGMP (Chung & Webster, 2006; Katayama & Chung, 2009;
Camacho-Jimenez et al., 2017), is the most important ion
transport enzyme in crustaceans that maintains and regu-
lates haemolymph osmotic pressure (Buranajitpirom et al.,
2010; Leone et al., 2015). One of the biogenic amines (BA),
dopamine (DA), acts as an endocrine neurotransmitter that
is involved in the regulation of CHH release to transmit
signals of osmoregulation in response to environmental
stress (Chung & Webster, 2006; Webster et al., 2012). For
instance, the concentration of CHH in L. vannamei
haemolymph increased dramatically after injection with
DA, which indicates that DA stimulates the secretion of
CHH in shrimp (Si et al., 2019b). When crustaceans are
exposed to environmental stress, such as low salinity, DA
activates the protein kinase A (PKA) signalling pathway to
phosphorylate FXYD2 (a type of Na
+
/K
+
-ATPase) and 14-
3-3 proteins (Zhang et al., 2008; Cortes et al., 2011; Silva
et al., 2012), which then increases Na
+
/K
+
-ATPase activity
(Wanga et al., 2008; Silva et al., 2012) to maintain osmotic
homeostasis. However, when levels of un-ionized ammonia
exceed 0.357 mg L
"1
, the immune system of shrimp could
be suppressed, thereby making shrimp more susceptible to
pathogens (Chang et al., 2015), since their oxidative and
immune systems would be destroyed under these condi-
tions of excessive oxygen free radicals, decreased PO activ-
ity and metabolic distortion (Spriggs et al., 2010; Qiu et al.,
2018; Zhang et al., 2018a; Xiao et al., 2019) (Table 4).
Effects of ammonia stress on prophenoloxidase-activating
system
Melanization is an important innate immune response
mechanism in invertebrates, mediated by the prophenolox-
idase (proPO)-activating system and catalysed by phe-
noloxidase (PO) (Amparyup et al., 2013). In shrimp,
melanization has been implicated in antiviral response
Figure 1 Proposed transport mechanism of ammonia in gill cells. Ammonia (NH
3
) diffuses through the lipid bilayers of shrimp gills, gets protonated
to NH
4+
, which can then be released into the surrounding environment through Na
+
/K
+
-ATPase transport. This ammonia transport mechanism could
also proceed via the dopamine (DA) and crustacean hyperglycaemic hormone (CHH)-mediated signalling pathway to activate Na
+
/K
+
-ATPase. 5-HT, 5-
hydroxytryptamine; cGMP, cyclic guanosine monophosphate (second messenger); CHH, crustacean hyperglycaemic hormone; CREB, cyclic AMP
response element-binding protein; D
4
, DA receptor; DA, dopamine; GC, guanylyl cyclase (CHH receptor); PKA, protein kinase A.
Reviews in Aquaculture, 1–18
©2020 John Wiley & Sons Australia, Ltd
6
M. Zhao et al.
Table 4 Effects of ammonia stress on physiological and biochemical changes in shrimp
Responding system Species Size/life stage Ammonia level
(mg L
"1
)
Tissue Increased factor Decreased factor References
Moulting Penaeus
monodon
21 !1 g 0, 40, 80, 100,
120, 140.
Hepatopancreas, gill –Chitinase, Chitinase-1,
Chitinase-5.
Zhou et al. (2017)
Penaeus
monodon
8.2 !1.0 g 78.15 Hepatopancreas –Chitinase Li et al. (2018b)
Litopenaeus
vannamei
Juveniles 32 Hepatopancreas Calreticulin Chitinase-5 Lu et al. (2018)
Litopenaeus
vannamei
–62.23 Hepatopancreas Ecdysteroid receptor
E75
Chitinase Lu et al. (2016)
Homeostasis Litopenaeus
vannamei
Postlarvae 0, 5, 10, 15, 20, 25,
30, 35, 40.
Gill Na+/K+-ATPase, Na+/
NH4 +transport
–Valencia-Castaneda
et al. (2018)
Portunus
pelagicus
Juveniles 0, 20, 40, 60, 80, 100. Gill Na
+
/K
+
-ATPase, Na
+
/
NH
4+
transport, K
+
.
–Romano and
Zeng. (2011)
Litopenaeus
vannamei
7.5 !0.5 cm 0.05, 2, 10,20. Gill, hindgut CHH, BA, 5-HT
7
, Na
+
/
K
+
-ATPase, CREB,
PKA, FXYD2, 14-3-3
proteins.
–Si et al. (2019a)
Prophenoloxidase-
activating
system
Litopenaeus
vannamei
63.54 mm 0, 2.5, 5, 7.5, 10. Plasma, haemolymph. PO, antibacterial,
bacteriolytic, glucose,
lactate levels
Oxy-haemocyanin Cui et al. (2017)
Litopenaeus
vannamei
3.68 !0.05 g 10 Cephalothorax actin 1, apoptosis
signal-regulating
kinase 1, O-
methyltransferase
CP Lu et al. (2019)
Litopenaeus
vannamei
5.5 !1.0 g 0.07, 2, 10, 20. Haemolymph DA, 5-HT, guanylyl
cyclase, 5-HT
7
, cAMP,
cGMP, CaM, PKA,
PKG, CREB, NF-jB,
CHH
D
4
,a
2
adrenergic
receptor, PKC, THC,
phagocytic,
antibacterial, PO
Zhang et al. (2018a)
Penaeus
monodon
18 !3 g 9, 90 Gill, hepatopancreas. GPCR. Zhu et al. (2018)
Antioxidant
defence system
Palaemon
serratus
Juvenile 10 –Oxygen consumption
rate
–Mehmet and
Osman. (2015)
Litopenaeus
vannamei
32 g 46 Hepatopancreas, gills CAT GST, GPx, PRDX Xiao et al. (2019)
Litopenaeus
vannamei
12.7 !1.5 g 5 Haemolymph –SOD, RBs Chen et al. (2012b)
Litopenaeus
vannamei
5.4 !0.3 g 20 Intestines O
2-
, LPO, MDA, SOD,
CAT, GPx, ferritin,
Trx, HSP70
–Duan et al. (2018b)
Ammonia-
metabolizing
enzymes
Litopenaeus
vannamei
3.8 !0.6 g 3.4, 13.8, 24.6 Muscle,
Hepatopancreas
GSase, GDH-bTGase Qiu et al. (2018)
Litopenaeus
vannamei
32 g 46 Hepatopancreas, gills Glycolysis, TCA GST Xiao et al. (2019)
Litopenaeus
vannamei
20.7 !0.5 g 0.001, 1.15, 5.11,
11.68
Haemocytes CP TGase Chang et al. (2015)
Others immune
responses
Penaeus
monodon
Juvenile 0, 10, 20, 30. Hepatopancreas,
muscle, gill
C-lysozyme,
antibacterial peptide
(crustin), anti-
lipopolysaccharide
factor
- Yang et al. (2015)
Litopenaeus
vannamei
6.5 cm 20 Hepatopancreas ATF4, XBP1, apoptosis - Liang et al. (2016)
Litopenaeus
vannamei
16.7 g !5.8 g 20 Haemocytes, serum Apoptosis THC Liu et al. (2020)
5-HT, 5-hydroxytryptamine; 5-HT
7
, 5-HT receptor; BA, biogenic amine; CaM, calmodulin; cAMP, cyclic adenosine monophosphate (second messen-
ger); CAT, catalase; cGMP, cyclic guanosine monophosphate (second messenger); CHH, crustacean hyperglycaemic hormone; CP, clottable protein;
CREB, cAMP response element-binding protein; D
4
, DA receptor; DA, dopamine; GDH-b, glutamate dehydrogenase-b; Gln, glutamine; GPCR, G pro-
tein-coupled receptor; GPx, glutathione peroxidase; GS, glutamine synthetase; GST, glutathione S-transferase; HSP70, heat shock protein 70; LPO,
lipid peroxidation; MDA, malondialdehyde; NF-jB: nuclear factor kappa-b; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PO,
phenoloxidase; PRDX, peroxiredoxin; RBs, respiratory bursts; SOD, superoxide dismutase; TCA, tricarboxylic acid; TGase, transglutaminase; THC, total
haemocyte count; Trx, thioredoxin.
Reviews in Aquaculture, 1–18
©2020 John Wiley & Sons Australia, Ltd 7
Ammonia stress and shrimp immunity
(Sangsuriya et al., 2018), while the PO enzyme is reported
to play a role in cellular defence by associating with factors
that aid phagocytosis, which is why PO is often used to
assess the impact of environmental stressors on invertebrate
immune system (Ellis et al., 2011). While ammonia stress
has been shown to reduce the immune response of aquatic
animals by inhibiting PO activity and phagocytosis (Wang
et al., 2012; Yang et al., 2015), as well as decreasing (up to
60%) proPO gene expression (Moullac, 2000), the question
is what regulates the proPO-activating system during
ammonia stress?
Bilateral eyestalk ablation in L. vannamei significantly
reduces the activities of proPO and PO, which suggest an
endocrine control of the effector immune response due to
the eyestalk ablation (Sainz-Hernandez et al., 2008). Crus-
taceans eyestalk includes an important neuroendocrine
organ, X-organ/sinus gland (XO-SG), which is part of the
neuroendocrine system involved in synthesis and control of
neuropeptide hormones, such as CHH, and biogenic ami-
nes (BAs) that are essential for regulating immune
response, physiology and metabolism (Zhao et al., 2016).
High ammonia exposure reduces PO, antibacterial and
bacteriolytic activities in shrimp, as well as decreases oxy-
haemocyanin levels in plasma, with concomitant increase
in glucose and lactate levels, which is synonymous with eye-
stalk ablation where levels of glucose and lactate decrease
dramatically (Cui et al., 2017). Given that CHH plays a
major role in carbohydrate metabolism during immune
response (Wanlem et al., 2011), it seems to suggest that
CHH is an eyestalk hormone involved in glucose metabo-
lism to meet energy requirements under ammonia stress
conditions. However, CHH is not a direct regulator of the
proPO-activating system, but directly participates in glu-
cose metabolism to generate enough energy for adaptation
to the external conditions. This view is consistent with pre-
vious studies where an increase in oxygen consumption
was observed under ammonia stress conditions (Mehmet &
Osman, 2015).
The neuroendocrine system in shrimp eyestalk may also
secrete neuroregulators such as CHH and biogenic amines
(e.g. dopamine (DA), noradrenaline (NE) and 5-hydrox-
ytryptamine (5-HT) (Zhao et al., 2016) in response to envi-
ronmental stress (Fig. 2). For instance, exposure of
L. vannamei to ammonia or low salinity stress results in the
Figure 2 Proposed cellular response induced by ammonia. During ammonia stress, 5-hydroxytryptamine (5-HT), dopamine (DA) and crustacean
hyperglycaemic hormone (CHH) transmit signals to the cell via their membrane receptors (e.g. D
4
, 5-HT
7
), which results in the regulation of phenoloxi-
dase (PO), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-jB), cyclic AMP response element-binding protein (CREB), etc., thereby
inhibiting superoxide dismutase (SOD), increasing malondialdehyde (MDA), inducing apoptosis and activating glutamine synthetase (GSase) to catal-
yse the conversion of NH
4+
into nontoxic glutamine (Gln). 5-HT, 5-hydroxytryptamine; 5-HT
7
, 5-HT receptor; cAMP, cyclic adenosine monophosphate
(second messenger); cGMP, cyclic guanosine monophosphate (second messenger); CHH, crustacean hyperglycaemic hormone; CREB, cyclic AMP
response element-binding protein; D
4
, DA receptor; DA, dopamine; GC, guanylyl cyclase (CHH receptor); Gln, glutamine; Glu, glutamate; GSase, glu-
tamine synthetase; MDA, malondialdehyde; NF-jB, nuclear factor kappa-light-chain-enhancer of activated B cells; PKA, protein kinase A; PKC, protein
kinase C; PKG, protein kinase G; PO, phenoloxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; TGase, transglutaminase; THC, total
haemocyte count; a2-M, alpha-2-macroglobulin.
Reviews in Aquaculture, 1–18
©2020 John Wiley & Sons Australia, Ltd
8
M. Zhao et al.
secretion of corticotrophin-releasing hormone (CRH) and
adrenocorticotropic hormone (ACTH) into the haemo-
lymph to stimulate the release of biogenic amines, such as
DA, NE and 5-HT (Jia et al., 2016; Si et al., 2019a). Follow-
ing the release of these biogenic amines, the membrane
receptors, such as D
4
receptor (for DA) and 5-HT
7
receptor
(for 5-HT), then transduce the signals onto haemocytes to
modulate the concentrations of cAMP and cGMP, thereby
regulating the levels of intracellular PKA and PKC (Zhao
et al., 2016; Zhang et al., 2018a). The biogenic amine recep-
tors are actually G protein-coupled receptors (GPCRs) that
activate signalling pathways such as the PKA and PKC
pathways to produce unique cellular signalling effects in
vertebrates and invertebrates (Chang & Mykles, 2011; Tort,
2011; Buckley et al., 2016). DA could therefore induce the
activation of proPO in haemocytes via the PKC signalling
pathways (Peng et al., 2013), while 5-HT decreases PKC
activity by increasing the concentration of Ca
2+
(Zhang
et al., 2018a). It has also been reported that protein kinase
G (PKG), which regulates Ca
2+
-CaM to indirectly control
exocytosis (Yamada et al., 2006; Ando et al., 2013), might
be involved in degranulation of haemocytes to release PO
(Xian et al., 2017).
When yellow catfish Pelteobagrus fulvidraco were exposed
to ammonia stress, the activity of alpha-2-macroglobulin
(a2-M) in plasma decreased initially (first 3 h) followed by
an increase at 6 h (Zhang et al., 2018a). On the other hand,
recombinant a2-M strongly and specifically inhibited the
activities of trypsin and PO in L. vannamei plasma dose-
dependently, while a2-M silencing increased PO activity in
shrimp plasma, despite a decrease in the expression of
proPO-activating enzyme (PPAE) and proPO (Ponprateep
et al., 2017). From the foregoing, it could be inferred that
in addition to the proPO system, there seems to be another
pathway that enhances PO activity. This alternative or
additional pathway might be related to haemocyanin,
which also has PO activity (Decker & Rimke, 1998; Mul-
laivanam Ramasamy et al., 2017).
Effects of ammonia stress on antioxidant defence system
Under normal physiological conditions, free radicals are
formed and cleared to maintain a dynamic balance (Ming
et al., 2012). Thus, the physiological effects induced by
environmental stress such as pH, temperature, salinity and
ammonia are related to the oxidation pathway of the body
(Richier et al., 2006; Ryter et al., 2007). In crustaceans, the
concentration of DA in haemolymph increased significantly
under salinity or ammonia stress (Pan et al., 2014). Ele-
vated levels of DA then stimulate the secretion of CHH
from the XO/SG complex in the eyestalk of shrimp to
induce high levels of glucose (Camacho-Jimenez et al.,
2017) that provides energy for respiratory burst. However,
when ammonia levels exceed the threshold of shrimp, the
antioxidant system could be damaged, impacting on
antioxidants and antioxidant enzymes, thereby decreasing
the ability to scavenge free radicals (Romano & Zeng,
2007b; Chen et al., 2012a). Superoxide dismutase (SOD), a
key antioxidant enzyme, constitutes an important part of
the first-line antioxidative defence system that eliminates
ROS from cells (Duan et al., 2016). High ammonia expo-
sure or nitrite stress could trigger the overproduction of
ROS to induce oxidative stress in juvenile yellow catfish
and L. vannamei (Duan et al., 2018b; Zhang et al., 2018b),
thereby increasing the level of malondialdehyde (MDA)
(Duan et al., 2018b). Thus, levels of MDA and antioxidant
enzymes serve as important indicators used for evaluating
the health status of marine organisms (Bebianno et al.,
2005). As an end product of lipid peroxidation, MDA is
therefore an important indicator of oxidative damage in
cell membranes (Liu et al., 2011) of damaged cells that are
to be eliminated via apoptosis (Mai et al., 2010). This sug-
gests that environmental stress and increased ROS levels
could cause extensive lipid peroxidation and DNA damage,
resulting in apoptosis as a cellular immune response (Flor-
ence et al., 2002). In shrimp, levels of oxy-haemocyanin
decrease under ammonia stress (Cheng et al., 2013; Cui
et al., 2017; Qiu et al., 2018), which provides basis to
believe that this could be an adaptive mechanism for respi-
ratory burst to counteract the ammonia stress condition.
However, since this conclusion is inferred from circum-
stantial evidence, more work is required to substantiate.
Effects of ammonia stress on ammonia-metabolizing
enzymes
Exposure to high levels of ammonia has been shown to
weaken metabolic functions of L. vannamei (Cui et al.,
2017), while elevated NH
4+
levels are reported to affect the
activity of ammonia-metabolizing enzymes and ammonia
excretion also in L. vannamei (Qiu et al., 2018). Low levels
of amino acids are reported in shrimp and fish during high
ammonia stress, which is believed to be due to increased
energy demand required to maintain homeostasis, as evi-
denced by an increase in the TCA cycle (Huang et al., 2017;
Xiao et al., 2019). Transglutaminase (TGase) is an essential
component of the immune system (Fagutao et al., 2012;
Zheng et al., 2018) and also involved in immunoregulation
(Yeh et al., 2009). In L. vannamei, high levels of ammonia
increase the activity of glutamine synthetase (GSase) but
inhibit TGase activity in muscle, hepatopancreas and
haemocytes, which suggest that shrimp are able to acceler-
ate the synthesis of glutamine from glutamate and NH
4+
to
alleviate ammonia stress (Chang et al., 2015; Qiu et al.,
2018) (see Table 4). Although free amino acids (FAAs) in
haemolymph are organic osmolytes that play a role in
Reviews in Aquaculture, 1–18
©2020 John Wiley & Sons Australia, Ltd 9
Ammonia stress and shrimp immunity
maintaining osmotic pressure, especially at higher salinities,
they seem to contribute less (Romano & Zeng, 2012).
Despite the fact that ammonia toxicity in shrimp is known,
ammonia stress response and tolerance mechanisms in
shrimp are currently not well understood. Figure 2 there-
fore shows a proposed model of how high ammonia levels
and ammonia stress are counteracted in shrimp.
Effects of ammonia stress on other immune responses
Increased levels of CHH activate guanylyl cyclase (GC), the
CHH receptor on membranes, to increase PKG activity via
cGMP activation (Chung & Webster, 2006; Nagai et al.,
2009; Wu et al., 2012). Upon this activation, PKG binds
and phosphorylates (activates) the transcription factor
CREB (cAMP response element-binding protein) in vascu-
lar smooth muscle cells, neuronal cells and haemocytes,
thereby regulating the transcription of target genes (Chung
& Webster, 2006; Johannessen & Moens, 2007; Si et al.,
2019b). The activation of CREB is also directly mediated by
PKA but not PKC (Johannessen & Moens, 2007), although
PKC can regulate cell phagocytosis (Plows et al., 2004). In
shrimp, high ammonia stress has been reported to cause
apoptosis in hepatopancreatic cells (Liang et al., 2016),
while recent transcriptomic analysis has revealed that
ammonia stress affects the expression of apoptotic genes in
shrimp haemocytes (Liu et al., 2020), promoting haemo-
cyte apoptosis and therefore decreasing THC. Given that
ammonia stress reduces the immune response of aquatic
animals by attenuating SOD and PO activity (Fig. 2), as
well as decreases phagocytosis (Wang et al., 2012; Yang
et al., 2015), this could partly explain the increased suscep-
tibility of shrimp to pathogens during ammonia stress con-
ditions (Lu et al., 2019) (Table 4).
Prevention and treatment of ammonia stress
A number of measures could be instituted in order to pre-
vent the accumulation of ammonia, especially at the source
of generation. To control the source of ammonia in aqua-
culture, the amount of feed used should be well balanced
and the levels of ammonia in the culture environment
monitored in real time. Ammonia levels in the culture envi-
ronment could also be controlled by balancing nutrient
salts in the water to allow algae to multiply rapidly to pro-
duce large amounts of oxygen. Once there is a large growth
of algae, they would directly absorb ammonia and convert
it into useful nutrients for their own use (Olowe & Kumar-
asamy, 2017). Similarly, zeolite powder can be added to the
water to help purify the water by adsorbing and settling the
ammonia (Wei et al., 2010; Xin et al., 2019). Most impor-
tantly, the level of ammonia should be monitored daily,
with prudent measures put in place to regulate ammonia
levels. One of the best ways to develop a healthy and sus-
tainable shrimp aquaculture industry is to breed and use
shrimp that have strong ammonia stress tolerance and dis-
ease resistance. For instance, the resistance of L. vannamei
shrimp could be quickly improved by breeding (Lu et al.,
2017). In addition, studies should be conducted to fully
understand the toxic effects and mechanism of action of
ammonia in shrimp. Such studies would allow for the
determination of the tolerance limit of shrimp to ammonia,
and therefore help inform measures that should be insti-
tuted to improve shrimp culture.
Shrimp haemocyanin and ammonia stress in
immune response
The proPO system is an enzyme cascade, similar to the
complement system of vertebrates, that plays an important
role in the non-specific immune defence of invertebrates
(Cerenius et al., 2010; Coates & Nairn, 2014; Sangsuriya
et al., 2018). In addition to its primary function in oxygen
transport, haemocyanin has been implicated in immune
response and therefore described as an important immune
defence molecule (Coates & Nairn, 2014). Since the first
report on the phenoloxidase activity of haemocyanin from
Eurypelma californicum (Decker & Rimke, 1998), a number
of studies have since revealed that haemocyanin and phe-
noloxidase are hexameric type 3 copper proteins (Masuda
et al., 2018), with the geometry and coordination environ-
ment of the active site of arthropodan PO very similar to
that of arthropodan haemocyanin (Masuda et al., 2014). In
addition, haemocyanin exhibits phenoloxidase activity
under some conditions, such as treatment with sodium
dodecyl sulphate (SDS), polysaccharide laminarin and
lipopolysaccharide (LPS) of Gram-negative bacteria, tryp-
sin or a trypsin-like protease in shrimp hepatopancreas
(Adachi et al., 2005; Kawabata et al., 2009; Fujieda et al.,
2010; Mullaivanam Ramasamy et al., 2017; Li et al., 2018a).
Numerous studies have shown that shrimp haemocyanin
has many immune-related functions, including antiviral
(Zhang et al., 2004; Chongsatja et al., 2007; Lei et al., 2008),
phenoloxidase (Coates et al., 2013; Bris et al., 2016), agglu-
tination (Fang et al., 2011), antibacterial (Zhang et al.,
2006; Fang et al., 2011; Zhang et al., 2017), haemolytic
(Zhang et al., 2009; Yan et al., 2011), anti-tumour (Zheng
et al., 2016; Liu et al., 2017) and many other immunological
functions, which could be one of the reasons for its high
abundance in shrimp haemolymph (Depledge & Bjerre-
gaard, 1989; Mullaivanam Ramasamy et al., 2017). Thus, it
is believed that when shrimp are invaded by pathogens
under high ammonia stress, some haemocyanin degrades
into antimicrobial peptides by proteases such as trypsin
(Destoumieux-Garzon et al., 2001; Li et al., 2018a) to pro-
tect shrimp from the pathogens. However, under these
Reviews in Aquaculture, 1–18
©2020 John Wiley & Sons Australia, Ltd
10
M. Zhao et al.
conditions it is not known whether the degraded haemo-
cyanin could still bind to oxygen or not. The relationship
between decreased levels of oxy-haemocyanin and ammo-
nia stress is therefore currently unknown. Some recent pre-
liminary work in our laboratory has revealed that shrimp
haemocyanin interacts with a2-M and TGase, with a posi-
tive correlation found between the expression levels of a2-
M and TGase (unpublished). This observation seems to
suggest the involvement of shrimp haemocyanin in ammo-
nia stress. Previous studies have also shown that haemo-
cyanin responds to multiple pathogen stimuli, but few
studies have explored this response under ammonia stress
conditions. It would therefore be of interest to further
explore how the immune defence mechanism of shrimp is
modulated by haemocyanin under various ammonia stress
conditions. Knowledge gained from this could be leveraged
for breeding new shrimp varieties that have robust immune
defence systems and stronger ammonia stress or other
stress resistance.
Conclusion
Some shrimp species such as L. vannamei and P. monodon
have different ammonia stress tolerance, which suggests
that different shrimp might have specific adaptive strategies
to adapt to high levels of ammonia stress (Chen et al., 2016;
Xiao et al., 2019). A strong positive correlation has been
found between body length, disease resistance and ammo-
nia tolerance (Li et al., 2016; Lu et al., 2017). However,
selection of disease resistance using the traditional selection
approach is very slow, because the heritability of disease-re-
sistant traits is very low (about 0.02) in shrimp (Gitterle
et al., 2007). On the other hand, the heritability of ammo-
nia tolerance is high (>0.5) in shrimp (Lu et al., 2017).
Thus, selective breeding for ammonia tolerance is a fast
and prudent selective breeding approach that could also
select for disease resistance shrimp and therefore one of the
best ways for selective breeding of shrimp for aquaculture.
Most importantly, some beneficial heritable traits could
result from environmental stress. For example, a new vari-
ety of shrimp (designated Huanghai No. 2) with strong tol-
erance to white spot syndrome virus (WSSV) was recently
bred by Chinese scientists after many generations of popu-
lation selection (Shi et al., 2016). This feat in selective
breeding illustrates the feasibility of such an approach in
producing new shrimp varieties under environmental
stress. Moreover, new shrimp varieties would enrich and
expand shrimp research as well as provide new opportuni-
ties to the shrimp breeding industry. However, the current
challenge is how to breed and culture new varieties of
shrimp with high yield and strong stress resistance. The
success in this quest can only be achieved if a clear and
detailed understanding of the molecular mechanisms of
stress resistance in shrimp is well understood. More focused
research could explore broad themes on ammonia stress
and its impact on shrimp oxy-haemocyanin (i.e. levels, oxy-
gen binding ability, degradation and role of the degraded
oxy-haemocyanin products in stress response), heritability
or transfer of ammonia tolerance in shrimp and the mecha-
nism of such an inheritance, as well as complementary ben-
efits of ammonia stress resistance and disease resistance in
shrimp.
Collectively, this review provides synthesis on ammonia
stress and its effects on shrimp pathophysiological response
and immunity. The research themes identified would pro-
vide strategies for enhancing stress and disease resistance in
shrimp as well as broaden our knowledge on the breeding
of stress disease-resistant shrimp.
Acknowledgements
JJA is currently Lecturer (Assistant Professor) at the Insti-
tute of Marine Sciences and College of Science, Shantou
University, Guangdong, China, and supported by Shantou
University Scientific Research Foundation for Talents (No.
NTF19005). YLZ is the head of our research team and sup-
ported by the National Natural Science Foundation of
China (No. 31872596), and Natural Science Foundation of
Guangdong Province (No. 2017A030311032). The authors
wish to apologize for important studies in this field that
could not be included in this review due to space limita-
tions.
Authors’ contributions
JJA and MMZ conceived the idea, performed the literature
search and wrote the paper with input and suggestions
from the other authors.
Competing interests
There are no competing interests.
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