Aquaculture research, tập 41, số 4, 2010

Aquaculture Research, 2010, 41, 451^467

doi:10.1111/j.1365-2109.2009.02339.x

REVIEW ARTICLE
Lactic acid bacteria vs. pathogens in the
gastrointestinal tract of fish: a review
Einar RingÖ1,2, Lisbeth LÖvmo1Ã, Mads Kristiansen1,Yvonne Bakken1w, Irene Salinas3,
Reidar Myklebust4, Rolf Erik Olsen2 & Terry M Mayhew5
1

Department of Marine Biotechnology, Norwegian College of Fishery Science, University of TromsÖ,TromsÖ, Norway

2

Institute of Marine Research, Bergen, Norway

3

Fish Innate Immune System, Department of Cell Biology, University of Murcia, Murcia, Spain

4

Institute of Anatomy and Cell Biology, University of Bergen, Bergen, Norway

5

School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, UK

Correspondence: E RingÖ, Department of Marine Biotechnology, Norwegian College of Fishery Science, University of TromsÖ, N-9037

TromsÖ, Norway. E-mail:
ÃPresent address: Lisbeth LÖvmo, GranÔsveien 34, 7048 Trondheim, Norway.
w

Present address: Yvonne Bakken, Skretting, 8450 Storkmarknes, Norway.

Abstract
Intensive ¢sh production worldwide has increased
the risk of infectious diseases. However, before any
infection can be established, pathogens must penetrate the primary barrier. In ¢sh, the three major
routes of infection are the skin, gills and gastrointestinal (GI) tract. The GI tract is essentially a muscular
tube lined by a mucous membrane of columnar
epithelial cells that exhibit a regional variation in
structure and function. In the last two decades, our
understanding of the endocytosis and translocation
of bacteria across this mucosa, and the sorts of cell
damage caused by pathogenic bacteria, has increased. Electron microscopy has made a valuable
contribution to this knowledge. In the ¢sh-farming
industry, severe economic losses are caused by furunculosis (agent, Aeromonas salmonicida spp. salmonicida) and vibriosis [agent, Vibrio (Listonella)
anguillarum]. This article provides an overview of
the GI tract of ¢sh from an electron microscopical
perspective focusing on cellular damage (speci¢c attack on tight junctions and desmosomes) caused by
pathogenic bacteria, and interactions between the
‘good’ intestinal bacteria [e.g. lactic acid bacteria
(LAB)] and pathogens. Using di¡erent in vitro methods, several studies have demonstrated that co-incubation of Atlantic salmon (Salmo salar L.) foregut
(proximal intestine) with LAB and pathogens can

r 2009 The Authors
Journal Compilation r 2009 Blackwell Publishing Ltd

have bene¢cial e¡ects, the cell damage caused by the
pathogens being prevented, to some extent, by the
LAB. However, there is uncertainty over whether or
not similar e¡ects are observed in other species such
as Atlantic cod (Gadus morhua L.). When discussing
cellular damage in the GI tract of ¢sh caused by
pathogenic bacteria, several important questions
arise including: (1) Do di¡erent pathogenic bacteria
use di¡erent mechanisms to infect the gut? (2) Does
the gradual development of the GI tract from larva to
adult a¡ect infection? (3) Are there di¡erent infection
patterns between di¡erent ¢sh species? The present
article addresses these and other questions.

Keywords: probiotics, pathogenic bacteria, gut
integrity, ¢sh

Introduction
With the development of commercial aquaculture, it
has become apparent that diseases can be a signi¢cant limiting factor. Major bacterial pathogens of ¢sh
include the Gram-negative species, Aeromonas salmonicida,Vibrio (Listonella) anguillarum,Vibrio (Aliivibrio)
salmonicida and Yersinia ruckeri, the aetiological
agents of furunculosis, vibriosis, cold-water vibriosis
and red mouth disease respectively. In addition, Aeromonas hydrophila may cause infections in ¢sh and is

451


Lactic acid bacteria vs. pathogens in the gastrointestinal tract of ¢sh E RingÖ et al. Aquaculture Research, 2010, 41, 451^467

generally associated with small surface lesions,
sloughing of scales, local haemorrhage and septicaemia. All these diseases are common worldwide and
produce considerable economic losses during intensive aquaculture of trout and salmon (Austin & Austin 1999).
The best way to avoid disease problems in a system
seems to be through e¡ective management practices,
i.e. management of stock, soil, water, nutrition and
environment. A number of other approaches have
been applied in an attempt to address the disease problem including sanitary prophylaxis, disinfection
and chemotherapy, with particular emphasis on the
use of antibiotics. The application of antibiotics and
other chemicals to pond culture is also quite expensive and undesirable and might lead to antibiotic resistance. In Norway, the use of antimicrobial drugs
has decreased from approximately 50 metric tonnes
in 1987 to 746.5 kg in 1997, measured as active components (Verschuere, Rombaut, Sorgeloos & Verstraete 2000). In 2007, use was still at the same low
level as in 1997 (Norwegian Scienti¢c Committee for
Food Safety 2009) and, recently, vaccinations against
speci¢c diseases have been used.
Intensive ¢sh production has increased the risk of
infectious diseases worldwide (Press & Lillehaug
1995; Karunasagar & Karunasagar 1999) but, to prevent microbial entry, ¢sh deploy protective mechanisms to hinder translocation of pathogens across the
primary barriers. These include production of mucus
by goblet cells, the apical acidic microenvironment of
the intestinal epithelium, cell turnover, peristalsis,
gastric acidity, lysozyme and antibacterial activity of
epidermal mucus (Birkbeck & RingÖ 2005). At the
same time, pathogenic microorganisms have evolved
mechanisms to penetrate these barriers.
Probiotics may reduce the incidence of disease or
lessen the severity of outbreaks. One of the proposed
de¢nitions of probiotics used in aquaculture is ‘live
microbial cultures added to feed or environment

(water) to increase viability (survival) of the host’
(Gram & RingÖ 2005). Probiotic mechanisms include
the production of inhibitory substances against
pathogens, competition for essential nutrients and
enzymes resulting in enhanced nutrition in the host
and the modulation of interactions with the environment and development of bene¢cial immune responses (RingÖ & Gatesoupe 1998; Verschuere et al.
2000; Balcazar, de Blas, Ruiz-Zarzuela, Cunningham,
Vendrell & Muzquiz 2006; Gomez & Balcazar 2008).
Today, it is generally accepted that lactic acid bacteria (LAB) form part of the normal intestinal micro-

452

biota of ¢sh from the ¢rst few days of life (RingÖ &
Gatesoupe 1998; RingÖ 2004; RingÖ, Schillinger &
Holzapfel 2005). One of the most important goals for
microbiologists has been to obtain a stabile indigenous microbiota in ¢sh. The practical e¡ect of this
activity is the exclusion of invading populations of
non-indigenous microorganisms, including pathogens that attempt to colonize the gastrointestinal
(GI) tract (RingÖ et al. 2005). The antagonistic e¡ects
of gut microbiota against pathogens and other organisms are possibly mediated by competition for nutrients and adhesion sites, formation of metabolites
such as organic acids and hydrogen peroxide and
production of bacterocins (for a recent review, devoted to antimicrobial activity of LAB isolated from
aquatic animals, see RingÖ et al. 2005). A fundamental question arises when discussing the protective
role of the GI tract microbiota: can the GI tract of ¢sh
serve as a port of entry for pathogens? During the last
25 years, numerous papers have suggested that the
alimentary tract is involved in Aeromonas and Vibrio
infections (Gro¡ & LaPatra 2000; Birkbeck & RingÖ
2005; Harikrishnan & Balasundaram 2005; RingÖ,
Salinas, Olsen, Nyhaug, Myklebust & Mayhew 2007;

RingÖ, Myklebust, Mayhew & Olsen 2007; Salinas,
Myklebust, Esteban, Olsen, Meseguer & RingÖ 2008).
Therefore, one can hypothesize that LAB and other
bene¢cial bacteria colonizing the GI tract by producing, for example, bacterocins may o¡er protection
against invading ¢sh pathogens.
The aim of this review is to present information on
the interaction between bene¢cial bacteria, in our
case LAB, and pathogenic agents in the digestive
tract of ¢sh using in vitro methods.
Pathogens and cell damage
Historically, Aeromonas salmonicida ssp. salmonicida
(A. salmonicida), the causative agent of furunculosis,
has been recognized as one of the most important
bacterial salmonid pathogens because of its severe
economic impact, especially on the aquaculture industry (Olivier 1997; Bricknell, Bron & Bowden
2006). As early as 1930, the Furunculosis Committee
suggested the intestine as a valuable site for isolating
the bacteria (Mackie, Arkwright, Pyrce-Tannatt,
Mottram, Johnston & Menzies 1930). Since then, controversy has existed as to whether or not the gut can
function as an infection route for this and other
pathogenic bacteria. The presence of A. salmonicida
in the intestine of some ¢sh species (RingÖ, Olsen,
Òverli & LÖvik 1997; LÖdemel, Mayhew, Myklebust,

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Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467


Aquaculture Research, 2010, 41, 451^467 Lactic acid bacteria vs. pathogens in the gastrointestinal tract of ¢sh E RingÖ et al.

Olsen, Espelid & RingÖ 2001; Pedersen & Dalsgaard
2003), together with evidence that salmonids fed
diets containing probiotic bacteria or soybean meal
showed changes in mortality rate after cohabitant
challenge with A. salmonicida (Krogdahl, BakkeMcKellep, RÖd & B×verfjord 2000; Robertson,
O’Dowd, Burrels, Williams & Austin 2000), indicates
that the intestine can be an important route of infection. It is known that pathogenic bacteria produce a
wide array of virulent factors (including haemolysins, cytotoxins, enterotoxins, endotoxins and adhesins), which can a¡ect intestinal barrier function and
facilitate translocation (Chopra, Xu, Ribardo, Gonzalez, Kuhl, Peterson & Houston 2000). Translocation
mechanisms include increased receptor-mediated
endocytosis (Skirpstunas & Baldwin 2002), increased paracellular permeability mediated by e¡ects
on junctional complexes and the cytoskeleton, and
direct damage to the intestinal cells (Fasano 2002).
In ¢sh, bacterial pathogens can enter the host by
one or more of three di¡erent routes: (a) skin, (b) gills
and (c) GI tract (Birkbeck & RingÖ 2005; RingÖ, Myklebust, et al. 2007). If the GI tract is involved as an infection route, mucosal adhesion is considered to be a
critical early phase in all infections caused by pathogenic bacteria (Knudsen, SÖrum, McLPress & Olafsen
1999; Namba, Mano & Hirose 2007). When the bacteria are able to colonize the intestinal mucus, they
can cross the GI tract lining by transcellular or paracellular routes. However, it is suggested that translocation of bacteria across the intestine, an essential
and prerequisite step for bacterial invasion, cannot
be studied e¡ectively using in vivo models. Table1presents an overview of in vivo and in vitro studies of cell
damage in the GI tract of ¢sh. Two di¡erent in vitro
methods, the Ussing chamber and intestinal sac, have
been used to evaluate translocation and cell damage
caused by pathogenic bacteria (RingÖ, Jutfelt, Kanapathippillai, Bakken, Sundell, Glette, Mayhew, Myklebust & Olsen 2004; Jutfelt, Olsen, Glette, RingÖ &
Sundell 2006; LÖvmo 2007a, b; RingÖ, Salinas, et al.
2007; Salinas et al. 2008).
The translocation of viable bacteria from the digestive tract into enterocytes has been reported in several investigations (for a review, see RingÖ, Olsen,
Mayhew & Myklebust 2003; RingÖ, Myklebust, et al.
2007). The phenomenon has mainly been observed

for non-pathogenic indigenous gut bacteria, but not
for pathogenic bacteria, and does not normally compromise cellular integrity. The situation for pathogenic bacteria is completely di¡erent, as severe
damage with loss of cellular integrity has been noted

in an in vitro study where the foregut of Atlantic salmon was exposed toA. salmonicida (RingÖ et al. 2004)
as well as in vivo infection of Atlantic salmon (Bakken
2002) and spotted wol⁄sh fry (Anarhichas minor Olafsen) by V. anguillarum (RingÖ, Mikkelsen, Kaino,
Olsen, Mayhew & Myklebust 2006). As no cell damage was observed in control ¢sh (not exposed to
pathogenic bacteria), we concluded that the indigenous bacteria do not a¡ect cellular integrity. RingÖ
et al. (2004) used the Ussing chamber technique and
observed detached, but almost intact, enterocytes in
the foregut lumen after exposure to A. salmonicida in
vitro. A similar result has been reported in the pyloric
caeca of Atlantic salmon in an in vivo challenge experiment (Fig. 1) and in an in vitro intestinal sac preparation (RingÖ, Salinas, et al. 2007; Salinas et al.
2008). However, in the in vitro experiment of RingÖ
et al. (2004), a quite di¡erent situation seems to occur
in the hindgut region (distal intestine) as no intact
enterocytes were found in the lumen, the microvilli
were disintegrating and some damage to intercellular tight junctions and desmosomes was observed.
The di¡erences between foregut and hindgut have
not been elucidated, but it is probable that enterocytes in di¡erent regions of the GI tract vary in their
susceptibility to pathogen-induced damage. This may
be linked to di¡erent regional rates of epithelial turnover or di¡erent mechanisms of enterocyte loss by
apoptosis or necrosis (Mayhew, Myklebust,Whybrow
& Jenkins 1999). Apoptosis-dependent processes tend
to preserve junctional integrity while necrosis-like
processes tend to be associated with junctional complex disruption and loss of microvillous morphology
(T. M. Mayhew, pers. comm.).
In vitro exposure of Atlantic salmon foregut to Vibrio anguillarum at two concentrations [6 Â 104 and
6 Â 106 colony-forming units (CFU) mL À 1] resulted

in clear changes in the intestinal epithelium compared with samples exposed only to Ringer solution
(control) (RingÖ, Salinas, et al. 2007). At the highest
dose there was an in£ammatory response of gut-associated lymphoid tissue involving leucocytes migrating from the lamina propria towards the lumen.
The di¡erence in bacterial translocation between
indigenous intestinal bacteria and pathogens might
be due to the production, by pathogens, of various
virulence factors such as extracellular enzymes, outer surface components such as S-layer or secretory
proteins and pore-forming toxins (Fivaz & van der
Goot 1999). These factors could result in severe cell
damage as demonstrated in the foregut of Atlantic
salmon (RingÖ et al. 2004).

r 2009 The Authors
Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467

453


454
FG
FG
HG and HGC
HG and HGC

In vitroÃ
In vitroÃ
In vitroÃ
In vitroÃ

Carnobacterium divergens

Vibrio anguillarum

Carnobacterium maltaromaticum

V. anguillarum

In vitro

Edwardsiella tarda

Intestine

In vitro

PC, MG, HG,
HGC

in vivo

HG

FG

In vitro/

In vivo

V. anguillarum

Different bacteria

In vivo

V. anguillarum

FG

NI

NI

NI

Yes

NI

NI

Yes

Yes

Yes

Yes

Yes

Yes

NI

NI

NI

Yes

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

to mucus

Adhesion

wAs only 13 autochthonous bacterial strains were isolated one can not draw a general conclusion.
zUssing chamber.
PC, pyloric cacea; FG, foregut; MG, midgut; HG, hindgut; HGC, hindgut chamber; NI, not investigated.

ÃIntestinal sac.

Several fish species

Spotted wolffish

Vibrio alginolyticus

Five probiotics, A. hydrophila and

In vitroz

A. salmonicida

Spotted grunter

HG

In vitroz

A. salmonicida

Intestine

In vitro

Three LAB strains and four pathogens

Intestine

Intestine

Rainbow trout

In vitro

In vitro

Listonella anguillarum and potential probiotics

Intestine

Lactobacillus L15

In virto

Seven LAB of human and animal orgin

Intestine

Intestine

Intestine

Flounder

Gilthead seabream

In vitro

Aeromonas hydrophila

In vivo

Common carp

FG

In vitroÃ

A. salmonicida

Three different LAB species

FG

In vitroÃ

Lactobacillus delbru¨eckii

Brown trout

FG

In vitroÃ

V. anguillarum

MG

FG

In vitroÃ

A. salmonicida

In vivo

FG

In vitroÃ

C. divergens

A. salmonicida

HG

FG

In vitroz

In vitroz

A. salmonicida

A. salmonicida

In vivo

Aeromonas salmonicida

Arctic charr

Atlantic salmon

PC

segment

Atlantic cod

GIT

used

species

in vivo

Bacteria

Fish

In vitro/

Yes

Yes

Yes

NI

Yes

No

NI

NI

NI

NI

NI

NI

NI

Yes

Yes

No

Yes

Yes

No

Less

Yes

Yes

No

No

No

No

damage

Cell

Yes

Yes

No

NI

No

No

NI

NI

NI

NI

NI

NI

No

Yes

No

No

No

No

No

No

No

No

No

No

No

No

location

Trans-

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

microbiota

Allochthonous

NI

NI

NI

NI

NI

NI

NI

NI

NI

Yes

Yes

microbiotaw

Autochthonous

microbiotaw

Autochthonous

microbiota

Effect on gut

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

Yes

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

response

Immune

Ringø et al. (2007b)

Ringø et al. (2006)

Ringø et al. (2006)

Vine et al. (2004b)

E. Ringø et al. (unpubl. obs.)

(unpubl. obs.)

E. Ringø, R. E.Olsen, K. Sundell & R. Myklebust

Balcazar et al. (2007)

Ying, Lei, Jiazhong, Zhantao & Liguo (2007)

Chabrillon, Arijo, Diaz-Rosales and Balebona (2006)

Chabrillon et al. (2006)

Van der Marel et al. (2008)

Van der Marel, Schroers, Neuhaus & Steinhagen (2008)

Balcazar et al. (2007)

Lødemel et al. (2001)

Salinas et al. (2008)

Salinas et al. (2008)

Ringø et al. (2007)

Ringø et al. (2007)

Ringø et al. (2007)

Ringø et al. (2004)

Ringø et al. (2004)

Bakken (2002)

Løvmo (2007b)

Løvmo (2007b)

Løvmo (2007a)

Løvmo (2007a)

References

Table 1 In vitro and in vivo investigations demonstrating adhesion to mucus, cell damage, translocation and e¡ect of gut microbiota caused by lactic acid bacteria and pathogenic bacteria in
the gastrointestinal tract (GIT) of ¢sh

Lactic acid bacteria vs. pathogens in the gastrointestinal tract of ¢sh E RingÖ et al. Aquaculture Research, 2010, 41, 451^467

r 2009 The Authors
Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 451^467


Aquaculture Research, 2010, 41, 451^467 Lactic acid bacteria vs. pathogens in the gastrointestinal tract of ¢sh E RingÖ et al.

Figure 1 Transmission electron microscopy micrograph
of the pyloric caeca from Atlantic salmon (Salmo salar L.)
challenge with Aeromonas salmonicida ssp. salmonicida.
The enterocytes are damaged and their organelles are exposed to the gut lumen. Several detached enterocytes are
seen in the gut lumen (arrows). After Bakken (2002). MV,
microvilli.

Human studies have shown that di¡erent bacteria
species colonize di¡erent parts of the GI tract (Madigan, Martinko & Parker 2000), and that di¡erent
pathogenic bacteria adhere to, and infect, di¡erent
parts of the GI tract. RingÖ et al. (2004) suggested
that the foregut of Atlantic salmon is an infection site
for A. salmonicida. However, as loosening of cell junctions was observed in the hindgut, this region is also
probably involved in A. salmonicida infection but to a
lesser extent than the foregut.
When discussing intestinal cellular damages, the
results presented for the foregut by RingÖ et al.
(2004) are quite similar to the severe epithelial damage associated with intracellular fat accumulation
from dietary linseed oil (Olsen, Myklebust, RingÖ &
Mayhew 2000). In these studies, cell debris was also
observed in the lumen, providing free access to the
epithelial basal membrane. As no information per se
is available about cell damage and dietary components and pathogens, this topic should be given high
priority in future studies, especially as there is increasing interest in substituting ¢sh oils and ¢sh
meal with vegetable products.

GI microbiota and probiotics in fish
Generally, probiotic strains have been isolated from
indigenous and exogenous microbiota of aquatic animals. Earlier publications stated that bacteria belonging to genus Photobacterium, Pseudomonas andVibrio

were retrieved among the dominant genera in the in-

testine of marine ¢sh (Cahill 1990; Sakata 1990;
RingÖ StrÖm & Tabachek 1995) while the indigenous
microbiota of freshwater ¢sh species tend to be
dominated by members of the genera Aeromonas,
Plesiomonas, representatives of the family Enterobacteriaceae, and obligate anaerobic bacteria of the genera Bacteroides, Fusobacterium and Eubacterium
(Cahill 1990; Sakata 1990; RingÖ et al. 1995). Recent
publications allow one to question whether this is
true, particularly the knowledge and experience
gained from bacteriological studies using, for example, polymerase chain reaction-denaturing gradient
gel electrophoresis (PCR-DGGE) (Gri⁄th, Melville,
Cook, Vincent, St Pierre & Lanteigne 2001; Jensen,
ÒvreÔs, Bergh & Torsvik 2004; Pond, Stone & Alderman 2006; Hovda, Lunestad, Fontanillas & Rosnes
2007; Kim, Brunt & Austin 2007; Liu, Zhou,Yao, Shi,
He, Benjamisen HÖlvold & RingÖ 2008; Zhou, Liu,
Shi, He,Yao & RingÖ 2009).
It is important to note that the population of
endogenous microbiota may depend on genetic, nutritional and environment factors. However, microorganisms present in the immediate environment of
aquatic species have a much larger in£uence on
health status than is the case with terrestrial animals
or humans. The gut microbiota of aquatic animals
probably comprise indigenous microbiota together
with arti¢cially high levels of allochthonous bacteria
maintained by their constant ingestion from the surrounding water (RingÖ & Birkbeck 1999).
While several studies on probiotics have been published during the last decade, the methodological and
ethical limitations of animal studies make it di⁄cult
to understand the mechanisms of action of probiotics, and only partial explanations are available.
Nevertheless, possible bene¢ts linked to administering probiotics have been suggested. They include: (i)
competitive exclusion of pathogenic bacteria (Moriarty 1997; Gomez-Gil, Roque & Turnbull 2000; BalcaŁzar, de Blas, Ruiz-Zarzuela, Vendrell & Muzquiz

2004; Vine, Leukes & Kaiser 2004; RingÖ et al. 2005;
Baccazar, Vendrell, de Blas, Ruiz-Zarzuela, Girones &
Muzquiz 2007); (ii) source of nutrients and enzymatic
contribution to digestion (Prieur, Nicolas, Plusquellec
& Vigneulle 1990; Sakata 1990; RingÖ & Birkbeck
1999); (iii) direct uptake of dissolved organic material
mediated by the bacteria (Moriarty 1997); (iv) enhancement of the immune response against pathogenic microorganisms (Andlid, VaŁzquez-JuaŁrez &
Gustafsson 1995; Scholz, Garcia-Diaz, Ricque, CruzSuarez, Vargas-Albores & Latchford 1999; Rengpipat,
Rukpratanporn, Piyatiratitivorakul & Menasaveta

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455


Lactic acid bacteria vs. pathogens in the gastrointestinal tract of ¢sh E RingÖ et al. Aquaculture Research, 2010, 41, 451^467

2000; Gullian & Rodr|¤ guez 2002; Irianto & Austin
2002a, b; Balcazar 2003; BalcaŁzar et al. 2004); and
(v) antiviral e¡ects (Kamei,Yoshimizu, Ezura & Kimura
1988; Girones, Jofre & Bosch 1989; Direkbusarakom,
Yoshimizu, Ezura, Ruangpan & Danayadol 1998).
Most probiotics proposed as biological control
agents in aquaculture belong to the LAB group (Lactobacillus, Lactococcus, Carnobacterium, Pediococcus,
Enterococcus and Streptococcus), genus Vibrio (Vibrio
alginolyticus), genus Bacillus, genus Pseudomonas,
genus Roseobacter although other genera or species
have also been mentioned (Aeromonas, Alteromonas
and Flavobacterium). Readers with special interest in

the di¡erent aspects of the use of probiotics in aquaculture are referred to the comprehensive reviews of
RingÖ and Gatesoupe (1998), Gatesoupe (1999),
RingÖ and Birkbeck (1999), Skjermo and Vadstein
(1999), Gomez-Gil et al. (2000), Irianto, Roberwen,
Austin and Pandalai (2000), Olafsen (2001),
Verschuere et al. (2000), Irianto and Austin (2002a),
RingÖ (2002, 2004), Burr, Gatlin and Ricke (2005),
Gram and RingÖ (2005), Hong, Duc and Cutting
(2005), RingÖ et al. (2005), Balcazar, de Blas, et al.
(2006), Balcazar, Decamp,Vendrell, de Blas and RuizZarzuela (2006), Farzanfar (2006), Vine, Leukes and
Kaiser (2006), Gatesoupe (2007, 2008), KesarcodiWatson, Kaspar, Lategan and Gibson (2008) and
Tinh, Dierckens, Sorgeloos and Bossier (2008).

LAB
It is well documented that LAB constitute a part of
the indigenous gut microbiota of several ¢sh species
(RingÖ & Gatesoupe 1998; RingÖ 2004; RingÖ et al.,
2005; Balcazar, de Blas, Ruiz-Zarzuela,Vendrell, Calvo,
Marquez, Girones & Muzquiz 2007; Michel, Pelletier,
Boussaha, Douet, Lautraite & Tailliez 2007). In most
of these studies, di¡erent species of Carnobacterium
have been isolated, but Lactobacillus species have also
been isolated from the digestive tract of ¢sh (for a review, see RingÖ & Gatesoupe 1998; Hagi,Tanaka, Iwamura & Hoshino 2004; RingÖ 2004; RingÖ et al. 2005;
Balcazar et al. 2007; Michel et al. 2007; Liu et al. 2008).

Carnobacterium divergens vs. A. salmonicida
and V. anguillarum in Atlantic salmon foregut
In two reviews devoted to LAB in ¢sh and ¢sh farming, RingÖ (2004) and RingÖ et al. (2005) suggested
that LAB and other bene¢cial intestinal bacteria
might be involved in the primary defence system

456

Table 2 Experimental treatments applied to Atlantic salmon (Salmo salar L.) intestine during in vitro exposure to various bacterial strains (colony-forming units, CFU)
Treatment
number
1
2
3
4
5
6
7
8
9
10
11

Bacterial strain and dose (CFU mL À 1)
Salmon Ringer solution
A. salmonicida 6 Â 106
V. anguillarum 6 Â 104
V. anguillarum 6 Â 106
C. divergens 6 Â 104
C. divergens 6 Â 106
A. salmonicida 3 Â 106 and C. divergens
3 Â 106
V. anguillarum 3 Â 104 and C. divergens
3 Â 104
V. anguillarum 3 Â 104 and C. divergens

3 Â 106
V. anguillarum 3 Â 106 and C. divergens
3 Â 104
V. anguillarum 3 Â 106 and C. divergens
3 Â 106

After RingÖ et al. (2007).
The estimate bacterial exposure of the foregut was measured by
plate counts. Stock solution was diluted in sterile 0.9% saline,
and 0.1mL volumes of appropriate dilutions were spread on the
surface of brainheart infusion agar (Merck, Darmstadt, Germany) (BHI) (Aeromonas salmonicida) and tryptic soy agar (TSA;
Oxoid, London, UK) added glucose (5 g L À 1) and NaCl (15 g L À 1)
TSA plates (V ibrio anguillarum and Carnobacterium divergens).

against pathogenic colonization and adherence of
pathogenic bacteria in the GI tract. Several LAB isolated from ¢sh and aquatic animals display antagonistic activity against ¢sh pathogenic agents (RingÖ
et al. 2005; RingÖ 2008). To test the hypothesis that
LAB can prevent pathogen-induced damage, RingÖ,
Salinas, et al. (2007) used C. divergens strain 6251, originally isolated from the foregut of the Artic charr
(Salvelinus alpinus L.) (RingÖ & Olsen 1999). This
strain has growth-inhibitory e¡ects against both
A. salmonicida and V. anguillarum (RingÖ, Sepploa,
Berg, Olsen, Schillinger & Holzapfel 2002; RingÖ
2008). The aim was to investigate, by means of light
and electron microscopy, the structural changes that
Atlantic salmon intestine underwent following in
vitro exposure to A. salmonicida, V. anguillarum and
C. divergens at two di¡erent doses (Table 2). In the
study by RingÖ et al. (2004), only foregut samples
were examined as this portion of the gut seems to be

a more likely infection route for pathogenic bacteria
in Atlantic salmon. Furthermore, the potentially protective role of C. divergens against pathogen-induced
damage has been evaluated by simultaneously exposing intestinal mucosa to one of the pathogenic

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Aquaculture Research, 2010, 41, 451^467 Lactic acid bacteria vs. pathogens in the gastrointestinal tract of ¢sh E RingÖ et al.

Table 3 Morphological description of Atlantic salmon
foregut treated with various types of bacteria (1^11; see
Table 2)
Bacterial treatment type
Morphology

1 2

3

4

5 6 7 8 9 10 11

Presence of cell debris in
lumen
Disorganized microvilli
Protruding epithelial cells
Oedema
Disintegrated tight junctions

Dark cellular bodies under LM
Loss of epithelial integrity
Phagolysosome-like vesicles
with bacteria
Bacteria-like particles close to
tight junctions
Column totals

0

3

3

3 0 0 0 2 0

2

2

0
0
0
0
0
0
0

2
0

2
2
0
2
0

3
3
0
3
3
3
2

3
3
0
3
3
3
0

1
0
1
0
2
1
0

3
3
0
3
3
3
0

3
3
0
3
3
3
0

0

0

0

0 0 0 3 0 0

0

3

1
0

0
0
0
0
0

1
0
0
0
0
0
0

0
0
3
1
0
2
0

1
2
0
2
0
2
0

0 11 20 18 1 1 9 9 5 17 20

After RingÖ et al. (2007).
Damage and tissue changes were assessed as follows: 0 5 not
observed, 1 5low frequency, 2 5 moderate frequency, 3 5 high
frequency.
Apparent di¡erences between treatments were signi¢cant
(Po0.01).

strains and to the probiotic strain. Both pathogens
used in the study of RingÖ, Salinas, et al. (2007) have
been shown previously to enter ¢sh through the GI
tract barrier (O’Toole, Hofsten, Rosqvist, Olsson &
Wolf-Watz 2004; RingÖ et al. 2004).
As shown in Table 3, clear di¡erences were observed between the pathogenic (A. salmonicida and
V. anguillarum) and the non-pathogenic (C. divergens)
strains. Treatment of the foregut with pathogens, at
both assayed concentrations, results in various damaging e¡ects: epithelial cells with altered microvilli
(Fig. 2), damaged tight junctions, protruding epithelial cells sloughing into the lumen and the presence
of cell debris in the gut lumen. Similar ¢ndings were
not observed following exposure to C. divergens. The
di¡erent morphological changes caused byA. salmonicida orV. anguillarum co-incubated with C. divergens
also suggest di¡erent invasive and pathogenetic mechanisms between the two pathogenic bacterial
strains. The foregut exposed to C. divergens was histologically similar to control samples showing an intact epithelial barrier. This observation is in
agreement with the results of RingÖ et al. (2004),
who suggested that the commensal microbiota do
not a¡ect gut cellular integrity.
Surprisingly, exposure to V. anguillarum at
3 Â 104 mL À 1 results in the presence of phagolysosome-like vesicles containing degraded bacteria in

Figure 2 Scanning electron micrograph of the foregut of
Atlantic salmon exposed to 6 Â 106 Vibrio anguillarum.
Note several detached/detaching enterocytes lacking uniform microvilli at the epithelial surface. After I. Salinas,
E. RingÖ and R. Myklebust (unpubl. obs.).

the cytoplasm of enterocytes. An immunohistochemical study with anti-V. anguillarum antibody
might support the nature of these degraded bacteria,
together with the observation of bacteria-like structures seen close to the tight junctions between enterocytes treated with V. anguillarum and C. divergens.
Whether these particles are the pathogenic or probiotic bacteria remains unclear, and identi¢cation of
the unknown bacteria-like particles by immunogold
labelling techniques, green £uorescence protein and
in situ £uorescence hybridization might elucidate
some of these questions and will be the subject of
further work. The ¢nding of phagolysosome-like
structures suggests that the enterocytes process the
bacteria and act as phagocytic cells or as antigenpresenting cells in the same way as M-cells in higher
vertebrates.
The histological changes described above were
present in most samples, and so C. divergens seems to
be unable to completely prevent tissue damage by
A. salmonicida in Atlantic salmon foregut when the
pathogen was present at the highest concentration.
In the case of V. anguillarum at the lowest concentration (3 Â 104 CFU mL À 1), reduced tissue damage was
observed when Carnobacterium was present at both a
high and a low concentration, indicating a possible
protective e¡ect of C. divergens. However, this observation might also be attributable to the low concentration of the pathogen in groups in which the
intestine was exposed to both the pathogen and the
probiont, compared with foregut exposed only to the
pathogen. To clarify this hypothesis, additional studies are necessary.

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Lactic acid bacteria vs. pathogens in the gastrointestinal tract of ¢sh E RingÖ et al. Aquaculture Research, 2010, 41, 451^467

Table 4 Outcome of comparison of in vitro e¡ects of experimental treatments on Atlantic salmon intestine (seeTable 2)
ranked by order of severity from 1 5control (Ringer) to
11 5V ibrio anguillarum at 6 Â 106 (Page’s L test, Po0.01)
Ranking

Bacterial strain and dose (CFU mL À 1)

1
2–3
4
5–6

Salmon Ringer solution
C. divergens 6 Â 104 or 6 Â 106
V. anguillarum 3 Â 104 and C. divergens 3 Â 106
V. anguillarum 3 Â 104 and C. divergens 3 Â 104;
A. salmonicida 3 Â 106 and C. divergens 3 Â 106;
A. salmonicida 6 Â 106
V. anguillarum 3 Â 106 and C. divergens 3 Â 104;
V. anguillarum 6 Â 106
V. anguillarum 3 Â 106 and C. divergens 3 Â 106;
V. anguillarum 6 Â 104

7
8
9
10–11

After RingÖ et al. (2007).

The partially reduced tissue damage observed
when the foregut was exposed to C. divergens in combination withV. anguillarum at the lowest population
level may be explained by C. divergens inhibiting the
growth of the pathogens in vitro (RingÖ et al. 2005;
RingÖ 2008) and, thereby, colonization. However,
the probiotic bacteria do not seem to reduce the tissue-damaging e¡ect when the gut was exposed to a
high concentration of V. anguillarum. The reason for
this has not been elucidated, although it may be attributable to the pathogen out-competing the probiotic
bacteria.
When C. divergens was present at the highest dose,
and the pathogen at the lowest dose, less damage was
seen (Table 4). To some extent, this observation might
be due to the antagonistic activity of the probiotic
bacteria against the pathogens, resulting in fewer
live pathogens being available to colonize the foregut.
Based on this hypothesis, more information on the
administration of appropriate levels of probiotic bacteria is needed.

Lactobacillus delbrueckii ssp. lactis vs.
A. salmonicida in Atlantic salmon foregut
It is generally accepted that Lactobacillus delbrueckii is
a heterogeneous group of bacteria that includes

three subspecies: ssp. delbrueckii, ssp. bulgaricus and
ssp. lactis (Weiss, Schillinger & Kansdler 1983). Jacobsen, Rosenfelt Nielsen, Hayford, MÖller, Michaelsen,
Paerregaard, Sandstr˛m, Tvede and Jakobsen (1999)
suggested that L. delbrueckii ssp. lactis is a good probiotic candidate according to its bacteriological prop-

458

erties in vitro. From an aquaculture point of view,
L. delbrueckii ssp. lactis has been administered in vivo
in the diet to gilthead seabream (Sparus aurata L.) and
increased the cellular innate immune responses (Salinas, Cuesta, Esteban & Meseguer 2005). The fate of
probiotic bacteria in the GI tract of ¢sh is less well
known, including the histological changes that
might take place in the gut following incubation with
LAB like L. delbrueckii ssp. lactis.
The aims of the work of Salinas et al. (2008) were,
by means of microscopy techniques, to (i) determine
whether an L. delbrueckii ssp. lactis (CECT 287,Valencia, Spain) of non-aquaculture origin was capable of
colonizing the Atlantic salmon GI tract during in vitro
incubation, (ii) describe the morphological changes
and cellular responses occurring in the intestinal
epithelium after in vitro exposure to this probiotic
strain and (iii) study the possible protective role of
L. delbrueckii ssp. lactis against tissue-damaging effects caused byA. salmonicida in the foregut of Atlantic salmon. This information is highly relevant as it is
well known that probiotic bacteria increase the host
health status and protect mucosal tissue against
pathogen-caused damage in mammalian models
(Fung, Woo, Wan-Abdullah, Ahmad, Easa & Liong
2009). Using confocal microscopy, their ¢ndings
showed that, following short-term in vitro incubation

of Atlantic salmon foregut with tetramethylrhodamine isothiocyanate (TRITC)-labelled L. delbrueckii
ssp. lactis, the probiont was able to colonize the enterocyte surface.When L. delbrueckii ssp. lactis were observed in the lumen, the bacteria were found in
groups or clumps (Fig. 3a). Moreover, labelled bacteria were also found at the villus surface, inside the mucosal epithelium or even in the lamina propria (Fig.
3b). As the intestines were thoroughly washed three
times before samples were taken, only those bacteria
able to adhere to the mucus or to the epithelial cells
were present. Moreover, foregut exposed to the probiotic bacteria only resulted in a healthy intestinal
barrier whereas exposure to A. salmonicida disrupted
its integrity. However, pre-treatment of salmon intestine with L. delbrueckii ssp. lactis prevented Aeromonas damaging e¡ects (Table 5). These results are
promising in the context of the use of non-autochthonous probiotic bacteria as prophylactic agents
against ¢sh bacterial infections in the GI tract.
Although Nikoskelainen, Salminen, Bylund and
Ouwehand (2001) studied the adhesion capacity of
di¡erent LAB strains to rainbow trout (Oncorhynchus
mykiss Walbaum) mucus in vitro, probiotic bacteria
had never been shown to cross the ¢sh gut before.

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Aquaculture Research, 2010, 41, 451^467 Lactic acid bacteria vs. pathogens in the gastrointestinal tract of ¢sh E RingÖ et al.

Whereas some authors (Fuller 1973; MÌyrÌ-MÌkinen,
Manninen & Gyllenberg 1983) have postulated that
probiotic bacteria are host speci¢c (and their e¡ects
are limited to their natural hosts), three studies have
reported the absence of speci¢city in LAB when binding host intestinal mucus (Gildberg & Mikkelsen
1998; Rinkinen, Westermarck, Salminen & Ouwehand 2003; Salinas et al. 2008). It was also evident
that the LAB species used by Salinas et al. (2008) ex(b) LP

(a)

V

L

M
N
L

LP

L

L

V
L
20µ

40µ

Figure 3 Confocal microscopy of the fate of tetramethylrhodamine isothiocyanate-labelled Lactobacillus
delbrueckii ssp. lactis in the foregut of Atlantic salmon. (a)
Fluorescent image of Atlantic salmon foregut cryosection
after incubation for 30 min with L. debrˇeckii subsp. lactis
[107 colony-forming units (CFU) mL À 1] labelled with
TRITC. L, lumen; M, mucus. Note the red £uorescence of
labelled bacteria associated with mucus in the gut lumen.

Scale bar 5 20 mm. (b) Fluorescent image of Atlantic salmon foregut cryosection after incubation for 30 min with
L. debrˇeckii subsp. lactis (107 CFU mL À 1) labelled with
TRITC. Note the red £uorescence of labelled bacteria located inside the villi (V) at the level of the lamina propria
(LP). Scale bar 5 40 mm. After Salinas et al. (2008). L, lumen; N, nuclei of enterocytes.

erted a pronounced local e¡ect on the GI tract lining.
The authors’ qualitative ¢nding, that the concentration of immune cells in the gut epithelium increased
following incubation with the probiotic strain, resembled the response that occurred when the intestine was exposed to A. salmonicida (RingÖ, Salinas,
et al. 2007). This is consistent with a recent study that
found increased numbers of acidophilic granulocytes
in the gut of seabream (S. aurata L.) larvae after a probiotic, a mixture of Lactobacillus fructivorans and Lactobacillus plantarum, was delivered through the diet
(Picchietti, Mazzini, Taddei, Renna, Fausto, Mulero,
Carnevali, Cresci & Abelli 2007).
Some information is available demonstrating that
A. salmonicida causes cell damage in the foregut of
Atlantic salmon (RingÖ et al. 2004; RingÖ, Salinas,
et al. 2007; Salinas et al. 2008), but these studies used
the pathogen without washing the culture supernatant. The e¡ects observed in these studies might be
due either to toxins, to the bacteria themselves or to
both toxins and bacterial cells. However, as a blocking e¡ect was observed when the intestine was exposed to the lactobacilli (Salinas et al. 2008), it can
be speculated that the lactobacilli compete with the
pathogen in adherence to the mucus layer, by producing bacteriocins, organic acid, H2O2, etc., which
would inhibit pathogenic colonization. The hypothesis proposed by Salinas et al. (2008) is in accord with
the results of Chabrillon, Ouwehand, Diaz-Rosales,
Arijo, Martinez-Manzanares, Balebona and Morinigo
(2006), who demonstrated that attachment of the
pathogenic bacteria (V. anguillarum, Photobacterium
damselae ssp. piscicida,V. alginolyticus and Vibrio harveyi) to intestinal mucus of Gilthead seabream was
signi¢cantly reduced by Lactobacillus rhamnosus and

Table 5 Morphological changes undergone byAtlantic salmon foregut following exposure toAeromonas salmonicida ssp. salmonicida, Lactobacillus delbrˇeckii ssp. lactis or both bacterial strains

Morphological observations

Control

Aeromonas
only

Lactobacillus
only

Lactobacillus1
Aeromonas

Presence of cell debris in the lumen
Disorganized microvilli
Oedema
Disintegrated tight junctions
Loss of epithelial integrity
Leucocyte regrouping
Presence of rodlet cells
Bacteria in lumen or between microvilli
Bacteria paracellularly

0
0
0
0
0

0
1
0
0

3
0
2
2
2
3
2
2
0

0
0
0
0
0
3
3
3
3

1
1
0
0
0

3
3
2
0

Tissue changes were assessed as follows: (0) not observed; (1) low frequency; (2) moderate frequency; (3) high frequency.
After Salinas et al. (2008).

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Lactic acid bacteria vs. pathogens in the gastrointestinal tract of ¢sh E RingÖ et al. Aquaculture Research, 2010, 41, 451^467

Bi¢dobacterium lactis intended for human use. These
results, together with those of Vine, Leukes, Kaiser,
Baya and Baxter (2004) working on the topic of attachment competition of probiotics and pathogenic
bacteria to ¢sh intestinal mucus, should stimulate
bacteriologists to carry out further studies on this
topic.
In spotted wolf ¢sh (A. minor Olafsen) fry exposed
toV. anguillarum, paracellular bacterial translocation
seems to occur as a secondary e¡ect of detachment
and loss of enterocytes that create large intercellular
spaces (RingÖ et al. 2006). In contrast to these results,
Salinas et al. (2008) reported paracellular bacteria between the enterocytes when the foregut was exposed
to L. delbrueckii ssp. lactis. This translocation occurred in virtually intact gut epithelia, which means
that translocation of the probiotic bacteria was not

associated with the creation of oedematous spaces, a
characteristic of pathogenic bacteria.
Carnobacteria vs. Vibiro (Listonella)
anguillarum in the GI tract of Atlantic cod
Atlantic cod was recently introduced into aquaculture and is common at the ¢shmonger and popular
across Northern Europe (Bowden, Adamson & Bricknell 2003). There are many reports on diseases in
Atlantic cod, on wild stocks and in aquaculture (for
a review, see Egidius 1987; Toranzo, Mamarinos &
Romalde 2005; Bricknell et al. 2006; Samuelsen, Nerland, JÖrgensen, SchrÖder, SvÔsand & Bergh 2006).
The major pathogenicVibrio of Atlantic cod in culture
is V. anguillarum serotype 02b, and there is evidence
that current salmonid vaccines, which contain serotypes 01 and 02a and V. ordalii, are not e¡ective in
protecting against infection with the 02b serotypes
(J. BÖgvald, pers. comm.). As culture of Atlantic cod
has become more common, the 02b serotype appears
to have become the dominant isolate (Santos, Pazos,
Bandin & Toranzo 1995).
Based on the available information that V. anguillarum serotype 02b is an important bacterial pathogen in gadoid culture, LÖvmo (2007a, b) conducted
studies using the intestinal sac method to evaluate
the potential protective role of carnobacteria against
V. anguillarum serotype 02b in the GI tract of Atlantic
cod. The GI tract was divided into six di¡erent parts:
stomach, pyloric caeca, foregut, midgut and hindgut
(distal intestine) and the hindgut chamber (fermentation chamber). LÖvmo (2007a) used the same carnobacteria strain previously used by RingÖ, Salinas,
et al. (2007) and exposed the foregut to 2 Â 106 V. an-

460

guillarum mL À 1 or 6.4 Â 106 C. divergens mL À 1. The
aims of this in vitro study were to investigate whether

exposure of the intestinal mucosa of the Atlantic cod
toV. anguillarum a¡ects the morphology of the intestinal epithelium in the foregut, and to compare these
results with those observed by exposing intestines to
a probiotic bacterium and another segment not exposed to bacteria. As shown in Table 6, some di¡erences were apparent when the foregut was exposed
to bacteria compared with the non-exposed control
group. All three experimental groups showed a normal-looking mucosa with an intact epithelium, consisting of undamaged enterocytes with numerous
normal-looking microvilli. However, the foregut exposed to bacteria showed paler epithelial cell nuclei,
disorganized microvilli and budding from the apices
of microvilli. Goblet cells were normally ¢lled when
the intestine was exposed to bacteria, both C. divergens and V. anguillarum, while the goblet cells were
empty when the intestine was exposed to the Ringer
solution. Based on the results that the foregut exposed to V. anguillarum was histologically similar to
the foregut exposed to C. divergens, it was suggested
that the foregut is not a major infection site forV. anguillarum. The result of Carnobacterium exposure is of
high importance as translocation and cell damage
have been proposed as important criteria when evaluating the use of probiotics in endothermic animals
as well as in ¢sh (Salminen, Deighton, Benno & Gorbach 1998; RingÖ, Myklebust, et al. 2007).

Table 6 Morphological description of Atlantic cod (Gadus
morhua L.) foregut exposed to no bacteria (control),Vibrio anguillarum and Carnobacterium divergens
Morphological
observations
Intraepithelial
lymphocytes
Disorganised microvilli
Filled goblet cells
Empty goblet cells
Loosening of
enterocytes from basal
membrane

Presence of rodlet cells
Macrophages
Granulocytes
Oedema
Terminal web (mm)

Control V. anguillarum C. divergens
0

2

3

0
0
2
0

2
2
0
1

2
2
0
1

0
0

0
0
1.0

1
1
2
0
1.0–1.5

1
0
1
0
1.5–2.0

Damage and tissue changes were assessed as follows: 0 ^ not observed, 1 ^ low; 2 ^ moderate and 3 ^ high frequency.
After LÖvmo (2007a).

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Table 7 Experimental treatments applied to Atlantic cod hindgut chamber during in vitro exposure to various bacterial
strains

Treatment

Bacterial strain and dose

Exposure
time (min)

1
2
3
4w

Sterile physiological saline
C. maltaromaticum 106
V. anguillarum 106
C. maltaromaticum 106
V. anguillarum 106
V. anguillarum 106
C. maltaromaticum 106

60
60
60
30
–
30
–

5z

WashedÃ
Yes

Yes
Yes
Yes
Yes

Exposure
time (min)

WashedÃ

–
30
–
30

Yes
Yes

Dose 5 colony forming units mL À 1. After LÖvmo (2007b).
ÃBefore sampling the hindgut chamber was washed times with 3 mL sterile saline.
wThe hindgut chamber was ¢rst exposed to Carnobacterium maltaromaticum (30 min), washed (three times with 3 mL sterile saline) and
thereafter exposed to V. anguillarum for 30 min.
zThe hindgut chamber was ¢rst exposed to Vibrio anguillarum (30 min), washed (three times with 3 mL sterile saline) and thereafter
exposed to C. maltaromaticum for 30 min.
The estimate bacterial exposure of the hindgut chamber was measured by plate counts. Stock solution was diluted in sterile 0.9%
saline, and 0.1mL volumes of appropriate dilutions were spread on the surface of and tryptic soy agar plates.

In a later study, LÖvmo (2007b) investigated the effect of bacterial exposure in both the hindgut and the
hindgut chamber. Here, only results obtained from
the hindgut chamber are presented, as this part of

the digestive tract seems to be more important, at
least from electron microscopic and bacteriological
perspectives. A detailed description of the di¡erent
experimental groups exposed to bacteria in the hindgut chamber is shown in Table 7. An adherent Carnobacterium originally isolated from the hindgut
chamber of Atlantic cod (Seppola, Olsen, Sandaker,
Kanapathippillai, Holzapfel & RingÖ 2006) and V. anguillarum serotype 02b was used. Previously, Carnobacterium has been shown to inhibit di¡erent
bacteria (Listeria monocytogenes, Enterococcus faecalis
and Staphylococcus aureus) in vitro (M. Seppola and
E. RingÖ unpubl. obs.), and the carnobacteria were
identi¢ed by 16S rRNA to Carnobacterium maltaromaticum (LÖvmo 2007b).
Log total viable counts detected in the hindgut
chamber of treatment groups 1, 4 and 5 yielded log
values of 4.6, 4.6 and 4.8 bacteria g À 1 respectively.
These results showed that the total bacterial population level was not a¡ected by bacterial exposure. A
total of 42, 44 and 39 isolates were identi¢ed by 16S
rRNA from experimental groups 1, 4 and 5, respectively, and these results demonstrated that the autochthonous bacterial communities were a¡ected (Fig.
4a^c). The dominant bacterial species identi¢ed in
the hindgut chamber of the control ¢sh (not exposed
to bacteria) wasVibrio wodanis (42%), followed byVibrio logei (25%) and Vibrio ¢scheri (24%) (Fig. 4a).

Photobacterium phosphoreum and Staphylococcus epidermis were also identi¢ed.When the hindgut chamber was ¢rst exposed to C. maltaromaticum, washed
three times and then exposed toV. anguillarum,V. wodanis was still the dominant autochthonous species
and accounted for 44% of the identi¢ed strains,
while V. anguillarum and C. maltaromaticum accounted for 22% and 20% respectively (Fig. 4b). In
comparison, when the hindgut chamber was ¢rst exposed toV. anguillarum, washed three times and then
exposed to C. maltaromaticum for 30 min, C. maltaromaticum accounted for 54% of the autochthonous
species but V. wodanis was still a dominant autochthonous species and accounted for 35% of the
identi¢ed strains. In this experimental group,V. anguillarum was not identi¢ed (Fig. 4c).
Based on the results of LÖvmo (2007b), it can be
concluded that V. wodanis belongs to the autochthonous microbiota in the hindgut chamber of Atlantic

cod and that its population level is not a¡ected by exposing the hindgut chamber to bacteria such as
C. maltaromaticum and V. anguillarum. As C. maltaromaticum is present in the hindgut chamber when the
chamber was ¢rst exposed to C. maltaromaticum and
thereafter toV. anguillarum, or ¢rst exposed toV. anguillarum and thereafter to C. maltaromaticum, this indicates that the carnobacteria are able to colonize the
hindgut chamber and out-compete V. anguillarum.
This is clearly demonstrated in experimental group
5, where LÖvmo (2007b) was not able to isolateV. anguillarum from the hindgut chamber post exposure
to the pathogen and thereafter to C. maltaromaticum.

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Lactic acid bacteria vs. pathogens in the gastrointestinal tract of ¢sh E RingÖ et al. Aquaculture Research, 2010, 41, 451^467

(a)

Fermentation chamber. Control fish
Vibrio wodanis

24 %
42 %

Photobacterium
phosphoreum
Staphylococcus
epidermis
Vibrio logei

25 %
2% 7%

Vibrio fischeri

Fermentation chamber. Fish first exposed to
C. maltaromaticum and thereafter to
V. anguillarum

(b)

Vibrio wodanis

20 %
3%

44 %

Photobacterium
phosphoreum
Staphylococcus
sp.
Vibrio anguillarum

22 %

Vibrio fischeri

3%8%

(c)

Carnobacterium
maltaromaticum

Fermentation chamber. Fish first exposed to
V. anguillarum and thereafter to
C. maltaromaticum
Vibrio wodanis

35 %

Photobacterium
phosphoreum
Shewanella
hanedai

54 %
3%
5%
3%

Vibrio logei
Carnobacterium
maltaromaticum

Figure 4 Relative abundance of adherent microbiota in
the hindgut chamber of Atlantic cod exposed to (a) sterile
physiological salt water, (b) Carnobacterium maltaromaticum and Vibrio anguillarum and (c) V. anguillarum and

C. maltaromaticum. A detailed description of the di¡erent
treatment is given in Table 7. After LÖvmo (2007b).

Moreover, the potential of this carnobacteria is also
illustrated by the fact the strain did not a¡ect morphology in the hindgut of Atlantic salmon. However,
as exposure of the hindgut chamber of Atlantic cod by
V. anguillarum did not cause cell damage, it is concluded that this part of the digestive tract is not involved as an infection route. Based on the results of
RingÖ et al. (2004), RingÖ, Salinas, et al. (2007), RingÖ,
Myklebust, et al. (2007) and Salinas et al. (2008) on
Atlantic salmon and the results of LÖvmo (2007a, b)

462

on Atlantic cod, we conclude that the intestinal infection route is di¡erent in these two species.

Future perspectives
In the studies of RingÖ, Myklebust, et al. (2007) and
Salinas et al. (2008), light and electron microscopy
were used. LÖvmo (2007a, b) also used light and electron microscopy, and isolation of culturable bacteria,
to evaluate cell damage and the ability to adhere to
the di¡erent parts of the digestive tract. As the GI
tract in some ¢sh species might be an important infection route (Gro¡ & LaPatra 2000; Birkbeck &
RingÖ 2005; Harikrishnan & Balasundaram 2005),
future studies on this subject have to include characterization using modern molecular biological DNAbased methods. In these studies, both microbes
attached to the epithelium and microbes associated
with intestinal digesta have to be assessed. Samples
should be subjected to isolation of bacteria from the
digesta and mucus material, lysing of the isolated
bacteria and DNA extraction using mechanical, chemical and enzymatic breakdown as described by Holben, SÌrkilahti, Williams, Saarinen and Apajalahti
(2002). In the exploratory phase, the microbial community should be assessed by amplifying the DNA

with universal primers for sequencing of the taxonomically important16S rRNA gene. Once the composition of the microbial community is covered, and the
most important microbiological clusters in relation to
the nutritional and health status of the salmon
are discovered, rapid tools based on quantitative
real-time PCR can be developed to routinely monitor
the important indicator organisms. However, the
threshold for bacterial cDNA may be a limiting factor in gut samples from ¢sh, except in acute-phase
infection. In addition to DNA-based techniques, important gut bacteria isolated by cultivation should
be investigated for their metabolic capabilities.
Furthermore, the metabolic pro¢les in the digesta
samples should be studied using gas and liquid
chromatography.
Future studies should also include di¡erent staining methods such as immunogold, green £uorescent
protein (GFP) and quorum sensing (QS). The introduction of GFP as an endogenous £uorescent tag provides a means of rendering the bacteria visible and
tracing their activity in living host cells (Valdivia,
Hromockyj, Monack, Ramakrishnan & Falkow 1996;
Lun & Willson 2004; Yin, Zhou, Li, Li, Hou & Zhang
2007). Green £uorescent protein is a small protein

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Aquaculture Research, 2010, 41, 451^467 Lactic acid bacteria vs. pathogens in the gastrointestinal tract of ¢sh E RingÖ et al.

(27 kDa) found in the jelly¢sh, Aequorea victoria. It
£uoresces when excited by ultraviolet (UV) light
(Chal¢e, Tu, Euskirchen, Ward & Prasher 1994). Bacteria tagged with the GFP gene can be easily identi¢ed as green £uorescing colonies under UV light.
The GFP £uorescence is independent of cofactors or
additional gene products, is sensitive, stable, speci¢c,

non-toxic and does not interfere with cell growth and
function (Chal¢e et al. 1994; Valdivia et al. 1996).
Green £uorescent protein-marked ¢sh pathogens
have been constructed to study the invasion pathways in vivo and in vitro (Ling, Xie, Lim & Leung
2000; O’Toole et al. 2004). Based on this information,
we recommend using GFP as a biomarker to illustrate
the infection kinetics and tissue localization of both
probiotic and pathogenic bacteria in future in vitro
studies.
In recent years, the ability of bacteria to communicate with one another using chemical signal
molecules has received considerable attention in
Gram-negative ¢sh pathogenic bacteria (Defoirdt,
Boon, Bossier & Verstraete 2004; Bruhn, Dalsgaard,
Nielsen, Buchholtz, Larsen & Gram 2005; Buchholtz,
Nielsen, Milton, Larsen & Gram 2006; Defoirdt, Boon,
Sorgeloos, Verstraete & Bossier 2008). The ability to
send, receive and process information allows unicellular organisms to act as multicellular entities and
increases their chances of survival in complex environments. Quorum sensing is commonly associated
with adverse health e¡ects such as bio¢lm formation,
bacteria pathogenicity and virulence, and it may provide a way of controlling infection in aquaculture
(Defoirdt et al. 2004, 2008). Recently, it was demonstrated that probiotics (Lactobacillus acidophilus La-5)
could a¡ect virulence-related gene expression in
Escherichia coli O157:H7 by secreting a molecule(s)
that either acts as a QS inhibitor or directly interacts
with genes involved in colonization (Medellina-Pena,
Wang, Johnson, Anand & Gri⁄ths 2007). As this information is not available in ¢sh, we recommend that
such studies are carried out.
It is generally accepted that probiotics block pathogenic bacterial e¡ects by producing bactericidal substances and competing with pathogens and toxins
for adherence to the intestinal epithelium. They also
regulate immune responses by enhancing innate immunity and modulating pathogen-induced in£ammation via Toll-like receptor-regulated signalling

pathways and regulate intestinal epithelial homeostasis by promoting intestinal epithelial cell survival,
enhancing barrier function and stimulating protective responses (Vanderpool, Yan & Polk 2008). As no

information is available in ¢sh, these topics should
be given high priority in future studies.
A fundamental question arises when discussing
the protective role of the GI tract microbiota: Can the
gradual development of the GI tract from larva to
adult a¡ect infection and are there di¡erent infection
patterns between di¡erent ¢sh species? Again, as no
information is available on these topics, scientists
might pro¢tably investigate the interactions between
LAB and pathogens in the digestive tract of ¢sh.

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doi:10.1111/j.1365-2109.2009.02334.x

Aquaculture Research, 2010, 41, 468^480

Changes to the histological gill structure and
haemolymph composition of early blue swimmer
crab Portunus pelagicus juveniles during elevated
ammonia-N exposure and the post-exposure recovery
Nicholas Romano & Chaoshu Zeng
School of Marine and Tropical Biology, James Cook University, Townsville, Qld, Australia
Correspondence: N Romano, School of Marine and Tropical Biology, James Cook University, Townsville, Qld 4811, Australia. E-mail:


Abstract

Introduction

It is yet unclear whether sub-lethal ammonia-N
levels cause irreparable damage to aquatic crustaceans, or if recovery is possible, the potential factors
involved. The aim was to investigate the e¡ect of
0.706 and 2.798 mmol L À 1 ammonia-N exposure on
the haemolymph osmolality, Na1, K1, Ca21, pH, ammonia-N, total haemocyte counts (THC) and gill histopathology of Portunus pelagicus juveniles at 0, 3, 6,
12, 24 and 48 h respectively. Following 48 h, crabs
were transferred to pristine seawater allowing a recovery period up to 96 h and similarly measured. In
addition moribund crabs, induced from lethal ammonia-N levels of 7.036 and10.518 mmol L À 1, were measured for haemolymph osmolality/ions and pH levels.
The results demonstrate that despite severe gill damage within 6- and 1h of 0.706 and 2.798 mmol L À 1
ammonia-N exposure, respectively, no signi¢cant
change (P40.05) in the haemolymph osmolality,
Na1, K1, Ca21 or pH levels occurred or by ammonia-N-induced morbidity. Although the gills can
completely recover within 24 and 48 h post exposure
to 0.706 and 2.798 mmol L À 1 ammonia-N, respectively, likely facilitated by signi¢cant haemocyte increases (Po0.05) within the haemolymph and gill
lamellae, dependent factors were the previous ammonia-N concentration and recovery duration while
individual variability was also noticed.

Ammonia is the ¢rst stage of the nitri¢cation cycle
and among the nitrogenous pollutants of nitrite
and nitrate, ammonia is often the most toxic to
aquatic animals (Meade & Watts 1995; Romano &
Zeng 2007a^c). On closed aquaculture systems that
often utilize high stocking densities with intensive
feeding, periodic spikes of sub-lethal ammonia-N
levels are a ubiquitous concern for aquaculture
farmers (Timmons, Ebeling, Wheaton, Summerfelt
& Vinci 2002; Kir & Kumlu 2006). Furthermore,

anthropogenic runo¡ and discharges of e¥uent
water from aquaculture systems also makes ammonia an ecologically relevant pollutant to aquatic
crustaceans (Biao, Zhuhong & Xiaorong 2004; Dave
& Nilsson 2005).
It has been previously demonstrated that elevated
ammonia-N can rapidly cause severe damage to the
gill structure of crustaceans including necrosis,
hyperplasia, epithelial damage, pillar cell (PC) disruption and lamellae collapse (Rebelo, Rodriguez,
Santos & Ansaldo 2000; Romano & Zeng 2007a).
The relative high vulnerability of the gills to potential pollutants may possibly be explained by their
constant contact with the external medium. This
can be of particular concern because the gills of
aquatic animals are multi-functional organs responsible for many crucial physiological processes,
including ion exchange, acid/base balance, ammonia-N excretion and respiration (Harrison & Humes
1992; Pe¤queux 1995; Freire, Onken & Mcnamara
2008). Indeed previous investigations detected

Keywords: gill histology, haemolymph ammonia,
osmoregulation, Portunus pelagicus, recovery, sublethal ammonia-N,THC

468

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Changes to the histological gill structure and haemolymph composition N Romano & C Zeng

signi¢cant decreases to haemolymph osmolality
and/or Na 1 ions at elevated ammonia-N levels
(Young-lai, Charmantier-daures & Charmantier
1991; Chen & Chen 1996; Harris, Coley, Collins &
Mccabe 2001; Romano & Zeng 2007d), which may
be linked to gill damage.
Because the gills of crustaceans clearly have important roles, are particularly vulnerable to external pollutants and the damage has been linked to
their mortality (Rebelo et al. 2000; Romano & Zeng
2007a), correlating such damage in sequence with
the physiological responses of crustaceans may
yield important information on the modes and progression of toxicity. However, to date, histological
examination of ammonia-N-induced gill damage,
and the associated physiological changes linked
with their function, have focused on a single sampling point of exposure (e.g. Rebelo et al. 2000;
Romano & Zeng 2007a; Miron, Moraes, Becker,
Crestani, Spanevello, Loro & Baldisserotto 2008).
Furthermore, it is yet unclear if sub-lethal ammonia-N levels will cause irreparable damage to
the gills of aquatic animals, or if healing is possible, the potential factors involved. One of these
healing processes may include the presence of
haemocytes, known to facilitate necrotic tissue
removal (Battistella, Bonivento & Amirante 1996;
Johansson, Keyser, Sritunyalucksana & S˛derhÌll
2000), within the haemolymph and gills of aquatic
crustaceans.
The blue swimmer crab, Portunus pelagicus,
commonly inhabits marine and estuarine systems,
although they are characterized as being weak osmoregulators (Romano & Zeng 2006). Increasingly
this species is becoming important to the ¢sheries
industry throughout the Indo-Paci¢c region and is
an emerging aquaculture species for pond and recirculating systems (Walker 2006; Romano & Zeng

2006). Because of the concerns of ammonia-N on
aquaculture systems, the experiment was hence
designed to continuously monitor the haemolymph
osmolality, Na 1, K 1, Ca21, pH, ammonia-N, total
haemocyte counts (THC), as well as the gill histopathology of early P. pelagicus juveniles over 48 h of
exposure to sub-lethal ammonia-N levels and the
subsequent post-exposure recovery period up to
96 h in pristine seawater (no added ammonia-N).
In addition, two lethal ammonia-N levels were used
to induce crab morbidity to determine whether haemolymph osmolality, Na 1, K 1, Ca21 and pH levels
were disrupted to potentially explain causes for
lethality.

Materials and methods
Source of crabs
The crabs used in the experiments were cultured
from newly hatched larvae in the laboratory at James
Cook University, Australia according to the methods
described by Romano and Zeng (2006). Brie£y, P. pelagicus broodstock crabs were collected from the wild
and kept in outdoor recirculating systems until
spawned. A few days before hatching, the berried female was transferred to an indoor 300 L tank for
hatching. The newly hatched Zoea I larvae were
stocked at approximately 500 larvae L À 1 in indoor
300 L tanks at temperature 29 Æ 1 1C and salinity of
25 Æ 1%. Larvae were initially fed rotifers (Branchionus sp.) with the daily addition of microalgae Nannochlorposis sp. However, from the Zoea II onwards,
newly hatched and enriched Artemia were added at
increasing densities of 1^5 Artemia mL À 1 until the
larvae ¢nal settled as juvenile crabs after approximately 2 weeks.
Upon metamorphosis to the ¢rst stage crabs (C1),
they were transferred outdoors and further cultured

until they reached the crab 5 (C5) stage, with mean
carapace width of 18.76 Æ 0.26 mm). This size was
chosen because it permitted a reliable and adequate
mount of haemolymph extraction. The juvenile crab
culture method is described by Romano and Zeng
(2007a) and the process took approximately 3 weeks.
When a su⁄cient number of crabs reached the C5
stage, similar-sized crabs were brought indoors for
the commencement of the experiment.

Experimental design and set-up
Test solutions
A 10 000 mg L À 1 ammonia-N stock solution was
made by adding 38.2 g of NH4Cl to 1L of distilled
water. This stock solution was then diluted in seawater to create the desired ammonia-N concentrations of the test solutions used for experiments 1 and
2. The salinity of the source seawater was 36%,
which was reduced to 30% using de-chlorinated
freshwater and the ammonia-N, nitrite-N and nitrate-N concentrations were measured and all were
below 0.01mg L À 1, which were previously analysed
at Australian Centre for Tropical Freshwater Research (ACTFR) according to APHA (1989). The pH
of each test solution was maintained at 8.1 through
the addition of sodium hydroxide pellets and the pH

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Changes to the histological gill structure and haemolymph composition N Romano & C Zeng

measured using a pH digital meter (WP-80; TPS,
Brisbane, Australia).
Experiment 1
A total of 288 crabs (C5 stage; mean weight 5
0.225 Æ 0.012 g), each individually kept within a separate container thus acting as a replicate, were used in
the experiment. Among these, 140 replicate crabs
were used for each of the two sub-lethal ammonia-N
treatments of 0.714 and 2.857 mmol L À 1 (or 10 and
40 mg L À 1 ammonia-N) respectively (based on values
from Romano & Zeng 2007a), while eight replicate
crabs were used for the control (no ammonia-N added).
Each crab was individually placed within a 5 L container, ¢lled with 2 L of the desired test solution and
gently aerated via a ¢ne-tipped pipette. Each container
received a daily100% water exchange according to the
‘static renewal method’described by the APHA (1989).
The majority of the crabs were daily fed formulated
crumble feed designed for the tiger prawn, Penaeus
monodon (43% protein,6% fat,3% ¢bre) in the morning
during the exposure and recovery phase. However,
each crab was starved 1 day before haemolymph sampling, which was facilitated by marking the containers
at di¡erent time intervals, to ensure1day starved crabs
were used. To prevent the decomposition of food a¡ecting the water ammonia-N levels, the feeds were siphoned out after 3 h, which was su⁄ciently long
enough for the crabs to cease eating. All containers
were held within six 1000 L freshwater baths and the
temperature was maintained at 28 Æ 0.5 1C through
submersible heaters and air conditioning.
At time intervals of 0,1,3,6,12, 24,36 and 48 h, the
haemolymph from eight intermoult replicate crabs
exposed to either 0.714 or 2.857 mmol L À 1 ammonia-N treatments (10 and 40 mg L À 1 ammonia-N

respectively) were obtained via a syringe inserted
through the proximal arthropodal membrane at the
base of the right second walking leg. Haemolymph
samples from ¢ve crabs were then measured for haemolymph osmolality and Na1, K1 and Ca21 levels
while haemolymph samples from the other three
crabs were used to measure the haemolymph pH,
THC and ammonia-N levels. To determine haemolymph Na1, K1 and Ca21 levels a haemolymph sample of 20 mL was immediately diluted with 2 mL of
distilled water and analysed on £ame photometer
(Sherwood 410, Cambridge, UK). To determine the
haemolymph osmolality, an aliquot of haemolymph
(50 mL) was immediately analysed on a cryoscopic
osmometer (Osmomat 030; Gonotec, Cambridge,

470

Aquaculture Research, 2010, 41, 468^480

UK). The haemolymph pH was measured using a pH
digital meter (WP-80;TPS) equipped with a micro-pH
electrode (MI-710, Microelectrodes, Bedford, NH,
USA) after a two-point calibration with precision buffers. Following the measurement of pH, the haemolymph was divided into two portions to be used for
either determining the haemolymph THC or ammonia-N levels. To measure the haemolymph THC, anticoagulant (citrate-concentrated solution, 4% w/v) was
added at a ratio of 1:9 (anticoagulant:haemolymph)
and the haemocytes were counted on a haemocytometer under a light microscope (magni¢cation
 10). After measuring for pH and a portion removed for THC measurements, the haemolymph
samples for measuring the ammonia-N were then diluted with distilled water, frozen at À 20 1C and analysed within 2 days at the ACTFR using the
Nesslerization method 4500 NH3^G according to
APHA (1989). To determine the actual ammonia-N
concentrations during the experiment, three samples from each sub-lethal ammonia-N treatment
were taken on the ¢rst and last day of the experiment

and similarly analysed for ammonia-N at the ACTFR.
The mean values of ammonia-N did not deviate from
the stated concentrations by 45%. Between the 2
days, the means of the actual concentrations in each
treatment did not deviate by 43% and the average of
these actual values, of 0.706 and 2.798 mmol L À 1
ammonia-N will be used throughout to indicate
these two sub-lethal ammonia-N treatments.
After each haemolymph sampling, four crabs were
immersion ¢xed in a 10% (v/v) FAACC formalin solution (4% formaldehyde, 5% acetic acid and 1.3% calcium chloride) for 3 days and transferred to 70% (v/v)
ethanol until further processing. For the histological
examination of the gills, the crabs were progressively
dehydrated at increasing concentrations of alcohol
and embedded in para⁄n wax. Sections (5 mm) were
cut using a rotary Leitz microtome (model 1512,
Netley, NJ, USA), stained with haematoxylin and eosin. The anterior and posterior gill structures were
examined and digitally photographed under a light
microscope.
All crabs subjected to haemolymph sampling were
removed from the experiment and not used a second
time. Following the 48-h exposure to either 0.714 or
2.857 mmol L À 1 ammonia-N, all remaining crabs
were immediately transferred to pristine seawater
with no added ammonia-N to initiate a 96-h recovery
period. To determine the duration required to stabilize haemolymph osmolality, Na1, K1 and Ca21, pH
and ammonia-N levels, as well as normalization of

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Changes to the histological gill structure and haemolymph composition N Romano & C Zeng

the gill structure post sub-lethal ammonia-N exposure, eight crabs were sampled at 1, 3, 6, 12, 24, 36, 48,
72, 84 and 96 h, respectively, adopting the same procedure mentioned above. However, during this recovery experiment, once the haemolymph ammonia-N
levels had returned to those of the pre-exposure level,
no further analysis was performed.
Experiment 2
To determine if osmo-ionoregulation and haemolymph pH regulation in early P. pelagicus juveniles is
interrupted by ammonia-N-induced morbidity, 16
crabs were exposed to lethal concentrations of 7.142
and 10.714 mmol L À 1 ammonia-N (or 100 and
150 mg L À 1 ammonia-N) (Romano & Zeng 2007a),
and another 16 crabs served as a control (no ammonia-N added). Similar experimental protocols and the
same crab stage/batch were used as those of experiment 1 (see Experiment 1). However, hourly observations for moribund crabs were made til 24 h only
(48 h in experiment 1). Morbidity was diagnosed as
the crabs exhibiting severe disorientation (e.g. an inability to remain upright) and/or little movement
when gently stimulated with a glass rod.When a moribund crab was observed, it was sampled immediately
for haemolymph osmolality, Na1, K1, Ca21 and pH
levels measurements using the methods described in
Experiment 1. For purposes of comparison, a control
crab was simultaneously sampled for analysis. To determine the ammonia-N levels of the water, three
samples from each treatment were measured using
the methods described in Experiment 1. The actual
values in the 7.142 and 10.714 mmol L À 1 ammoniaN treatments were 7.036 and 10.518 mmol L À 1 ammonia-N, respectively, and these values will be used
throughout the text.

Data analysis

The haemolymph ammonia-N, Na1, K1 and Ca21
levels are expressed as mmol L À 1. To convert
mmol L À 1 to mg L À 1, multiply mmol L À 1 by the respective molecular weights. In the case of ammoniaN conversions to mg L À 1, the molecular weight of
nitrogen is used.
Because the damage to the anterior and posterior
gills following elevated ammonia-N exposure appeared similar, only the anterior gill structural
changes (pairs 2^4) were quanti¢ed. To quantify
these histopathological changes, from each ammonia-N treatment and exposure/recovery duration, 30
lamellae were randomly chosen from each of the four
replicate crabs for measurements of lamellae width
(based on the distance from each epithelia, see line
on Fig. 1a, which de¢nes lamellae width, to quantify
swelling or collapse) and number of haemocytes present within the lamellae. The lamellae width was
measured using a micrometer (1 mm) and the number
of haemocytes was counted within each lamellae.
The data from the 30 lamellae, of each replicate crab,
were then pooled for statistical analysis.
To determine any signi¢cant e¡ects (Po0.05) of the
ammonia-N levels and exposure/recovery duration on
the haemolymph composition or quanti¢ed gill histological measurements either a one-way or a two-way
analysis of variance (ANOVA) was used. One-way ANOVAs
were used to determine signi¢cant di¡erences over
time within each ammonia-N treatment as well as signi¢cant di¡erences between ammonia-N treatments
at the same time interval. A two-way ANOVA was used
to determine any signi¢cant time, ammonia-N concentration or interaction with the haemolymph composition or gill histopathology. To determine any
signi¢cant e¡ects (Po0.05) of ammonia-N-induced

Figure 1 The anterior gills of early Portunus pelagicus juveniles showing the lamallae of control crabs. Note the presence
of intact pillar cells (PC), low incidences of haemocytes (HAE) within the lamellae and undamaged epithelium. The unlabelled line refers to how the lamellae with was measured. (a) Magni¢cation  20 and (b) magni¢cation  10; (a) scale
bar 5 30 mm and (b) 60 mm.

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Changes to the histological gill structure and haemolymph composition N Romano & C Zeng

morbidity on the haemolymph osmolality, Na1, K1,
Ca21 and pH levels a one-way ANOVA was used. Di¡erences between treatments were determined using
Duncan’s multiple range test (Duncan 1955) using the
SPSS statistical software version 16.0.

Results
Haemolymph osmolality, Na1, K1, Ca2, pH,THC
and ammonia-N
In both experiments1and 2, the haemolymph osmolality and Na1, K1, Ca21 levels of the crabs remained
above that of the external medium at a salinity of
30% (composition of seawater given in the ¢rst line

Aquaculture Research, 2010, 41, 468^480

of Table 1) indicating hyperosmo-ionoregulation
(Fig. 2a^d; Table 1). The results of experiment 1
showed that exposure to sub-lethal ammonia-N
levels of 0.706 and 2.789 mmol L À 1 up to 48 h had
no signi¢cant e¡ect (P40.05) on the haemolymph osmolality, Na1, K1, Ca21 or pH levels. The haemolymph osmolality and pH of the crabs from the
experiment 1 ranged from 896 to 915 mOsm kg À 1
(Fig. 2a) and 7.4 to 7.9 (Fig. 3), respectively, while

mean haemolymph Na1, K1 and Ca21 levels £uctuacted from 508 to 516, 10 to 15 and 10.8 to
14 mmol L À 1, respectively, throughout the 48-h exposure and subsequent 96-h recovery period (Fig. 2b^d).
Similarily in experiment 2, moribund crabs induced
by exposed to subtantially higher lethal ammonia-N

Table 1 Osmolality, Na1, K1, Ca21 and pH levels of the seawater (30%) and in the haemolymph (Æ SE) of the control and
moribund early Portunus pelagicus crabs induced by exposure to lethal concentrations of ammonia-N (7.142 and
10.714 mmol L À 1) within 24 h
Osmolality (mOsm kg À 1), ionic composition (mmol L À 1) and pH of seawater at a salinity of 30%

Ammonia-N
treatment
Control
7.036 mmol L À 1
10.518 mmol L À 1

Osmolality

Sodium

Potassium

Calcium

pH

840

393.1

8.5

9.5

8.1

Osmolality (mOsm kg À 1), ionic composition (mmol L À 1) and pH of the crab haemolymph
Osmolality

Sodium

Potassium

Calcium

pH

908 Æ 5.96
903 Æ 9.5
907 Æ 8.3

510.2 Æ 5.2
513.0 Æ 6.3
512.8 Æ 7.3

12.0 Æ 0.3
12.8 Æ 2.16
12.5 Æ 1.76

12.0 Æ 0.4

12.2 Æ 0.9
12.2 Æ 1.0

7.59 Æ 0.23
7.69 Æ 0.20
7.61 Æ 0.18

No signi¢cant ammonia-N e¡ect on the haemolymph were detected (P40.05).

Figure 2 The mean haemolymph (a) osmolality (mOsm kg À 1), (b) Na1, (c) K1 and (d) Ca21 levels (mmol L À 1) ( Æ SE) of
early Portunus pelagicus juveniles exposed to two sub-lethal ammonia-N levels of 0.706 mmol L À 1 (dashed line) and
2.798 mmol L À 1 (solid line) over 48 h and post-exposure recovery period. No signi¢cant time or ammonia-N e¡ect were
deteced (P40.05).

472

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Changes to the histological gill structure and haemolymph composition N Romano & C Zeng

Figure 3 The mean haemolymph pH (Æ SE) of early Portunus pelagicus juveniles exposed to two sub-lethal ammonia-N
levels of 0.706 mmol L À 1 (dashed line) and 2.798 mmol L À 1 (solid line) over 48 h and post-exposure recovery period. No
signi¢cant time or ammonia-N e¡ect were deteced (P40.05).

levels of 7.036 and 10.518 mmol L À 1 also showed no
signi¢cant change (Po0.05) on their haemolymph

Na1, K1, Ca21 and pH when compared with the control crabs (Table1). A two-way ANOVA detected no significant time (of exposure and recovery duration),
ammonia-N level or interaction e¡ect (P40.05) on
the haemolyph osmolality, Na1, K1, Ca21 or pH levels.
In contrast to the haemolymph osmolality and
ions, both the haemolymph ammonia-N levels and
THC of the crabs exposed to the sub-lethal ammonia-N levels showed an increasing trend with both
the ammonia-N concentration and duration of exposure. The mean haemolymph ammonia-N levels of
the control crabs (no added ammonia-N) was low at
0.69 Â 10 À 3 mmol L À 1. However, within1h of exposure to 0.706 and 2.798 mmol L À 1 ammonia-N the
haemolymph ammonia-N levels signi¢cantly increased (Po0.01) (Fig. 4). The highest haemolymph
ammonia-N levels of 0.361 and 1.692 mmol L À 1 occurred at 48 h, which was the longest exposure duration, at 0.706 and 2.798 mmol L À 1 ammonia-N
respectively (Fig. 4). However, within1h of post-exposure recovery from 2.798 mmol L À 1 ammonia-N, the
haemolymph ammonia-N signi¢cantly decreased
(Po0.01) from the 48-h exposure peak. By 12 h post
exposure to both sub-lethal ammonia-N levels the
haemolymph ammonia-N of the crabs was not significantly di¡erent (P40.05) from those in the control
(Fig. 4). A two-way ANOVA detected a signi¢cant time
(of exposure and recovery duration) and ammonia-N
concentration e¡ect (Po0.01) on the haemolymph
ammonia-N levels, however, no signi¢cant interaction was detected (P40.05).
The mean haemolymph THC of the control crabs
was 29.51 ( Â 10 4 cells mL À 1), however, by 12 and
6 h to f exposure to 0.706 and 2.798 mmol L À 1 ammonia-N, respectively, the haemolymph THC signi¢-

cantly increased (Po0.01) (Fig. 5). Once this
signi¢cant THC increase occurred, it continued
throughout the exposure to both 0.706 and
2.798 mmol L À 1 ammonia-N. At the 84-h recovery
from post exposure to both sub-lethal ammonia-N levels, the haemolymph THC of the crabs signi¢cantly
decreased (Po0.05) to levels that were not signi¢cantly di¡erent (P40.05) from those in the control

(Fig. 5). A two-way ANOVA detected a signi¢cant time
e¡ect (of exposure and recovery duration) (Po0.05)
on the haemolymph THC, however, no signi¢cant
ammonia-N level or interaction e¡ect was detected
(P40.05).

Gill histopathological changes
The anterior lamellae of the control crabs showed intact PC, occasional presence of haemocytes (HAE)
within the lamellae and a thin epithelium (Fig. 2a
and b). Although no mortalities occurred during the
48-h exposure to 0.706 and 2.798 mmol L À 1 ammonia-N, at 3 and 1h, respectively, the gill lamellae
width signi¢cantly decreased (Po0.01) and the number of haemocytes (HAE) signi¢cantly increased
(Po0.05) within the gill lamellae of these crabs. Such
gill histopathological changes lasted throughout the
rest of the 48-h exposure period at both sub-lethal
ammonia-N levels although the degree of such
changes was greater at the higher ammonia-N level
of 2.798 mmol L À 1 (Table 2). Other histological
changes, which were di⁄cult to quantify, also occurred throughout the exposure duration, which included epithelial damage (e.g. sloughing, thickening
and detachment), disrupted/necrotic pillar cells
(DPC) leading to a complete breakdown in the intralamellar septum and lamellae distortion (Fig. 6a^f).

r 2009 The Authors
Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 468^480

473


Changes to the histological gill structure and haemolymph composition N Romano & C Zeng

Aquaculture Research, 2010, 41, 468^480

Figure 4 The mean haemolymph ammonia-N levels (mmol L À 1) (Æ SE) of early Portunus pelagicus juveniles exposed to
two sub-lethal ammonia-N levels of 0.706 mmol L À 1 (dashed line) and 2.798 mmol L À 1 (solid line) over 48 h and postexposure recovery period. Di¡erent lower and upper case letters indicate signi¢cant di¡erences (Po0.05) within 0.706
and 2.798 mmol L À 1 ammonia-N respectively.

Figure 5 The mean haemolymph total haemocyte count (THC) (Æ SE) of early Portunus pelagicus juveniles exposed to
sub-lethal ammonia-N levels of 0.706 mmol L À 1 (dashed line) and 2.798 mmol L À 1 (solid line) over 48 h and post-exposure recovery period. Di¡erent lower and upper case letters indicate signi¢cant di¡erences (Po0.05) within 0.706 and
2.798 mmol L À 1 ammonia-N respectively.

When the recovery period was initiated, an increase to the gill lamellae width and decrease in the
haemocyte number within the gill lamellae occurred
and these were more rapid for the crabs exposed to
0.706 mmol L À 1 ammonia-N than those exposed to
2.798 mmol L À 1 ammonia-N (Table 2). At 24 and
36 h of recovery, the lamellae width of the crabs previously exposed to 0.706 mmol L À 1 ammonia-N signi¢cantly increased (Po0.01) while the haemocyte
number decreased (Po0.05) when compared with
the 48-h exposed crabs. However, for the crabs previously exposed to 2.798 mmol L À 1 ammonia-N, a

474

substantially longer recovery period of 48 and 72 h
was required for signi¢cantly increased lamellae
width (Po0.01) and decreased haemocyte number
(HAE) (Po0.05) to occur respectively (Table 2). In addition, coinciding with signi¢cant increases to the lamellae width and decrease in number of haemocytes
within the lamellae, other signs of gill normalization
also occurred, which included PC restructuring,
epithelial healing, and a clearing of the intralamellae septum (Fig. 7a^f). A two-way ANOVA detected a
signi¢cant treatment and time e¡ect (of exposure
and recovery duration) on the lamellae width and

r 2009 The Authors
Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 468^480