Download diodon nicthemerus

Aust. J. Mar. Freshw. Res., 1986, 37, 587-93
Haemosiderosis in Platycephalus bassensis
and Diodon nicthemerus in South-east Australian Coastal Waters
Jeremy S. Langdon
Australian Fish Health Reference Laboratory, Regional Veterinary Laboratory, P.O. Box 388,
Benalla, Vic. 3672.
Abstract
The degree of haemosiderin deposition in the spleen, liver, and kidney of P. bassensis and D, nicthemerus
was compared in specimens from Port Phillip Bay, and Lakes Entrance, Victoria, Bass Strait, and the
Derwent estuary, Tasmania. D. nicthemerus displayed extensive visceral haemosiderosis and fatty infiltration
of the liver at all sites, apparently as normal conditions. P. bassensis from Port Phillip Bay displayed
severe visceral haemosiderosis, whereas specimens from the remaining sites had only low background
levels of haemosiderin deposition. It is suggested that high levels of visceral haemosiderin in fish species
normally displaying low levels are indicative of a suboptimal health status in fish populations. No infectious
cause of the haemosiderosis in Port Phillip Bay fish has been identified, and toxic or pollution-related
causes are thus considered likely to be responsible.
Introduction
Fish health problems including mass mortalities and cutaneous ulceration, primarily in
bottom-dwelling species, have been recognized in Port Phillip Bay, Victoria, for many years
(Anon. 1984). Investigations subsequent to large mortalities in early 1984 revealed in addition
high levels of haemosiderin in the viscera of several bottom-dwelling species in the Bay.
Haemosiderin is an iron-containing storage pigment produced by catabolism of haemoglobin,
and is deposited in the viscera in syndromes involving accelerated breakdown of erythrocytes
(Robbins and Cotran 1979). Haemosiderosis has been reported in association with both
infectious diseases (Agius 1979) and pollutant exposure (Kranz 1984; Kranz and Peters 1984;
Malins et al. 1984; Scarano et al. 1984) and has potential as an indicator of the health status
of fish populations (Wolke et al. 1985).
The degree of hepatic, renal and splenic haemosiderosis in two common bottom-dwelling
species, the sand flathead Platycephalus bassensis Cuvier and the globefish Diodon nicthemerus
Cuvier was thus compared in specimens from Port Phillip Bay and near Lakes Entrance in
Victoria, in Bass Strait, and in the Derwent estuary in Tasmania. The investigation aimed
to assess these species as indicator organisms in studies of haemosiderosis, and to determine
if the haemosiderosis observed in Port Phillip Bay fish was a normal feature or an abnormal
syndrome.
Materials and Methods
Fish samples were collected by trawl netting in Port Phillip Bay near Point Cook (38"S.,144"501E.;
15 m depth) and St Leonards (38"1O'S.,144"44'E.; 15 m), in an off-shore area near Lakes Entrance
(37°50'-38020'S.,147030'-148030'E.;
22-43 m), at an off-shore site in Bass Strait (38°22'S.,144039'E.;
48 m), and in the Derwent estuary (42°55'S.,147025'E.; 20-25 m), on the dates given in Table 1.
Blood was collected into heparinized syringes from the caudal vessels or heart of fish anaesthetized
with quinaldine. Two blood smears were prepared from each fish and stained with Geimsa stain at
0067-1940/86/050587$02.00
Jeremy S. Langdon
p H 7 . 2 for 90 min. Haematocrit and leucocrit (percentage of blood volume occupied by erythrocytes
and leucocytes respectively) values were determined in the Port Phillip Bay fish according to Wedermeyer
et al. (1983).
Samples of kidney, liver and spleen tissue were fixed in 10% (v/v) neutral buffered formalin and
processed by conventional paraffin embedding and sectioning. The sections were stained by Perls' Prussian
blue method to differentiate haemosiderin from other pigments by its intense blue reaction (Fig. 1)
(Drury and Wallington 1973). The degree of splenic haemosiderosis was assessed semi-quantitatively
on a scale of 0-10 as the area of a splenic cross-section occupied by haemosiderin in the melano-macrophage
centres. Increases in the degree of haemosiderosis resulted from increases in the size and number of the
melano-macrophage centres and the intensity of staining within the centres (Fig. 1). Samples of kidney,
liver, spleen, gill, intestine, brain and heart from the Port Phillip Bay fish were processed similarly, but
stained with haematoxylin and eosin.
Kidney tissue from Port Phillip Bay samples was inoculated o n sheep blood agar and incubated at
28°C in an atmosphere of air for 7 days to culture bacterial pathogens.
Fork length and state of gonadal development were noted for each fish.
Fig. 1. Haemosiderosis in melano-macrophage centres (mmc) of spleen of D. nicthemerus.
(a) Haematoxylin and eosin staining. (b) Perls' Prussian blue staining, indicating the presence of
haemosiderin as the dark-staining patches in the photograph. Scale bar, 100 pm.
Results
D. nicthemerus
D. nicthemerus from all sites displayed variable but usually high levels of splenic haemosiderin
(Table 1). The specimens ranged in fork length from 15.3 to 26.3 cm and included male
and female fish in all stages of sexual maturity except ripe spawning fish, but there were
no apparent relationships between size or sexual development and haemosiderin deposition.
The kidney and liver contained smaller and variable amounts of haemosiderin.
The intra-erythrocytic schizonts of the protozoan Haemogregarina sp. (cf. Hoffman 1967)
were detected in blood smears from six out of 19 specimens from Port Phillip Bay, and
seven out of nine specimens from Bass Strait. The parasite was present in less than 0 . 1 %
of the erythrocytes, and no lesions typical of clinical haemogregariniasis or other diseases
were detected histopathologically, although all specimens displayed marked fatty infiltration
of the liver.
Haemosiderosis in P. bassensis and D. nicthemerus
Activation of the haematopoietic tissue of the anterior kidney was evident in D. nicthemerus
from Port Phillip Bay compared with Bass Strait specimens. The haematocrit levels were normal,
but the leucocrit levels (Table 1) were elevated several-fold, compared to other teleosts
(Wedermeyer et al. 1983). No bacterial pathogens were isolated from the kidney of Port Phillip
Bay specimens.
Table 1. Comparison of splenic haemosiderosis in fish at four sampling sites
n, Number of fish. Haematocrit and leucocrit data are means i s.e. (n = 5)
Sampling
location
and date
Port Phillip Bay
(Point Cook)
10.x.84
Port Phillip Bay
(St Leonards)
19.i.85
Bass Strait
21 i i . 8 5
Lakes Entrance
21.x.85
n
Fork
length
(cm)
Degree of
haemosiderosis
(1-10) ( 4
Diodon nicthemerus
16.0-24.2
6-8
Derwent estuary
2O.xii.85
Other observations (n)
Haemogregarina infection (2)
Mature females (1)
Mature males (6)
Haemogregarina infection (4)
Haematocrit: 39.8 k 3 . 3
Leucocrit:
4 . 7 t 1. 6
Haemogregarina infection
Haemogregarina infection (6)
Mature female
Immature female
Mature females (3)
Mature females (5)
Mature males (3)
Platycephalus bassensis
Port Phillip Bay
(Point Cook)
22.ii.84
10.x.84
Port Phillip Bay
(St Leonards)
19.iii.85
Bass Strait
21 i i . 8 5
Lakes Entrance
21.x.85
Derwent estuary
2O.xii.85
Collected following high fish
mortalities in Bay
Haemogregarina infection (1)
Haematocrit: 28.4 k 4.8
Leucocrit:
2 . 8 k 1.1
Haemogregarina infection (1)
Mature females (5)
Mature females (2)
Mature males (1)
P. bassensis
All P. bassensis specimens sampled from Port Phillip Bay between February 1984 and
April 1985 displayed severe splenic haemosiderosis, except two specimens from Point Cook
which had only low background levels (2) (Table 1, Fig. 2). Specimens of a similar size from
Bass Strait, Lakes Entrance and the Derwent estuary had uniformly low levels (0-3) similar
to those seen in healthy specimens of most teleost species (Agius 1979). The specimens ranged
from immature males and females to spawning females and varied in fork length from 16.7
to 47.0 cm, with no apparent correlation between haemosiderosis and size or sexual
development. The bulk of the haemosiderin was contained in the numerous splenic melanomacrophage centres, but small amounts of haemosiderin were detectable in the splenic red
pulp and ellipsoids, and as a fine stippling of pigment in the liver, and occasional small deposits
Jeremy S. Langdon
in the kidney. The amount of hepatic and renal haemosiderin was also greater in Port Phillip
Bay specimens than in those from the other sites.
Haemogregarina sp. infection was detected in one out of nine Port Phillip Bay fish,
and in one out of seven Bass Strait fish, in March 1985. The parasite was again present
in less than 0.1% of the erythrocytes, and no lesions typical of haemogregariniasis or other
diseases were detected. Only mature or spawning females displayed fatty infiltration of
the liver.
Activation of the renal haematopoietic tissue was evident in histological sections from Port
Phillip Bay specimens compared with those from Bass Strait. Haematocrit levels (Table 1)
were normal, but leucocrit levels were higher than those regarded as normal in other species
(Wedermeyer et al. 1983). No bacterial pathogens were isolated from the kidney of Port Phillip
Bay specimens.
Fig. 2. Comparison of severity of splenic haernosiderosis in Port Phillip Bay and Bass Strait.
(a) P. bassensis, Port Phillip Bay (degree of haernosiderosis judged as 'S', cf. Table 1). (b) P. bassensis,
Bass Strait (degree of haernosiderosis judged as '2', cf. Table 1). Perls' Prussian blue. Scale bar, 200 prn.
Other Species
Severe splenic haemosiderosis (6 or greater) was also seen in Ammotretis rostratus Giinther
(22 February 1984, n = 3) and Sardinops neopilchardus (Steindachner) ( 1 5 May 1985,
n = 3) but only low levels were seen in Mugil cephalus L. or Thyrsites atun (Euphrasen)
(27 August 1984, n = 2) from Point Cook in Port Phillip Bay.
Discussion
D. nicthemerus appears to have variable but usually extensive haemosiderin deposits
in the splenic melano-macrophage centres, and smaller deposits in the liver and kidney,
as a normal feature. Agius (1979) reported similar findings in Sarotherodon mossambicus
(Peters). Haemosiderosis in D, nicthemerus is thus of little value in evaluating the environment
and health status of fish populations, as it was impossible to define a normal range in
this species.
P. bassensis on the other hand had only low background levels (0-3) of haemosiderin
at all sites except Port Phillip Bay, where severe visceral haemosiderosis, graded 6-10 in
Haemosiderosis in P. bassensis and D. nicthemerus
Table 2, was present in all but two specimens. This suggests that the level of haemosiderosis
in this species has potential as an indicator of fish health, with levels of 0-3 representing
the normal range, 4-5 a suspect range, and 6-10 a clearly abnormal degree of haemosiderosis.
Port Phillip Bay would then appear to represent a suboptimal environment for fish (Wolke
et al. 1985). The severe haemosiderosis seen in A. rostratus and S. neopilchardus from
Port Phillip Bay supports this conclusion, but its significance in these species was not fully
investigated.
Table 2. Described and possible causes of haemosiderosis in fish
Type of
haemosiderosis
described
Possible cause
Physiological
In Sarotherodon mossambrcus
In Diodon nicthemerus
Infectious
References
Agius (1979)
Present author
Vibriosis
Haemogregariniasis
Piscine erythrocytic necrosis
Ulcer syndrome
Agius (1979)
Agius (1979)
Reno et al. (1985)
Kranz (1984)
Toxic (methaem~globinaemia)~ Nitrite, aliphatic nitrites,
arninopheno!~,
phenylhydroxylamine,
aniline, nitrobenzene,
N-hydroxyarylamines
Robbins and Cotran
(1979); Smith
(1980); Scarano
et al. (1984)
Toxic (other)
Lead
Elemental phosphorus
Hodson et al. (1978)
Zitko et al. (1970)
General pollution
Metals, aromatic
hydrocarbons, etc.
Unspecified
Malins et al. (1984)
Dietary
Rancid lipids
Moccia et a[. (1984)
Metabolic
Starvation
Agius and Roberts
(1981)
Vascular congestion,
interstitial haemorrhage
Robbins and Cotran
(1979)
A
Described in mammals also.
Kranz (1984); Kranz
and Peters (1984)
'Described only in mammals.
The cause of the haemosiderosis in Port Phillip Bay fish remains unknown. The
haematopoietic activation and maintenance of normal haematocrits suggest that the erythrocyte
turnover rate was elevated, with the haemosiderin deposition representing increased erythrocyte
breakdown and haemoglobin catabolism. Reported other and possible infectious, toxic and
metabolic causes of haemosiderosis are listed in Table 2. In view of the highly urbanized and
industrialized environs of Port Phillip Bay compared to the other sites, pollutant causes, or
pollutants interacting with infectious agents, may well be involved.
The Haemogregarina sp. infections were present in Bass Strait fish showing little
haemosiderosis as well as in Port Phillip Bay fish, which together with the low erythrocyte
Jeremy S. Langdon
infection rate, and the absence of anaemia or the visceral and muscle tumours seen in clinical
haemogregariniasis (Kirmse 1980), suggests that the parasites were not of pathogenic significance.
Haemogregarines are common parasites of marine fish and rarely cause disease, and were
reported in Australian fish as early as 1925 (Mackerras and Mackerras 1925).
Chronic vibriosis is a frequently observed cause of haemosiderosis in marine fish (Agius
1979), but Vibrio spp. or other pathogenic bacteria were not detected in the Port Phillip Bay
samples. The elevated leucocrits in P. bassensis and D. n i c t h e m e r u s in the Bay thus do not
appear t o represent leucocytosis in response to bacterial infection, and may be due to a
generalized haematopoietic activation.
The virus of piscine erythrocytic necrosis damages erythrocytes (Reno ef al. 1985) and so
could lead to haemosiderosis, but the intra-erythrocytic inclusion bodies characteristic of the
virus were not detected in blood smears in this study.
Several pollutants such as lead and elemental phosphorus damage erythrocytes in fish and
so could in theory produce haemosiderosis (Table 2). Nitrite is the best-known toxic cause
of haemosiderosis in fish (Scarano ef al. 1984), and other nitro and amino compounds known
to induce methaemoglobinaemia in other vertebrates could also produce haemosiderosis
(Table 2). There is no evidence to date which implicates specific pollutants in the Port Phillip
Bay syndrome. Tissue lead levels, although low, were found to be several-fold higher in
Port Phillip Bay P. bassensis and D. n i c t h e m e r u s than in Bass Strait specimens (Fabris
and Gibbs 1985), so further investigations of lead exposure and haemosiderosis may be
rewarding.
Haemosiderosis and fatty infiltration of the liver have also been reported in association
with exposure to mixed industrial pollutants including aromatic hydrocarbons (Malins ef al.
1984). D. nicthemerus appeared to have extensive fatty infiltration of the liver as a normal
condition, whereas fatty livers were found only in mature or spawning female P. bassensis,
without relation to the degree of haemosiderosis, in the present work.
The contribution of specific or general pollution to the haemosiderosis syndrome in
Port Phillip Bay fish thus remains unknown, and no infectious aetiologies have been identified.
It remains possible that natural toxins from organisms such as dinoflagellates or molluscs
are involved. The prime aim of this paper is to highlight the suboptimal health status of
certain fish populations of Port Phillip Bay. It is suggested that P. bassensis or other
platycephalids would be a good indicator species in studies of haemosiderosis within its
range, being readily captured and normally displaying only low background levels of visceral
haemosiderin.
Acknowledgments
I thank J. Andrews, J. Wankowski, M. Hortle, C. Gibbs, L. Williams and P. Hodson
for valuable discussions and assistance in fish sampling.
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Manuscript received 17 February 1986, accepted 7 May 1986