AN ABSTRACT OF THE THESIS OF
Jaires A. Anderson for the degree of Master of Science in Fisheries
and Wildlife presented on May 2, 1985.
Title:
A Morphological
and Histological Study of Kudoa spp. (Myxozoa, Myxosporea) in
Four Pacific Marine Fishes.
Abstract approved:
Redacted for privacy
Specirrens of English sole (Parophrys vetulus Girard), pacific
hake (Merluccius productus Ayres), rock sole (Lepidopsetta bilineata
Ayres) and speckled sanddab (Citharichthys stiginaeus Jordan and
Gilbert) collected of f Oregon were examined for histozoic n'xosporean
parasites.
pacific hake were found to be infected with Kudoa
thyrsites (Myxozoa, Myxosporea) and K. paniformis.
Three previously
undescribed species of the genus Kudoa were observed, one each from
English sole, rock sole and the speckled sanddab.
Rock sole were
also infécted with an undescribed species of the genus Unicapsula
(Myxozoa, Myxosporea).
Many, although not all, myxosporeans have been associated with
rapid, post-mortem host tissue degradation.
The host-parasite
interface of members of the genus Kudoa infecting English sole,
Pacific hake and speckled sanddabs was examined using both light and
electron microscopy to determine if t3ifferences between hosts occur
at this boundary which might account for the effects that various
species have on their respective hosts.
observed.
No such differences were
The precise mechanism by which some members of the genus
Kudoa are involved in host tissue degradation remains unclear.
Electron microsccy revealed the parasite pseudocyst in speckled
sanddabs, and probably in English sole and Pacific hake, to be
surrounded only by a cell membrane of parasite origin.
In
photomicrographs, the parasite cell membrane appeared niich thicker in
cross section than in longitudinal section.
Examination of electron
micrographs failed to explain this apparent inconsistency.
A Morphological and Histological study of Kudoa spp.
(Myxozoa, Myxosporea) in Four pacific Marine Fishes
by
Janes A. Anderson
A THESIS
-
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed May 2, 1985
Coninencement June 1985
Redacted for privacy
Associate Professor of Fisheries and Wildlife in charge of major
Redacted for privacy
Head of Departuent 0
Redacted for privacy
Dean of Graduate
Date thesis is presented
Typed by J.A. 1\nderson
May 2, 1985
AcKNOWLEDGE1ENTS
Countless individuals were instrunental to the successful
coirpletion of this thesis.
I am, of course, deeply indebted to Dr.
Robert Olscn, my major professor, for his faith in me and his
patience and encouragement to see this project through to the end.
Thank you, Bob, for being my mentor and my friend.
Many other people extended invaluable assistance.
Thanks are
especially due to:
James Seavers, commercial fisherman from Newport, OR and to Ric
Brodeur of Oregon State University for providing fish for
study;
Al Soeldner and Chris Weiss of the Oregon State University
Electron Microscopy Facility for their technical expertise;
Marilyn Gum
and Lucia Murray for always finding that one more
obscure but vitally important reference;
Dave Mitchell for translating the Russian journals and Alan Heres
for his help with Spanish;
Range Bayer, Ric Brodeur and Jon Shenker for their word
processing wizardry and generous loan of equipment;
Joe Choromanski for his guidance in photography and photoprocessing; and, finally,
Kathy Sercu for sacrificing many hours in technical assistance.
TABLE OF CONTENTS
IiISJilSL*i*(IJ1
LITERATURE REVIEW
Life cycle and transmission studies
Taxonon7
Pathology
4
4
6
11
14
MATERIALS AND METHODS
14
Collection data
15
Observations of infected material
16
Parasite description
17
Histological observations
17
Light microscopy
19
Electron microscopy
Relationship between Kudoa spp. and tissue breakdown
19
in English sole and speckled sanddabs
RESULTS
21
Parasite occurrence
Observations of infected material
Parasite description
Kudoa sp. from English sole
Kudoa thyrsites from Pacific hake
Kudoa paniformis from Pacific hake
Kudoa sp. from rock sole
Unicapsula sp. from rock sole
Kudoa sp. from speckled sanddabs
Histological observations
Light microscopy
Electron microscopy
English sole
Pacific hake
Speckled sanddabs
Relationship between Kudoa spp. and tissue breakdown
in English sole and speckled sanddabs
English sole
Speckled sanddabs
21
22
27
27
33
34
36
36
38
38
38
48
48
55
58
64
64
76
DISCUSSION
81
LITERATURE CITED
94
LIST OF FIGURES
Page
Figure
a) Stellate;
b) Quadrate.
18
1
Kudoa terminology,
2
Kudoa sp. pseudocyst in fresh English sole with
skin removed, blind side.
23
Kudoa sp. pseudocysts in fresh, intact speckled
sanddab, blind side.
23
Pacific hake. a) Non-melanized Kudoa sp.
pseudocysts; b) Melanized Kudoa sp. pseudocysts; C) Uninfected fillet.
25
Rock sole. a) Heavy Kudoa sp. infection;
b) Light Unicapsula sp. infection; c) Uninfected fillet.
28
3
4
5
6
7
8
,9
a) and b).
contrast.
Kudoa sp. from English sole. Phase
30
a) and b). Kudoa thyrsites from Pacific hake.
Phase contrast.
30
a), b) and c). Kudoa paniforinis from pacific
hake. Phase contrast.
30
a) and b).
contrast.
Kudoa sp. from rock sole. Phase
30
Phase contrast.
30
10
Unicapsula sp. fran rock sole.
11
a) and b). Kudoa sp. from speckled sanddab.
phase ccntrast.
30
12
Kudoa sp. from English sole.
32
13
Kudoa thyrsites from pacific hake.
33
14
Kudoa paniformis from pacific hake.
15
Kudoa sp. from rock sole.
35
16
Unicapsula sp. fran rock sole.
37
17
Kudoa sp. from speckled sanddabs.
Page
FIGURE
18
19
20
21
22
a) and b). Kudoa thyrsites spores free within
body musculature of Pacific hake. Wheatleys
modified Goriori trichrome stain.
39
a) and b). Kudoa sp. spores free within body
musculature of rock sole. Giernsa stain.
39
Swollen muscle fiber (arrow) of English sole
infected with Kudoa sp. Wheatleys modified
Gomori trichrome stain.
42
Swollen muscle fiber (arrow) of Pacific hake
infected with Kudoa thyrsites. Giemsa stain.
42
Swollen muscle fiber (arrow) of speckled sanddab
infected with Kudoa sp. Giemsa stain.
42
23
Swollen muscle fiber of English sole infected
with Kudoa sp. Giemsa stain, a) Cross section.
b) Longitudinal section.
24
Swollen muscle fiber of pacific hake infected with
K. thyrsites. Giemsa stain, a) Cross section.
b) Longitudinal section.
25
Swollen muscle fiber of speckled sanddab infected
with Kudoa sp. Wheatleys modified Gomori
trichrome stain, a) Cross section. b) Longitudinal section.
44
a) and b). A single Kudoa sp. pseudocyst (P)
in English sole sectioned in both the cross and
longitudinal planes. Wheatley's modified Gomori
trichrome stain.
46
Kddoa sp. pseudocysts in Pacific hake.
toxylin and eosin stain.
46
26
27
28
29
30
a) and b). Kudoa sp. in rock sole.
modified Gonori trichrome stain.
Heina-
Wheatley's
a) and b). Unicapsula sp. pseudocyst in rock
sole. Wheatley"s modified Gomori trichrome stain.
a) and b). Unicapsula sp. in rock sole.
Wheatleys modified Gornori trichrome stain.
46
49
Figure
31
32
33
34
35
36
37
Page
Transmission electron micrograph of Kudoa sp.
in English sole. Longitudinal section of
pseudocyst and adjacent host tissue.
51
Transmission electron inicrograph of Kudos sp.
spore in English sole.
51
Transmission electron inicrograph of Kudoa sp. in
English sole. Cross section of parasite pseudocyst and adjacent host tissue.
53
Transmission electron inicrograph of Kudos sp. in
English sole. Cross section of parasite pseudocyst and adjacent host tissue.
53
Transmission electron inicrograph of Kudos sp. in
Pacific hake. Longitudinal section of a parasite
pseudocyst, possibly inelanized, and adjacent host
tissue.
56
Transmission electron micrograph of Kudos sp.in
Pacific hake. Longitudinal section of a parasite
pseudocyst, possibly rnelanized, and adjacent host
tissue.
56
Transmission electron inicrograph of Kudoa sp. in
Pacific hake. Cross section of parasite pseudocyst and adjacent host tissue.
59
38
Transmission electron rnicrograph of KUC3Oa sp in
Pacific hake. Cross section of parasite pseudocyst and adjacent host tissue.
39
Transmission electron inicrograph of Kudoa sp. in
speckled sanddab. Longitudinal section of parasite pseudocyst and adjacent host tissue.
61
Transmission electron micrograph of Kudos sp. in
speckled sanddab. Cross section of parasite
pseudocyst and adjacent host tissue.
61
Transmission electron micrograph of Kudoa sp. in
speckled sanddab. Longitudinal section of parasite pseudocyst and adjacent host tissue.
63
Kudoa sp. in English sole, 7 h post-mortem.
Wheatley's modified Gornori trichrome stain.
66
40
41
42
Figure
43
Page
a) and b). English sole control, 7 h postmortem. Wheatleys modified Gorrori trichrome
stain.
44
45
46
47
48
49
50
5L
52
53
66
a) and b). English sole control, 7 h postmortem. Wheatleys modified Goirori trichrome
stain.
66
a) and b). Kudoa sp. in English sole, 30 h
post-mortem. Wheatley"s modified Gomori tnchrome stain.
68
a) and b). English sole control, 30 h postmortem. Wheatleys modified Gorroni tnichrome
stain.
68
a) and b). English sole control, 30 h postmortem. Wheatley's modified Gorroni tnichroire
stain.
68
a) and b). Kudoa sp. in English sole, 48 h
post-mortem. Wheatleys modified Gomoni tnchrome stain.
70
a) and b). English sole control, 48 h postmortem. Wheatley's modified Gonori trichrorre
stain.
70
a) and b). Kudoa sp. in English sole, 60 h
post-mortem. Gieirsa stain.
72
a) and b). English sole control, 60 h postmortem. Wheatleys modified Gornori trichrome
stain.
72
Kudos sp. in English sole, 72 h
post-mortem. Wheatley's modified Gomoni tnchrome stain.
74
a)- and b).
a) and b). English sole control, 72 h postmortem. Wheatleys modified Gononi trichrorne
stain.
54
Kudoa sp. in speckled sanddab, 30 h postmortem. Wheatley's modified Gomoni tnichrome
stain.
55
Kudoa sp. in speckled sanddab, 60 h postmortem. Giemsa stain.
74
77
Figure
56
57
58
59
Page
Speckled sanddab control, 30 h post-mortem.
Wheatley's modified Gomori trichrorne stain.
77
Speckled sanddab control, 60 h post-mortem.
Wheatley's modified Gomori trichroii stain.
77
a) and b). Kudoa sp. in speckled sanddab, 72
h post-mortem. Wheatleys modified Gomori tnchrane stain.
79
a) and b). Speckled sanddab control, 72 h
postmortem. Wheatleys modified Gomori tnchrome stain.
79
LIST OF TABLES
Table
1
2
Page
Dimensions and hosts of the quadrate spores of
the species of Kudoa which locate within host
skeletal muscle.
8
Dimensions and hosts of the stellate spores of
the species of Kudoa which locate within host
skeletal raiscie.
10
Dimensions of fresh Kudoa sp. spores from
English sole.
33
Dimensions of Kudoa thyrsites from previously
frozen Pacific hake.
34
Dimensions of Kudoa paniformis fran previously
frozen Pacific hake.
34
6
Dimensions of Kudoa sp. spores from previously
frozen rock sole.
36
7
Dimensions of fresh Kudoa sp. spores from
speckled sanddabs.
38
3
4
5
A MORPHOLOGICAL AND HISTOLOGICAL STUDY OF KUDOA SPP.
(MYXOZOA, MYXOSPOREA) IN POUR PACIFIC MARINE FISHES
INTJDUCTION
Several tissue dwelling (or histozoic) myxosporean genera have
been associated with rapid post-rrortem host tissue degradation.
These are Unicapsula (Davis 1924; Lester 1982), Hexacapsula (Arai and
Matsumoto 1953), Neochloromyxum (Matsumoto 1954) and Kudoa (Meglitsch
1947). Myxosporeans, particularly those infecting hake (Merluccius
spp.), have been the subject of nunrous investigations because of
this association with tissue degradation and the so-called milkiness
of host flesh.
Merrbers of the genus Kudoa have been irrplicated in
the milky condition of Mauretanean hake (M. capensis Castlenau)
(Fletcher, Hodgkiss and Shewan 1951) and K. peruvianus may cause
liquifaction of Peruvian hake (M. gayi peruanus Gingsburg) (Salas
1972). Kudoa rosenbuschi occurs in Chilean hake (M. gayi Guichenot)
(Gelormini 1944) and K. thyrsites and K. paniformis have both been
described from Pacific hake (M. productus Ayres) (Kabata and Whitaker
1981).
Several studies have reported the effect of infection by species
of the genus Kudoa on the flesh of Pacific hake (Dassow, patashnik
and Koury 1970;
nonyrnous 1978; Patashnik, Groninger, Barnett, Kudo
and Koury 1982; Tsuyuki, Williscroft, Kabata and Whitaker 1981).
Dassow et al.
(1970) found 100% of the pacific hake in some sample
2
areas were infected.
They also found, as did Patashnik et al.
(1982), that these parasites adversely affect the keeping quality of
processed Pacific hake and its proximate analysis.
Tsuyuki et al.
(1982) concluded that K. paniformis is responsible for tissue
degradation in Pacific hake and that uninfected fish or those
infected with K. thyrsites alone do not exhibit abnormal tissue
texture.
Spores identified as members of the genus Kudoa have been
implicated in the milkiness of English sole (Parophrys vetulus
Girard) off Vancouver Island, British Columbia, Canada (Margolis
1953; Forrester 1956). Forrester (1956) concluded that the prevalence
of these parasites in English sole decreased when fishing pressure on
the population increased.
This conclusion was based on his findings
from a 4 year special conuirrcial fishery designed to assess possible
stock utilization.
Pacific hake from California, Oregon and
Washington were examined over a 10 year period and it was concluded
that the prevalence of infection by the genus Kudoa in pacific hake
is not related to fishing pressure (Anonymous 1978).
Myxosporeans in the genus Kudoa have also been found in English
sole, rock sole (Lepidopsetta bilineata Ayres) and speckled sanddabs
(CitharichtFys stiginaeus Jordan and Gilbert) from Oregon (Olson,
personal communication).
These myxosporeans have not been studied so
their relationship to those in pacific hake and their effects on host
flesh are not known.
The present study was undertaken to: 1)
describe the morphological characteristics of myxosporean spores from
3
English sole, Pacific hake, rock sole and speckled sanddabs and to
compare these characteristics to those of previously described
species, 2) determine if post-mortem flesh degradation is associated
with myxosporean infection in English sole and speckled sanddabs and
3) examine the host-parasite interface in English sole, Pacific hake
and speckled sanddabs, using both light and electron microscopy.
Possible variations between hosts at this interface may account for
observed differences in the effects of myxosporean infection on host
tissue degradation.
4
LITERATURE REVIEW
Life cycle and transmission studies
Myxosporeans (Myxozoa, Myxosporea) are obligate parasites
occurring primarily in fresh and saltwater fishes.
The mature spore
is the most frequently observed and the best understood stage.
Until
recently, it has been widely believed that the inyxosporean life cycle
is direct, although none has been successfully followed through all
stages in the laboratory (Mitchell 1977; Wolf and Markiw 1984). There
has been much uncertainty about the role of spores in parasite
transmission.
Simple exposure of susceptible rainbow trout to mature
Myxosoma cerebralis spores is not sufficient to induce infection
(Hoffman, Dunbar and Bradford 1962). Hoffman and Putz (1969) reported
that only after aging the spores in cool running water for four to
five months could they infect rainbow trout fry with M. cerebralis.
They did not know v/nat factors, living or dead, present in the water
or in the mud, may have exerted an influence upon the infectivity of
the aged spores.
Atterrpts to transmit Ceratornyxa shasta from infected to
uninfected fish under laboratory conditions have also failed (Fryer
and Sanders 1970). Unsuccessful conditions include direct contact of
infected and uninfected fish, aging spores as for M. cerebralis and
feeding fish viscera containing the parasites to uninfected fish
(Johnson, Sanders and Fryer 1979). Fryer and Sanders (1970) were able
to infect trout by exposing them to mud removed from a lake known to
5
contain the infectious stage of the disease.
Salmonids have
routinely been infected with C. shasta by holding them in cages in
rivers where the parasite naturally occurs (Sanders, Fryer and Gould
1970; Zinn, Johnson, Sanders and Fryer 1977; Johnson, Sanders and
Fryer 1979; Ching and Munday 1984). Again, the infectious agent was
not identified.
The inability to transmit myxosporeans directly between fish
hosts in the laboratory has lead to speculation that an intermediate
host may be involved (Johnson 1975). Sindermann (1970) suggested that
certain zooplankton may act as intermediate hosts of nyxosporeans
infecting pelagic fishes.
Meyers, Scala and Sirnrrons (1970) retrieved
viable M. cerebralis spores from blue heron feces, thereby possibly
implicating avian vectors.
Markiw and Wolf (1983) demonstrated the
role that tubificid annelids play in the transmission of N.
cerebralis and sought to complete their explanation of the life cycle
by identifying the infective agent of the disease (Wolf and Markiw
1984). They provided evidence that Actinosporea and Myxosporea,
thought to be distinct classes of the phylum Myxozoa (Levine et al.
1980), are actually different life stages of the same organism.
Spores of N. cerebralis are released from infected fish at death or
after ingestion by predators and tubificid annelids are infected by
the spores.
Myxosporean conversion to actinosporean occurs in the
annelid gut and mature actinosporeans are released or the annelid is
ingested by a susceptible trout in which mature N. cerebralis spores
develop.
Wolf and Markiw (1984) postulated that comparable
alterations of form and host will be found among other Myxozoa.
Taxonorry
Myxosporeans have traditionally been classified on the basis of
spore morphology (Noble 1944). However, the validity of the many
myxosporean species separated solely on the basis of spore structure
is questionable (Mitchell 1977). Meglitsch (1957) was the first to
suggest that variations in the spore alone should not be sufficient
to confer new species status on myxosporeans.
Geographic
distribution, host-specificity, organ-specificity and host tissue
response should all be taken into account in taxonomic work.
In the
absence of successful infectivity experiments, however, spore
morphology has been heavily relied upon.
Morphological details that
have been used to distinguish and describe myxosporeans include polar
capsules, polar filaments, sutural markings, shell valves and
sporoplasm constituents (Arai and Matsumoto 1953; Lom 1969; Yoshino
and Moser 1974; Schubert, Sprague and Reinboth 1975; Moser, Love and
Jensen 1976; Sankurathri 1977; Lester 1982).
Electron microscopic studies have added greatly to the knowledge
of myxosporean biology over the past 25 years.
Most of these studies
have been either of myxosporean sporogenesis (Current and Janovy
1977; Hulbert, Komourdjian, Moon and Fenwick 1977; Desser and
Patterson 1978; Current 1979; Current, Janovy and Knight 1979;
Uspenskaya 1982; Desser, Molnar and Horvath 1983; Desser, Molnar and
Weller 1983) or the surface patterns and cross sections of mature
spores (Lunger et al.
1975; Nakajima and Egusa 1978; Voelker et al.
1978; EguSa and Nakajima 1980; Lester 1982; Egusa and Shiomitsu
7
1983). Several authors have stressed the need for critical ultrastructural observations in taxonomic studies of rr'xosporeans
(Schubert et al.
1975; Lunger, Rhoads, Wolf and Markiw 1975; Current
and Janovy 1976; Voelker, Kassel, Weinber and McKee 1978). Spore
surface patterns (Lunger et al.
1975; Egusa and Nakajiina 1980),
plasinodium structure (Desser and Paterson 1978; Pulsford and Matthews
1982) and host tissue reaction (Current and Janovy 1978; Voelker et
al.
1978), all visualized at the ultrastructural level, have been
valuable observations in myxosporean taxonomic studies.
Meglitsch (1947) established the genus Kudoa with eight species
formerly in the genus Chiororryxum. This removed all Chloroiryxuin with
four shell valves instead of two and all histozoic forms to the new
genus.
The diagnosis of the new genus also included spores quadrate
(valves rounded to a spherical to subspherical outline) or stellate
(valves drawn out into pointed processes) in anterior view.
The type
species is K. clupeidae (Hahn 1917). The genus Kudoa now contains 29
described species, 22 that exhibit quadrate morphology and 7 with
stellate morphology.
Of these, 14 quadrate and all stellate species
parasitize the skeletal musculature of fish (Tables 1 and 2). The
remaining species of Kudoa localize specifically in fish nervous
tissue, pericardial cavity, gall bladder or gills.
Polar capsule length ratios have also been used to differentiate
iirjxosporean species (Kovaleva, Shulman and Yakovlev 1979). Kudoa
thyrsites is the only species of the genus described as having polar
capsules of unequal size.
Gilchrist (1924) observed one large polar
Table
1.
Host
Species
K.
Measurements in kim.
Dimensions and hosts of the quadrate spores of the species of Kudoa which locate within host skeletal muscle.
alliaria
Length mean
Spore
Width mean
Thickness mean
Polar Capsules
Length mean Width mean
(range)
(range)
2.4
1.8
(range)
(range)
(range)
(7 - 8)
(9 -10)
(8
(6.3-7.4)
(6.3-7.4)
(6.0-6.3)
(2.1-2.6)
(4.5-5.5)
(5.0-6.0)
(5.0-6.0)
(1.5-2.0)
(1.0-1.2)
5.5
1.8
(8.0-8.5)
(11-12)
(11-12)
Strongylura
strongylura
5.5
7.2
5.8
7.5
(6.8-8.2)
Micromesiscius
australis
Reference
Kovaleva et al.
979
9)
Macruronus
magellanicus
IC.
alliaria
Notothena
ramzay
2.0
Kovaleva et al.
1979
N. conina
K.
amamiensis
Abudefduf
sexfasciatus
Egusa and
Nakajima 1980
A. vagiensis
Chromis
isharai
C. notatus
Chrysiptera
ass imil is
Serbia
uinqueradiata
K. bora
K.
chblkaensis
cephalis
K.
crumena
Scomberomorus
maculatus
K.
funduli
Fundulus
heteroclitus
K.
insolita
Seriola
dumerli
Fujita 1930
Tripathi 1951
3.5
(1.0-1.5)
9.9
(9.3-10.4)
Iversen and Van
Meter 1967
9.0
(8.2-9.7)
4.0
(3.2-4.6)
2.5
(2.1-2.9)
1.5
Meglitsch 1948
2.0
Kovaleva et al.
6.7
6.7
7.4
3.3
(4.2-5.3)
(6.4-7.5)
(5.3-6.4)
(3.23.7)
1979
Table
Continued
1.
Species
K. iwatal
K. musculoliguefaciens
Host
Length mean
Spore
Width mean
(range)
(range)
7.2
(6.7-8.0)
9.1
(8.8-9.6)
6.2
(5.3-7.3)
Thickness mean
(range)
Polar capsule
Length mean Width mean
(range)
(range)
10.1
(9.7-10.7)
4.0
(3.8-4.5)
2.2
(2.0-2.4)
7.9
(7.4-9.9)
8.3
(7.0-9.0)
2.1
(1.7-2.8)
(1.7-2.5)
(5.3-6.5)
(7.5-8.0)
(8.5-9.8)
(2.7-3.2)
Merluccius
productus
5.0
(4.5-6.0)
5.9
(5.0-6.5)
6.7
(6.0-7.0)
2.1
(2.0-2.4)
(1.5-2.0)
Merluccius
productus
5.7
(5.6-7.0)
6.4
(5.6-7.0)
7.0
(7.0)
2.0
(1.4-2.1)
(1.4-2.1)
Merluccius
peruanus
(4.7-5.1)
(5.6-6.5)
(2.2-2.7)
(1.3-1.8)
Pagurus major
Xiphias gladius
K. nova
K. paniformis
K. paniformis
K. peruvianus
K.
4m
K. rosenbuschl
Egusa and
Shiomitsu 1983
Matsumoto 1954
2.0
Najdenova 1975
1.7
Kabata and
Whitaker 1981
1.5
This paper
Salas 1972
Syngnathus acus
Merluccius
1.8
Reference
5.0
7.0
6.0
Thelohan 1895
2.5
Gelormini 1943
Table 2.
Dimensions and hosts of the stellate spores of the species of Kudoa which locate within host skeletal muscle.
Dimensions in pm.
Spore
Species
Host
Width I
Length
K. bengalensis
Tachysurus
platvstomus
K. clupeidae
C1upea
K. histolyticum
Scomber
scomber
K. kabatal
K. lunata
Zeugopterus
punctatus
posus
thori
(range)
a
(range)
7.9
(7.0-8.5)
8.4
(7.0-11.0)
5
b
(range)
Width II
(range)
Thickness
(range)
Polar cpasule length
Large
Medium
Small
(range)
(range)
(range)
Sarkar and
Mazunder 1983
3.8
(3.0-4.8)
7
7
2
(12-15)
(7-9)
(3-6)
(4.0-5.0)
(5.0-7.7)
(5.0-7.7)
(1.5-2.0)
5.3
(4.5-6.2)
10.0
(9.0-11.4)
Reference
Hahn 1917
Perard 1928
Kovaleva et al.
1979
tom et al. 1983
2.5
(2.0-3.0)
A. imperialis
A. laterna
K. thyrsites
Thyrsites
8.0
12.0
3
2
1.5
Gilchrist 1924
at un
K. thyrsites
Merluccius
productus
7.1
16.7
8.0
(6.0-10.0) (14.0-19.0)
12.7
(6.0-8.0) (10.0-14.0)
K. thyrsites
Merluccius
productus
Kudoa sp.
Citharichthys
7.1
st I gmaeus
(5.6-8.4)
6.4
10.5
8.2
9.4
(8.4-11.2) (7.0-9.8)
Kudoa sp.
Lepidopsetta
bilineata
Kudoa sp.
Parophrys
vetulus
7.2
(7.0-8.4)
Neochloromyxun
cruciformum
Lateolabrax
japonicus
(7.0-9.1) (11.2-15.4)
13.9
(11.2-16.8)
7.0
9.8
7.0
(5.6-8.4)
12.1
8.7
(9.8-15.4) (7.0-11.2)
9.8
(7.0-12.6)
12.6
7.4
7.1
(5.6-8.4)
9.7
7.4
(8.4-11.2) (6.3-8.4)
13.9
5.4
(4.9-5.9)
4.1
(2.9-4.9)
4.9
3.4
Kabata and
Whitaker 1981
This
paper
12.5
(9.8-14.0)
5.4
(4.2-7.0)
3.9
(2.8-5.6)
2.8
(1.4-4.2)
This paper
18.3
(14.0-22.4)
5.9
(5.6-7.0)
4.2
(2.8-5.6)
3.2
(1.4-4.2)
This paper
13.9
(8.4-15.4)
4.8
(4.2-5.6)
3.5
(2.8-4.2)
2.8
(2.8-4.2)
This
16.0
(14.0-18.2)
6.3
(5.6-7.8)
4.0
(2.8-4.9)
Matsumoto 1954
paper
11
capsule, one small polar capsule and two polar capsules of equal,
intermediate length in K. thyrsites, whereas, Kabata and Whitaker
(1981) reported one large polar capsule and three smaller ones of
equal size in the same species. Perard (1928) reported the polar
capsules of K. histolyticum to be "a little unequal" and his drawings
suggest one large and three smaller, equal polar capsules, but he
apparently pooled the dimensions and indicated only one average polar
capsule length and one average width. The only species in the genus
Neochloromyxuni, N. cruciformum, has also been reported with unequal
polar capsules, one large and three smaller ones of equal size
(Matsuinoto 1954). Although Kudo (1966) included the stellate
Neochlororriyxuni in the genus Kudoa, others have continued to refer to
it as Neochloromyxuin (Salas 1972; Kabata and Whitaker 1981; Ainandi
1984).
Pathology
Many myxosporeans, particularly non-tissue dwelling (or
coelozoic) forme, cause no obvious damage to the host (Sindermann
1970). Histozoic forms are more often harmful to their hosts.
Myxosoma cerebralis is a serious pathogen which infects cartilage in
salmonids and may cause severe whirling disease. Epizootics of
whirling disease have occurred in trout hatcheries in Europe, Asia,
South Africa and North America (Halliday 1976). Ceratoinyxa shasta
produces a highly lethal disease in trout and salmon and has caused
massive epizootics in the northwestern United States and Canada
(Schafer 1968; Margolis and Evelyn 1975; McDonald 1983; Ching 1984).
12
Other histozoic niyxosporeans, including those in the genus
Kudoa, have been associated with rapid post-mortem host tissue
degradation.
Willis (1949), P3nonyinous (1978) and Konagaya (1980) all
suggested that increased proteolytic activity may be associated with
these myxosporean infections.
Willis (1949) further suggested that
while the host is alive, the enzyme is carried away by the blood
stream and after host death, the cessation of circulation allows the
enzyme to accumulate and tissue breakdown follows.
A protease has
recently been identified from Henneguya salminicola, a inyxosporean
associated with hydrolysis of sockeye salmon (Onchorhynchus nerka
Walbauin) muscle (Bilinski, Boyce, Jonas and Peters 1984).
uyuki et
al (1982) have demonstrated proteolytic activity in the acid and
neutral pH ranges in Pacific hake infected with K. thyrsites and K.
paniformis, but this enzyme has not been characterized completely.
Patashnik et al.
(1982) measured proteolytic activity in
spore-filled pseudocysts in Pacific hake and in the host blood but
not in the normal appearing muscle fibers surrounding the
pseudocysts.
The level of proteolytic activity per unit of muscle
tissue after storage did not increase significantly over the levels
in the fresh dead host.
It appeared that a fixed amount of protease
diffused through the host tissue with storage.
Patashnik et al.
(1982) concluded that an active proteolytic enzyme diffuses out of
the pseudocyst after host death and, as host cells are broken down,
the released sarcoplasniic and vascular fluids act as carriers.
Aggregations of spores in the genus Kudoa have been referred to
as either cysts or pseudocysts.
In most instances, increased
13
proteolytic activity has been reported only in the latter.
The term
"cyst" is usually restricted to those species which do not localize
within the host somatic muscle fibers and are isolated by a
host-derived collagenous capsule, e.g., K. branchiata (Joy 1972), K.
cerebralis (Paperna and Zwerner 1974), K. tetraspora (Narasimhamurti
and Kavalati 1979a), K. sphyraeni (Narasimhamurti and Kavalati 1979b)
and K. amainiensis (Egusa and Nakajima 1980). parasite-induced tissue
breakdown has not been associated with any of the above species.
Kudoa clupeidae has been variously described as occurring in
interrnuscular cysts with a host-derived collagen capsule (Heckmann
and Jensen 1978) and in intramuscular cysts (Meglitsch 1947) or
intermuscular pseudocysts (Hahn 1917) with no mention of the spore
aggregation boundary in either of these last reports.
In each
instance, whether in a cyst or pseudocyst, K. clupeidae has been
associated with post mortem host tissue breakdown.
The term "pseudocyst" has been used for those species which
localize within host muscle fibers and for which there is no host
cell envelope surrounding the spores, e.g., K. paniformis, K
thyrsites (Kabata and Vhitaker 1981), K. lunata and K. alliaria (Lom,
Dykova and Lhotakova 1983). Kudoa paniformis has been associated with
host tissue degradation.
Pseudocysts are usually described as
occurring within a muscle fiber (Kabata and Whitaker 1981; Lom et
al.
1983), but Tsuyuki et al.
(1983) referred to the entire
infected muscle fiber as the pseudocyst.
be used in the present study.
The former definition will
14
MATERIALS AND METHODS
Collection data
Fish were obtained for this study opportunistically and caine
from three sources.
Rock sole were caught by a cojintercial trawler
off the central Oregon coast in May 1982 and delivered to a fish
processing plant in Newport, Oregon. Both whole fish and fillets from
this catch were forwarded to the Hatfield Marine Science Center Fish
Disease Laboratory in Newport, Oregon because the flesh texture was
found to be coimnercially unacceptable.
pacific hake were caught
incidentally by commercial fishermen of f the northern Oregon coast in
May 1983 and the southern Oregon coast in September 1983. English
sole and speckled sanddabs were collected irregularly from March 1983
through August 1984 by trawling in Yaquina Bay, Oregon and the nearby
ocean.
English sole and speckled sanddabs were held alive in tanks of
running seawater at the Hatfield Marine Science Center and were
examined fresh.
Pacific hake were frozen at -20 'C within one hour of
capture by a blast freezer on board the fishing vessel.
They were
transferred without thawing to the laboratory where they were
maintained at -20'C. Rock sole were iced on board the fishing vessel
and stored on ice for approximately 3 days before reaching the
laboratory where they were stored at -20 'C.
15
Observations of infected tissue
None of the Pacific hake was frozen for longer than 4 weeks
prior to intial examination.
The rock sole fillets were examined
when they arrived at the laboratory and the infecting parasite was
identified as a rnerrber of the genus Kudoa. Both the fillets and the
whole rock sole were then frozen for 12 to 24 nonths before they were
examined in the present study.
Pacific hake and rock sole were
dissected immediately after thawing to allow detection of parasites
and for observations on muscle texture and infection levels.
The
live English sole and speckled sanddabs were small and thin enough to
allow detection of parasite pseudocysts in whole fish by back
lighting from the eyed side.
specimens of English sole, Pacific hake and speckled sanddabs
were examined for parasites both macroscopically and using a
dissecting microscope (60
120x). Infection intensity levels were
characterized as follows: Noneno macrosccpic pseudocysts observed
and no free spores found by macerating cubes of tissue (approximately
0.5 cm on each side) in 0.7% saline and examining two drops/sample at
400x from three sample areas/fish; Trace--no macroscopic pseudocysts
observed but spores found free within body musculature; Light--up to
25 macroscopic pseudocysts/fish in English sole and speckled sanddabs
and up to 50 macroscopic pseudocysts/fish in Pacific hake;
Heavy--more than 25 macroscopic pseudocysts/fish in English sole and
speckled sanddabs and more than 50 macroscopic pseudocysts/fish in
16
Pacific hake.
No macroscopic pseudocysts were observed in any specimen of rock
sole.
Infection levels, therefore, could not be determined as
above.
A qualitative assessment of flesh texture was used to
characterize the tissue condition, retaining the category terms used
for the other host species: Trace--normal texture but myxosporean
spores found free within body musculature; Light--opaque, milky-white
areas with some softening of tissue; Heavy--nearly liquified flesh.
When infections were light, the apparently uninfected tissue was
sampled from each host species.
These samples were macerated and
examined to detect spores not confined to macroscopic pseudocysts or
to areas of softened tissue.
Parasite description
Spores for morphological study were obtained according to the
technique of Lom (1969). Pseudocysts were teased from English sole,
Pacific hake and speckled sanddab muscle fibers under a dissecting
microscope (120
500x), punctured with needles on coverslips and
embedded in 2% agar on glass microscope slides.
Drops of tissue
slurries from rock sole were also embedded on agar slides to reduce
Brownian movement during observation of spores.
Measurements of fresh spores from each host were made using an
ocular micrometer.
For spores with stellate morphology, length was
defined as the anterioposterior diameter, width I as the narrowest
diameter in the sutural plane (measured from the tip of one valve to
17
the tip of each adjacent valve), width II as the distance tip to tip
in the sutural plane and thickness as the capsular plane diameter
(Matsuinoto 1954) (Figure la). For spores with quadrate morphology,
length was defined as the anterioposterior diameter, width as the
sutural plane diameter and thickness as the capsular plane diameter
(Kabata and 1'hitaker 1981) (Figure ib). Polar capsule ratios have
also been used to differentiate species of the genus Kudoa (Kovaleva
et al.
1979). Polar capsule relationships used in this study were:
1) the four polar capsules all equal in length; 2) one polar capsule
large and the other three smaller than the one and equal to each
other; or 3) one polar capsule large, one small and the other two of
an equal intermediate length.
The decision to place the polar
capsules into one of the above groups was based on a Student's t of
>= 95%. Drawings of fresh spores were made with the aid of a camera
lucida.
Histological observations
Light microscopy
Tissue blocks (1 cm3) from both fresh and frozen specimens were
fixed in Bouins fixative, dehydrated in a graded ethanol series,
cleared in toluene and embedded in paraplast. Sections (5 - 7 jim)
were cut on an American Optical Co. 820 microtome and stained with
hematoxalin and eosin, Giemsa or Wheatley's modified Gomori trichrome
stain (Alger 1966). Photograph of spores and histological sections
were taken with a 35 irurt camera mounted on a Zeiss con!pound
microscope.
18
4
Wib
PCL.]
ThN\ 2
I
WIl
(WIa); Width lb
Figure la. Stellate Kudoa terininology: Width Ia
(L);
Thickness
(T); polar
(WIb); Width II (Wil); Length
Capsule Length (PCL).
PCL
2
Figure lb. Quadrate Kudoa terminology: Width (W); Length (L);
Thickness (T); Polar Capsule Length (PCL); Polar Capsule
Width (Pcw).
19
Electron microscopy
Individual pseudocysts were teased from muscle fibers of English
sole, pacific hake and speckled sanddabs and prepared for
transmission electron microscopy.
They were fixed in 2.5%
glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.4) or in 3.5%
formaldehyde in 0.066 M Sorenson's phosphate buffer with 2.5% w/v
sucrose (pH 7.2). Pseudocysts fixed in glutaraldehyde were post-fixed
in 1% osmium tetroxide, dehydrated in a graded acetone series with 1%
uranyl acetate in the 70% acetone step and embedded in Spurr's medium
in plastic weighing dishes or in gelatin capsules.
Those fixed in
formaldehyde were dehydrated in a graded ethanol series which
included 1% phosphotungstic acid in the first absolute ethanol step
and embedded in L.R. White medium in gelatin capsules.
Embedded
samples were sectioned (10 nm) with glass or diamond knives on a
Sorval MT-2 ultramicrotome and viewed in cross and longitudinal
section on a Philips 300 transmission electron microscope operated at
60 kv.
Photographs were taken at magnifications of 6,500x and
lO,000x with an additional enlargement of l.8x made of the
micrographs.
Relationship between Kudoa spp. and tissue breakdown in
English sole and speckled sanddabs
Heavily infected specimens of English sole and speckled sanddab
and an uninfected control of each species were killed quickly with a
blow to the head and held at room terrperature
(21CC)
for 72 h.
20
Tissue blocks (approximately 5 mm on each side) were excised from the
infected and the control fish at 0.0, 0.5, 7, 30, 48, 60 and 72 h
after host death and fixed in Bouin's fixative.
least two macroscopically visible pseudocysts.
Each sauple had at
Sanpies were
dehydrated in a graded ethanol series, cleared in toluene and
embedded in Paraplast. After sectioning at 5 kim, the blocks were
broken apart without melting the Paraplast and remounted to allow
sectioning of the same pseudocyst in both the cross and the
longitudinal sectional planes.
stain for 60 mm
Sections were stained with Giemea
or in Wheatleys modified Gomori trichrome stain.
21
RESULTS
Parasite occurrence
Although samples of fish for this study could not be obtained
according to a plan that allowed quantitative conclusions about the
characteristics of myxosporean infection within a fish population,
some general observations on parasite occurrence were made.
Over the
course of 18 mo, approximately 250 English sole ranging from 3.0 to
12.5 cm, total length (TL), were examined for ixosporean infection.
Yaquina Bay, Oregon is a nursery ground for English sole (Olson and
Pratt 1973) and all fish were in the 0 year class.
During the same
period, approximately 500 juvenile and adult speckled sanddabs
ranging from 3.5 to 13.0 cm TL were also examined for pseudocysts.
There is some evidence to suggest that Yaquina Bay is also a nursery
ground for speckled sanddabs, but this is inconclusive (Pearcy and
Myers 1974). In the present study, juvenile to gravid adult speckled
sanddabs were found both within Yaquina Bay and immediately
offshore.
Adults infected with myxosporeans were found at all
collection sites.
Prevalences of infection in each host were low
(less than 10%) but infected individuals often contained more than 25
parasite pseudocysts.
The Pacific hake examined in this study were host to K.
thyrsites and K. paniformis as described by Kabata and Whitaker
(1981). Five specimens of Pacific hake were examined from the
collection off the northern Oregon coast and 24 from the southern
22
Oregon coast and the resulting data on myxosporean infections were
pooled.
The fish ranged from 30 to 53 cm TL. Five fish (17.2%) had
no infection, five (17.2%) had trace infections, ten (34.5%) had
light infections and in nine fish (31.1%), the infections were
heavy.
Six whole rock sole and five fillets which had been cut before
the material reached the laboratory were examined.
The whole fish
ranged from 30 to 41 cm TL and the fillets appeared to have come from
fish of similar size.
Two histozoic myxosporeans were found, Kudoa
sp.
and unicapsula sp.
sp.
only, two were infected with tjnicapsula sp.
one fish was heavily infected with Kudoa
only (one trace and
one light infection), one had a heavy mixed infection and two were
uninfected.
The fillets were infected only with Kudoa sp.
One had a
trace infection, three had light infections and one fillet was
apparently uninfected.
Observations of infected tissue
The texture of fresh English sole and speckled sanddab muscle
was similar, appeared normal, and in neither species was it affected
by the presence of the parasite.
Spores in each host species were
confined to swollen but intact muscle fibers.
Infected fibers
containing pseudocysts varied from white to pale yellow and measured
0.1 by 1.5 mm (Figures 2 and 3). All spores in each host were
associated with pseudocysts, no free spores were observed.
Pseudocysts in infected Pacific hake were similar in appearance
23
Figure 2.
Kudoa sp. pseudocyst (arrow) in fresh English sole with
skin removed, blind side. Bar = 0.1 cm.
Figure 3.
Kudea sp. pseudocvsts (arrows) in fresh, intact speckled
sanddab, blind side. Bar = 1.0 cm.
24
to those of English sole and speckled sanddabs, but were much longer,
up to 15 nun (Figure 4a). Some pseudocysts in Pacific hake were
surrounded by melanin deposits, giving them a mottled brown to black
appearance.
Melanized pseudocysts were smaller (0.2 by 5.0 mm) and
much less common than the non-melanized pseudocysts (Figure 4b). 1n
uninfected fillet of Pacific hake is shown in Figure 4c. pacific hake
was the only host in which melanized pseudocysts were found.
Non-melanized pseudocysts in all pacific hake examined were K.
paniformis.
Most melanized pseudocysts were K. thyrsites although
some K. paniformis pseudocysts were also melanized.
Of the five fish
with only a trace infection, four had mixed infections and one was
infected with K. thyrsites only.
Five (17.2%) of the 29 Pacific hake examined exhibited abnormal
muscle texture.
rwo of the five individuals with trace infections
(one infected with K. thyrsites and one with a mixed infection) each
had a single area of softened musculature approximately 4 cm in
diameter near the nape.
Only three of the nine fish with heavy
infections exhibited any softened somatic tissue.
In each of these
latter instances, the muscle in approximately the anterior half of
the fillet did not respond with normal resiliency when depressed with
a blunt instrument.
No macroscopic pseudocysts were found in any rock sole examined
and the tissue was characterized on the basis of texture.
whole fish infected with Kudoa sp.
Flesh of
ranged from firm with normal
color to softened and marked by opaque, milky-white areas to nearly
25
pseudocysts
Figure 4. Pacific hake, a) Non-melanized KudOa sp.
(arrows); b) Melanized Kudoa sp. pseudocysts (arrows); C)
Uninfected fillet. Bars = 2 cm.
4
..
/
,
-
27
liquified (Figure 5a). Tissue slurries taken to determine if
myxosporean spores were associated with obvious milky areas revealed
spores throughout the musculature of all infected fish, regardless of
whether or not the muscle appeared normal.
Three specimens of rock sole were parasitized by Unicapsula sp.
When the aiiunt of tissue for slurries from these fish was reduced to
a few muscle bundles, a massive number of spores was still observed.
The macroscopic appearance of the tissue was the same as that
infected with Kudoa sp.: white, opaque areas of softened muscle
surrounded by tissue of normal appearance and texture (Figure 5b). An
uninfected fillet of rock sole is shown in Figure Sc.
Parasite description
The only myxosporean stage observed in this study was mature
spores.
Each flatfish species was infected with a stellate species
of the genus Kudoa (Figures 6, 9, 11, 12, 15 and 17). Pacific hake
was infected with the stellate K. thyrsites and the quadrate K.
paniformis (Figures 7, 8, 13 and 14). The spores infecting each
flatfish host were measured and the dimensions were found to differ
significantly (Students t >=95%) between hosts and from any
previously described species.
Unicapsula sp., found only in rock
sole, also differed from any previously described species (Figures 10
and 16).
Kudoa sp.
from English sole (Figures 6 and 12)
Spores stellate; four equal valves tapering distally into rays
Figure 5. Rock sole, a) Heavy Kudoa sp.
infection, flesh exhibits
marked degradation; b) Light Unicapsula sp. infection,
note milky white areas (arrows); c) Uninfected fillet.
Bars = 2 cm.
29
30
Figure 6. Kudoa sp. from English sole, a) Polar view; b) Extruded
polar filament (arrow). Phase contrast. Bars = 10 pm.
Figure 7. Kudoa thyrsites from Pacific hake. a) Polar view. Some
polar capsules are disoriented (arrow); b) Lateral view.
Phase contrast. Bars = 10 pin.
Figure 8. Kudoa paniformis from pacific hake. a) Polar view. Bar =
10 urn; b) Note loops of polar filament (arrow) visible
within polar capsule and c) extruded polar filaments
(arrow). Phase contrast. Bars = b) and C) 5 pm.
Figure 9. Kudoa sp. from rock sole, a) and b) Same spore at
different focal planes. Phase contrast. Bars = 10 pIn.
Figure 10. Unicapsula sp.
from rock sole.
Phase contrast.
Bar = 5
pm.
Figure 11. Kudoa sp. from speckled sanddab. a) Polar view; b)
Lateral view (arrow). Phase contrast. Bars = 10 pm.
(...
(.
p
0
1S
I
[1
'a
I
I
32
Figure 12. Kudoa sp.
frai' English sole.
Figure 13. Kudoa thyrsites from pacific hake.
33
in polar view; one pyriform polar capsule in each valve, one large,
two of medium length and one small; polar filaments visible within
polar 4psules but number of turns unclear; sutural lines fine;
sporoplasrn granular; position of nuclei not observed.
Spore
dimensions are given in Table 3.
Table 3. Dimensions of fresh Kudoa sp. spores from English sole.
Measurements are in micrometers, based on 50 spores.
Character
Length
Width I
Width II
Thickness
Large polar
capsule length
Medium polar
capsule length
Small polar
capsule length
Mean
7.22
9.76
7.06
13.86
Standard
Error
Range
7.0
7.0
5.6
8.4
- 8.4
- 12.6
- 8.4
- 15.4
0.52
0.95
0.35
0.70
4.84
4.2 -
5.6
0.70
3.53
2.8 -
4.2
0.77
2.83
2.8 -
4.2
0.20
Kudoa t1yrsites from Pacific hake (Figures 7 and 13)
Spores stellate; four unequal valves tapering distally into rays
in polar view, one ray larger and its diagonal opposite smaller than
the other two rays which are equal to each other; one pyriformn polar
capsule in each valve, one large and the other three equal to each
other in length; polar filaments not easily visible within polar
capsules, however, they are straight when extruded; sutural lines
fine; sporoplasm granular; position of nuclei not observed.
dimensions are given in Table 4.
Spore
34
Table 4. Dimensions of K. thyrsites from previously frozen Pacific
hake. Measurements are in micrometers, based on 20 spores.
Character
Mean
Length
Width I a)
6.40
10.50
9.80
7.35
13.86
b)
Width II
Thickness
Large polar
capsule length
Small polar
capsule length
Standard
Error
Range
5.6
8.4
7.0
7.0
12.6
- 7.0
- 12.6
- 12.6
- 9.8
- 15.4
0.61
1.40
1.11
0.77
1.00
4.90
4.2 -
7.0
0.99
3.40
1.4 -
5.6
1.74
Kudoa paniformis from Pacific hake.
(Figures 8 and 14)
Spores quadrate, almost round in polar view with slight
constrictions at sutural lines; sutural lines distinct; spore surface
smooth; four equal polar capsules, one in each valve; polar filaments
visible within polar capsules but number of turns unclear; polar
filaments straight when extruded; sporoplasm granular; position of
nuclei not observed.
Spore dimensions are given in Table 5.
Table 5. Dimensions of K. paniformis from previously frozen Pacific
hake. Measurements in micrometers, based on 20 spores.
Standard
Error
Character
Mean
Range
Length
Width
Thickness
Polar capsule
length
Polar capsule
width
5.74
6.37
7.00
5.6 - 7.0
5.6 - 7.0
2.05
1.4
2.1
0.02
1.52
1.4 - 2.1
0.01
7.0
0.04
0.23
0.00
35
5j.m
Figure 14. Kudoa paniformis from Pacific hake.
5im
Figure 15. Kudoa sp.
from rock sole.
36
Kudoa sp.
from rock sole (Figures 9 and 15)
Spores stellate; four unequal valves tapering distally into rays
in polar view; one ray larger and its diagonal opposite smaller than
the other two rays which are equal to each other; one pyriform polar
capsule in each valve, one large, two of medium length and one small;
polar filaments visible within polar capsules but number of turns
unclear; sutural lines fine; sporoplasm granular; position of nuclei
not observed.
Spore dimensions are given in Table 6.
Table 6. Dimensions of Kudoa sp. spores from previously frozen
rock sole. Measurements in micrometers, based on 50 spores.
Character
Width I
Mean
a)
b)
Width II
Thickness
Large polar
capsule length
Medium polar
capsule length
Small polar
capsule length
Unicapsula sp.
Range
Standard
Error
-
16.8
15.4
11.2
22.4
1.36
1.20
1.36
2.57
13.88
12.08
8.70
18.28
11.2
9.8
7.0
14.0
5.86
5.6
7.0
0.55
4.21
2.8
5.6
0.42
3.16
1.4 -
4.2
0.68
from rock sole (Figures 10 and 16)
Spores round; three valves, two of which wrap around the third;
spore diameter 7.7 im (range of 10 measurements, 6.9 - 8.8 pin); only
the center valve has a polar capsule; polar capsule diameter 2.3gm
(1.9 -2.5 pm); polar filament makes between two and three turns
within polar capsule; sutural lines distinct.
37
V
Li
2.5jm
Figure 16. Unicapsula sp.
from rock sole
5pm
Figure 17. Kudoa sp.
from speckled sanddab.
Kudoa sp.
from speckled sanddabs (Figures 11 and 17)
Spores stellate; four unequal valves tapering distally into rays
in polar view; one ray larger and its diagonal opposite smaller than
the other two rays which are equal to each other; one pyriforin polar
capsule in each valve, one large, two of medium length and one small;
polar filaments visible within polar capsules but number of turns
unclear; sutural lines fine; sporoplasm granular; position of nuclei
not observed.
Spore dimensions are given in Table 7.
Table 7.
Dimensions of fresh Kudoa sp. spores from speckled
sanddabs. Measurements in micrometers, based on 50 spores.
Character
Length
Width I
Mean
a)
b)
Width II
Thickness
Large polar
capsule length
Medium polar
capsule length
Small polar
capsule length
Standard
Error
Range
9.8
- 8.4
- 14.0
0.51
0.73
0.74
0.49
0.94
4.2
7.0
0.66
3.94
2.8 -
5.6
0.58
2.83
1.4 -
4.2
0.35
7.09
9.36
8.18
6.97
12.50
5.6
8.4
7.0
5.6
9.8
5.37
-
8.4
- 11.2
Histological observations
Light microscopy
Spores of the genus Kudoa were found free within muscle tissue
in histological sections of Pacific hake and rock sole only (Figures
18 and 19). In infected Pacific hake, the irusculature near the free
spores had started to break down and tissue debris was visible
39
Figure 18. a) and b). Kudoa thyrsites spores (arrows) free within
body musculature of pacific hake. Note tissue debris (D)
between disrupted muscle fibers. Wheatley's modified
Gomori trichrome stain. Bar = a) 100 pin; b) 25 pm.
Figure 19. a) and b). Kudoa sp. spores (arrows) free within body
musculature of rock sole. Note tissue debris (D) between
disrupted muscle fibers. Giemsa stain. Bar = a) 100 pin;
b) 25 pin.
i.
ff
.Ir
t,
r
i!:
;#
J
1._P
(
'
.;
.,
:
1; '
D
b
/
1
'
'
4
41
between the fibers.
Muscle degradation was even greater in infected
rock sole tissue in which few of the muscle fibers near the spores
maintained any integrity.
Whether the spores caused the muscle
degradation or collected in areas were deterioration was advanced
could not be determined from the histological sections.
Pseudocysts in English sole, pacific hake and speckled sanddabs
were similar in histological section.
The distended muscle fibers,
viewed in cross section, were up to five times larger than nearby
uninfected fibers (Figures 20
22). In cross section, infected
muscle fibers in each host had a darkly staining, unidentified band
or zone between the spores and the muscle fiber (Figures 23a, 24a and
25a). This structure was not observed in longitudinal section, where
it appeared as if parasite spores were in direct contact with host
muscle fibers (Figures 23b, 24b and 25b). The presence of an apparent
structure between the parasite and the host tissue in cross section
and its apparent absence in longitudinal section was not a result of
sectioning different pseudocysts.
When the same pseudocyst was
sectioned in both planes, this observation was still made (Figure
26).
Melanized pseudocysts were observed only in Pacific hake.
Spores in these pseudocysts were often completely disrupted and the
pseudocyst was filled with spore fragments.
Frequently, no intact
spores were observed (Figure 27).
Rock sole were infected with both genera, Kudoa and Unicapsula.
Although no macroscopic pseudocysts were detected in any infected
42
Figure 20. Swollen muscle fiber (arrow) of English sole infected with
Kudoa sp. Wheatleys modified Gomori trichrome stain.
Bar = 100 )IJT1.
Figure 21. Swollen muscle fiber (arrow) of Pacific hake infected with
Kudoa thyrsites. Giemsa stain. Bar = 100 pin.
Figure 22. Swollen muscle fiber (arrow) of speckled sanddab infected
with Kudoa sp. Giemsa stain. Bar = 100 pm.
;-_
%: 1'.
S.
'1
S
,(
:..
..I-
k
i'.
----.
;(
F"
[221
r&i
Figure 23. Swollen muscle fiber of English sole infected with Kudoa
sp. Giemsa stain, a) Cross section. Note darkly stained
zone (arrow) at host/parasite interface; b) Longitudinal
section. No structure visible at host/parasite interface
(I). Bars = 25 pin.
Figure 24. Swollen muscle fiber of Pacific hake infected with K.
thyrsites. Giernsa stain, a) Cross section. Note darkly
stained zone (arrow) at host/parasite interface; b)
Longitudinal section. No structure visible at
host/parasite interface (I). Bars = 25 ,uJn.
Figure 25. Swollen muscle fiber of speckled sanddab infected with
Kudoa sp. Wheatleys modified GoimDri trichrome stain, a)
Cross section. Note darkly stained zone (arrow) at
host/parasite interface; b) Longitudinal section. No
structure visible at host/parasite interface (I). Bars
25 pin.
45
Figure 26. a) and b). A single Kudoa sp. pseudocyst (P) in English
sole sectioned in both the cross and longitudinal planes.
Note darkly stained zone (arrows) at host/parasite
interface in cross section which is not visible in
longitudinal section (I). Wheatleys modified Gomori
trichrorne stain.
Bar = 25 pm.
Figure 27. Kudoa sp. pseudocysts in pacific hake; melanized (M) and
non-melanized (N). Hematoxylin and eosin stain. Bar = 100
pm.
Figure 28. a) and b). Kudoa sp. in rock sole. Some tissue
degradation (D) is visible away from the pseudocyst (P)
but no structure is visible at the host/parasite interface
(I). Wheatley's modified Gomori trichrome stain. Bar = a)
100 pm; b) 25 pin.
¶t
i
-
:
-
'i1
--'
cI_
:iiàd
:,-
.'.
"dl
A.
.'
A
;
/
'
i'
L. ..
/1
'I
____
I
'\
\ \' 'V
I
ø'.
I -_
--
-. -----
:
-
ri4a
-
48
specimens, typical pseudocysts were observed in histological sections
(Figure 28). Sections were longitudinal and there was no detectable
host or parasite membrane between the spores and the host tissue.
Some tissue degradation was observed, but not immediately adjacent to
the myxosporean spores.
The muscle fiber surrounding a Unicapsula sp.
pseudocyst was
fragmented extensively (Figure 29). However, tissue breakdown was not
visible in nearby fibers and few parasite spores had moved away from
the pseudocyst into the nearby muscle tissue.
In lightly infected
rock sole, in which opaque, milky-white areas were visible in the
flesh, there was no evidence of pseudocysts (Figure 30). Rather, the
spores were spread throughout the musculature and degradation was
extensive.
Electron microscopy
English sole.
Muscle fibers of infected English sole viewed in
longitudinal section in the electron microscope appeared disoriented
and fragmented.
The Z lines were uneven and, because of the
separated filaments, the I bands were almost indistinguishable.
The
sarcoplasmic reticulum was misaligned and free ribosomes dotted the
fibers.
No membrane was visible between the parasite cytoplasm and
the host tissue (Figure 31). A section through two polar capsules of
an intact spore showed three turns of the polar filament (Figure 32).
Infected nuscle fibers also appeared disrupted when viewed in
cross section (Figure 33), although not as greatly as in longitudinal
Figure 29. a) and b). tinicapsula sp. pseudocyst in rock sole.
Infected muscle fiber surrounding pseudocyst (P) has
fragmented although adjacent fibers (F) appear normal.
Spores (arrows) are starting to spread. Wheatley S
Bar = a) 100 pm; b) 25
modified Gorrori trichrome stain.
jim.
Figure 30. a) and b). Unicapsula sp. in rock sole. Aggregation of
spores (S) is not confined to a pseudocyst but are
spreading throughout the body musculature (arrows) and
tissue degradation (D) is pronounced. Wheatleys modified
Gomori trichrome stain. Bar = a) 100 yin; b) 25 )irrn.
c!
-
(I)
-
:E!*
1
-.
A
IiLI
r
-
51
Figure 31. Transmission electron micrograph of Kudoa sp. in English
sole. Longitudinal section of pseudocyst and adjacent
host tissue. Note disorientation of myofibrilar Z lines
(arrows) and I bands (I); cisternae of the sarcoplasrnic
reticulum (SR), free ribosoines (FR), free polar capsules
(PC). Bar = l.0)im.
in English
sole. Three turns of the polar filament (PF) are visible
within the polar capsule (PC). Note spore valve (SV),
Figure 32. Transmission electron inicrograph of Kudoa sp.
sporoplasm (S) and nucleus (N). Bar = 1.0 pin.
,-,,
-, $
4..
:
'L.
-
4
,-
52
a
53
Figure 33. Transmission electron micrograph of Kudoa sp.
in English
sole. Cross section of parasite pseudocyst and adjacent
host tissue. Note cisternae of the sarcoplasrnic reticulum
(C) is misaligned around the myofibrils (M); parasite
ectoplasm (PE). Bar = 1.0 pm.
Figure 34. Transmission electron micrograph of Kudoa sp.
in English
sole.
Cross section of parasite pseudocyst and adjacent
host tissue. Parasite cell membrane (cM) is separated
from host tissue (M) by a zone of cisternae (C), free
ribosomes (FR) and globular particles (GP). Bar = 1.0 jim.
54
(. I
r4I.--
.è..:
I-
L
:
:.
..*
4:
.r'
'.
.4
J a
11.
'%
::
.
..
.
i
;....-..
j
.4
9.J
S
55
section.
In some areas, the sarcoplasmic reticulum did not uniformly
surround the ir'ofibrils and divisions between adjacent myofibrils
were indistinct.
A zone approximately 1.3 pin wide separated the
rrryxosporean from the host nuscle tissue (Figure 34). This zone, not
evident in longitudinal section, was made up of cisternae, free
ribosornes and globular particles.
This tissue was sanpled at the
same time from the same fresh host as the sample that was sectioned
longitudinally which suggests the nore extensive disruption of the
muscle and pseudocyst in longitudinal section may have been
artifacts.
pacific hake.
Ultrastructural examination of infected pacific
hake revealed the host-parasite interface to consist of a thin (< 0.2
pin), darkly staining structure.
membrane.
This did not appear to be a
In longitudinal section, this structure was irregular,
sometimes folding back on itself, and incomplete (Figures 35 and 36).
It appeared to be the only feature separating the host muscle fibers
from the myxosporean cytoplasm.
In some areas the muscle tissue was
only slightly disoriented with the sarcoplasmic reticulum visible
between the myofibrils (Figure 35). Elsewhere, the muscle was highly
disorganized (Figure 36). The thin structure normally separating the
parasite cytoplasm and the host tissue was also broken at this
point.
In longitudinal section, no cell organelles, generative cells or
mature spores were identifiable within the parasite pseudocyst.
The
cytoplasrnic area was filled with darkly staining, granular material
56
Figure 35. Transmission electron inicrograph of Kudoa sp. in pacific
Longitudinal section of a parasite pseudocyst,
hake.
possibly melanized, and adjacent host tissue. Note darkly
staining boundary (B) between myofibrils (M) and
pseudocyst (p). pseudocyst is filled with much cellular
debris (D) and no identifiable spores or polar capsules.
Bar = 1.0 pin.
Figure 36. Transmission electron micrograph of Kudoa sp.in Pacific
hake. Longitudinal section of a parasite pseudocyst,
possibly melanized, and adjacent host tissue. Boundary
(B) between host and parasite is inconplete; myofibrils
(M) are highly disorganized. Pseudocyst is filled with
much cellular debris (D) and no identifiable spores or
polar capsules. Bar = 1.0 pin.
'
-
p
-IuIpI.p
a
,v
2
0.
V
4
-
-
I,
,4v
2
L
irc
I
'
V
(Figures 35 and 36). In contrast, cell organelles and mature spores
were observed in cross section (Figures 37 and 38).
The host-parasite interface in pacific hake in cross section was
similar in appearance to that of longitudinal section.
A thin,
darkly staining structure was in contact with host muscle tissue on
one side and myxosporean cytoplasm on the other.
The riuscie tissue
varied from only slightly disrupted (Figure 37) to very disorganized
(Figure 38).
Speckled sanddabs.
In longitudinal section, the speckled
sanddab pseudocyst cell membrane was only 0.6 pm thick (Figure 39). A
zone of cisternae and free ribosomes separated the cell membrane from
the host tissue.
This was particularly evident in cross section
(Figure 40). Also in cross section, the cell membrane, up to 2.5 pitt
in some places, was deeply folded into what may have been pinocytotic
channels.
The end sacs of some of these channels were pinched off
into vesicles.
The rnyofibrils adjacent to the parasite pseudocyst were
predominately intact.
misaligned.
Some of the sarcomeres, however, were
At one point, it appeared as though a protrusion of the
parasite cell membrane caused a break in the muscle fibers (Figure
41). occasionally, where the myofibrils met the zone of cisternae and
ribosomes, the muscle was raggedly sheared as though it had been
torn.
The sarcoplasmic reticulum was intact in most areas with the
transverse tubules and terminal cisternae in proper orientation.
In
general, it appeared that speckled sanddab muscle tissue was little
Figure 37. Transmission electron niicrograph of Kudoa sp. in Pacific
hake. cross section of parasite pseudocyst and adjacent
host tissue. similar boundary (B) between host and
parasite to that observed in longitudinal section.
Myofibrils (M) are well defined; a spore (SP) is visible
within the pseudocyst. Bar = 1.0 JIm.
Figure 38. Transmission electron inicrograph of Kudoa sp in Pacific
hake. Cross section of parasite pseudocyst and adjacent
host tissue. Boundary is incomplete (arrows); inyofibrils
(M) are disorganized ; mature spores (SP) are visible
within the pseudocyst. Bar = 1.0 pm.
.l
A
j4
4'
J1
,
"uur
p7
Lw
-:
ç;
..
I
I
...
-
'*it
61
in speckled
Figure 39. Transmission electron micrograph of Kudoa sp.
sanddab. Longitudinal section of parasite pseudocyst and
adjacent host tissue. Parasite cellular membrane (CM) is
separated from ir'ofibrils CM) by a zone (z) of cisternae
and free ribosomes. Note generative cell (GC). Bar = 1.0
pm.
in speckled
Figure 40. Transmission electron micrograph of Kudoa sp.
sanddab. Cross section of parasite pseudocyst and
adjacent host tissue. A broad zone of cisterne (C) and
free ribosomes (FR) separate the parasite cell membrane
(cM) from the rnyofibrils (M). Cell membrane is invaginated
with some channels forming pinocytotic vessicles (PV). Bar
= 1.0 pm.
/
..-
-S..
r
.'
S
I
)
-
.1
I.
4
_______
______
l..
I
I'
7'f
I
I
I
__
__
-'I
-
-
4
r
-
Ak
4
-
if
/
'.4
.
63
Figure 41.
Transmission electron micrograph of Kudoa sp. in speckled
Longitudinal section of parasite pseudocyst and
sanddab.
Myofibrils (M) are predominately
adjacent host tissue.
intact with some sarcoplasmic reticulum cisternae (C) and
T-tubules (T) misaligned. Myofibrils appear normal almost
up to point of contact with parasite cell membrane (CM).
Torn myofibrils (arrow) may be caused by physical
displacement by the pseudocyst or may be an artifact of
histological preparation. Bar = 1.O1um.
influenced by the presence of the parasite pseudocyst except for
physical displacement.
Relationship between Kudoa spp. and tissue breakdown in
English sole and speckled sanddabs
There was no macroscopic difference in muscle texture observed
between myxosporean-infected and control samples of English sole or
speckled sanddabs left at room temperature for 72 h following host
death.
Microscopic examination of histological sections, however,
revealed abnormal tissue breakdown in infected English sole.
Speckled sanddab tissue appeared to be more disrupted than English
sole tissue, but in speckled sanddabs the tissue breakdown in the
infected samples was comparable to that in the control samples.
Unevenly stained muscle fibers, i.e., those with lightly stained,
swirled centers, were taken as indicative of muscle deterioration.
They were observed in only 1 of 14 control samples and in most of the
infected samples in which the muscle fibers surrounding the infected
fiber appeared altered.
Although the results of this experiment were
inconclusive, they suggested that tissue degradation following host
death was more pronounced in infected English sole than in uninfected
samples.
The degree of post-mortem tissue breakdown in infected
speckled sanddabs was similar to that in the control samples.
English sole
Individual pseudocysts were intact through 7 h post-mortem but
some of the muscle fibers in this sample showed fragmentation at this
65
stage (Figure 42). Most tissue in the 7 h control sample showed
little muscle fragmentation (Figure 43). Areas that did have
indications of muscle breakdown did not contain muscle fibers with
lightly staining centers which possibly indicated muscle disruption,
as seen in the infected sample (Figure 44).
At 30 h post-Iiortem, the pseudocysts were broken open, spores
were free within the surrounding tissue and muscle tissue breakdown
increased (Figure 45). Most of the tissue in the 30 h control sample
showed very little breakdown (Figure 46). Some unevenly staining
muscle fibers were observed in the 30 h control samples (Figure 47),
but not in any later samples.
After 48 h, spores had spread further
into the surrounding tissue (Figure 48) and tissue debris was visible
between damaged muscle fibers.
Similar debris was not evident in the
48 h control sample (Figure 49). At 60 h, a pseudocyst was completely
disrupted with some spores in close proximity and many diffused into
the surrounding tissue (Figure 50). The control sample exhibited very
little breakdown at 60 h (Figure 51).
Tissue degradation appeared to be related to spore maturity as
well as to the length of time following host death.
In the 72 h
infected sample, spores were confined to an intact pseudocyst and the
surrounding muscle fibers appeared normal (Figure 52). The 72 h
control sample was similar (Figure 53). In earlier samples, the
spores had started to diffuse away from the disrupted pseudocyst into
the adjacent, deteriorating tissue.
The spores within pseudocysts in
these earlier samples also exhibited variations in staining, possibly
Figure 42. Kudoa sp. in English sole, 7 h post-riortem. Pseudocyst
(P) is intact but nearby muscle fibers (M) exhibit lightly
staining centers, possibly indicative of degradation.
Wheatleys modified Gomori trichrome stain. Bar = 100
pm.
Figure 43. a) and b). English sole control, 7 h post-mortem. Muscle
fibers (M) exhibit no uneven staining. Wheatleys
modified Gomori trichrome stain.
Bar = a) 100 pm; b) 25
pm.
Figure 44. a) and b). English sole control, 7 Ii post-mortem. Muscle
fibers (M) exhibit extensive fragmentation but none have
lightly staining centers. Wheatleys modified Gonhori
trichrorne stain.
Bar = a) 100 pm; b) 25 pin.
67
in English sole, 30 h post-mortem. a) Muscle
fibers (M) near pseudocyst (P) and sorre not in the
immediate vicinity of the pseudocyst are disrupted.
Wheatleys modified Gomori trichrome stain. Bar = 100 pin;
b) Spores (arrows) free within host tissue. 'Wheatley's
modified Gomori trichrome stain. Bar = 25 jUn.
Figure 45. Kudoa sp.
Figure 46. a) and b). English sole control, 30 h post-mortem.
Muscle fibers (M) exhibit no uneven staining. Wheatley
modified Goirori trichrorne stain.
5
Bar = a) 100 pin; b) 25
pm.
Figure 47. a) and b). English sole control, 30 h post-mortem. Some
muscle fibers CM) exhibit lightly staining centers,
possibly indicative of degradation. Muscle disruption is
not as extensive as observed in 30 h Kudoa-infected
Wheatleys modified Gorrori trichronie stain. Bar
= a) 100 pm; b) 25 pin.
sample.
;;
a.'
c
-
-
.,'.:
-
1
/..
w: %
'
1
'Eá
h(
k;-
..:
-
_
-
,..
-:
Vi
:
Li
:'
70
a)
Figure 48. Kudoa sp. in English sole, 48 h post-mortem.
Pseudocyst (P) is broken and muscle fibers (M) are geatly
disrupted. wheatleys modified Gomori trichrome stain.
Bar = 100 pin; b) Spores (arrows) are spreading away from
the pseudocyst (p) and tissue debris (D) is visible
between muscle fibers. wheatleys modified Gomori
trichrome stain. Bar = 25 pm.
Figure 49. a) and b). English sole control, 48 h post-mortem.
Muscle fibers (M) are intact with no tissue debris between
Bar =
them. Wheatley's modified Gomori trichrome stain.
a) 100 mi; b) 25 ).lm.
'r
'1.
ac. Tk
II
I
M L 1
fc
-
a
v;i
r t
y
I
I
MhiJ
J
J.
72
Figure 50. a) and b). Kudoa sp. in English sole, 60 h post-mortem.
Pseudocyst (P) is completely disrupted and spores (arrows)
are free within the muscle tissue. Giemsa stain. Bar =
a) 100 pin; b) 25 pin.
Figure 51. a) and b). English sole control, 60 h post-mortem.
Muscle fibers (M) are intact with no tissue debris between
them. Wheatleys modified Gomori trichrome stain. Bar =
a) 100 ).in; b) 25 jim.
73
ii
5Ob
'51b
JuII-.'
74
Figure 52. a) and b). Kudoa sp. in English sole, 72 h post-mortem.
Pseudocyst (P) is intact; although some muscle fibers (M)
stain unevenly, rio tissue debris is visible between them.
Qheatley's modified Gomori trichronie stain. Bar = a) 100
J.lm; b) 25 pm.
Figure 53. a) and b). English sole control, 72 h post-mortem.
Muscle fibers (M) are intact with no tissue debris visible
between them. Wheatley's modified Gomori trichrorne
stain. Bar = a) 100 pm; b) 25 pm.
75
76
related to the development of the spores.
Speckled sanddabs
Although minor muscle fragmentation was evident in infected
speckled sanddabs at 30 1-i (Figure 54) and at 60 h post-mortem (Figure
55), individual pseudocysts were intact through 60 h post-mortem
(Figure 55). In neither sample was the deterioration substantially
different from the control samples (Figures 56 and 57).
Only at 72 h post-mortem was the pseudocyst broken apart and
spores free within the surrounding muscle (Figure 58). Muscle
degradation within the vicinity of the spores was pronounced.
However, the appearance of the infected tissue did not differ
substantially from that of control samples (Figures 59). Most of the
tissue breakdown observed, whether in infected or control sanples,
was in close proximity to the speckled sanddab skin.
Speckled sanddab myxosporeans also exhibited a range of spore
development as suggested by their staining properties.
Nevertheless,
spore development alone cannot account for the differences observed
between the tissue breakdown in English sole and speckled sanddabs.
Many, if not most, of the pseudocysts in speckled sanddabs contained
what appeared to be fully mature spores.
77
Figure 54. Kudoa sp.
in speckled sanddab, 30 h post-mortem. Muscle
fibers (M) exhibit degradation near intact pseudocyst (P)
and skin (S). Wheatley's modified Gomori trichrome stain.
Bar = 100 pin.
Figure 55. Kudoa sp. in speckled sanddab, 60 h post-mortem. Muscle
fibers (M) near the intact pseudocyst (P) appear normal,
but elsewhere (arrows), they exhibit degradation. Giemsa
stain. Bar = 100 pm.
Figure 56. Speckled sanddab control, 30 h post-mortem. Muscle fibers
vary from intact (I) to extensively disrupted (D).
Wheatley's modified Gomori trichrome stain. Bar = 100
pm.
Figure 57. speckled sanddab control, 60 h post-mortem. Muscle fibers
are extensively disrupted (D). wheatley's modified Gomori
trichrome stain. Bar = 100 pm.
F
I
/J(I I
fi
7 ric
d1
.(
I
_./
'/' -
A
I
1
Ii
F'
j
:
rs)
,gç
II.
!'A'
j/: 111!
..IYAf L'i
J
I
g
&
t
:
/
d
,.rt
t'4
I
79
in speckled sanddab, 72 h
Kudoa sp.
Pseudocyst (P) has lost all integrity,
post-IrKrtent.
muscle fibers (M) in the vicinity of the pseudocyst are
disrupted. Most tissue near the skin is broken down, but
muscle fibers away from the pseudocyst and the skin are
intact (I). Spores are free within the musculature
(arrows). Wheatleys modified Gomori trichrome stain. Bar
Figure 58. a) and b).
= a) 100 pin; b) 25 pin.
Figure 59. a) and b). Speckled sanddab control, 72 h post- mortem.
Fibers away from the skin (S) are intact (I). Muscle
fibers (N) near the skin are disrupted. Wheatleys
a) 100 pin; b) 25
modified Gomori trichrome stain. Bar
Ptm.
:.
'
'':
Sv
i
\
: 'a1"
'
DISCUSSION
The quadrate species of the genus Kudoa that parasitized pacific
hake in this study was determined to be K. paniformis, a irtyxosporean
recently described from the same host by Kabata and Whitaker (1981).
¶Lo other quadrate Kudoa species have been described from hosts
closely related to Pacific hake, K. rosenbuschi from Chilean hake
(Gelormini 1944) and K. peruvianus from Peruvian hake (Salas 1972).
Kudoa rosenbuschi differs from K. paniformis by having larger spores
of a different shape and by having intermuscular rather than
intramuscular pseudocysts.
Although Kabata and Whitaker (1981) made
no reference to K. peruvianus in their discussion of K. paniformis,
the dimension ranges and other characteristics of K. peruvianus are
similar to Kabata and Whitaker's description of K. paniformis.
However, Salas (1972) described K. peruvianus as having only two
valves.
The relationship between K. peruvianus and K. paniformis is
not clear.
According to Kabata and Whitaker (1981), the stellate species of
Kudoa in Pacific hake is K. thyrsites.
Although they acknowledged
that the polar capsule size ratio and the arrangement of the
filaments within the polar capsules that they observed differed from
the original K. thyrsites description given by Gilchrist (1924), they
nevertheless considered the parasite in Pacific hake to be K.
thyrsites.
The basis for this determination was the general
similarity of the spores, the observation that the parasite forms
pseudocysts without involving the hosts connective tissue in a
manner similar to K. thyrsites, and the occurrence of K. thyrsites in
another hake species, Merluccius capensis.
The observations made in
this study were consistant with those of Kabata and Whitaker (1981)
and the stellate myxosporean in pacific hake was considered to be K.
thyrsites.
The stellate spores from the flatfish hosts in this study
differed from all described species of the genus Kudoa, except K.
thyrsites, by having polar capsules of unequal length.
Each flatfish
uyxosporean had three distinctly different sized polar capsules, one
large, one small and two of equal intermediate length.
Polar capsule
size ratio, spore dimensions, spore shape and hosts distinquish the
parasites in English sole, rock sole and speckled sanddabs from all
previously described species.
The Kudoa most similar to those found in flatfishes in this
study is K. thyrsites.
However, K. thyrsites spores were
significantly different in size from those found in flatfishes and K.
thyrsites has one large and three smaller, equal polar capsules
rather than the three distinctly different sized polar capsules
observed in spores from each flatfish host.
Finally, K. thyrsites
has not been reported from either the pleuronectidae or the Bothidae.
Because of these spore and host differences, it was concluded that
English sole, rock sole and speckled sanddabs are each infected by a
different parasite and all of these species were previously
undescribed.
The ecology and habitats of English sole and speckled
sanddabs overlap extensively but the uyxosporeans infecting each host
are apparently different.
This suggests a strong host specificity on
the part of these rnyxosporeans.
Species in the genus Unicapsula are differentiated on the basis
of the spore dimensions, the number of turns made by the polar
filaments within the polar capsule, the microscopic appearance of the
infected host muscle fiber and the host infected (Lester 1981). The
Unicapsula sp.
observed in rock sole in this study was much larger
than all other described species of the genus.
The fraction of the
third turn made by the polar filament could not be determined without
electron microscopy, but based on spore dimensions and the host
in rock sole was
infected, it was concluded that the Unicapsula sp.
previously undescribed.
The relationship between riyxosporean infection and rapid
post-mortem host tissue degradation has never been clearly defined.
Many investigators have inferred a cause and effect relationship
(Gilchrist 1924; Fletcher et al.
1951; Matsurnoto 1954; Forrester
1956; Patashnik and Groninger 1964; Dassow et al.
1970; Salas 1972;
Konagaya 1980; Kabata and Whitaker 1981; Okada et al.
Patashnik et al.
1982; Tsuyuki et al.
1981;
1982) but no
parasite-produced enzyme has been isolated from a myxosporean
pseudocyst or adjacent host tissue.
spores of the genus Kudoa spp.
Neither have all studies of
occurring in fish musculature
reported host tissue degradation (Iversen and van Meter 1967; Egusa
and Nakajirna 1980). The ultrastructure of the host-parasite interface
was examined in the present study to determine if differences occur
at this boundary which might account for the various effects that the
parasites have on their respective hosts.
In light microscopic cross sections of English sole, pacific
hake and speckled sanddabs, infected muscle fibers had a darkly
staining, unidentified band or zone between the spores and the muscle
fibers.
This zone was not observed in longitudinal section where it
appeared as though parasite spores were in direct contact with host
muscle fibers.
It was hoped that electron microscopy could unravel
this perplexing observation and determine if there is a melTbrane or
other structure of either host or parasite origin at the interface.
Few investigators have mentioned the characteristics of the
host-parasite interface in their descriptions of the microscopic
appearance of myxosporean infected tissue.
Voelker et al.
(1978)
observed a "space of uniform width" that they believed represented a
cyst wall between the pseudocyst and the surrounding sarcolemma.
All
light micrographs were cross sections of infected muscle fibers and
were similar to those observed in the present study, but no further
discussion of the "space" was given.
patashnik et al.
(1982)
included both cross and longitudinal sections in their investigation
of myxosporeans in Pacific hake but it is difficult to discern much
detail about the host-parasite interface from their figures.
The
appearance of the host-parasite interface in cross section was
similar to that observed in the present study but the authors made no
mention of the interface in their discussion.
Lom et al.
(1983) used both light and electron microscopy to
examine the host-parasite interface of various species of the genus
Kudoa and their hosts.
They concluded that pseudocysts locate within
muscle fibers, are encased only in their own plasmalemma and elicit
no host tissue reaction.
Each light micrograph shown was of a
longitudinal section of infected material where spores appeared to be
in direct contact with the host muscle fibers.
When discussing the
various parasite species that were studied, numerous references were
made to the trophozoite cell membrane although there was no
description of the appearance of this structure in cross section nor
was any explanation given for its apparent absence in
photomicrographs of longitudinal section of infected muscle fibers.
Electron micrographs of the host-parasite interface of K. lunata
revealed the boundary between host and myxosporean to be an
extensively folded trophozoite plasmalenirna which is in direct contact
with the myofilainents and with the vesicles of the sarcoplasmic
reticulum.
These vesicles are drawn into the deep folds of the
plasinalemma indicative of pinocytotic activity of the trophozoite.
The myofilaments maintain structural integrity in the vicinity of the
pseudocyst, exhibiting only minor disorganization in limited areas
(Lom et al.
1983).
In the present study, electron microscopy did not resolve the
dilemma of the appearance of the host-parasite interface in cross
versus longitudinal section.
In speckled sanddabs, the thickness of
the pseudocyst cell membrane was consistent in longitudinal section.
It was also consistent in cross section but the measurements in this
In
plane were four times as thick as those in longitudinal section.
longitudinal section, the cell membrane appeared to be only 0.3 to
1.0 pin wide in the electron microscope.
The limit of resolution in
light microscopy at 400x is approximately 0.3 pin, yet higher
magnification failed to reveal the parasite cell membrane in
longitudinal section in the light microscope.
It was not possible to
compare electron micrographs of cross and longitudinal sections of
the host-parasite interface in English sole and Pacific hake.
In
longitudinal sections from each host, the pseudocysts were not
intact, they contained much cellular debris and adjacent myofibrils
were greatly disrupted.
This may have been a result of the condition
of the tissue prior to histological preparation or it may have been
an artifact of fixation.
Unfortunately, no control muscle samples
from any host were prepared for electron microscopy in this study.
It was concluded that in speckled sanddabs and probably in English
sole and Pacific hake, the myxosporean pseudocyst is surrounded only
by a membrane of parasite origin.
This agrees with the descriptions
of K. lunata and various unidentified species of the genus (Lom et
al.
1983). Neither light nor electron microscopy, however, explained
why the parasite cell membrane appears much thicker in cross section
than it does in longitudinal section.
Evaluations of myxosporean-infected fish muscle texture have
traditionally been macroscopic observations and have been qualitative
assessments of raw fish (Davis 1923; Gilchrist 1924; Matsumoto 1954;
Forrester 1956; Arai and Matsuinoto 1957; Heckinann and Jensen 1978) or
cooked fish (Dassow et al.
1970; Anonymous 1978; Okada et al.
1981;
87
1982) or they have been quantitative tests of the
Patashnik et al.
force necessary to shear cooked infected muscle fibers (Lester 1982;
Tsuyuki et al.
1982). Seldom have these evaluations been combined
with histological observations (Patashnik et al.
1982). The above
investigators who examined raw fish obtained their samples from
coirmercial fish markets, so whether the fish had been frozen or not,
they had been dead for at least several hours prior to examination.
This may have coirplicated the interpretation of host tissue texture.
The English sole and speckled sanddabs examined in this study to
chronicle possible myxosporean-associated tissue degradation were
examined without previous freezer storage and without cooking and the
evaluation of tissue condition was based on observations of
histological sections.
Only one other report has included photomicrographs of
histological sections of myxosporean-infected material (K. paniforinis
in Pacific hake) fixed over time to document tissue degradation
(Patashnik et al.
samples.
1982). There was, however, no reference to control
Patashnik et al.
(1982) pointed out tissue damage that was
some distance away from the pseudocyst although the pseudocyst
nearest the damaged tissue may not have been included in the figure.
Similar observations were made in the present study.
Tissue
breakdown occurred in English sole muscle fibers not irrmediately
adjacent to the pseudocysts.
It is uncertain why, if the myxosporean
pseudocyst is producing a proteolytic enzyme, the tissue degradation
would not radiate out from the infected fiber.
Softened muscle tissue of fish infected with inyxosporeans has
been related to the length of time between host death and examination
(Willis 1949), the method of cooking infected fillets (Dassow et al.
1970; Patashnik et al 1982), the parasite species (Tsuyuki et al.
1982) and the degree of melanization of the pseudocysts (Okada et
al.
1982; Tsuyuki et al.
1982). The greater length of time between
host death and examination may have contributed to the observed
differences in tissue texture between the previously frozen infected
Pacific hake and rock sole and the English sole and speckled sanddabs
examined fresh.
Most reports of abnormal flesh texture in fish
parasitized by irtyxosporeans have been from observations made on hosts
which had been dead for several hours to several days (Fletcher et
al.
1951; Matsumoto 1954; Patashnik and Groninger 1964; Dassow et
al.
1970; Okada et al.
Tsuyuki et al.
1981; Lester 1982; patashnik et al.
1982;
1982). The Pacific hake and rock sole examined in
this study had been dead for at least several days before
examination, although specimens were frozen soon after capture.
Hoffman and Putz (1969) showed that Myxosoma cerebralis spores can
survive -20°C by infecting rainbow trout (Salmo gairdneri Richardson)
fry with spores frozen for 18 days and aged for four months.
Tissue
degradation has also been associated with myxosporean infection in
previously frozen hosts (Matsuinoto 1954; patashnik and Groninger
1964; Patashnik et al.
1982; Tsuyuki et al.
1982).
Freezing and thawing could affect the integrity of the
peudocysts, thereby allowing the spores to diffuse into the
musculature as observed in this study in pacific hake and rock sole.
Physical handling of the host, either during commercial harvest or,
in the case of rock sole, at the fish processing plant, could also
rupture the pseudocyst.
English sole and speckled sanddabs were
transported to the laboratory in tanks of sea water and held alive
until examination.
They were not subjected to the same treatment as
the Pacific hake or rock sole (coiruiercial harvesting or processing, a
period of days between host death and examination or freezing and
thawing) which may account for the observation of no spores free
within the muscle tissue of English sole or speckled sanddabs.
Many investigators report flesh mushiness only after the
infected material has been slowly cooked (Dassow et al.
et al.
1981; Lester 1982; Patashnik et al.
1970; Okada
1982; Tsuyuki et al.
1982). Some investigators have reported that apparently uninfected
hosts may become unacceptably soft after cooking (Okada et al.
1981). Without establishing a positive cause and effect relationship
between myxosporean infection and tissue breakdown, they
characterized the levels of myxosporean infection on the degree of
tissue mushiness after cooking rather than on direct observation of
spores or pseudocysts.
Host tissue mushiness may be related to the parasite species
(Tsuyuki et al.
1982). Of the five pacific hake with softened
musculature examined in this study, one was infected with K.
thyrsites only, one was infected with K. paniformis only and three
were infected with both species.
There appeared to be no difference
between the softened texture of the pacific hake with K. thyrsites
only and those with either a mixed infection or K. paniformis alone.
The specimen with K. thyrsites only had a trace infection (no visible
pseudocysts) so it is possible that K. paniformis may have been
present but not observed.
Although one individual infected with K.
thyrsites alone with softened muscle texture does not support any
generalized conclusions, these results appear to conflict with those
of Tsuyuki et al.
(1982). Their results indicated mushiness
associated only with K. paniformis.
Uninfected Pacific hake or those
infected with only K. thyrsites did not exhibit unacceptably soft
cooked flesh texture, based upon quantitative evaluations of flesh
texture.
Investigators who did not specifically identify the parasite
species present, related tissue texture to pseudocyst melanization
and found inelanized pseudocysts to be less enzymatically active than
non-melanized pseudocysts (Anonymous 1978; Kabata and Whitaker 1981;
Patashnik et al.
Okada et al.
1982). In marked contrast to all other accounts,
(1982) indicated that yellow or black (melanized)
pseudocysts were most enzymatically active.
They referred to the
parasite as a myxosporean without identifying the genus, but it was
probably in the genus Kudoa.
Although melanin deposition is commonly associated with
parasitic infections in which the host attempts to isolate the
parasite (Gamble and Drew 1911; Hsiao 1941; Chapman and Hunter 1954;
MacQueen, MacKenzie, Roberts and Young 1973), no rnelanized
pseudocysts were observed in any flatfish host in this study.
In
J1
Pacifc hake, however, melanized pseudocysts were relatively common.
Melanization is either a host defense response (Smith 1931; Chapman
and Hunter 1954) or the result of abnormal tissue metabolism (Chapman
and Hunter 1954) and it has been reported to increase with the age of
parasite infection (Hsiao 1941; Chapman and Hunter 1954; MacQueen et
al.
1973). Similarly, pseudocysts and spores appear to be destroyed
by the deposition of melanin (Kabata and Whitaker 1981; Patashnik et
al.
1982) and melanized pseudocysts are believed to be older than
non-melanized pseudocysts (Kabata and Whitaker 1981; Tsuyuki et al.
1982).
In the present study, most melanized pseudocysts in pacific hake
were K. thyrsites and all non-melanized pseudocysts were K.
paniforinis.
A similar relationship between parasite species and
melanization in pacific hake was found by Kabata and Whitaker (1981)
and Tsuyuki et al.
(1982) although they did report a few
non-rnelanized K. thyrsites pseudocysts.
They interpreted this host
response of pseudocyst melanization as an indication that Pacific
hake and K. thyrsites have an old, well established relationship and
that K. paniformis is a relatively recent parasite of Pacific hake.
This interpretation was based on the assumption that one of the
hallmarks of an old and well established host-parasite system is a
moderate or slight virulence of the parasite in that system.
A
parasite newly acquired by the host tends to be much more harmful.
Kabata and Whitaker (1981) also assumed that the relative abundance
of new (non-melanjzed) and old (jnelanized) infections with K.
paniformis and K. thyrsites can be taken as an indication of the rate
92
of infection, the level of development of the host's defensive
systems or both.
Another interpretation could be the reverse of Kabata and
Whitakers (1981). Many other host-parasite relationships also
involve melanin deposition (Roberts 1975). In arthropods, potential
parasites invading the tissues of unsuitable hosts may be treated as
foreign objects and be melanized.
In the appropriate hosts, they may
remain unencapsulated or a non-inelanized capsule of a different kind
may form around them (Nicholas 1973). Crompton (1967) found that
healthy acanthocephalan larvae do not stimulate the haemocytic
reactions in their arnphipod hosts, but wounded or damaged parasites
are encapsulated.
Apparently a living parasite within its envelope
is treated like healthy host tissue.
Nicholas (1973) noted
exceptions to his generalizations and Crompton (1967) cautioned
against extending his findings to other taxa without further studies,
but these conclusions suggest a possible alternative explanation of
the melanized and non-inelanized pseudocysts in Pacific hake.
The
relative abundance of parasites and the lack of host reaction in the
form of melanization of pseudocysts may indicate that K. paniforinis
is a long time parasite of Pacific hake.
Kudoa thyrsites may have
recently been introduced to Pacific hake which results in the
foreign body" reaction of melanization.
The appearance of infection by the genus Kudoa in English sole,
rock sole and speckled sanddabs is similar to that of in other hosts
as reported in the myxosporean literature.
pseudocysts in each host
are surrounded only by a cell membrane of parasite origin.
The
precise mechanism by which members of the genus Kudoa are involved in
host tissue degradation remains unclear.
94
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