Quantitative assessment of Azumiobodo hoyamushi distribution in the tunic of soft tunic syndrome–affected ascidian Halocynthia roretzi using real-time polymerase chain reaction
© Shin et al.; licensee BioMed Central Ltd. 2014
Received: 21 August 2014
Accepted: 13 November 2014
Published: 26 November 2014
The kinetoplastid parasite, Azumiobodo hoyamushi, is the causative agent of soft tunic syndrome (STS) in ascidians and leads to their mass mortality in Korean waters. This study was conducted to quantify A. hoyamushi density during the development of STS in the tunics of ascidians (Halocynthia roretzi) using real-time polymerase chain reaction (qPCR).
The infection intensity of A. hoyamushi, as measured by qPCR, varied depending on the part of the tunic analyzed, as well as the stage of STS development. The highest infection intensity was recorded in the tunics of the siphons. The infection intensity of A. hoyamushi in the siphons was only 2.9 cell/tunic (area, 0.25 cm2) or 106.0 cell/gram tunic (GT) in the early phase of STS, but this value increased dramatically to 16,066 cells/tunic (0.25 cm2) or 617,004 cell/GT at the time of death. The number of A. hoyamushi parasites increased gradually and their distribution spread from the siphons to the other parts of the tunics.
qPCR enabled the quantitation of A. hoyamushi and the results revealed that parasite density increased as STS progressed. In addition, our results suggested that the siphons might function as the portal of entry for A. hoyamushi during infection.
Ascidians (Halocynthia roretzi) belong to class Ascidiacea, order Pleurogona, and family Pyuridae, and have been commercially important marine invertebrates in Korea for decades . However, over the past 20 years, soft tunic syndrome (STS) has been plaguing the Gyeongnam Province area on the southern coast of Korea, which has most of the country’s ascidian farms, causing mass mortality of the ascidians. In Japan, outbreaks of ascidian STS have been reported in farms located in the Miyagi Prefecture since 2007 .
A flagellated protozoan was isolated from an ascidian affected with STS, and the flagellate was subsequently identified as Azumiobodo hoyamushi by Japanese investigators ,. Recently, our group also isolated a kinetoplastid protozoan from STS-affected ascidians cultured in the East Sea of Korea and found that the 18S rRNA sequence of the organism was highly homologous to that of the A. hoyamushi isolated in Japan, suggesting a common causative pathogen for STS in both Korea and Japan .
STS causes the tunic fiber bundle to disintegrate by diminishing the rigidity and integrity of the tunic fiber. Due to low fiber density, the tunics become thin and soft and finally rupture leading to leakage of internal tissues and death -. Breakdown of the tunics is mediated by a metalloprotease enzyme secreted by A. hoyamushi.
Diagnostic methods for the pathogen include histology and conventional polymerase chain reaction (PCR), and these tools have helped understand STS pathogenesis ,. A couple of reports have suggested that the siphons are the possible entry routes for the parasites in the ascidians, although the disease is not easily identifiable until apparent symptomatic changes occur in the central tunic of the STS ascidians ,. According to this hypothesis, if the parasite initially infects the tissues of the siphon and then subsequently propagates to the central tissues, there will be a sequential distinction in the number of parasites at different tunic tissues of the same symptomatic stage. It is also likely that progressively higher parasite numbers will be observed at the same site as the severity of symptom heightens. In this manner, parasite numbers will be closely correlated with the progression of soft syndrome in a given tunic tissue. This study was designed to evaluate the severity of the syndrome at different tunic sites with enhanced quantitative accuracy, employing qPCR for parasite enumeration. This experimental design will help understand the anatomical progression of the disease. Additionally, the study design might aid early parasite detection in the ascidians. Furthermore, to our knowledge, no study has examined the changes in parasite numbers in different portions of the diseased tunic.
Findings and discussion
The number of A. hoyamushi in tunics in various KS phases, as measured using qPCR
# of A. hoyamushi/ 0.25cm2
2.9 ± 0.4
160.1 ± 16.7
2,994.6 ± 1,783.5
16,066.9 ± 2,609.9
2.9 ± 0.6
46.6 ± 30.3
2,337.6 ± 424.1
4,121.0 ± 281.7
0.2 ± 0.2
0.4 ± 0.5
1,912.9 ± 650.3
5,053.2 ± 2,560.8
0.0 ± 0.0
38.0 ± 1.1
2,339.4 ± 257.5
5,599.2 ± 915.1
0.2 ± 0.0
3.6 ± 0.0
1,362.0 ± 462.1
5,396.1 ± 507.3
# of A. hoyamushi/ GT
106.0 ± 1.3
7,939.6 ± 1,241.9
39,093.3 ± 18,334.2
617,004.1 ± 10,949.2
85.5 ± 1.8
3,012.5 ± 2,607.6
70,866.4 ± 6,961.5
131,835.5 ± 37,352.4
6.0 ± 2.6
12.4 ± 13.7
49,108.0 ± 6,987.4
169,404.2 ± 94,002.7
0.3 ± 0.4
1,005. 2 ± 44.8
59,569.7 ± 6,555.5
205,291.7 ± 24,037.0
3.1 ± 0.6
114.9 ± 23.6
31,702.4 ± 7,770.8
211,555.4 ± 86,528.5
A high density of live A. hoyamushi was visible in the tunics from ascidians with KS-3 phase STS upon observation under a light microscope (data not shown). Currently, microscopic enumeration using a hemocytometer is commonly used to quantify in vitro-cultured A. hoyamushi. However, because A. hoyamushi is present within the tunic fiber tissue, which consists of a rigid cellulose structure ,,, accurate quantification of A. hoyamushi cannot be achieved using the simple microscopic method. Accordingly, qPCR can provide a more accurate method for the quantitative evaluation of A. hoyamushi in ascidian tunics.
In conclusion, we developed a method to quantify A. hoyamushi using qPCR and found that the density of A. hoyamushi varied from a few individuals to several thousands of the pathogen depending upon the phase of STS development. Our results also suggest that the siphons may serve as the portal of entry for A. hoyamushi. The quantification of A. hoyamushi in ascidians and their environments may provide more information about the etiology of STS and help predict and monitor outbreaks of STS in the future.
This work was financially supported by the National Fisheries Research and Development Institute of Korea (NFRDI, RP-2014-AQ-038). We also thank the Marine Biology Education Center of the Kunsan National University for providing ascidian culture facilities during the experiment.
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