Emergency cases in Molluscs and Ascidian. Tomoyoshi Yoshinaga Graduate School of Agricultural and Life Sciences The University of Tokyo

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1 Emergency cases in Molluscs and Ascidian Tomoyoshi Yoshinaga Graduate School of Agricultural and Life Sciences The University of Tokyo

2 Two cases Akoya oyster disease The soft tunic syndrome (of sea squirt)

3 Akoya pearl production Collection of natural spats Implantation of shell beads into the gonad of a recipient akoya oyster Production in hatcheries Seed Production of Akoya oyster Raising of pearl mother oysters (ca. 2 years) Raising (1 year)

4 Akoya oyster disease Susceptible hosts: Pinctada fucata martensii (The Japanese pearl oyster, or akoya oyster) *The Chinese pearl oyster, one geographical type of Pinctada fucata in China, is tolerant to the disease. Pathogen: Unknown infectious agent filterable at 0.45 μm filter Diagnosis: Macroscopically, red-brown adductor muscle Histologically, degenerated loose connective tissue in the mantle and infiltration of hemocytes in the mantle connective tissue Environmental factor Progression of the disease at high temperature (>20 o C) Subchronic progression of disease (2-3 months were needed for significant mortalities in challenge experiments) Natural occurrences in late summer and autumn Mortality up to 80 % in one-year-old oysters and lower mortality rates in older oysters

5 Western Japan Major area for Production of pearl mother oysters Ehime Prefecture Nagasaki Mie Kumamoto Ehime Major areas for pearl production Transportation of pearl mother oysters from Ehime to pearl-producing areas Ehime Prefecture Mie Prefecture Nagasaki Prefecture Kumamoto Prefecture

6 The beginning High mortalities (up to 70 %) characterized with red-brown adductor muscle occurred in some local areas in Ehime Prefecture and Oita Prefecture, but did not attract concerns of researchers and officers in local governments. Oita Prefecture Ehime Prefecture

7 Autumn 1996 High mortalities occurred in most areas producing pearl mother oysters and pearls.

8 The Sea of Pearl Becomes a Graveyard Nothing could be done to control the disease; the disease was novel, little information was available, and the disease was epidemic in most akoya oyster farms by Asahi Shimbun (11/10/1996)

9 Efforts to find the etiology of the disease many theories on the etiology Infectious or non-infectious Pollution with formalin used as antiparasitics for fish aquaculture Low phytoplankton density in the sea others National Research Institute of Aquaculture (NRIA), Ministry of Agriculture, Forestry and Fisheries (MAFF) organized a research unit and meetings in response to the mortalities. The research unit for the etiology of the akoya oyster mortalities (researchers in NRIA and those in local governments) The emergency meetings in response to the akoya oyster mortalities (officers of local and central governments, university researchers, pearl industry associations)

10 Healthy naïve akoya oysters were needed for research. Fortunately, a healthy wild population of akoya oysters was available in spite of the wide and rapid spread of the disease.

11 Jan NRIA proved that the disease was infectious by implantation of the mantle piece of affected oysters to naïve oysters and by cohabitation of naïve oysters with affected oysters. Mar MAFF announced The disease is an epidemic or 1999 A joint group of Ehime Prefecture and NRIA demonstrated that injection with filtered (at 0.45 μm) hemolymph of affected oysters caused the disease. Mar MAFF announced The etiological agent of the disease is an unknown filterable pathogen. However, the pathogen is not known yet. The confirmatory and rapid diagnosis of the disease are still difficult.

12 The pathogen came from China. Low pathogenicity in Chinese akoya oysters Simultaneous and sympatric occurrence of the disease with the beginning of importation of Chinese akoya oysters. Site A 1994 Chinese akoya oysters were imported from China and implanted into site A in 1994, where the disease first occurred At least 400x10 3 mother oysters and 250x10 3 juvenile oysters were imported from China. Fisheries Agency, MAFF, requested refrainment of importation of Chinese akoya oysters (non-mandatory request) At least 1.5x10 6 oysters were imported 1998 Akoya oysters were imported from China but the amount was not clear.

13 Control measures Raising akoya oysters in low-temperature areas Deep immersion of akoya oyster ( 2m 5-10m) Cross-bred akoya oysters between Chinese oysters and Japanese oysters

14 Annual Production of Akoya Pearls in Japan(in values) million US$ National Ehime Pref

15 Factors responsible for the spread of Akoya oyster disease Novel epidemic (no information) Low awareness and concern on epidemics of akoya oyster farmers Late response by government and researchers Delay in recognition of the infectivity of the disease Spread by transportation of akoya oyster Absence of laws and rules for mandatory orders for unspecified epidemics

16 The Soft Tunic Syndrome Healthy sea squirt Affected sea squirt

17 Sea squirt culture in Japan Substrates (oyster shells) Collection of natural larvae by immersing the collectors in the sea in Dec. Substrates are separately fixed in ropes and immersed in the sea in autumn anchor Rope length: 10~20m Raising on natural phytoplanktons for 3-4 years

18 Hokkaido Prefecture 58 t Aomori Prefecture 291 t Iwate 1,187 t Miyagi Prefecture 8,268 t Production of Sea Squirt in metric ton in total

19 The soft tunic syndrome (of sea squirt) Susceptible hosts: Sea squirts Halocynthia roretzi (mainly 2-3 year-old individuals) Styela clava possibly some other sea squirts Pathogen: The flagellate Azumiobodo hoyamushi (Class Kinetoplastea; Order Neobodonida) Diagnoses Microscopic or PCR detection of causative flagellates in tunic Environmental factor Seasonal occurrence (Dec Aug) Progression below 20 o C Mortality cumulative mortality : % in three-year-old sea squirts % in two-year-old sea squirts

20 Histology of affected sea squirt Tunic of healthy sea squir Tunic of affected sea squirt (1) Lesions limited to the tunic. (2)Coarse fibrous tissue and partially destroyed tunic (3)Flagellates in lesions in the tunic

21 The disease first occurred in Korea. (Metric ton) Sea squirt production in Korea First mass mortality in 1995 Nation-wide spread by 2000 Etiology suspected in Korea environmental changes pollution infectious pathogens others The etiology was unclear, when the disease was found first in Japan in 2007.

22 Actions of the local government, fishermen cooperative association and sea squirt farms in Miyagi Prefecture before the first outbreak of the disease In 2004 May (association) : A inquiry commission sent to Korea found out about the presence of the disease in Korea. July (association): The commission reported the presence of the disease publicly. Sep (farms): Korean sea squirt seedlings were introduced in the northern area in Miyagi. Most of the introduced seedlings died soon but there were some survivors left in farms. In 2005 Sep (local government): The first warning on the disease of sea squirt with non-mandatory request for self-refrainment of the introduction of foreign sea squirt seedling. In 2006 Feb (local government): The second warning with non-mandatory request was issued. Autumn(farms) : Mass mortality occurred in the survivors of sea squirt introduced from Korea in Sep(farms): Korean seedlings were introduced into the central area in Miyagi. In 2007 Jan (local government): The third warning with non-mandatory request was issued. Jan (farms): Diseased sea squirts appeared for the first time in Japan. Feb (local government): The occurrence of the disease was confirmed.

23 Studies on Etiology Aug 2008 (17 months after the first occurrence of the disease in Japan) Infectivity of the disease was proven by challenge experiments using pieces of tunic from affected sea squirts as pathogen source. (the left photo) Apr 2010 (39 months after the first occurrence) The pathogen (a parasitic flagellate) was specified by challenge experiments using flagellates isolated from affected sea squirts and cultured in a cell-culture medium. (the right picture); the researches fulfilled the Koch s postulates for determination of pathogens..

24 Spread of the disease in Miyagi Prefecture 2007 Korean seedling in 2004 Korean seedling in No occurrence Occurrence areas (bays or inlets) 6 areas 14 areas >90% Seedlings were produced here. 15 areas In 2007 and 2008, outbreaks occurred in the areas where Korean seedlings were introduced. After 2009, outbreaks occurred in locations where Korean seedlings were not introduced as well. Feb 2009, the disease occurred in Samenoura (the green circle), where more than 90% of seedlings were produced. In 2011, a devastating tsunami hit Miyagi Prefecture and destroyed aquaculture industry there.

25 Presence of affected sea squirts in wild populations Survey after the TSUNAMI in 2011 Visual observations by divers Areas St. Mo. No. abnormal /No. examined Laboratory examinations Flagellates No. positive / No. abnormal PCR No. positive / No. abnormal Contaminated St 1 Jul 5/500 0/5 1/5 St 2 Jul 6/500 1/6 1/6 St 3 Aug 1/700 0/1 0/1 St 1 Aug 7/500 0/7 2/7 Uncontaminated St 4 Aug 8/500 0/8 0/8 St 5 Aug 3/400 0/3 0/3 The disease was established in wild populations of sea squirts in Japan, even though at very low levels.

26 Factors responsible for the invasion and spread of the soft tunic syndrome Sea squirt culture was prosperous and farmers needed seedlings. Low awareness and concern of farmers on epidemics Low communication between farmers and government All requests from local government and central government were not mandatory; some farmers did not comply to the repeated warnings and requests.

27 Characteristics of epidemics in Molluscs Non-containable and non-eradicable Presence of wild populations Open culture environment Human factors Non-predictable High diversity in host species Host switch Non-treatable No acquired immunity Difficulty in drug administration

28 How should we fight? Emphasis on prevention of invasion of exotic disease, not on containment, eradication, and treatment/vaccine after invasion. Active use of measures based on scientific justifications and risk assessment and temporary precautionary measures Use of different SPS measures according to risks (live seedlings for aquaculture/live animals for food/fresh animals/frozen animals)

29 Acknowledgements I thank Dr. Akira Kumagai, who gave me many PPT slides that I used for the explanation of the soft tunic syndrome of sea squirt.

30 Thank you for your attention.