BORKHANUDDIN Hafiz, CECH Gábor, OSTOROS Györgyi, MOLNÁR Kálmán, SZÉKELY Csaba Veterinary Medical Research Institute of the Hungarian Academy of Sciences, 1143. Budapest. Hungária krt. 21.
Introduction Phylum Myxozoa myxosporean stage in fish alternates with an actinosporean stage in an annelid worm It is estimated that 136 types of actinospores belonging to different collective groups have been described recently 34 actinospores have been identified as developmental stages of myxospores (Hallett et al., 2002; Lom & Dykova, 2006)
The actinospores are mostly classified according to spore morphology. However, spore morphology on its own may be fallacious (Hallet et al., 2002) proved that a single aurantiactinomyxon genotype might have different phenotypes The processes of one were swollen, leaf-like and had either pointed or rounded ends The process of the second type were elongated, finger like, and terminated in a point at the ends
Eszterbauer et al., (2006) presented the morphological and molecular characterization of actinospores collected from the oligochaetes of Fish Farm in Szazhalombatta & River Tisza, Tiszafured 14 different morphotypes; 10 genotypes
Sampling Activities 2011 Tihany: 10th May, 1st June, 7th July, 3rd August Siofok/Balatonvilagos: 18th May & 16th June Keszthely: 4th April, 5th May, 8th June & 3rd August Balatonszemes: 26th April, 8th June & 3rd August Kis-Balaton: 13th July Zala River: 31st August
Decantation / Sieving Cell well-plate Photographed (Olympus BH12) Oligochaetes - examined (Zeiss Axiovert-25 )
Molecular methods DNA isolation & purification PCR 18S rdna amplification Sequencing
Isochaetides michaelseni (Lastočkin, 1937) Branchiura sowerbyi (Beddard, 1892)
a Small oligochaetes. a) Nais sp.; b) Aeolosoma sp.; c) Dero sp. b c
Descriptions of the Actinosporean types El-Mansy et al. (1998) has collected 10 types of actinospores : - 5 triactinomyxons - 2 raabeias - 3 aurantiactinomyxons Kelemen et al. (2009) have isolated 13 actinospore types: - 5 triactinomyxons - 3 echinactinomyxons - 5 aurantiactinomyxons
As of April to August 2011, nine types of actinospores: aurantiactinomyxons (4 types) echinactinomyxons (1 type) raabeia (1 type) triactinomyxon (3 types)
Aurantiactinomyxon Janiszewska, 1952 Type 1 Spore in ventral /apical view Spore in side view Host: Isochaetides michaelseni Localities: Keszthely, Hungary Date of collection: May-June, 2011 Genetic remarks: 1645bp ~ Myxobolus cultus (92%)
=16.16 (13.34-18.68) μm = 22.48 (19.12-38.68) μm = 55.55 (48.02-63.37) μm Aurantiactinomyxon Type 1 = 4.71 (4.67-6.23) μm a = diameter of spore body b = length of caudal processes c = width of caudal processes from side d = width of caudal processes from apical view e = largest span f = length of polar capsule g = width of polar capsule
Aurantiactinomyxon Janiszewska, 1952 Type 2 Spore body, sporoplasm containing secondary cells and with polar capsules at the apical end. Host: Isochaetides michaelseni Localities: Keszthely, Hungary Date of collection: May-June, 2011 Genetic remarks: Sequence in progress
65.41 (68.7-64.03) μm = = 5.77 (4.67-7.11) μm =10.32 (8.00-13.34) μm Aurantiactinomyxon Type 2 = 32.53 (29.35-38.69) μm 3.39 μm (approx)= = 2.61 μm (approx) a = diameter of spore body b = length of caudal processes c = width of caudal processes from side d = largest span e = length of polar capsule f = width of polar capsule
Aurantiactinomyxon Janiszewska, 1952 Type 3 Secondary cells Spore in ventral view Host: Branchiura sowerbyi Localities: Kis Balaton, Hungary Date of collection: July 2011 Genetic remarks: Sequence in progress
Aurantiactinomyxon Type 3 10.08 (9.12-11.34) μm= = 124.78 (98.05-191.43) μm 58.24 (51.8-65.59) μm= = 20.68 (19.34-24.68) μm P.C = 3.79 (3.34-4.00) μm a = diameter of spore body b = length of caudal processes c = width of caudal processes from apical view d = largest span
Aurantiactinomyxon Janiszewska, 1952 Type 4 Spore body insert, 3 polar capsules at the apical end. Host: Branchiura sowerbyi Localities: Kis Balaton, Hungary Date of collection: July, 2011 Genetic remarks: 1294bp ~ Myxobolus intimus (97%)
Aurantiactinomyxon Type 4 12.46 (9.78-14.23) μm= = 64.23 (46.69-129.40) μm = 23.99 (19.12-29.68) μm = 20.94 (19.34-24.01) μm a = diameter of spore body b = length of caudal processes c = width of caudal processes from apical view d = largest span
Raabeia Janiszewska, 1955 Spore in side view Host: Isochaetides michaelseni Localities:Keszthely, Hungary Date of collection: May-June, 2011 Genetic remarks: Sequence in progress
Raabeia spore body in side view.
= 5.33 (4.57-5.72) μm = 5.22 (4.18-8.0) μm = 63.25 (59.44-67.44) μm = 3.40 (2.61-4.57) μm = 6.94 (6.48-8.0) μm = 185.1 (136-209.5) μm Raabeia a = length of spore body b = width of spore body c = length of caudal processes d = width of caudal processes e = length of polar capsule f = width of polar capsule
Echinactinomyxon Janiszewska, 1957 Spore in ventral view Spore in side view Host: Isochaetides michaelseni Localities: Keszthely, Hungary Date of collection: May June, 2011 Genetic remarks: Sequence in progress
Spore body in side & ventral view.
= 8.98 (8.00-11.43) μm = 6.37 (4.57-8.00) μm = 19.19 (16.0-22.86) μm = 3.02 (2.29-3.43) μm = 5.44 (4.57-6.86) μm = 125.97 (94.87-144.4) μm Echinactinomyxon a = length of spore body b = width of spore body c = length of caudal processes d = width of caudal processes e = length of polar capsule f = width of polar capsule
Triactinomyxon Štolc, 1899 Type 1 Host: Isochaetides michaelseni Localities:Keszthely, Balatonszemes, Tihany, Siofok, Hungary Date of collection: May-August, 2011 Genetic remarks: 610bp ~ M. pseudodispar (99%) Spore in side view
Spore body of the actinospore with sporoplasm containing secondary cells and with polar capsules at the apical end.
= 11.5 (10.4-15.6) μm 5.2 (5.34-6.67) μm = = 41.9 (31.2-57.2) μm 3.5 (3.34-5.34) μm = 15.6 (13.0-26.0) μm = = 188.8 (145.6-202.8) μm M. pseudodispar = 239.2 (178.1-302.9) μm 447.7 (369.2-618.8) μm = a = length of spore body b = width of spore body c = width of style d = length of style e = length of caudal processes f = largest span g = length of polar capsule h = width of polar capsule
Triactinomyxon Štolc, 1899 Type 2 Spore in side view with deformed caudal processes of the spore in side view. Host: Isochaetides michaelseni Localities: Tihany, Hungary Date of collection: June, 2011 Genetic remarks: 632bp~ M. pseudodispar (99%)
Triactinomyxon Štolc, 1899 Type 3 Host: Isochaetides michaelseni Localities:Keszthely, Balatonszemes, Tihany, Siofok, Hungary Date of collection: May-August, 2011 Genetic remarks: Sequence in progress Spore in side view
Spore body of the actinospore with sporoplasm containing secondary cells and with polar capsules at the apical end.
= 18.36 (13.0-23.4) μm 8.35 (7.8-12.0) μm = = 46.15 (36.4-59.8) μm 4.46 (2.6-5.34) μm = 23.56 (20.8-26.0) μm = = 260.81 (221.0-296.4) μm M. shaharomae 426.08 (358.8-566.8) μm = a = length of spore body b = width of spore body c = width of style d = length of style = 252.5 (182-283.4) μm e = length of caudal processes f = largest span g = length of polar capsule h = width of polar capsule
General Discussion This study represented 9 different morphotypes. However, only 4 have been studied & partially sequenced for their molecular biology information. 1294bp ~ Myxobolus intimus (97%) The DNA sequence data assigned the actinospores to three myxosporean 1645bp ~ Myxobolus cultus (92%)
Present and previous study showed that, identification based solely on the traditional morphological features may be false Thus sequence analysis of 18S rdna is recommended to facilitate in the process if species classifications (Hallett et al., 2002; Eszterbauer et al., 2006) 632bp~ M. pseudodispar (99%) However, morphological features still have a strong influences to distinguished the spores of some groups 610bp ~ M. pseudodispar (99%)
The work by collecting and storing actinospore stages is only a first phase of myxosporean research in the effort to reveal the infection of fishes and oligochaetes. Completed sequences of both the actinosporeand myxospore stages might help us to select identical spores of the same species, thus completing new life cycles of myxozoans from the Balaton.
Acknowledgement Thanks for the members of the Fish Pathology & Parasitology Group (VMRI), Aquaculture Group at Georgikon Faculty, Pannon University, and the Institute of Limnology Tihany for the help in collecting oligochaetes. Financial support: OTKA K 71837 project and Malaysian Governmental Scholarship of the junior author.
thank you
References El-Mansy, A., Székely, Cs. and Molnár, K. 1998a. Studies on the Occurrence of the Actinosporean Stages of Myxosporeans in Lake Balaton, Hungary, with the Description of the Triactinomyxon, Raabeia, and Aurantiactinomyxon Types. Acta Veterinaria Hungarica. Vol. 46 (4): 437-450. El-Mansy, A., Székely, Cs. and Molnár, K. 1998b. Studies on the Occurrence of the Actinosporean Stages of Fish Myxosporeans in The Fish Farms of Hungary, with the Description of the Triactinomyxon, Raabeia, Aurantiactinomyxon and Neoactinomyxon Types. Acta Veterinaria Hungarica. Vol. 46 (2): 259-284. Eszterbauer, E., Marton, S., Racz, O. Z., Letenyei, M. and Molnar, K. 2006. Morphological and Genetic Differences among Actinosporean Stages of Fish-Parasitic Myxosporeans (Myxozoa): Difficulties of Species Identification Gregory, R.D. and Blackburn, T.M. 1991. Parasite Prevalence and host Sample Size. Parasitology Today. Vol. 7: 316-318.
Kelemen, O., Ostoros, G., Cech, G. and Székely, Cs. 2009. Study of the development of fish-parasitic myxosporeans in oligochaete alternative hosts in Lake Balaton. Abstract Book. p. 269. EAFP 14th International Conference Diseases of Fish and Shellfish, 14-19 September, 2009 Prague, Czech Republic. Lom, J. and Dyková, I. 2006. Myxozoan Genera: Definition and Notes on Taxonomy, Life-cycle Terminology and Pathogenic Species. Folia Parasitologica. Vol. 53: 1-36. Morris, D.J. and Freeman, M.A. 2010. Hyperparasitism has wide-ranging implications for studies on the invertebrate phase of myxosporean (Myxozoa) life cycles. International Journal for Parasitology. Vol. 40: 357-369.
Özer, A., Wootten, R. and Shinn, P. 2002. Infection prevalence, seasonality and host specificity of actinosporean types (Myxozoa) in an Atlantic salmon fish farm located in Northern Scotland. Folia Parasitologica. Vol. 49: 263-268. Székely, Cs. Yokoyama, H., Urawa, S., Timm, T. and Ogawa, K. 2003. Description of Two New Actinosporean Types from a Brook of Fuji Mountian, Honshu, and from Chitose River, Hokkaido, Japan. Vol. 53: 127-132. Szekely, Cs. Hallett, S. L., Al-Samman, A. and Dayoub, A. 2007. First Description of Myxozoans from Syria: Novel records of Hexactinomyxon, Triactinomyxon and Endocapsa actinospore Types
Class Myxosporea
Figure 1. Actinospores clutch inside the body cavity of infected oligochaete
Actinospores released from infected oligochaete
General Morphology Polar capsule Sporoplasm 2 nd -dary cells Style
Style Caudal processes
Overall morphology distinct between the Aurantiactinomyxons: Spore Features Morphotype Type I Type II Type III Type IV Spore body 16.16 (13.34-18.68) 10.32 (8.00-13.34) 20.68 (19.34-24.68) 20.94 (19.34-24.01) Caudal processes (L) 22.48 (19.12-38.68) 32.53 (29.35-38.69) 58.24 (51.8-65.59) 23.99 (19.12-29.68) (W) 4.71 (4.67-6.23) 5.77 (4.67-7.11) 10.08 (9.12-11.34) 12.46 (9.78-14.23) Largest span 55.55 (48.02-63.37) 65.41 (68.7-64.03) 124.78 (98.05-191.43) 64.23 (46.69-129.40) Remarks Elongated processes; finger-like; curve downwards; rounded ends Elongated processes; finger-like; curve downwards; rounded ends; spherical spore body in side an apical views Elongated processes; straight; rounded almost pointed tips; spore body mainly spherical in apical view Swollen, leaf-like; curve downwards; pointed or rounded ends
Results Table 1: No. of collected annelids & no. of actinospores infected according to location Location Annelids No. of Annelid Collected No. of annelid Infected Keszthely Isochaetides michaelseni 2232 67 (49-TAM, 11-AAM, 4-RB, 3-ECM) Tihany I. michaelseni 288 24 (16-TAM, 8-AAM) Balatonszemes I. michaelseni 96 9 (7-TAM, 2-AAM) Siofok I. michaelseni 838 20 (19-TAM, 1-AAM) Balatonvilagos I. michaelseni 74 0 Kis-Balaton Zala River - Balaton Branchiura sowerbyi 42 2 (2-AAM) B. sowerbyi 103 1 (AAM) A total of 3673 annelids, collected from the Lake Balaton & Kis Balaton during April and August of 2011
Prevalence Of the 3528 oligochaetes, I. michaelseni, monitored from the Lake Balaton, only 120 worms (3.4%) infected. While 145 branchiura worms, B. sowerbyi, studied 3 infected (2.0%) Each actinospores morphotypes showed prevalence range between 0.1 % - 7.3%.
Percentage (%) 100 9 8 Triactinomyxon Raabeia 7.3 Aurantiactinomyxon Echinactinomyxon 7 6 5.5 5 4 2.8 3 2.2 2.4 2 2 Aurantiactinomyxon 1 0.4 0.1 0.1 Raabeia Echinactinomyxon 0 Triactinomyxon Keszthely Tihany Balatonszemes Siofok Balatonvilagos Figure 1: Prevalence (in percentage, %) of different actinospores in oligochaetes from Lake Balaton
Table 4: Comparison of prevalence of actinospores collected during the present study and some previous study. Locality Annelid host No. of Oligochaetes Collected No. of Oligochaetes Infected Prevalence (%) References Hungary, Lake Balaton Isochaetides michaelseni (Lastockin) 3528 120 3.4 Present study Hungary, Lake Balaton Tubifex tubifex (Muller) & Limnodrilus hoffmeisteri (Claparede) 1164 293 25.17 El-Mansy et al., 1998 Scotland, Atlantic Salmon farm T. tubifex, L. hoffmeisteri & Lumbriculus variegatus (Muller) 28, 387 823 2.9 Ozer et al., 2002 Japan, Mena River Rhyacordrilus komarovi (Timm) 620 16 2.6 Japan, Chitose River L. variegatus 680 55 8.1 Szekely et al., 2003 Szekely et al., 2003 Japan, Fuji Mountain T. tubifex, Rhyacodrilus coccineus (Vejdovsky), Dero digitata (, L. hoffmeisteri 130 1 0.77 Szekely et al., 2003 Hungary, River Tisza T. newaensis (Michaelsen) 117 1 0.85 Eszterbauer et al., 2006 Hungary, Fish Farm (Szazhalombatta) T. tubifex (Muller) 2924 4 0.13 Syria, Orontes River Psammoryctides albicola (Michaelsen) 80 6 7.5 Eszterbauer et al., 2006 Szekely et al., 2007 Syria, Euphrates River (Lake Assad) P. albicola 150 2 1.33 Szekely et al., 2007
The findings from El-Mansy et al. (1998a), Kelemen et al., (2009) & current study showed a relatively high diversity of the spores in Lake Balaton We also collected one new type of raabeia collective group (morphologically).
Genetic remarks: 1645bp ~ Myxobolus cultus (92%) This was the first detection of this parasite in Hungary. The myxospore of M. cultus has not so far been identified in fish from the examined habitats in Hungary, probably because of the small size and unusual localisation of the cysts in the fish host. The raabeia type examined shared 99.4% base pairs with Myxobolus cultus that develops cysts in the gill-arches of goldfish Carassius auratus auratus (Eszterbauer et al., 2006).
Gills of the roach Rutilus rutilus (L.) from the Lake Balaton (Racz et al., 2004) Genetic remarks: 1294bp ~ Myxobolus intimus (97%)
The present molecular biological investigation reveals that for actinosporeans, different morphotypes might belong to the same genotype. Genetic remarks: 632bp~ (99%) 610bp ~ M. pseudodispar (99%) Two different spore morphotypes belonged to the same genotype (TAM1 & TAM2) = M. pseudodispar Parasite of the muscles of the roach Rutilus rutilus (L.) (Szekely et al., 2001)
Objectives: This study specifically embarks on the following objectives: To determine the myxozoan species parasitizing fish and invertebrate alternate hosts. To characterize the myxozoan seasonal occurrence between ecological and geographical difference. To compare the DNA structure of the species found by molecular techniques.
Table 2: Prevalence (in percentage, %) of different actinospores in oligochaetes from Lake Balaton, collected in 2011 Prevalence (%) Location Triactinomyxon Aurantiactinomyxon Raabeia Echinactinomyxon Keszthely 2.2 0.4 0.1 0.1 Tihany 5.5 2.8 / / Balatonszemes 7.3 2.0 / / Siófok 2.4 0.1 / / Balatonvilágos / / / /
Table 3: Infection prevalence (%) for each actinosporean collective group Actinospore group No. of Oligochaetes Infected Prevalence (%) Triactinomyxon 91 2.5 Aurantiactinomyxon 22 0.6 Raabeia 4 0.1 Echinactinomyxon 3 0.08
It is estimated that 136 types of actinospores belonging to different collective groups have been described recently (Lom & Dykova, 2006). With an increasing attention of the their pathogenicity and continually emerging threats to the development of pisciculture; new findings is estimated.
Prevalence rate of the actinospores infecting I. michaelseni from this study could be considered low with 3.4% prevalence. However, El-Mansy (1998a) recorded prevalence up to 25% from the oligochaetes (T. tubifex & L. hoffmeisteri) collected from Lake Balaton.
The current study embark to present the morphometric and molecular characterization of actinospores collected in Lake Balaton and Kis Balaton, Hungary.
Actinospore stages - from annelid Myxospore stages - from fishes