Occurrence of Anguillicola crassus (Nematoda: Dracunculoidea) in Japanese eels Anguilla japonica from a river and an aquaculture unit in SW Taiwan

INTRODUCTION

Anguillicola crassus Kuwahara, Niimi & Itagaki, 1974, is an endemic swimbladder parasite of the Japanese eel Anguilla japonica in East Asia (Kuwa-hara et al. 1974, Nagasawa et al. 1994). The parasite has successfully spread to Europe, North Africa and North America, where it has attained high prevalence and intensities within populations of the European eel A. anguilla and the American eel A. rostrata, respec-tively (Neumann 1985, Taraschewski et al. 1987, Køie 1988, Moravec & Taraschewski 1988, Moravec 1992, Johnson et al. 1995, Fries et al. 1996, Maamouri et al. 1999, Sures et al. 1999b, Kirk 2003). The parasite was obviously introduced to Europe with live-eel imports from Taiwan to the German harbor of Bremerhaven (see Koops & Hartmann 1989, Køie 1991).

In Europe, this alien parasite has created interest among ichthyoparasitologists and individuals involved in fisheries and aquaculture because of its high patho-genicity in the economically important European eel, which had not coevolved with the parasite (Boon et al. 1990, Van Banning & Haenen 1990, Molnár 1993, 1994, Hartmann 1989, Molnár et al. 1993, 1995, Würtz et al. 1996, 1998, Knopf et al. 1998, Kelly et al. 2000, Würtz & Taraschewski 2000, Kirk 2003).

According to the available literature, the European and the Asian populations of Anguillicola crassus seem to differ in several aspects of their life-cycle and their host –parasite relations. In Asia as well as in Europe different copepods and ostracods are known to act as intermediate hosts (Hirose et al. 1976, De Charleroi et al. 1990, Kennedy & Fitch 1990, Thomas 1993, Moravec & Konecny 1994, Nagasawa et al. 1994, Ooi et

© Inter-Research 2006 · www.int-res.com *Email: marcel.muenderle@gmx.de

Occurrence of

Anguillicola crassus (Nematoda:

Dracunculoidea) in Japanese eels

Anguilla japonica

from a river and an aquaculture unit in SW Taiwan

M. Münderle

*, H. Taraschewski

_{, B. Klar}

_{, C. W. Chang}

_{, J. C. Shiao}

_,

K. N. Shen

, J. T. He

, S. H. Lin

, W. N. Tzeng

1_{Zoologisches Institut, Ökologie/Parasitologie and,} 2_{Institute for Mathematical Stochastics, Universität Karlsruhe,}

76131 Karlsruhe, Germany

3_{Institute of Fisheries Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan 106, ROC}

ABSTRACT: The infection by swimbladder nematodes of the genus Anguillicola (Dracunculoidea: Anguillicolidae) was examined in 2 populations of the Japanese eel Anguilla japonica in SW Taiwan. Wild eels from the Kao-Ping river were compared with cultured eels from an adjacent aquaculture unit. Only the cosmopolitan species Anguillicola crassus was present. Among wild eels, prevalence of infection varied between 21 and 62%, and mean intensity between 1.7 and 2.7 for adult worms. Similar intensity values (1.3 to 2.8) were recorded for the larvae. In cultured eels, prevalence as well as mean intensities were higher. In the cultured hosts, mean larval intensities exceeded those of adult worms 2-fold, and maximum larval intensities were 4- to 5-fold higher than in eels from the river. In cultured eels, dead larvae were also more abundant than in wild eels. We conclude that infrapopula-tions of A. crassus in Japanese eels are regulated by the defense system of this host, intraspecific den-sity-dependent regulation being less likely as the major regulatory mechanism. No influence of the parasite on eel condition was found in either wild or cultured eels, indicating a low or moderate path-ogenic effect of A. crassus on this host. This study shows that A. crassus is moderately common in cul-tured and wild Japanese eels in Taiwan, where the parasite is endemic.

KEY WORDS: Anguillicola crassus · Anguilla japonica · Swimbladder · Taiwan · Aquaculture Resale or republication not permitted without written consent of the publisher

al. 1997, Kirk et al. 2000a,b, Moravec et al. 2005). However, transmission via paratenic hosts has not been described from Asia, whereas in Europe various species of fishes as well as amphibians and even larvae of aquatic insects have been found to carry L3 (and

partly L4) -larvae in their viscera or their swimbladders

(Haenen & van Banning 1990, Thomas & Ollevier 1992, Moravec & Konecny 1994, Székely 1994, Moravec & 2koríková 1998, Sures et al. 1999a). These differences might be due to the more intensive research activity related to A. crassus in Europe in comparison to Asia. However, parallel laboratory infections with A. japon-ica and A. anguilla have revealed a more severe host reaction directed against A. crassus in Japanese eels than in European eels (Nielsen & Buchmann 1997, Nielsen 1999, Knopf & Mahnke 2004).

The present study is the first report on Anguillicola crassus in wild and cultured populations of Japanese eels in Taiwan. Our aim was to study population struc-tures of A. crassus in its Asian heartland and compare them with those described from Germany and Europe, where this invasive species has established a dense mesh of huge metapopulations over the last 20 yr. Fur-thermore, our investigation was also meant to have practical relevance. Eel farming hFas become a pros-perous industry in Taiwan, China and other east and southeast Asian countries (Lee et al. 2003a, b). The aquaculture plants are run with A. japonica and other Asian or non-Asian eel species (Ooi et al. 1996, 1999, Shin & Chen 2000, Chang et al. 2002). Unfortunately, these eel farms do not publish their experiences with A. crassus, and so the otherwise well-organized fishery management in Asia still depends on published data collected from Japanese eel farms more than 25 yr ago (Egusa 1979).

MATERIALS AND METHODS

Wild eels were collected from the Kao-Ping River in SW Taiwan from December 2000 to March 2003. The sampling locations and the methods of eel capture have been described in detail by Tzeng et al. (2002). Farm eels were purchased from 2 ponds of an aquaculture system at Bu-Dai in SW Taiwan in February and March 2003. In the years 2000 to 2002, live eels were trans-ported to the laboratory, killed by decapitation, and deep-frozen until parasitological examination. In the year 2003, eels were investigated in fresh condition.

The length and weight of the eels were measured to the nearest 1.0 mm and 0.1 g, respectively. The sex of each eel was determined according to external mor-phology and visual examination of the gonads. The developmental stage of the fish was classified as either a yellow or silver eel according to skin and

fin coloration, eye diameter and gonad development (Pankhurst 1982, Han et al. 2003). The condition (c- or corpulence-factor) of the eels was calculated as described by Schäperclaus (1990).

The adult parasites were removed from the swim-bladder lumen of the eels by forceps; their species, sex and number were recorded. The walls of the swim-bladders were checked for larval stages (L3and L4) by

squash preparation using 2 Perspex plates. In accor-dance with the results of Blanc et al. (1992), all larvae with a body length >1.5 mm were considered Stage L4

larvae. The prevalence, mean intensity and abundance of the parasites in the eels were calculated as described by Bush et al. (1997). Additionally, we also counted encapsulated dead larvae and adults or rem-nants of both stages, but without determining the sex. For taxonomic studies, adult worms were cleaned carefully with distilled water and then fixed and stored in 70 % alcohol until morphometrical investigation. We measured the following features: length/width of body, length/width of oesophagus, length/width of buccal capsule and number of peribuccal teeth. Ratios of length and width of the body, oesophagus and buccal capsule were calculated.

For statistical analysis of the infection (p ≤ 0.05) and condition (p ≤ 0.01) data, a Mann-Whitney U-test was employed. Spearmans correlation coefficient was used to determine the correlations between the dif-ferent parameters (Sachs 1992). All results were checked for outliers according to the outlier test n·SD > 4, p < 0.05.

RESULTS

Eel data

Sex, developmental stage, body length, weight and condition of the investigated Japanese eels are summarized in Table 1. The sex ratios of the eels were skewed towards females in wild eels but towards males in cultured eels, similarly to results from previ-ous studies eels in Taiwan (Tzeng et al. 1995, 2002).

The cultured eels had higher C-factors than the wild eels sampled in spring 2003, which could indicate that the availability of food or temperature are more favor-able under culture conditions than in the river.

Identification and measurement of Anguillicola crassus

All adult nematodes collected from the lumen of the swimbladders of all wild and cultured Japanese eels were identified as Anguillicola crassus Kuwahara, Niimi & Itagaki, 1974, using the key of Moravec &

Tara-schewski (1988). A. globiceps Yamaguti, 1935 was not found in any of the eels investigated. The morphological features of 133 specimens are summarized in Table 2.

Prevalence and intensity of Anguillicola crassus infection

Quantitative data on the swimbladder nematodes are presented in Table 3. Eels from the river had a lower prevalence (P) of Anguillicola crassus (Pmax =

62%) than cultured eels (Pmax= 88 %) The intensity of

larval (p < 0.01) and adult worms (p < 0.05) in the wild

eels collected in early 2003 was signifi-cantly lower than in the cultured eels. The most striking differences were the mean larval intensities and especially the maximum larval number per eel (Table 3).

We classified 4 groups of eels har-boring dead Anguillicola crassus: (1) no dead; (2) 1 to 10 dead; (3) 11 to 20 dead; (4) > 20 dead; (Fig. 1). The fre-quency distribution of dead Anguilli-cola crassus differed significantly be-tween wild and cultured eels. We found dead A. crassus in 22% of the wild eels, whereas 56% of the cultured eels harbored at least 1 dead nema-tode (Fig. 1).

Seasonal occurrence of Anguillicola crassus infection

The prevalence of infection with Anguillicola crassus in wild eels was somewhat lower in the winter sample (December) than in the other seasons (Table 3), but the mean intensity of larval and adult A. crassus did not dif-fer significantly among seasons except between September 2001 compared to December 2000 (p < 0.05). No compari-son could be made for cultured eels since these were only available for 1 season.

Frequency distribution of Anguillicola crassus infection

The frequency distribution of adult and larval Anguillicola crassus per eel was calculated for wild and cultured eels, and approached a negative binomial distribution in the wild eel population, indicating a high degree of overdispersion (Fig. 2). In cultured eels, the extent of over dispersion was more pronounced, corresponding with the overall higher prevalence of infection in the cul-tured eels.

Condition factor of eels

We could not detect any significant difference in condition between uninfected and infected eels, either in the river or in the aquaculture population (Fig. 3). Locality/ date n Percentage Weight Lenght C-factor

Females Silver (g) (cm) R/ Dec 2000 14 50 43 385.2 ± 168.2 62.0 ± 6.3 0.15 ± 0.02 R/ Mar 2001 20 100 10 289.4 ± 125.5 58.4 ± 7.8 0.14 ± 0.01 R/ Jun 2001 20 75 0 229.4 ± 66.5 52.0 ± 4.3 0.16 ± 0.02 R/ Sep 2001 21 86 0 178.0 ± 46.1 51.4 ± 4.4 0.13 ± 0.01 R/ Aug 2002 20 78 0 205.3 ± 138.2 49.8 ± 9.0 0.15 ± 0.02 R/ Mar 2003 73 82 0 136.5 ± 176.1 43.8 ± 11.5 0.12 ± 0.02 C/ Feb 2003 25 4 0 248.0 ± 49.6 53.9 ± 3.0 0.16 ± 0.01 C/ Mar 2003 46 15 0 237.3 ± 26.7 54.8 ± 2.4 0.14 ± 0.02 Table 1. Anguilla japonica. Percentage of females and silver eels in samples,

and mean (± SD) weight, length and condition factor of all eels examined (including males). R: river, C: cultured eels

Characteristic Sex Culture (m=7; f=8) River (m=71; f=47)

min.–max. x ± SD min–max x ± SD No. of teeth m 22 – 28 25.4 ± 2.4 18 – 33 24.1 ± 3.0 f 24 – 30 27.1 ± 2.1 20 – 30 23.7 ± 2.1 Dry weight m 0.0 – 0.3 0.1 ± 0.1 0.0 – 2.6 0.4 ± 0.5 f 0.0 – 2.2 0.6 ± 0.9 0.0 – 20.8 4.1 ± 4.9 Body length (mm) m 5.83 – 9.01 7.1 ± 1.4 2.29 – 23.21 9.4 ± 3.8 f 3.3 – 19.7 9.0 ± 5.9 4.54 – 31.45 16.6 ± 7.7 width (mm) m 0.24 – 0.52 0.3 ± 0.1 0.14 – 1.55 0.6 ± 0.3 f 0.22 – 1.30 0.6 ± 0.4 0.21 – 3.59 1.5 ± 0.9 length:width m 17.2–25.0 21.1 ± 3.0 9.3 – 36.1 16.6 ± 4.4 f 13.9 – 21.2 16.6 ± 2.5 7.5 – 26.2 12.9 ± 4.3 Oesophagus length (µm) m 495 – 653 611.0 ± 58.1 361 – 871 621.5 ± 85.3 f 540 – 921 682.5 ± 140.7 554 – 1040 781.6 ± 118.1 width (µm) m 119 – 198 160.5 ± 26.6 69 – 248 167.9 ± 42.6 f 119 – 287 177.6 ± 61.7 129 – 347 218.2 ± 54.2 length:width m 3.3 – 4.3 3.9 ± 0.4 2.7 – 6.5 3.9 ± 0.8 f 3.2 – 5.3 4.0 ± 0.7 2.7 – 5.0 3.7 ± 0.5 Buccal capsule length (µm) m 18 – 22 19.8 ± 1.7 13 – 22 18.3 ± 2.2 f 14 – 24 20.7 ± 3.3 15 – 24 19.9 ± 1.8 width (µm) m 46 – 60 51.8 ± 5.4 38 – 61 48.2 ± 5.3 f 45 – 69 60.9 ± 7.7 39 – 69 53.1 ± 5.6 width:length m 2.1 – 3.2 2.6 ± 0.3 2.2 – 3.3 2.6 ± 0.2 f 2.8 – 3.3 3.0 ± 0.2 2.3 – 3.2 2.7 ± 0.2 Table 2. Anguillicola crassus. Morphometric features of parasites from wild

(n = 168) and cultured (n = 71) Japanese eels Anguilla japonica. Data for all

DISCUSSION

The investigated populations of the Japanese eel Anguilla japonica in Taiwan harbored only 1 species of swimbladder nematode, Anguillicola crassus. This par-asite has been recorded from East Asia within the cen-tral distributional range of A. japonica. The respective data from Japan, Korea and China have been reviewed by Nagasawa et al. (1994). Anguillicola glo-biceps, another swimbladder nematode occuring in Japanese eels, was not found in the present study. This latter parasite, originally described by Yamaguti (1935) from Japan, differs in several morphological features (oesophagus, buccal capsule, number of peribuccal teeth) from all other congeneric species (Moravec & Taraschewski 1988), and is thus easily distinguishable from A. crassus. Nevertheless, in many reports from Japan, China and Taiwan, A. crassus and A. globiceps

were not properly differentiated (see Kuo 1994, Nagasawa et al. 1994). Thus, due to lack of recent data, the distribu-tion of these 2 species in East Asia is uncertain.

The pattern of seasonal occurrence of Anguillicola crassus described herein corresponds with reports for cultured Japanese eels in Japan (Egusa et al. 1969), with a lower prevalence in late winter and spring (February to April) than in summer (June to August). Decreased prevalence in winter was also noted in eels from Pusan, Korea (Kim et al. 1989). This could arise from, several factors such as lower availabil-ity of copepods during the cold or dry season in East Asia (November to April). During the rainy season, which begins in subtropical East Asia in May (Yen et al. 1990), the eels become more active (Tesch 1999).

The frequency of adult and larval nematodes approaches a negativly binominal distribution in wild

Locality/ P A Mean intensities

date % x ±SE (larvae) (adults) larvae + adults x ±SE max. x ±SE max x ±SE max. R/ Dec 2000 21 0.7 ± 0.5 2 ± 0 2 2.7 ± 1.7 6 3.3 ± 1.5 6 R/ Mar 2001 55 1.1 ± 0.4 1.3 ± 0.3 2 1.8 ± 0.5 6 2 ± 0.6 8 R/ Jun 2001 60 1.2 ± 0.3 1.3 ± 0.2 2 1.7 ± 0.3 6 2 ± 0.4 8 R/ Sep 2001 62 2.0 ± 0.8 2.8 ± 1.0 5 2.6 ± 0.9 12 3.2 ± 1.2 17 R/ Aug 2002 60 2.1 ± 0.9 2.7 ± 1.0 8 2.6 ± 0.9 9 3.5 ± 1.3 17 R/ Mar 2003a _{51 1.2 ± 0.2 2.8 ± 1.0 12 1.7 ± 0.2 4 2.3 ± 0.4 12} C/ Feb 2003 88 6.7 ± 2.9 6.8 ± 3.4 64 2.9 ± 0.6 10 7.6 ± 3.2 73 C/ Mar 2003 65 4.4 ± 1.3 5.4 ± 2.2 42 2.9 ± 0.6 9 6.7 ± 1.8 47

a_{In March 2003, 1 eel did not fit statistically (n·sigma = 8.44, p = 0) and} was omitted from the table. We detected 157 larval stages and 1 adult female in its swimbladder. It is likely that this fish had escaped from an aquaculture farm

Table 3: Anguilla japonica infected with Anguillicola crassus. Prevalence

(P), abundance (A) and mean intensity (MI) of parasites in wild (R) and of cultured (C) eels 11–20 dead A. crassus (2%) >20 dead A. crassus (2%) no dead A. crassus (78%) 1–10 dead A. crassus (18%) >20 dead A. crassus (21%) no dead A. crassus (44%) 11–20 dead A crassus (17%) _{1–10 dead} A. crassus (18%) River Aquaculture

Fig. 1. Anguilla japonica infected with Anguillicola crassus.

Frequency distribution of dead parasites (larvae and adults) found in wild (n = 168) and cultured (n = 71) Japanese eels

a) b) 0 10 20 30 40 50 60 70 80 90 100 0 1 3 4 6 7 1 3 4 6 7 2 5 >7

No. adult A. crassus per swimbladder

No. eels (%) wild culture 0 10 20 30 40 50 60 70 80 90 100 0 2 5 >7

No. larval A. crassus per swimbladder

No. eels (%)

wild culture

Fig. 2. Aguilla japonica infected with Anguillicola crassus.

Observed frequency distribution of (a) larval and (b) adult parasites in wild (n = 168) and in cultured (n = 71)

and cultured eels, revealing a high degree of overdis-persion. This is characteristic of macroparasite infec-tions in hosts (Anderson & May 1978, Shaw & Dobson 1995) and thus expected. Experimental infection of European eels with Anguillicola crassus revealed that a few hosts were responders in terms of antibody pro-duction, while others showed almost no reaction (Knopf et al. 2000a, b). Probably, Japanese eels (which have a stronger defense against A. crassus than Euro-pean eels: Knopf & Mahnke 2004) also differ markedly in their response. This heterogeneity might be the major causative mechanism behind the observed aggregation.

The results of our study, revealing prevalence rates of 21 to 88%, contrast with data from Japan, where lower prevalences (10 to 40 %) of Anguillicola crassus have been described from wild and pond-reared Japanese eels (Moravec 1994). The river eels from Kao-Ping had a maximum prevalence of 62%, which is within the range of data for European eels in Europe. Sures & Streit (2001) reported 94% of eels examined from the river Rhine in Germany to be infected with A. crassus, whereas very low prevalences (6.7 to 8.9%) were reported for the Butroe river in Spain (Gallastegi et al. 2002). These data show that infection rates of

Japanese as well as European eels can vary extremely between different habitats, obviously reflecting vari-ous ecological characteristics such as the availability of intermediate and/or paratenic hosts and the popula-tion density of the hosts. Nevertheless, the prevalence of 80 to 100% recorded for Europe (Kirk 2003) are unknown for East Asia (Moravec 1994, Nagasawa et al. 1994). The same applies to the intensity of infection (Kirk 2003): 30 yr after arrival of the parasites, eels in the river Rhine still harbor more than twice as many adult A. crassus (Sures & Streit 2001) than Japanese eels in the Kao-Ping river.

Data on other host –parasite systems involving a native host and its indigenous Anguillicola species indicate lower abundances than in our study. From dif-ferent populations of the Australian eel Anguilla aus-tralis in New Zealand Lefebvre et al. (2004) reported, very low prevalence (<12 %) and mean intensity infected with Anguillicola novaezelandiae (1 to 2 nematodes infected eel–1_{). Taraschewski et al. (2005)}

detected similar low prevalence and intensity of A. papernai in Anguilla mossambica in South Africa. These observations suggest that A. crassus has very efficient modes of transmission and persistence, not only in recently adapted hosts such as the European eel, but also in its natural (East Asian) final and inter-mediate hosts. In Europe it quickly colonized the conti-nent, whereas A. novaezelandiae introduced into a lake in Italy failed to spread, and finally disappeared (Paggi et al. 1982, Moravec et al. 1994).

Comparison of the 2 sampling areas in the present study revealed interesting differences in the host para-site interaction. The higher prevalence and intensity of infection in the cultured eels is probably related to the higher density of the final host (eel). In addition, inter-mediate hosts (copepods) seem to be sufficiently avail-able in culture. Under aquaculture conditions, the high infection pressure is reflected by the 2-fold higher intensity of larvae compared to adults. Furthermore, the maximum larvae intensity was many times higher in cultured eels than in the eels from the river, where larvae and adults occurred at about the same intensity levels. However, mortality of the parasites in the cul-tured eels was considerably higher than in river eels. Thus, it appears that in the Japanese eel the infrapop-ulations of the parasite regulate themselves in a density dependent fashion or (more likely) become regulated by the immune system of this host (con-comitant immunity).

In our field study on Anguilla japonica and its para-site Anguillicola crassus, high infection pressure was accompanied by high mortality among the parasites, obviously arising from the host’s immune response to parasite-density pressure. Apparently, this host has evolved effective mechanisms for parasite recognition

a)

Adult nematodes per swimbladder

C-factor of wild eels

y = 0.13235 – 5.14077E-04x

b)

Fig. 3. Aguilla japonica infected with Anguillicola crassus.

Condition factor (c-factor) of (a) wild (n = 168) and (b) cultured (n = 71) Japanese eels as a function of infection intensity

and defense during a long host-parasite coevolution. In the European eel, this has not been the case. Knopf & Mahnke (2004) conducted comparative experimental infection of A. anguilla and A. japonica with A. crassus. In the Japanese eel, the recovery rates of the parasites were lower, their mortality higher and their individual weight lower than in the European eel. Knopf et al. (2000a,b) reported that the specific humoral immune response of European eels against A. crassus was char-acterized by a late onset and mainly directed against antigens in the body wall of adult nematodes. In com-parison with European eels, Japanese eels showed an enhanced humoral immune response against antigens of A. crassus (Nielsen & Buchmann 1997, Nielsen 1999), which might partly explain the differences in susceptibility between the 2 eel species.

We do not know whether the different survival rates of Anguillicola crassus in the 2 eel species is connected with the differential rise in antibodies, or whether the antibodies are just ‘markers’. Nevertheless, the different host parasite relations, leading to different cellular alter-ations of the swimbladder-wall (Würtz & Taraschewski 2000), are obviously connected with the different abun-dance and pathogenicity of the parasite in populations of the Japanese compared to the European eel.

The western part of Taiwan is still one of the fastest developing industrial zones of the world. However, it has no legislative restrictions on pollution (Chi 1994, Tsai et al. 2003). The consequences of rapid economic growth are nowadays ascertainable. The Kao-Ping River basin is not only the largest and most intensively used river in Taiwan, it is also heavily polluted (Kao et al. 2003). Livestock wastewater from hog farms, as well as municipal, domestic and industrial sewage repre-sent the main sources of water pollution (Kao et al. 2003). Under these circumstances, it is surprising that sufficient suitable copepods are available to allow suf-ficient transmission of Anguillicola crassus to achieve the high prevalence (approx. 50%) and intensity (~2 adult worms per infected eel) reported here. Thus, our results support the hypothesis that A. crassus is a gen-eralist which can persist under various environmental conditions.

Our findings show that Anguillicola crassus is still pre-sent in eel aquaculture in East Asia. In Bu-Dai aquacul-ture, no losses of eels associated with infection by A. crassus are known and the condition-factors calculated herein do not support high pathogenicity in the Japan-ese eel. Obviously the parasite is not a major problem in aquaculture systems based on the indigenous eel species Anguilla japonica. Thus far, all economic losses of cul-tured eels have occurred only when European (Egusa 1979) or American (Ooi et al. 1996) eels were involved as hosts. Accordingly A. crassus is not a major target in aquacultures with A. japonica. However, many countries

in South and Southeast Asia, Oceania and Africa (South Africa, Moçambique, Madagascar, Réunion) are pre-sently running pilot projects on establishing eel farms with local or imported eels (H. Taraschewski unpubl.). These projects should pay special attention to prevent-ing the further spread of A. crassus.

Acknowledgements. The authors thank the National Science

Council of the Rupublic of China for the financial support for specimen collection; ‘Landesgraduiertenförderung’ and ‘Hochschulvereinigung’ of the University Karlsruhe for sup-porting the first author’s travel expenses from Germany to Tai-wan; G. H. Chen, H. Y. Teng and H. Chu for help in field and laboratory work; Dr. T. Petney, Dr. C. H. Wang, Dr. C.L. Lin and Dr. C.W. Shin for valuable comments on the manuscript.

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Editorial responsibility: Wolfgang Körting, Hannover, Germany

Submitted: July 26, 2005; Accepted: January 30, 2006 Proofs received from author(s): July 15, 2006