Two new species of threadlike blood flukes (Aporocotylidae), with a molecular revision of the genera Ankistromeces Nolan & Cribb, 2004 and Phthinomita Nolan & Cribb, 2006.

Ankistromeces Nolan & Cribb, 2004 and Phthinomita Nolan & Cribb, 2006 are sister genera of threadlike blood flukes (Trematoda: Aporocotylidae) infecting teleost fishes of the tropical Indo-west Pacific. Here, we report new collections of these genera from Australia, Indonesia, and Japan. A new species of Ankistromeces, Ankistromeces kawamurai n. sp., is described from Siganus spinus (Linnaeus) off Okinawa, Japan, and a new species of Phthinomita, Phthinomita abdita n. sp., from Choerodon cephalotes (Castelnau), in Moreton Bay, Australia; the new species are morphologically cryptic within their respective genera and are delineated by molecular and ecological data. Ankistromeces olsoni Nolan & Cribb, 2006 is reported from Siganus fuscescens (Houttuyn) off Heron Island (southern Great Barrier Reef), Lizard Island (northern Great Barrier Reef), and Okinawa and Wakayama Prefectures, Japan and from Siganus spinus (Linnaeus) from off Bali, Indonesia. Ankistromeces mariae Nolan & Cribb, 2004 is re-reported from the type-host, Meuschenia freycineti (Quoy & Gaimard), from a new location, Gypsy Bay, Tasmania. Phthinomita poulini Nolan & Cribb, 2006 is re-reported from its type-locality, Lizard Island, from a range of mullids, including five new host species, and its range is extended to include Moreton Bay. Phthinomita symplocos Nolan & Cribb, 2006 is reported from Bali and P. hallae Nolan & Cribb, 2006, P. jonesi Nolan & Cribb, 2006, P. littlewoodi Nolan & Cribb, 2006, and P. munozae Nolan & Cribb, 2006 are each re-reported from their type-host and type-localities. New cox1 mtDNA data were generated for all known species of these two genera from new and archival material. Analyses of these data enabled an evaluation of all known Phthinomita species; P. robertsthomsoni Nolan & Cribb, 2006 is synonymised with P. adlardi Nolan & Cribb, 2006, and P. brooksi Nolan & Cribb, 2006 is synonymised with P. sasali Nolan & Cribb, 2006. We highlight the failure of ITS2 data to delineate closely related aporocotylid species. In contrast, cox1 sequence data are proving reliable and effective in this context and we recommend their incorporation in future studies of blood fluke taxonomy.

Insemination of the monogenean Neobenedenia girellae (Capsalidae, Benedeniinae).

In vitro spermatophore formation and insemination of Neobenedenia girellae (Monogenea: Capsalidae, Benedeniinae) were recorded on video and described for the first time. Upon contact of two individuals, the anterior adhesive discs of the donor firmly attached to the dorsal tegument of the recipient and the donor's fore body strongly contracted such that the genital pore region protruded and the penis was pushed anteriorly to protrude through the genital pore. It is hypothesised that the donor penis mechanically damaged the tegument of the recipient. The sperm and spermatophore matrix were released together through the penis, which was placed under the left anterior attachment disc immediately behind the adhesive pad. The spermatophore matrix containing the spermatozoa became solid and attached to the dorsal surface of recipient's body. When observed under scanning electron microscopy, the spermatophores were irregularly shaped, with a diameter of 52-83µm. Under light microscopy they consisted of a proximal eosinophilic matrix portion and a distal thin-walled portion containing spermatozoa. Both parts were enclosed with a thin outer casing. Insemination occurred during and after spermatophore formation. Three types of insemination were recorded, unilateral and mutual insemination and self-insemination. The presence of self-insemination indicates that even a single N. girellae on a cultured fish may cause a significant parasite infection in the entire aquaculture system.

Kudoa ogawai n. sp. (Myxozoa: Multivalvulida) from the trunk muscle of Pacific barrelfish Hyperoglyphe japonica (Teleostei: Centrolophidae) in Japan.

Kudoa ogawai n. sp. (Myxozoa; Multivalvulida) is described from the trunk muscle of Pacific barrelfish Hyperoglyphe japonica caught in Japan. Oval to ellipsoidal cysts, white to yellowish in colour, 1-2 mm in size, were filled with numerous spores having four polar capsules. Spores were stellate with rounded peripheral edges in apical view, and garlic shaped with four prominent apical projections in side view. Average (range) spore size was 13.3 (12.0-14.2) µm in width and 9.0 (8.3-9.7) µm in length. The spore dimensions and the apical projections of K. ogawai n. sp. were clearly distinguishable from the other muscle-infecting species. Molecular analyses of 18S rDNA and 28S rDNA sequences showed that K. ogawai n. sp. is closely related to Kudo iwatai and Kudoa hemiscylli (95% and 92% genetic similarity, respectively), but the present species is morphologically distinct from the two species. Phylogenetic analysis of 18S and 28S rDNA placed K. ogawai n. sp. sister to all Kudoa spp. available in GenBank.

Cardicola opisthorchis n. sp. (Trematoda: Aporocotylidae) from the Pacific bluefin tuna, Thunnus orientalis (Temminck & Schlegel, 1844), cultured in Japan.

A new aporocotylid blood fluke is described, based on specimens from the ventricle of the Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel), cultured in Wakayama and Nagasaki Prefectures, Japan. The new species is morphologically similar to the members of the genus Cardicola Short, 1953, but shows distinct differences in the body form, location of the testis and the orientation of the ootype. The body of the new species is long and slender, whereas other Cardicola species are small and generally lanceolate. The testis is mostly located posterior to the caeca and anterior to the ovary, occupying 31-45% of body length, in contrast to the known Cardicola species, whose testis is typically intercaecal. The ootype is oriented anteriorly, while in most congeners, it is directed posteriorly or horizontally. Phylogenetic analyses of this aporocotylid, together with Cardicola orientalis Ogawa, Tanaka, Sugihara et Takami, 2010 from the same host, were conducted based on DNA sequences of the ITS2 rDNA and the 28S region of ribosomal RNA. The analyses revealed that the new blood fluke belongs to the genus Cardicola despite the marked morphological differences. Thus, this aporocotylid is named Cardicola opisthorchis n. sp. and the generic diagnosis is emended in this paper. In addition, 100% identity among the ITS2 sequences from the present species, Cardicola sp. from T. orientalis in Mexico and Cardicola sp. from the northern bluefin tuna, Thunnus thynnus (Linnaeus) in Spain suggests that C. opisthorchis n. sp. has a broad geographical distribution and that it infects both the Pacific and northern bluefin tuna.

Kudoa prunusi n. sp. (Myxozoa: Multivalvulida) from the brain of Pacific bluefin tuna Thunnus orientalis (Temminck & Schlegel, 1844) cultured in Japan.

Kudoa prunusi n. sp. (Myxozoa; Multivalvulida) is described from the brain of Pacific bluefin tuna Thunnus orientalis cultured in Japan. Numerous white cysts, up to 0.5mm in size, were found on and in the brain. Spores having typically five spore valves and five polar capsules resembled a five-petal cherry blossom in apical view and were conical shape with a round bottom in side view. Average spore size was 9.63 (8.5-10.3) µm in width and 7.50 (6.7-8.6) µm in length. The spore dimensions of K. prunusi overlapped with those of Kudoa yasunagai ex Sillago ciliata having five to six spore valves, but they were clearly distinct in spore shape, 18S rDNA and 28S rDNA sequences (0.3% and 1.7% differences, respectively). Phylogenetic analysis of 18S rDNA revealed that K. prunusi grouped with the brain-infecting multivalvulid species, K. yasunagai, K. chaetodoni, K. lethrini and K. neurophila, rather than five-valved Kudoa spp. Combined with morphological, molecular and biological differences, K. prunusi was proven to be a new species.

Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease, reduces fecundity and feeding activity of Tubifex tubifex (Oligochaeta).

Myxobolus cerebralis is the causative agent of whirling disease that has significant economical and ecological impacts on trout populations. Although intensive studies have been conducted to understand its effects on and interactions with its fish host, only limited information is available about how and to what extent M. cerebralis affects its oligochaete host, Tubifex tubifex. We investigated the effects of M. cerebralis on survival, growth, reproduction, and feeding activity of T. tubifex. Mature, immature and juvenile worms were exposed to myxospores and their infection prevalence, mortality, sexual development, reproduction and spore production were compared with unexposed worms. The parasite affected neither survival nor growth but inhibited clitellar development and reduced cocoon production by over 80%. Numbers of actinospores released from mature worms were nearly 9-fold higher than that of immature worms. When non-clitellated infected worms were kept at 30 degrees C for 4 days, spore release ceased and they re-developed a clitellum. These results suggest parasite-induced castration. Comparative monitoring of defecation rate revealed that M. cerebralis reduced feeding activity of T. tubifex by approximately 40%. Low energy intake and impaired energetic allocation may be the underlying mechanism behind reduced fecundity of infected T. tubifex.

Infection dynamics of Neoheterobothrium hirame (Monogenea) on juvenile olive flounder, Paralichthys olivaceus (Temminck & Schlegel), in coastal waters of Japan.

A recent epidemic of the invasive monogenean Neoheterobothrium hirame is suspected to have caused the significant decline of the commercial catch of olive flounder, Paralichthys olivaceus, in Japan. To clarify the causal link between parasite invasion and host depletion, we monitored the infections of N. hirame on juvenile flounder for 2 years at two widely separate bays, Obama Bay, experiencing a devastating reduction in flounder catches, and Miyako Bay with a stable catch. Prevalence, mean intensity and abundance of developmental stages of N. hirame were compared between the bays and between fish year classes. Fishes captured at Obama Bay harboured three times more worms than those at Miyako Bay and had a significantly higher overall prevalence of infection. In addition, there was a negative correlation between parasite intensity and host condition factor at Obama Bay, suggesting a causal link between the N. hirame epidemic and the reduction of local flounder populations.

Chronology of parasite-induced alteration of fish behaviour: effects of parasite maturation and host experience.

We monitored temporal changes in the magnitude of altered host behaviour in minnows (Pimephales promelas) experimentally infected with metacercariae of a brain-encysting trematode (Ornithodiplostomum ptychocheilus). This parasite develops and then encysts in a region of the brain that mediates the optomotor response (OMR), an innate behaviour that links visual stimuli with motor performance. The OMR of infected and uninfected minnows was evaluated between 0 and 10 weeks post-infection (p.i.), an interval spanning the development period of metacercariae to infectivity in birds. Trials involved monitoring the time an individual minnow spent following a spinning drum that had been painted with alternating black and white stripes. At 2 and 4 weeks p.i., infected minnows followed the drum 40% less often than controls. Differences between controls and infected fish declined thereafter, and were undetectable by 10 weeks p.i. Both control and infected fish habituated equally rapidly to the spinning drum. However, the difference in performance between controls and infected fish was 29% for experienced fish and 48% for fish that had never experienced the drum. Because maximum parasite-induced reduction in OMR coincided with the period of maximum parasite development, the behavioural effects are most likely due to unavoidable pathology in the brain associated with developing larvae.