Hematozoa of teleosts from Lizard Island, Australia, with some comments on their possible mode of transmission and the description of a new hemogregarine species

Little is known of the blood parasites of coral reef fishes and nothing of how they are transmitted. We examined 497 fishes from 22 families, 47 genera, and 78 species captured at Lizard Island, Australia, between May 1997 and April 2003 for hematozoa and ectoparasites. We also investigated whether gnathiid isopods might serve as potential vectors of fish hemogregarines. Fifty-eight of 124 fishes caught in March 2002 had larval gnathiid isopods, up to 80 per host fish, and these were identified experimentally to be of 2 types, Gnathia sp. A and Gnathia sp. B. Caligid copepods were also recorded but no leeches. Hematozoa, found in 68 teleosts, were broadly hemogregarines of 4 types and an infection resembling Haemohormidium. Mixed infections (hemogregarine with Haemohormidium) were also observed, but no trypanosomes were detected in blood films. The hemogregarines were identified as Haemogregarina balistapi n. sp., Haemogregarina tetraodontis, possibly Haemogregarina bigemina, and an intraleukocytic hemogregarine of uncertain status. Laboratory-reared Gnathia sp. A larvae, fed experimentally on brushtail tangs, the latter heavily infected with the H. bigemina-like hemogregarine, contained hemogregarine gamonts and possibly young oocysts up to 3 days postfeeding, but no firm evidence that gnathiids transmit hemogregarines at Lizard Island was obtained.     

Host specificity of two species of Gnathia (Isopoda) determined by DNA sequencing blood meals

Host specificity data for gnathiid isopods are scarce because the parasitic stages are difficult to identify and host-parasite contact is often brief. We examined two common nocturnal species, Gnathia falcipenis and Gnathia sp. C, collected in light traps from two locations at Lizard Island on the Great Barrier Reef, Australia. Engorged third stage gnathiids were photographed and permitted to moult into adults to allow identification. We compared approximately 580 bp sequences of 16S mtDNA from blood meals with host sequences available on GenBank using BLASTn. Where homology was <98%, familial identity was investigated with neighbour-joining trees. All blood meal sequences (n=60) and homologous fish sequences (n=87) from GenBank were used in a Bayesian analysis, which identified all but three sequences to family. The host frequency distributions used by each species were significantly different; only four host families were shared. No gnathiids fed on elasmobranchs, blennies or apogonids, and most fed on host families whose representatives are typically large. Gnathia sp. C showed a distinct predilection for nemipterids. Gnathia falcipenis often parasitised sand-dwelling families, and unlike sympatric diurnal gnathiid species, it also frequently parasitised pomacentrids. We conclude that G. falcipenis and Gnathia sp. C operate as generalist micropredators with preferences.     

Live coral repels a common reef fish ectoparasite

Coral reefs are undergoing rapid changes as living corals give way to dead coral on which other benthic organisms grow. This decline in live coral could influence habitat availability for fish parasites with benthic life stages. Gnathiid isopod larvae live in the substratum and are common blood-feeding parasites of reef fishes. We examined substrate associations and preferences of a common Caribbean gnathiid, Gnathia marleyi. Emergence traps set over predominantly live coral substrata captured significantly fewer gnathiids than traps set over dead coral substrata. In laboratory experiments, gnathiids preferred dead coral and sponge and tended to avoid contact with live coral. When live gnathiids were added to containers with dead or live coral, significantly fewer were recovered from the latter after 24 h. Our data therefore suggest that live coral is not suitable microhabitat for parasitic gnathiid isopods and that a decrease in live coral cover increases available habitat for gnathiids.     

Impact of micropredatory gnathiid isopods on young coral reef fishes

The ecological role of parasites in the early life-history stages of coral reef fish, and whether this varies between fish with and without a pelagic phase, was investigated. The susceptibility to, and effect of reef-based micropredatory gnathiid isopods on larval, recently settled, and juvenile fishes was tested using two damselfishes (Pomacentridae): Neopomacentrus azysron, which has pelagic larvae, and Acanthochromis polyacanthus, which does not. When larval and recently settled stages of N. azysron and very young A. polyacanthus juveniles (smaller than larval N. azysron) were exposed to one or three gnathiids, the proportion of infections did not vary significantly among the three host types or between the number of gnathiids to which the fish were exposed. The overall infection was 35%. Mortality, however, differed among the three gnathiid-exposed host types with most deaths occurring in larval N. azysron; no mortalities occurred for recently settled N. azysron exposed to one or three gnathiids, and A. polyacanthus exposed to one gnathiid. Mortality did not differ significantly between larval N. azysron and A. polyacanthus juveniles, failing to provide support for the hypothesis that reef-based A. polyacanthus juveniles are better adapted to gnathiid attack than fish with a pelagic phase. The study suggests that settling on the reef exposes young fish to potentially deadly micropredators. This supports the idea that the pelagic phase may allow young fish to avoid reef-based parasites.     

Brooding and embryonic development in the crustacean Paragnathia formica (Hesse, 1864) (Peracarida: Isopoda: Gnathiidae)

The crustacean family Gnathiidae Leach, 1814 (Peracarida: Isopoda) comprises 12 genera known mostly from marine environments. Juvenile gnathiid isopods are fish ectoparasites, feeding on blood and tissue fluids in order to complete their life cycles. Gnathiid juvenile development generally includes three moults, the last involving metamorphosis to non-feeding, adult stages. The blood meal ingested by juveniles provides resources for adult survival, reproduction and embryological development. Reproductive biology in the brackish water gnathiid, Paragnathia formica (Hesse, 1864), is unusual amongst crustaceans, since brooding females have paired internal uterine sacs, rather than an external brood pouch. Known embryological development for P. formica includes three post gastrulation stages. In the current study, brooding and embryological development in this gnathiid were reexamined using histological and fluorescence methods, and by scanning electron microscopy. Novel observations were made of the blastodisc and germ cell migration within developing eggs, release of Stage 2 embryos by rupture of embryonic membranes, the in utero moult of Stage 2 to Stage 3 embryos, and the asynchronous development of the brood within the paired uterine sacs. These findings highlight the remarkable nature of brooding in P. formica and expand the paucity of knowledge of embryological development in gnathiids in general.     

Further Studies on Haemogregarina bigemina Laveran & Mesnil,the Marine Fish Blennius pholis L., and the Isopod Gnathia maxillaris Montagu1

Haemogregarina bigemina is redescribed from the blood of the marine fish Blennius pholis, and stages presumed to be those of the haemogregarine are recorded from the hematophagous praniza larva of the isopod Gnathia maxillaris. At College Rocks, Aberystwyth, Wales, the main study area, a high incidence of infection occurred in B. pholis. No exoerythrocytic stages were observed in these fish, nor was sexual dimorphism of the gametocyte evident. As in an earlier study, ecological evidence favored transmission by G. maxillaris rather than by leeches. Gametocytes, syzygy, oocysts, sporoblasts, and sporozoites were identified in the anterior hindgut of the isopod. The stages observed in G. maxillaris are compared with those of other haemogregarines described from the digestive tract of leeches. Mention is made of an intraleucocytic haemogregarine of another fish, Crenilabrus melops.     

Diurnal variation in the abundance of juvenile parasitic gnathiid isopods on coral reef fish: implications for parasite-cleaner fish interactions

 The dynamics of parasitic gnathiid isopod infestation on the fish Hemigymnus melapterus were examined at Heron Island, Great Barrier Reef, by measuring the abundance and feeding state of gnathiids on fish collected between dawn and sunset and by estimating the time required for gnathiids to become engorged on host fluids. A model was developed to estimate gnathiid abundance on fish for any given time of day and host size. Fish at dawn had 2.4 times as many gnathiids compared with fish at sunset, indicating that some gnathiids infest fish overnight. Most gnathiids had engorged guts (72–86%); the proportion of empty guts and engorged guts did not differ in three time periods of collection (<0800 h, 0800 to 1100 h, and >1100 h). In the laboratory, gnathiids fed quickly with 75% of gnathiids exposed to fish for 4 h having engorged guts. The short time required for gnathiids to become engorged and the presence of gnathiids with empty guts throughout the day suggests that gnathiids also infest fish during the day. Thus gnathiids eaten by cleaner fish during the day may be replaced by other gnathiids during the day or night suggesting that interactions between gnathiids and cleaner fish are highly dynamic. Accepted: 15 April 1999     

A simple molecular technique for identifying marine host fish by sequencing blood-feeding parasites

Gnathiid isopods are common ectoparasites of fish on the Great Barrier Reef, Australia. While screening for appropriate markers for phylogenetic studies of gnathiids, we found that primers for 12S and 16S rDNA preferentially amplified the host fish DNA instead of gnathiid DNA. This amplification occurred even when using gnathiids that were not engorged with host blood and adult gnathiids that do not feed on fish blood. This method could be used in host-parasite studies to identify hosts without having to sample parasites directly from the host (which can be costly and requires considerable skill in a marine environment). Target ribosomal DNA sequences can be amplified from total DNA extracted from parasites that are captured in funnel traps or plankton tows. Sequence data from these can be used to identify the hosts that gnathiids were feeding on before capture.     

Haemogregarina georgianae n. sp., a new haemogregarine parasite from the Antarctic teleost Parachaenichthys georgianus

Haemogregarina georgianae n. sp. occurs frequently in the bathydraconid teleost Parachaenichthys georgianus from the western Antarctic portion of the Southern Ocean. The haemogregarine and its developmental stages in the vertebrate have been found in erythrocytes of the fish: both microschizogony and macroschizogony have been seen in fish caught during the austral summer. Morphological evidence suggests the merozoites from macroschizogony give rise to the microschizont, and that the microschizont merozoites give rise to gametocytes. Comparison of H. georgianae with other haemogregarines known from teleosts shows that it has a previously undescribed morphology.     

Phylogenetic analysis of the genus Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina)

A phylogenetic analysis of species of Hepatozoon Miller, 1908 was performed using 16 morphological, morphometric and developmental characters. An adeleorin parasite of gastropod molluscs, Klossia helicina Schneider, 1875 and four haemogregarines of other genera, Karyolysus lacertae (Danilewsky, 1886), Cyrilia lignieresi (Laveran, 1906), Desseria myoxocephali (Fantham, Porter & Richardson, 1942) and Haemogregarina balli Paterson & Desser, 1976 were used as the outgroup to 12 species of Hepatozoon. A single most parsimonious interpretation of the data was found, with the resulting cladogram having 28 transformations and a consistency index of 0.75. The proposed phylogeny revealed Hepatozoon as a paraphyletic group. One monophyletic lineage contained 10 of the 12 species of Hepatozoon, including all of those species that undergo gametogenesis and sporogony in the haemocoel of arthropods. Within this lineage, gamonts of those species found in vertebrate leucocytes instead of erythrocytes formed a clade basal to the remainder of the species. Species of Hepatozoon which undergo gametogenesis and sporogonic development in the gut epithelium of acarines, and which produce aflagellate microgametes, as well as the four outgroup taxa of haemogregarines, formed a monophyletic group and were the sister group to the remainder of the species of Hepatozoon. The biological and morphological diversity of these parasites suggests that species of the genus Hepatozoon, which are members of the paraphyletic haemogregarine complex, could be partitioned into at least two genera of adeleorin parasites.     

Habitat degradation drives increased gnathiid isopod ectoparasite infection rate on juvenile but not adult fish

Widespread coral mortality is leading to coral reef degradation worldwide. Many juvenile reef fishes settle on live coral, and their predator-avoidance behaviour is disrupted in seawater exposed to dead corals, ultimately increasing predation risk. Gnathiid isopods are micropredatory fish ectoparasites that occur in higher abundances in dead coral. However, the effect of seawater associated with dead coral on the susceptibility of fish to micropredators has never been investigated. We tested whether the infection rate of cultured gnathiid ectoparasites on individual damselfish, Pomacentrus amboinensis Bleeker 1868, from two different ontogenetic stages (juveniles and adults) was influenced by seawater exposed to three different treatments: dead coral, live coral, or no coral. Seawater treatments were presumed to contain different chemical properties and are meant to represent environmental changes associated with habitat degradation on coral reefs. Gnathiid infection of juvenile fish in seawater exposed to dead coral was twice as high as that of fish in live coral or no coral. Infection rates did not significantly differ between live coral and no coral treatments. In contrast to juveniles, the susceptibility of adults to gnathiids was not affected by seawater treatment. During experiments, juvenile fish mortality was relatively low, but was higher for infected fish (9.7%), compared to fish held without exposure to gnathiids (1.7%). No mortality occurred in adult fish that became infected with gnathiids. Our results suggest that chemical cues released from dead corals and/or dead coral colonisers affect the ability of juvenile, but not adult fish to avoid parasite infection. Considering increased habitat degradation on coral reefs and that gnathiids are more abundant in dead coral substrate, it is possible that wild juvenile fish may experience increased susceptibility to parasitic infection and reduced survival rate. This highlights the importance of including parasitism in ecological studies of global environmental change.

     

Low Susceptibility of Invasive Red Lionfish (Pterois volitans) to a Generalist Ectoparasite in Both Its Introduced and Native Ranges

Escape from parasites in their native range is one of many mechanisms that can contribute to the success of an invasive species. Gnathiid isopods are blood-feeding ectoparasites that infest a wide range of fish hosts, mostly in coral reef habitats. They are ecologically similar to terrestrial ticks, with the ability to transmit blood-borne parasites and cause damage or even death to heavily infected hosts. Therefore, being highly resistant or highly susceptible to gnathiids can have significant fitness consequences for reef-associated fishes. Indo-Pacific red lionfish (Pterois volitans) have invaded coastal habitats of the western tropical and subtropical Atlantic and Caribbean regions. We assessed the susceptibility of red lionfish to parasitic gnathiid isopods in both their native Pacific and introduced Atlantic ranges via experimental field studies during which lionfish and other, ecologically-similar reef fishes were caged and exposed to gnathiid infestation on shallow coral reefs. Lionfish in both ranges had very few gnathiids when compared with other species, suggesting that lionfish are not highly susceptible to infestation by generalist ectoparasitic gnathiids. While this pattern implies that release from gnathiid infestation is unlikely to contribute to the success of lionfish as invaders, it does suggest that in environments with high gnathiid densities, lionfish may have an advantage over species that are more susceptible to gnathiids. Also, because lionfish are not completely resistant to gnathiids, our results suggest that lionfish could possibly have transported blood parasites between their native Pacific and invaded Atlantic ranges.     

Gnathia maxillaris infestation in exhibition aquaria: control and treatment strategies

An outbreak of gnathiids in a captive fish population in a large exhibition aquarium is described, including a method for enumeration of the causative agent. This procedure for harvesting and counting the parasites was based on the positive phototropism of their larvae (zuphea and praniza). Water pumps with spotlights allowed capture of larva using a 200‐micron plankton net. Harvested samples were rinsed with fresh water in the laboratory to immobilise the zuphea and praniza and thus facilitate the enumeration process by direct observation under a stereoscopic glass. The sampling method was found to be representative of the degree of infestation experienced by the entire aquarium system; this finding is very important as it allows the enumeration of different life‐cycle stages (zuphea, praniza and adults). The outbreak was studied and the infestation monitored over a period of many years. Several substrates were assayed for the capture of gnathiid males, with identification of the causative parasite by such morphological characteristics as Gnathia maxillaris. Physical and chemical treatments were applied to mitigate the infestation. Selected to control the infestation and its lifetime in closed marine‐water systems, the Trichlorfon was evaluated by chromatography.     

An unusual intraerythrocytic parasite of Parablennius cornutus from South Africa

An intertidal horned blenny, Parablennius cornutus, captured at De Hoop Nature Reserve, South Africa, was found to harbor an unusual blood parasite and the haemogregarine Haemogregarina bigemina. In Giemsa-stained blood films, the enigmatic parasite occurred primarily as intraerythrocytic ringlike stages, with unstained centers and peripheral bands of beaded chromatin, not unlike Haemohormidium spp. Larger forms of the same organism stained pink with Giemsa, with nuclei occurring as 4-8 minute structures around the parasite body or distributed within it. These larger parasites apparently segmented into up to 8 individuals that were rounded or oval with deep-stained, comma-shaped or polar regions surrounding blue cytoplasm. Extracellular, binucleate, sporelike structures in clusters of as many as 16 individuals were also seen in blood films. Praniza larvae of the isopod Gnathia africana were seen in histological sections of gill tissue. Examination of spleen tissue by transmission electron microscopy showed intraerythrocytic organisms with ultrastructural characteristics like those of Haematractidium scombri, namely, a single boundary membrane, sometimes closely apposed nuclei with nucleoli, and profiles of dense material of variable structure. It is concluded that the parasite is probably related to Haemohormidium spp. and H. scombri, but it also shares features with some Microsporida.     

Evolutionary divergence in common marine ectoparasites Gnathia spp. (Isopoda: Gnathiidae) on the Great Barrier Reef: phylogeography, morphology, and behaviour

The blood‐feeding juvenile stages of gnathiid isopods are important ectoparasites of marine fishes on the Great Barrier Reef (GBR), and are a major component of the diet of cleaner fishes. We report here that these gnathiids have undergone evolutionary diversification, both geographically and temporally (into diurnally and nocturnally active taxa), which has been accompanied by changes in their morphology and behaviour. To perform this analysis, we sequenced a portion of the nuclear ribosomal ITS2 for 47 gnathiids collected from 29 host fishes of 11 species at three locales spanning 2000 km on the GBR. Maximum parsimony and Bayesian phylogenetic analyses both revealed four major clades. There was some degree of geographical structuring in these clades, but there was no evidence supporting host fish specialization, as gnathiids collected from the skin of different teleost taxa did not resolve into distinct clades. The topology of the phylogeny also implied some structuring that was dependent upon collection time (day or night), so we investigated whether there were also behavioural and morphological differences between taxa active at these different times. Nocturnal gnathiids had significantly longer antennules and larger eyes than diurnal gnathiids – two traits presumably adaptive for nocturnal activity. Behavioural tests showed that both nocturnal and diurnal gnathiids use olfaction and vision while foraging, but that nocturnal gnathiids used olfaction more often in dark conditions, and that they were able to perceive movement under extremely low levels of light. Diurnal gnathiids used vision more effectively when there was some ambient light. Our results thus suggest that both phenotypic and genotypic divergence in gnathiids may be influenced by natural selection acting on ecological traits, such as predator avoidance and host detection. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94 , 569–587.     

Male dimorphism in the harem-forming gnathiid isopod Elaphognathia discolor (Crustacea: Isopoda)

Previously unreported males of a gnathiid isopod were found in reproductive aggregations of the harem-forming gnathiid Elaphognathia discolor. Although the male gnathiids were small in size and morphologically different from E. discolor males, the male sexual organ, appendix masculina, was similar to that of E. discolor males, and possible conspecific larvae and females of the small male gnathiid were never found. In the laboratory, the small male gnathiids as well as male E. discolor successfully copulated with female E. discolor, and the development of embryos in female brood pouches was observed. Offspring of small male gnathiids develop to adults of E. discolor after molting three times, or small male gnathiids after molting two times. Thus, the small male gnathiid was concluded to be an alternative male form compared to the regular large male form of E. discolor. This male polymorphism was thought to have a genetic basis, since no small male specimens appeared in offspring of regular E. discolor males. Field sampling showed that a regular large male formed a harem composed of one large male and several females and never coexisted with other large males as previously reported. However, small males were often found together with large males. Therefore, small males are thought to be sneakers intruding into harems dominated by large males.     

Interactions between gnathiid isopods,cleaner fish and other fishes on Lizard Island,Great Barrier Reef

The rate of emergence of micropredatory gnathiid isopods from the benthos, the proportion of emerging gnathiids potentially eaten by Labroides dimidiatus, and the volume of blood that gnathiids potentially remove from fishes (using gnathiid gut volume) were determined. The abundance (mean ±s.e .) of emerging gnathiids was 41·7 ± 6·9 m^?2 day^?1 and 4552 ± 2632 reef^?1 day^?1 (reefs 91–125 m²). The abundance of emerging gnathiids per fish on the reef was 4·9 ± 0·8 day^?1; but excluding the rarely infested pomacentrid fishes, it was 20·9 ± 3·8 day^?1. The abundance of emerging gnathiids per patch reef was 66 ± 17% of the number of gnathiids that all adult L. dimidiatus per reef eat daily while engaged in cleaning behaviour. If all infesting gnathiids subsequently fed on fish blood, their total gut volume per reef area would be 17·4 ± 5·6 mm³ m^?2 day^?1; and per fish on the reefs, it would be 2·3 ± 0·5 mm^?3 fish^?1 day^?1 and 10·3 ± 3·1 mm³ fish^?1 day^?1 (excluding pomacentrids). The total gut volume of gnathiids infesting caged (137 mm standard length, L_S) and removed from wild (100–150 mm L_S) Hemigymnus melapterus by L. dimidiatus was 26·4 ± 24·6 mm³ day^?1 and 53·0 ± 9·6 mm³ day^?1, respectively. Using H. melapterus (137 mm L_S, 83 g) as a model, gnathiids had the potential to remove, 0·07, 0·32, 0·82 and 1·63% of the total blood volume per day of each fish, excluding pomacentrids, caged H. melapterus and wild H. melapterus, respectively. In contrast, emerging gnathiids had the potential of removing 155% of the total blood volume of Acanthochromis polyacanthus (10·7 mm L_S, 0·038 g) juveniles. That L. dimidiatus eat more gnathiids per reef daily than were sampled with emergence traps suggests that cleaner fishes are an important source of mortality for gnathiids. Although the proportion of the total blood volume of fishes potentially removed by blood‐feeding gnathiids on a daily basis appeared to be low for fishes weighing 83 g, the cumulative effects of repeated infections on the health of such fish remains unknown; attacks on small juvenile fishes, may result in possibly lethal levels of blood loss.     

Fish mucous cocoons: the 'mosquito nets' of the sea

Mucus performs numerous protective functions in vertebrates, and in fishes may defend them against harmful organisms, although often the evidence is contradictory. The function of the mucous cocoons that many parrotfishes and wrasses sleep in, while long used as a classical example of antipredator behaviour, remains unresolved. Ectoparasitic gnathiid isopods (Gnathiidae), which feed on the blood of fish, are removed by cleaner fish during the day; however, it is unclear how parrotfish and wrasse avoid gnathiid attacks at night. To test the novel hypothesis that mucous cocoons protect against gnathiids, we exposed the coral reef parrotfish Chlorurus sordidus (Scaridae) with and without cocoons to gnathiids overnight and measured the energetic content of cocoons. Fish without mucous cocoons were attacked more by gnathiids than fish with cocoons. The energetic content of mucous cocoons was estimated as 2.5 per cent of the fish's daily energy budget fish. Therefore, mucous cocoons protected against attacks by gnathiids, acting like mosquito nets in humans, a function of cocoons and an efficient physiological adaptation for preventing parasite infestation that is not used by any other animal.