Life cycle of Hepatozoon affluomaloti sp. n. (Apicomplexa: Haemogregarinidae) in crag lizards (Sauria: Cordylidae) and in culicine mosquitoes from South Africa.

A new haemogregarine species Hepatozoon affluomaloti sp. n. is described from erythrocytes in the peripheral blood of crag lizards Pseudocordylus melanotus (Smith) and Pseudocordylus subviridis (Smith) (Sauria: Cordylidae) from mountainous regions in the Eastern Free State, South Africa. This species can be distinguished from all other congeners based on its large size, staining properties and life cycle development in its vector, Culex (Afroculex) lineata (Theobald) (Diptera: Culicidae). Mature gamonts stain mostly uniformly pinkish-purple with Giemsa, sometimes containing darker azurophilic granules anterior and posterior to the nucleus. The reflexed posterior extremity of the gamont stage sometimes stains slightly deeper purple and the nucleus is dense and placed in the posterior third of the parasite body. Merogonic stages of this haemogregarine occur in the liver tissues of P. melanotus with dizoic meronts. Macromeronts contains 2-7 macromerozoites and micromeronts contains 9-24 micromerozoites. Sporogonic developmental stages found in the proposed final host and vector, C. lineata, include large oocysts, measuring 54 × 48 µm on average. Sporulating oocysts with 8 nuclei are present in mosquitoes 6-7 days post-feeding on infected lizards. Sporocysts with mature sporozoites measure 31.0 × 21.8 µm on average and each contains 2-8 large sporozoites. It is suggested that transmission of infective sporozoites is achieved through predation of lizards on mosquitoes.

Reassignment of the land tortoise haemogregarine Haemogregarina fitzsimonsi Dias 1953 (Adeleorina: Haemogregarinidae) to the genus Hepatozoon Miller 1908 (Adeleorina: Hepatozoidae) based on parasite morphology, life cycle and phylogenetic analysis of 18S rDNA sequence fragments.

SUMMARY Research was undertaken to clarify the true taxonomic position of the terrestrial tortoise apicomplexan, Haemogregarina fitzsimonsi (Dias, 1953). Thin blood films were screened from 275 wild and captive South African tortoises of 6 genera and 10 species between 2009-2011. Apicomplexan parasites within films were identified, with a focus on H. fitzsimonsi. Ticks from wild tortoises, especially Amblyomma sylvaticum and Amblyomma marmoreum were also screened, and sporogonic stages were identified on dissection of adult ticks of both species taken from H. fitzsimonsi infected and apparently non-infected tortoises. Parasite DNA was extracted from fixed, Giemsa-stained tortoise blood films and from both fresh and fixed ticks, and PCR was undertaken with two primer sets, HEMO1/HEMO2, and HepF300/HepR900, to amplify parasite 18S rDNA. Results indicated that apicomplexan DNA extracted from tortoise blood films and both species of tick had been amplified by one or both primer sets. Haemogregarina  fitzsimonsi 18S rDNA sequences from tortoise blood aligned with those of species of Hepatozoon, rather than those of species of Haemogregarina or Hemolivia. It is recommended therefore that this haemogregarine be re-assigned to the genus Hepatozoon, making Hepatozoon fitzsimonsi (Dias, 1953) the only Hepatozoon known currently from any terrestrial chelonian. Ticks are its likely vectors.

Hepatozoon langii n. sp. and Hepatozoon vacuolatus n. sp. (Apicomplexa: Adele-orina: Hepatozoidae) from the crag lizard (Sauria: Cordylidae) Pseudocordylus langi from the North Eastern Drakensberg escarpment, Eastern Free State, South Africa.

Two new haemogregarine species, Hepatozoon langii n. sp. and Hepatozoon vacuolatus n. sp., are described from the pe-ripheral blood of the high altitude crag lizard, Pseudocordylus langi, collected between October 2006 and April 2009 from the North Eastern Drakensberg, Eastern Free State. Hepatozoon langii n. sp. has maturing and mature gamonts that appear encapsulated and have narrow, curved tails. Their cytoplasm stains pinkish-purple with Giemsa, while their nuclei are pur-ple stained with stranded chromatin. Mature gamonts measure 19.1 ± 1.0 (15.4-28.1) µm long by 6.2 ± 1.1 (3.5-7.9) µm wide. Hepatozoon vacuolatus n. sp. gamonts are mostly broader at one pole than the other, have bluish-pink cytoplasm characterised by distinctive rounded and oval vacuoles, and demonstrate pink granules with Giemsa staining. Nuclei stain purple and are mainly coarsely granular. Mature gamonts measure 16.5 ± 1.0 (14.7 - 17.6) µm long by 5.9 ± 1.2 (4.0 - 7.7) µm wide. Both species parasitize erythroblasts, as well as erythrocytes and can dehaemoglobinize the cytoplasm of their host cells. Hepatozoon langii n. sp occurred in the absence of H. vacuolatus n. sp., but the latter haemogregarine always formed mixed infections with the former; no stages intermediate between the two haemogregarine types were observed.

Novel, panzootic and hybrid genotypes of amphibian chytridiomycosis associated with the bullfrog trade.

Global amphibian declines are linked with the presence of specific, highly virulent genotypes of the emerging fungal disease chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) known as the global panzootic lineage (Bd-GPL). The global trade in amphibians for human consumption is suspected to have facilitated emergence of the disease, but evidence to support this is largely lacking. Here, we investigated the role the Lithobates catesbeianus (North American bullfrog) trade in spreading Bd genotypes by comparing strains associated with L. catesbeianus to a global panel using 36 sequenced loci from multiple chromosomal regions. Most bullfrogs were infected with Bd-GPL genotypes, but we also detected novel, highly divergent Bd genotypes (Bd-Brazil) from a live bullfrog in a US market and from native Brazilian anurans in the Atlantic Forest where bullfrogs are widely farmed. Sexual reproduction was also detected for the first time in Bd in the form of a hybrid genotype between the Bd-GPL and Bd-Brazil lineages in the Atlantic Forest. Despite the demonstration that ribosomal RNA types in Bd fail to undergo concerted evolution (over 20 sequence types may be found in a single strain), the Bd-GPL and Bd-Brazil lineages form largely separate clusters of related internal transcribed spacer (ITS) RNA sequences. Using ITS sequences, we then demonstrate the presence of Bd-Brazil in Japan, primarily on invasive L. catesbeianus. The finding that Bd is capable of sexual reproduction between panzootic and endemic genotypes emphasizes the risk of international wildlife trade as a source of additional Bd epizootics owing to hybridization.

Haematozoans from deep water fishes trawled off the Cape Verde Islands and over the Porcupine Seabight, with a revision of species within the genus Desseria (Adeleorina: Haemogregarinidae).

Archived blood smears from 32 of 113 fishes in 18 families and 12 orders, trawled from deep North Atlantic waters off the Cape Verde Islands in 1999 and over the Porcupine Seabight in 2001 were found to harbour haematozoans. These included four species of haemogregarines (Adeleorina, Haemogregarinidae) and a species of trypanosome (Trypanosomatina, Trypanosomatidae) located in Porcupine Seabight fishes. Also present were Haemohormidium-like structures of uncertain status found in samples from this location and from the Cape Verde Islands. Although material was limited, two of the haemogregarines were provisionally named Desseria harriottae sp. n. from Harriotta raleighana Goode et Bean (Chimaeriformes, Rhinochimaeridae), and Haemogregarina bathysauri sp. n. from Bathysaurus ferox Günther (Aulopiformes, Bathysauridae). The two remaining haemogregarines were identified as Desseria marshalllairdi (Khan, Threlfall et Whitty, 1992) from Halosauropsis macrochir (Günther) (Notacanthiformes, Halosauridae), and Haemogregarina michaeljohnstoni (Davies et Merrett, 2000) from Cataetyx laticeps Koefoed (Ophidiformes, Bythitidae). The name H. michaeljohnstoni was proposed to replace Haemogregarinajohnstoni Davies et Merrett, 2000 from C. laticeps and to avoid confusion with Hepatozoon johnstoni (Mackerras, 1961) Smith, 1996 from varanid lizards, originally named Haemogregarina johnstoni Mackerras, 1961. The trypanosome formed a mixed parasitaemia with D. harriottae in H. raleighana and was provisionally named Trypanosoma harriottae sp. n. No blood parasites had been described previously from cartilaginous fishes of the Holocephali, making the finds in H. raleighana unique. Haemohormidium-like structures were located in erythrocytes in one fish, Coryphaenoides armatus (Hector), among the Cape Verde Islands samples and in 12 species of fishes from the Porcupine Seabight; all these hosts were bony fishes. Finally, the haemogregarine species listed in the genus Desseria Siddall, 1995 were reassessed. Of the original list of 41 species, 30 were retained and 5 species added, including D. harriottae, so that the genus now contains 35 species.

Spreading by snail (Lymnaea stagnalis) defence cells is regulated through integrated PKC, FAK and Src signalling.

Cell adhesion and spreading are vital to immune function. In molluscs, haemocytes (circulating phagocytes) are sentinels and effectors of the internal defence system; however, molecular mechanisms that regulate integrin-mediated spreading by haemocytes have not been characterised in detail. Visualisation of Lymnaea stagnalis haemocytes by scanning electron microscopy revealed membrane ruffling, formation of lamellipodia and extensive filopodia during early stages of cell adhesion and spreading. These events correlated with increased phosphorylation (activation) of protein kinase C (PKC) and focal adhesion kinase (FAK), sustained for 60 min. Treatment of haemocytes with the PKC inhibitors GF109203X or Gö 6976, or the Src/tyrosine kinase inhibitors SrcI or herbimycin A, attenuated haemocyte spread by 64, 46, 32 and 35%, respectively (P

A redescription of Haemogregarina fitzsimonsi Dias, 1953 and some comments on Haemogregarina parvula Dias, 1953 (Adeleorina: Haemogregarinidae) from southern African tortoises (Cryptodira: Testudinidae), with new host data and distribution records.

Blood films were examined from 154 wild and captive tortoises from four provinces of South Africa, including Gauteng, Kwazulu-Natal, North West and Western Cape. The five species ofchelonians studied were Chersina angulata (Schweigger), Kinixys belliana belliana (Gray), K. lobatsiana Power, K. natalensis Hewitt, and Stigmochelys pardalis (Bell). Two species of haemogregarines, previously reported from Mozambique, were identified in blood films, namely Haemogregarina fitzsimonsi Dias, 1953 and Haemogregarina parvula Dias, 1953. Additional stages of development (trophozoites and probable meronts, merozoites and immature gamonts) in blood preparations from South Africa warranted the redescription of H. fitzsimonsi. A variety of hosts and broad host distribution range were observed for this haemogregarine, with all five species of tortoises parasitized, wild and captive, from all four provinces, in all seasons. In contrast, only two individuals of K. b. belliana and one S. pardalis, all three captive in Kwazulu-Natal, contained H. parvula with encapsulated stages resembling those of Hemolivia mauritanica (Sergent et Sergent, 1904). For H. fitzsimonsi, parasite prevalences, but not parasitaemias, were significantly higher in captive than wild S. pardalis; captive female S. pardalis also showed a significantly greater prevalence of infection than males, but younger, lighter hosts were not significantly more heavily parasitized than older, heavier individuals. The ticks, Amblyomma marmoreum Koch, 1844 and A. sylvaticum (De Geer, 1778), found attached to some tortoises, may prove to be definitive hosts for the two species of haemogregarines observed.

Gnathia trimaculata n. sp. (Crustacea: Isopoda: Gnathiidae), an ectoparasite found parasitising requiem sharks from off Lizard Island, Great Barrier Reef, Australia.

Gnathia trimaculata n. sp. is described from one black tip reef shark Carcharinus melanopterus Quoy & Gaimard and four grey reef sharks C. amblyrhynchos Bleeker collected off Lizard Island, Great Barrier Reef, Australia. Third-stage juveniles (praniza 3) were maintained in fresh seawater until they moulted into adults. Male adults emerged seven days post-removal (d.p.r) of pranizae from host fishes, whereas the female pranizae completed their moult into adult females 24 d.p.r. Distinctive features include the relatively large size of all stages and the unique mediofrontal process of the male, which is divided into two lobes forming a key-hole shape between them. The female frontal border is characterised by paired simple, pappose setae on the sides of the mid-dorsal area, as well as four long, pappose setae on the mid-dorsal region. The pranizae have eight teeth on each mandible. Live pranizae have stripes and three pairs of distinctive black spots within yellow circles on the sides of the pereonites and this pigmentation pattern persists in the adults. This represents the second description of a gnathiid parasitising elasmobranchs off Australia.

Disruption of ERK signalling in Biomphalaria glabrata defence cells by Schistosoma mansoni: implications for parasite survival in the snail host.

Biomphalaria glabrata is an intermediate snail host for the human blood fluke Schistosoma mansoni. To survive in B. glabrata, S. mansoni must suppress the snail's haemocyte-mediated defence response; the molecular mechanisms by which this is achieved remain largely unknown. We report here that S. mansoni excretory-secretory products (ESPs) attenuate phosphorylation of extracellular signal-regulated kinase (ERK) in haemocytes from a B. glabrata strain susceptible to S. mansoni. Whole S. mansoni sporocysts also impair ERK signalling in these cells. In striking contrast, ERK signalling in haemocytes from a B. glabrata strain refractory to schistosome infection is unaffected by ESPs or sporocysts. Effects of ESPs on ERK are similar in the presence or absence of snail plasma, thus ESPs seem to affect haemocytes directly. These findings reveal novel schistosome interference mechanisms; as ERK regulates various haemocyte defence reactions, we propose that disruption of ERK signalling in haemocytes facilitates S. mansoni survival within susceptible B. glabrata.

A new gnathiid (Crustacea: Isopoda) parasitizing two species of requiem sharks from Lizard Island, Great Barrier Reef, Australia.

Third-stage juveniles (praniza 3) of Gnathia grandilaris n. sp. were collected from the gill filaments and septa of 5 requiem sharks, including a white tip reef shark, Triaenodon obesus, and 4 grey reef sharks, Carcharhinus amblyrhynchos, at Lizard Island, Great Barrier Reef, Australia, in March 2002. Some juvenile gnathiids were then maintained in fresh sea water until they molted to adults. Adult males appeared 19 days following detachment of juveniles from host fishes, but no juveniles molted successfully into females. The current description is based, therefore, on bright field and scanning electron microscopy observations of adult males and third-stage juveniles. Unique features of the male include the triangular-shaped inferior medio-frontal process, 2 areolae on the dorsal surface of the pylopod, and a slender pleotelson (twice as long as wide) with lateral concavities. The third-stage juvenile has distinctive white pigmentation on the black pereon when alive, while the mandible has 9 triangular backwardly directed teeth. This species has the largest male and third-stage juvenile of any Gnathia spp. from Australia and of any gnathiid isopods associated with elasmobranchs.

Potential environmental and host gender influences on prevalence of Haemogregarina platessae (Adeleorina:Haemogregarinidae) and suspected Haemohormidium terraenovae (incertae sedis) in Brazilian flounder from the Patos Lagoon Estuary, southern Brazil.

Flounder, Paralichthys orbignyanus (Valenciennes), were captured in polluted and non-polluted sites within the Patos Lagoon Estuary, southern Brazil, over four seasons. Blood films showed a high prevalence of infection with a haemogregarine, or mixed parasitaemias of this and an organism resembling Haemohormidium terraenovae So, 1972. Haemogregarine gamont stages conformed to existing descriptions of Desseria platessae (Lebailly, 1904) Siddall, 1995 from flatfishes, but intraerythrocytic division of meronts was observed, leading to the recommendation for nomenclatural correction, placing the haemogregarine in the genus Haemogregarina (sensu lato) Danilewsky, 1885. Statistical analyses suggested that although sample sizes were small, infections with meront stages, immature and mature gamonts were all influenced by site, and possibly therefore, by pollution. Season also appeared to determine likelihood of infection with meronts and immature gamonts, but not mature gamonts, while adult fish gender apparently affected infection with immature and mature gamonts, but not meronts. The H. terraenovae-like organism exhibited unusual extracellular forms and did not match closely with the type description of H. terraenovae; precise identification was therefore difficult. Data analyses suggested that parasitism by this organism was influenced by site and fish gender, since females and males from non-polluted water were infected, but only females from the polluted site. Season was also important and significantly more adult fish of both sexes were infected with this parasite in the Brazilian summer and autumn, compared with winter and spring. Finally, these appeared to be the first observations of Haemogregarina platessae, and possibly H. terraenovae, from the southern hemisphere.

Integrin engagement modulates the phosphorylation of focal adhesion kinase, phagocytosis, and cell spreading in molluscan defence cells.

Integrins play a key role in cellular immune responses in a variety of organisms; however, knowledge of integrins and their effects on cell signalling and functional responses in molluscan defence reactions is poor. Using integrin-mediated cell adhesion kits, alphaVbeta3 and beta1 integrin-like subunits were identified on the surface of Lymnaea stagnalis haemocytes. Haemocyte binding via these integrins was found to be dependent on Ca2+/Mg2+. Western blotting with an anti-phospho (anti-active) focal adhesion kinase (FAK) antibody revealed a 120-125 kDa FAK-like protein in these cells; this protein was transiently phosphorylated upon haemocyte adhesion over 90 min, with maximal phosphorylation occurring after 30 min binding. Also, integrin engagement with the tetrapeptide Arg-Gly-Asp-Ser (RGDS) resulted in a rapid increase in phosphorylation of the FAK-like protein; however, RGDS did not affect the phosphorylation of extracellular signal-regulated kinase. Treatment of haemocytes with RGDS (2 mM) inhibited phagocytosis of E. coli bioparticles by 88%. Moreover, at this concentration, RGDS reduced cell spreading by 61%; stress fiber formation was also impaired. Taken together, these results demonstrate a role for integrins in L. stagnalis haemocyte adhesion and defence reactions and, for the first time, link integrin engagement to FAK activation in molluscs.

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.

A new fish haemogregarine from South Africa and its suspected dual transmission with trypanosomes by a marine leech.

Twenty two percent (22/98) of intertidal fishes of 10 species captured in South Africa at Koppie Alleen, De Hoop Nature Reserve (south coast) and Mouille Point, Cape Town (west coast), harboured single or combined infections of haemogregarines, trypanosomes and an intraerythrocytic parasite resembling a Haemohormidium sp. The haemogregarines included the known species Haemogregarina (sensu lato) bigemina (Laveran et Mesnil, 1901) Siddall, 1995 and Haemogregarina (sensu lato) koppiensis Smit et Davies, 2001, while Haemogregarina (sensu lato) curvata sp. n. was observed in Clinus cottoides Valenciennes and Parablennius cornutus (L.) at Koppie Alleen. This last haemogregarine is characterised particularly by its distinctly curved gamonts. Also at Koppie Alleen, squash and histological preparations of 9/10 leeches, Zeylanicobdella arugamensis De Silva, 1963, taken from infected C. cottoides and P. cornutus contained developmental stages of H. curvata and/or trypanosomes, but these were absent from haematophagous gnathiid isopods (Gnathia africana Barnard, 1914) taken from infected fishes. It is suspected that Z. arugamensis transmits the haemogregarine and trypanosomes simultaneously between fishes, a double event unreported previously from the marine environment.

Activation of extracellular-signal regulated kinase is required for phagocytosis by Lymnaea stagnalis haemocytes.

Haemocytes are the primary defence cells of molluscs. In the present study, extracellular-signal regulated kinase (ERK) 1/2-like proteins were identified within Lymnaea stagnalis haemocytes, with apparent molecular weights of 44 and 43 kDa, respectively. Mitogen-activated protein kinase (MAPK) activity assays have confirmed that the L. stagnalis ERK possesses kinase activity towards Elk-1. Challenge of haemocytes with bacterial lipopolysaccharide (LPS) resulted in a transient activation of ERK, and immunocytochemistry revealed that phospho-ERK was present in both the perinuclear region and the nucleus following challenge. MAPK/ERK kinase (MEK) inhibitors blocked ERK activation confirming that MEK lies upstream of ERK in haemocytes. Moreover, phagocytosis assays, using various inhibitors, showed that ERK activity was vital for efficient phagocytosis and that ERK may be activated by both Ras-dependent and Ras-independent mechanisms. Overall, this study has furthered knowledge of ERK signalling in molluscan immunity and has shown that the ERK pathway regulates the phagocytic activity of molluscan haemocytes.

Reproductive and feeding ecology of parasitic gnathiid isopods of epaulette sharks (Hemiscyllium ocellatum) with consideration of their role in the transmission of a haemogregarine.

Epaulette sharks Hemiscyllium ocellatum were surveyed on Heron Island, Great Barrier Reef, Australia for gnathiid isopods and protozoan (haemogregarine) parasites to determine the prevalence and intensity of infection and to investigate the potential role of gnathiids as vectors of these haemogregarines, the first such study carried out on elasmobranchs. Juvenile gnathiids were collected and quantified using a novel non-invasive and chemical-free technique and gnathiid squashes were examined for haemogregarine developmental stages. The feeding and reproductive ecology of the Gnathia spp. was investigated to better understand the relationship between gnathiids and haemogregarines. Gnathiids were found on all sharks and intensities ranged between two and 66. Only third-stage gnathiid juveniles were found, which fell into two size groups (A and B). These juveniles remained attached to H. ocellatum for up to 17 days, the longest period of attachment yet recorded for gnathiids. Group A female gnathiids produced broods of 45-187 (median =120) first stage juveniles from between 54 and 82 days (median=63 days) after detachment. First stage juveniles survived for an average of 15.8+/-0.1 (SEM) days without feeding. The prevalence (6.7%) and parasitaemia (usually <0.1% infected erythrocytes) of infections of the haemogregarine Haemogregarina hemiscyllii were relatively low and most stages were immature gamonts. Two undescribed Gnathia spp. were identified by examining adult male gnathiids that metamorphosed from juveniles from each of the two size groups. Our hypothesis that Gnathia spp. transmit H. hemiscyllii is neither supported or refuted, as although intact H. hemiscyllii gamonts were detected in squashes of gnathiids that had engorged on haemogregarine-positive H. ocellatum 24-57 days previously, no further developmental stages were detected.

Carbohydrates that mimic schistosome surface coat components affect ERK and PKC signalling in Lymnaea stagnalis haemocytes.

Molluscs are intermediate hosts for helminth parasites such as Schistosoma spp. that possess an immunogenic surface coat of high carbohydrate content, with fucose as the predominant saccharide. More than a decade ago, it was postulated that such components could block receptors on snail haemocytes thus preventing recognition of intra-molluscan schistosome stages. Although more recent studies have shown that carbohydrates can suppress processes such as phagocytosis by haemocytes, interference of the haemocyte cell signalling pathways that regulate immunity by saccharides has not yet been investigated. We have recently reported the presence of extracellular-signal regulated kinase and protein kinase C in Lymnaea stagnalis haemocytes. Here we show that extracellular-signal regulated kinase and protein kinase C activities are down-regulated when haemocytes are exposed to albumin-linked fucose and galactose in the absence of haemolymph. Moreover, we demonstrate that phagocytosis is reduced under these conditions. Interestingly, in the presence of haemolymph, only protein kinase C activity is down-regulated and only galactose suppresses phagocytosis, implying a role for serum factors in the preservation of haemocyte function following exposure. We therefore propose that the establishment of a compatible relationship between a schistosome and its snail host is at least in part due to down-regulation of cell signalling events in haemocytes.

Two genotypic groups of morphologically similar fish trypanosomes from the Okavango Delta, Botswana.

Blood smears and blood lysate samples from freshwater fishes captured in the Okavango Delta, Botswana, were examined to determine whether their trypanosomes were all Trypanosoma mukasai, a species of supposed broad host specificity and widespread existence across Africa. Trypanosomes and/or babesiosomes occurred in 20/32 blood smears, and morphometric analysis of trypanosomes from 13/32 smears showed features suggestive of T. mukasai, including nuclear indices consistently >1. In 16/32 blood lysate samples from which DNA was extracted, trypanosome DNA was detected in 12/16 by PCR (polymerase chain reaction), using trypanosome-specific ssu rRNA gene primers. Two samples positive for trypanosomes in blood smears yielded no amplifiable trypanosome DNA, but 4 samples with no detectable infection in blood smears were positive for trypanosome DNA, suggesting an overall trypanosome prevalence rate of 17/32 (53%) among fishes and demonstrating the value of PCR in trypanosome recognition. Cloning and sequencing of the 12 amplified fragments revealed 2 genotypic groups among these fish trypanosomes. Group 1 trypanosomes were from cichlids and 3 families of catfishes, Group 2 from 2 types of catfishes. Sequence comparison showed that the consensus Group 1 sequence was most similar to that of Trypanosoma cobitis, representing European fish trypanosomes of the carassii type, while the consensus Group 2 sequence showed similarity with a trypanosome sequence from another African catfish, Clarias angolensis. It was concluded that the identification of T. mukasai remains a problem, but at least 2 genotypic groups of trypanosomes occur in Okavango Delta fishes, and catfishes in this region appear to contain both types.

Intraerythrocytic merogony in Haemogregarina koppiensis (Apicomplexa: Adeleorina: Haemogregarinidae).

During October 2003, a specimen of Amblyrhynchotes honckenii (Bloch, 1795) was captured at low tide, with a hand net, in a rock pool at Koppie Alleen, De Hoop Nature Reserve, South Africa. This fish was heavily parasitized by unidentified gnathiid praniza larvae, caligid copepods identified as Caligus tetrodontis Barnard, 1948, cymothoid isopods identified as Cinusa tetrodontis (Schioedte et Meinert, 1884), and the blood protozoan Haemogregarina koppiensis Smit et Davies, 2001. Giemsa-stained blood smears from this fish revealed new and unusual stages of merogony for H. koppiensis that included small, rounded, likely intraerythrocytic merozoites arranged in circles of eight around the host nucleus. Host cells appeared ghost-like and enlarged compared with normal erythrocytes. Identical merozoites, usually in clusters of up to 16, were also observed free of host cells. The pattern of merogony seen in H. koppiensis is unusual for a fish haemogregarine.

Taxonomic re-evaluation of the South African fish haemogregarine, Desseria fragilis.

Development stages of a haemogregarine were found in Giemsa-stained heart blood smears of 3 of the 4 horned blennies (Parablennius cornmutus) captured at De Hoop Nature Reserve, South Africa. Gamonts of this haemogregarine conformed to an existing description of Desseria (Haemogregarina) fragilis from P. cornmutus, but intraerythrocytic trophozoites, as well as meronts undergoing division, were reported for the first time for this species. A detailed redescription of D. fragilis allowed its taxonomic re-evaluation. Intraerythrocytic division excluded D. fragilis from Desseria, whereas morphometric similarities and identical patterns of development lead to the conclusion that D. fragilis is the cosmopolitan haemogregarine, Haemogregarina bigemina. Nomenclatural correction synonymizing the 2 species is recommended.