Sea lice (Copepoda: Caligidae) from South Africa, with descriptions of two new species of Caligus.

Thirteen species of sea lice (family Caligidae) are reported from a range of elasmobranch and actinopterygian fishes caught off South Africa or obtained from public aquaria in South Africa. Two new species of Caligus Müller, 1785 are described: C. linearis n. sp. from Pomatomus saltatrix (Linnaeus) and C. tumulus n. sp. from Chrysoblephus cristiceps (Valenciennes). A supplementary description is provided for both sexes of Caligus tetrodontis Barnard, 1948 taken from Amblyrhynchotes honckenii (Bloch) and previous records of this parasite from South African fishes are critically reviewed. It is concluded that Caligus material from Arothron hispidus Linnaeus was previously misidentified as C. tetrodontis and is in urgent need of re-examination. Morphological and molecular observations on Caligus furcisetifer Redkar, Rangnekar & Murti, 1949 indicate that this copepod is phenotypically and genetically identical to Lepeophtheirus natalensis Kensley & Grindley, 1973, and the latter becomes a junior subjective synonym of C. furcisetifer. We include new geographical distribution records for Caligus longipedis Bassett-Smith, 1898, C. rufimaculatus Wilson, 1905 and Lepeophtheirus spinifer Kirtisinghe, 1937, extending into South African waters, as well as both new distribution and host records for Alebion gracilis Wilson, 1905, Caligus dakari van Beneden, 1892 and Lepeophtheirus acutus Heegaard, 1943. The molecular analysis confirmed the monophyly of the genus Caligus. The South African species of Caligus did not cluster together, but the two included South African species of Lepeophtheirus were recovered as sister taxa.

Morphological and molecular characterization of an African freshwater fish trypanosome, including its development in a leech vector.

Trypanosomes are ubiquitous blood parasites of fishes and at least 16 species were originally described infecting African freshwater fishes. This number was later reduced to six and in the late 1990s it was proposed that most records of freshwater fish trypanosomes across Africa are Trypanosoma mukasai Hoare, 1932. Recently, results from a molecular analysis of fish trypanosomes from the Okavango Delta, Botswana, reported the presence of at least two genotypic groups and concluded that the identification of T. mukasai remains problematic. The aims of the present study were thus to elucidate the life cycle of a freshwater fish trypanosome from southern Africa and to do a morphological and molecular characterization of this parasite from both the fish host and leech vector. To locate trypanosome stages, leeches were removed from fishes captured in the Phongolo River, South Africa, and fish blood films and leech squashes were Giemsa-stained and screened. To determine whether trypanosome stages in fishes and leeches were of the same genotype, DNA was extracted and fragments of the 18S rDNA gene were amplified and sequenced. Trypanosomes were detected in the fish families Cichlidae, Clariidae, Mochokidae and Schilbeidae. Sequence data showed that the trypanosome from one of the leeches, identified as Batracobdelloides tricarinata (Blanchard, 1897), was highly similar to those obtained from the plain squeaker, Synodontis zambezensis, with 0.7% difference recorded between them. From morphological and molecular data presented here, it is clear that the trypanosomes from Phongolo are closely related to those of the Okavango and should be considered as a single diverse species with genetic differentiation between 0.4-2.9%, under the 3-5% differences expected to be seen between true distinct species within the rRNA. Developmental stages of the trypanosome found in the leech B. tricarinata supports its status as the vector and the molecular evidence shows the relationship between the trypanosome in the fish and leech, but also illustrates the exceptional genetic and morphological diversity of a single species of trypanosome between host species. The work presented here provides us with clear information to take further steps in resolving the taxonomy and systematics of African freshwater fish trypanosomes.

Molecular evidence for resurrection of Plesiochorus elongatus (Digenea: Gorgoderidae): An urinary bladder parasite of sea turtles.

Trematodes of the genus Plesiochorus were recovered from the urinary bladder of a stranded female adult loggerhead sea turtle, Caretta caretta, on a beach in Rio de Janeiro State, Brazil. Morphological analysis of the specimens revealed characteristics resembling the sub-species Plesiochorus cymbiformis elongatus rather than the recently synonymised Plesiochorus cymbiformis. Molecular phylogenetic analysis of the ITS2 region also showed that P. c. elongatus was distinct from P. cymbiformis and related taxa. Further analysis of the ITS2 revealed substantial differentiation between P. cymbiformis from the USA and Brazil and the newly sequenced P. c. elongatus from Brazil, while a previously unspecified Plesiochorus sp. from the USA closely related to the novel Brazilian P. c. elongatus was reconciled as a USA isolate of P. c. elongatus. Based on both the morphological and molecular data it is suggested that P. c. elongatus should be referred to as Plesiochorus elongatus and be considered as the second species in the genus.

Molecular insights into the identification and phylogenetics of the cosmopolitan marine fish blood parasite, Haemogregarina bigemina (Adeleorina: Haemogregarinidae).

Haemogregarina bigemina is one of the most prevalent haemogregarines of marine fishes and has long been considered an enigmatic and cosmopolitan species. However, to determine whether H. bigemina truly represents a single global species, or whether it should be partitioned into several species or subspecies, and to confirm its taxonomic status among the Haemogregarina, molecular analysis is required. Here, we provide the first molecular characterisation of H. bigemina from one of its type hosts, Lipophrys pholis, in the UK using 18S rDNA sequences. Phylogenetic and p-distance comparisons of the newly generated H. bigemina sequences with those published from other haemogregarine taxa and related apicomplexans suggest that H. bigemina falls outside of the "Haemogregarina" clade as well as the Adeleorina altogether, forming a separate apicomplexan marine clade, and appears to be in fact more closely related to other non-haemogregarine Apicomplexa genera. Further work including sequences of H. bigemina and H. bigemina-like parasites observed from fishes in other localities is now needed to investigate this further.

DNA barcoding of the medically important freshwater snail Physa acuta reveals multiple invasion events into Africa.

The medically important freshwater snail Physa acuta is highly invasive and has been reported in several freshwater environments across Africa. To identify species and provide initial insights into the origins of P. acuta into African freshwater environments standard molecular barcoding analyses, using the mitochondrial cytochrome c oxidase subunit I gene (COI), was performed on P. acuta isolates from Angola, Burundi and South Africa. Phylogenetic analyses of isolates from Africa could not be distinguished from P. acuta populations from other countries. Comparisons of COI sequences between isolates of P. acuta showed there to be no geographically specific clusters and the African isolates were distributed across four distinct unrelated clades suggesting several independent invasion events. Haplotype analyses indicated that there were a high number of haplotypes with low variation between them, which led to significant differences in AMOVA analyses between countries. This was further evidence of multiple invasion events suggesting multiple novel haplotypes being continually and independently introduced to each country. This approach not only provides initial insight into the invasion of Africa by P. acuta but a molecular method to monitor and manage the use of an agent of biological control.

Morphological and molecular characterization of a marine fish trypanosome from South Africa, including its development in a leech vector.

BACKGROUND: Trypanosomes are ubiquitous blood parasites of marine and freshwater fishes, typically transmitted by aquatic leeches. Phylogenetic studies have been dominated by examples derived from freshwater fishes, with few marine representatives. Furthermore, life cycle studies on marine fish trypanosomes have focused on those of the northern hemisphere. In this investigation, we have examined the life cycle and molecular taxonomy of a marine fish trypanosome from South Africa. METHODS: To locate trypanosome stages, leeches were removed from fishes captured on the west and south coasts of South Africa, and fish blood films and leech squashes were Giemsa-stained and screened; leeches were also examined histologically. To determine whether trypanosome stages in fishes and leeches were of the same genotype, DNA was extracted from Giemsa-stained fish blood films and leech squashes, and from fish whole blood. Fragments of the 18S rRNA gene were amplified by PCR using trypanosome-specific primers and sequenced. Resulting sequence data were compared with each other and with published trypanosome 18S rDNA sequences, and used for phylogenetic analysis. RESULTS: Trypanosomes were detected in blood films from fishes of the families Clinidae, Blenniidae and Gobiidae. The flagellates ranged in size and staining properties within the films and across fish hosts. In squashes and histological sections of adult and juvenile leeches, identified as Zeylanicobdella arugamensis, trypanosome developmental stages were predominantly slender epimastigotes. Sequence data showed that trypanosomes derived from fishes were identical, irrespective of whether they were small or large forms; sequences derived largely from leech epimastigotes were also identical to those obtained from fish trypanosomes. Fish and leech trypanosome sequences fell into a marine fish aquatic clade, and aligned most closely with two trypanosome sequences from marine fishes off Norway. CONCLUSIONS: Combined morphological and molecular methods indicate that the trypanosomes examined here represent a single pleomorphic species, rather than the three species described originally. This species is identified as Trypanosoma nudigobii Fantham, 1919 with the leech Z. arugamensis as its vector, and T. capigobii Fantham, 1919 and T. blenniclini Fantham, 1930 are regarded as junior synonyms of the species. Phylogenetic analysis establishes its affinity with marine fish trypanosomes off Norway.

Three-dimensional visualisation of developmental stages of an apicomplexan fish blood parasite in its invertebrate host.

BACKGROUND: Although widely used in medicine, the application of three-dimensional (3D) imaging to parasitology appears limited to date. In this study, developmental stages of a marine fish haemogregarine, Haemogregarina curvata (Apicomplexa: Adeleorina), were investigated in their leech vector, Zeylanicobdella arugamensis; this involved 3D visualisation of brightfield and confocal microscopy images of histological sections through infected leech salivary gland cells. FINDINGS: 3D assessment demonstrated the morphology of the haemogregarine stages, their spatial layout, and their relationship with enlarged host cells showing reduced cellular content. Haemogregarine meronts, located marginally within leech salivary gland cells, had small tail-like connections to the host cell limiting membrane; this parasite-host cell interface was not visible in two-dimensional (2D) light micrographs and no records of a similar connection in apicomplexan development have been traced. CONCLUSIONS: This is likely the first account of the use of 3D visualisation to study developmental stages of an apicomplexan parasite in its invertebrate vector. Elucidation of the extent of development of the haemogregarine within the leech salivary cells, together with the unusual connections between meronts and the host cell membrane, illustrates the future potential of 3D visualisation in parasite-vector biology.

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.

Haemogregarina bigemina (Protozoa: Apicomplexa: Adeleorina)--past, present and future.

This paper reviews past, current and likely future research on the fish haemogregarine, Haemogregarina bigemina Laveran et Mesnil, 1901. Recorded from 96 species of fishes, across 70 genera and 34 families, this broad distribution for H. bigemina is questioned. In its type hosts and other fishes, the parasite undergoes intraerythrocytic binary fission, finally forming mature paired gamonts. An intraleukocytic phase is also reported, but not from the type hosts. This paper asks whether stages from the white cell series are truly H. bigemina. A future aim should be to compare the molecular constitution of so-called H. bigemina from a number of locations to determine whether all represent the same species. The transmission of H. bigemina between fishes is also considered. Past studies show that young fish acquire the haemogregarine when close to metamorphosis, but vertical and faecal-oral transmission seem unlikely. Some fish haemogregarines are leech-transmitted, but where fish populations with H. bigemina have been studied, these annelids are largely absent. However, haematophagous larval gnathiid isopods occur on such fishes and may be readily eaten by them. Sequential squashes of gnathiids from fishes with H. bigemina have demonstrated development of the haemogregarine in these isopods. Examination of histological sections through gnathiids is now underway to determine the precise development sites of the haemogregarine, particularly whether merozoites finally invade the salivary glands. To assist in this procedure and to clarify the internal anatomy of gnathiids, 3D visualisation of stacked, serial histological sections is being undertaken. Biological transmission experiments should follow these processes.