Experimental Parasite Infection Causes Genome-Wide Changes in DNA Methylation.

Parasites are arguably among the strongest drivers of natural selection, constraining hosts to evolve resistance and tolerance mechanisms. Although, the genetic basis of adaptation to parasite infection has been widely studied, little is known about how epigenetic changes contribute to parasite resistance and eventually, adaptation. Here, we investigated the role of host DNA methylation modifications to respond to parasite infections. In a controlled infection experiment, we used the three-spined stickleback fish, a model species for host-parasite studies, and their nematode parasite Camallanus lacustris. We showed that the levels of DNA methylation are higher in infected fish. Results furthermore suggest correlations between DNA methylation and shifts in key fitness and immune traits between infected and control fish, including respiratory burst and functional trans-generational traits such as the concentration of motile sperm. We revealed that genes associated with metabolic, developmental, and regulatory processes (cell death and apoptosis) were differentially methylated between infected and control fish. Interestingly, genes such as the neuropeptide FF receptor 2 and the integrin alpha 1 as well as molecular pathways including the Th1 and Th2 cell differentiation were hypermethylated in infected fish, suggesting parasite-mediated repression mechanisms of immune responses. Altogether, we demonstrate that parasite infection contributes to genome-wide DNA methylation modifications. Our study brings novel insights into the evolution of vertebrate immunity and suggests that epigenetic mechanisms are complementary to genetic responses against parasite-mediated selection.

New species and new records of camallanid nematodes (Nematoda, Camallanidae) from marine fishes and sea snakes in New Caledonia.

Recent examinations of camallanid nematodes (Camallanidae) from marine fishes off New Caledonia, collected in the years 2003-2011, revealed the presence of the following five new species of Procamallanus Baylis, 1923, all belonging to the subgenus Spirocamallanus Olsen, 1952: Procamallanus (Spirocamallanus) dispar n. sp. from the common ponyfish Leiognathus equulus (type host) and the striped ponyfish Aurigequula fasciata (both Leiognathidae, Perciformes); Procamallanus (Spirocamallanus) bothi n. sp. from the leopard flounder Bothus pantherinus (Bothidae, Pleuronectiformes); Procamallanus (Spirocamallanus) hexophtalmatis n. sp. from the speckled sandperch Parapercis hexophtalma (Pinguipedidae, Perciformes); Procamallanus (Spirocamallanus) synodi n. sp. from the sand lizardfish Synodus dermatogenys (Synodontidae, Aulopiformes); and Procamallanus (Spirocamallanus) thalassomatis n. sp. from the yellow-brown wrasse Thalassoma lutescens (Labridae, Perciformes). These are described based on light and scanning electron microscopical (SEM) studies. An additional three congeneric nematodes unidentifiable to species are reported from perciform fishes and a shark: Procamallanus (Spirocamallanus) sp. 3 of Moravec et al., 2006, Procamallanus (Spirocamallanus) sp. 1, and Procamallanus (Spirocamallanus) sp. 2. Ten fish species are recorded as new hosts for Camallanus carangis Olsen, 1954. Two camallanids, Procamallanus (Spirocamallanus) sp. 3 (subgravid female) and Camallanus carangis (fourth-stage larva) were also found in the digestive tract of the New Caledonian sea krait Laticauda saintgironsi, serving apparently as postcyclic and paratenic hosts, respectively, for these fish nematodes.

TITLE: Nouvelles espèces et nouveaux signalements de nématodes camallanidés (Nematoda, Camallanidae) provenant de poissons marins et de serpents de mer en Nouvelle-Calédonie. ABSTRACT: L'étude récente de nématodes camallanidés (Camallanidae) de poissons marins capturés au large de la Nouvelle-Calédonie, recueillis de 2003 à 2011, a révélé la présence des cinq nouvelles espèces suivantes de Procamallanus Baylis, 1923, toutes appartenant au sous-genre Spirocamallanus Olsen, 1952 : Procamallanus (Spirocamallanus) dispar n. sp. de Leiognathus equulus (hôte-type) et de Aurigequula fasciata (tous deux Leiognathidae, Perciformes) ; Procamallanus (Spirocamallanus) bothi n. sp. de Bothus pantherinus (Bothidae, Pleuronectiformes) ; Procamallanus (Spirocamallanus) hexophtalmatis n. sp. de Parapercis hexophtalma (Pinguipedidae, Perciformes) ; Procamallanus (Spirocamallanus) synodi n. sp. de Synodus dermatogenys (Synodontidae, Aulopiformes) ; et Procamallanus (Spirocamallanus) thalassomatis n. sp. de Thalassoma lutescens (Labridae, Perciformes). Ces espèces sont décrites sur la base d'études réalisées au microscope électronique à balayage et au microscope photonique. Trois autres nématodes congénères non identifiables au niveau spécifique sont signalés chez des poissons perciformes et un requin : Procamallanus (Spirocamallanus) sp. 3 de Moravec et al., 2006, Procamallanus (Spirocamallanus) sp. 1 et Procamallanus (Spirocamallanus) sp. 2. Dix espèces de poissons sont rapportées comme nouveaux hôtes pour Camallanus carangis Olsen, 1954. Deux camallanidés, Procamallanus (Spirocamallanus) sp. 3 (femelles subgravides) et Camallanus carangis (larve du quatrième stade) ont également été trouvés dans le tube digestif du serpent marin de Nouvelle-Calédonie, Laticauda saintgironsi, qui semble servir respectivement d'hôte post-cyclique et d'hôte paraténique pour ces nématodes de poissons.

New information on morphology and molecular data of camallanid nematodes parasitising Xenopus laevis (Anura: Pipidae) in South Africa.

Three species of nematodes from the Camallanidae that are known to infect Xenopus laevis Daudin (Anura: Pipidae) were collected from several localities across South Africa. New data on morphology, partial 28S and cox1 genes, infection levels and distribution are presented herein. The most common species, Batrachocamallanus slomei Southwell et Kirshner, 1937, from the stomach and less often oesophagus, was found in eight localities. Camallanus kaapstaadi Southwell et Kirshner, 1937, also from the oesophagus, was found in two localities and C. xenopodis Jackson et Tinsley, 1995, from the intestine, at a single locality. New localities for both C. kaapstaadi and C. xenopodis provide a geographical range extension. Males of C. xenopodis are described for the first time herein. The existence of a left spicule in the males of both the species of Camallanus Railliet and Henry, 1915 is confirmed and measurements are provided. Although C. xenopodis is distinguished from C. mazabukae Kung, 1948 in the present study, we suggest greater sampling effort in other African amphibians to confirm the species status of the latter taxon. Finally, the new molecular data showed distant relationships between collected species of Camallanus and species parasitising fish and freshwater turtles.

Inter- and intraspecific conflicts between parasites over host manipulation.

Host manipulation is a common strategy by which parasites alter the behaviour of their host to enhance their own fitness. In nature, hosts are usually infected by multiple parasites. This can result in a conflict over host manipulation. Studies of such a conflict in experimentally infected hosts are rare. The cestode Schistocephalus solidus (S) and the nematode Camallanus lacustris (C) use copepods as their first intermediate host. They need to grow for some time inside this host before they are infective and ready to be trophically transmitted to their subsequent fish host. Accordingly, not yet infective parasites manipulate to suppress predation. Infective ones manipulate to enhance predation. We experimentally infected laboratory-bred copepods in a manner that resulted in copepods harbouring (i) an infective C plus a not yet infective C or S, or (ii) an infective S plus a not yet infective C. An infective C completely sabotaged host manipulation by any not yet infective parasite. An infective S partially reduced host manipulation by a not yet infective C. We hence show experimentally that a parasite can reduce or even sabotage host manipulation exerted by a parasite from a different species.

A new species of camallanidae from Ageneiosus ucayalensis (Pisces: Siluriformes) from Pará State, Brazil.

Procamallanus (Spirocamallanus) belenensis n. sp. is described using light and scanning electron microcopy, based on specimens taken from Ageneiosus ucayalensis, Castelnau, 1855 (Pisces: Siluriforme), a freshwater fish commonly called mandubé, in Pard State, Brazil; Procamallanus (Spirocamallanus) rarus is recorded from the same host. To our knowledge there are no previous reports of nematodes from this fish species. Procamallanus (S.) belenensis is characterized by the presence of 8-13 cuticular spiral thickenings coating 2/3 of posterior region of the buccal capsule of males and females (the anterior third is smooth); small sub-equal spicules; 3 and 4-5 pairs of pre- and post-cloacal papillae, respectively; the proportion of muscular and glandular esophagus is 1:1.5-2; males present 2 spike-like projections at the tail tip, which are absent in females by light microscopy. The most closely related species are Procamallanus (Spirocamallanus) inopinatus and Procamallanus (Spirocamallanus) pintoi. The new species differs from the most closely related species by the presence of 8-10 cuticular spiral thickenings in the buccal capsule of males and 9-13 in the females, 8 pairs of male clocal papillae (3 pre- and 5 post-cloacal, respectively), and only males bearing 2 minute, spike-like projections at the posterior end.

Genetic variation of fish parasite populations in historically connected habitats: undetected habitat fragmentation effect on populations of the nematode Procamallanus fulvidraconis in the catfish Pelteobagrus fulvidraco.

Habitat fragmentation may have some significant effects on population genetic structure because geographic distance and physical barriers may impede gene flow between populations. In this study, we investigated whether recent habitat fragmentation affected genetic structure and diversity of populations of the nematode Procamallanus fulvidraconis in the yellowhead catfish, Pelteobagrus fulvidraco. The nematode was collected from 12 localities in 7 floodplain lakes of the Yangtze River. Using 11 intersimple sequence repeat markers, analysis of molecular variance showed that genetic diversity occurred mainly within populations (70.26%). Expected heterozygosity (He) of P. fulvidraconis was barely different between connected (0.2105) and unconnected lakes (0.2083). Population subdivision (Fst) between connected lakes (0.2177) was higher than in unconnected lakes (0.1676). However, the connected and unconnected lakes did not cluster into 2 clades. A Mantel test revealed significant positive correlation between genetic and geographic distances (R = 0.5335, P < 0.01). These results suggest that habitat fragmentation did not cause genetic differentiation among populations or a reduction of diversity in isolated populations of P. fulvidraconis. At least 2 factors may increase the dispersal range of the nematode, i.e., flash flooding in summer and other species of fish that may serve as the definitive hosts. Moreover, lake fragmentation is probably a recent process; population size of the nematode in these lakes is large enough to maintain population structure.

Molecular characteristics of Camallanus Spp. (Spirurida: Camallanidae) in fishes from China based on its rDNA sequences.

In the paper, we explored the intra- and interspecific evolutionary variation among species of Camallanus collected from different fish species in various regions of China. We determined the internal transcribed spacers of ribosomal DNA (ITS rDNA) sequences of these nematodes. The divergence (uncorrected p-distance) of ITS1, ITS2, and ITS rDNA data sets confirmed 2 valid species of Camallanus in China, i.e., C. cotti and C. hypophthalmichthys. The 2 species were distinguished not only by their different morphologies and host ranges but also by a tetranucleotide microsatellite (TTGC)n present in the ITS1 region of C. cotti. Phylogenetic analyses of the nematodes disclosed 2 main clades, corresponding to different individuals of C. cotti and C. hypophthalmichthys from different fish species in various geographical locations, although the interior nodes of each clade received poor support.

Camallanus cotti Fujita, 1927 (Nematoda, Camallanoidea) in ornamental aquarium fishes: pathology and morphology.

The pathology induced by the nematode Camallanus cotti in the aquarium fishes Beta splendens (beta fish) and Poecilia reticulata (guppy) consisted of gross and microscopic lesions, the former characterized by abdominal swelling with reddish parasites protruding from the anus in both fish hosts and the latter, similar in the beta fishes and guppies, by hemorrhage, congestion, edema, a few glandular elements, and extensive erosion areas in the rectum mucosa, with a marked thickening of the wall and absence of inflammatory infiltrate. Lesions were associated with the presence of several worms attached to the wall or free in the rectal lumen. This is the second reference of the parasite in Brazil and the first report of pathological findings related to this nematode species that is also briefly redescribed and illustrated for the first time on the basis of Brazilian samples.

Procamallanus (Spirocamallanus) anguillae sp. n. (Camallanidae) and some other nematodes from the Indonesian shortfin eel Anguilla bicolor in Thailand.

A new species of parasitic nematode, Procamallanus (Spirocamallanus) anguillae sp. n. (family Camallanidae), is described based on specimens recovered from the intestine of the Indonesian eel Anguilla bicolor McClelland (type host) from southern Thailand (type locality Phuket Island). It is characterized mainly by the presence of 10-13 spiral ridges in the buccal capsule, length of spicules (366-372 microm and 198-216 microm), presence of a gubernaculum, arrangement of caudal papillae, and by the broad female tail with a digit-like projection bearing two cuticular spikes. In addition, two species of larval nematodes, Physalopteridae gen. sp. and Anisakis cf. simplex (Rudolphi 1809), were recorded from A. bicolor. All species are briefly described and illustrated.

Ultrastructure of Procamallanus (Spirocamallanus) halitrophus (Nematoda: Camallanidae) parasite of flounder.

The ultrastructure of the camallanid nematode Procamallanus (Spirocamallanus) halitrophus, a parasite of flounder, is described for the first time by the use of transmission electron microscopy. The body wall is composed of an outer cuticle, a hypodermis, and a muscular layer. The cuticle comprises the epicuticle, the cortical, median, fibrous, and basal layers. The cortical layer is subdivided into an outer zone and an inner zone; the median layer is subdivided into an outer layer, rich in electrondense fibrils, and an inner layer, which does not contain these fibrils; the fibrous layer is subdivided into three regions delimited by electrondense lines; the basal layer presents electrondense sustaining structures. Underlying the basal layer is the hypodermis where many organelles are observed. The musculature is striated, and each muscle cell consists of individualized contractile and non-contractile regions. Inclusion bodies are present in the muscle fibers, hypodermis, hypodermal chord, and in the intestine.

Redescription of Camallanus hypophthalmichthys Dogel and Akhmerov, 1959 (Nematoda: Camallanidae) and its first record from fishes in China.

The nematode Camallanus hypophthalmichthys Dogel and Akhmerov, 1959 is redescribed from specimens collected from the intestine of the bighead carp Aristichthys nobilis, from Liangzihu Lake (Yangtze River basin), Hubei Province, central China. The light and scanning electron microscopical examination made it possible to study in detail the morphology of this so far little-known species and to confirm its validity. The main specific features of C. hypophthalmichthys distinguishing it from the most similar Camallanus spp. is the presence of 3 small caudal processes on the male tail tip, 13-16 longitudinal ridges on the inner surface of the valve of the buccal capsule, and the arrangement of preanal and postanal genital papillae in the male. This finding represents a new host record, the first record of this parasite in the Yangtze River basin, and the first documented record of C. hypophthalmichthys from China. Camallanus hypophthalmichthys is considered a specific intestinal parasite of fishes of the cyprinid Hypophthalmichthyinae.

Seasonal and yearly population dynamics of two exotic helminths, Camallanus coti (Nematoda) and Bothriocephalus acheilognathi (Cestoda), parasitizing exotic fishes in Waianu Stream, O'ahu, Hawaii.

Exotic poeciliid fishes introduced into Hawaiian freshwaters are responsible for the introduction of several exotic parasites, of which the most important are Camallanus cotti and Bothriocephalus acheilognathi in terms of potential disease threat to native stream fishes. This roundworm and tapeworm are the most prevalent and abundant freshwater fish helminths in Hawaiian streams. This study examined the seasonal and yearly population structure of C. cotti and B. acheilognathi to determine if the tropical Hawaiian environment characterized by low climatic variability permits continuous opportunities for parasite transmission regardless of time of year. Camallanus cotti displayed seasonal differences in prevalence and mean abundance, whereas B. acheilognathi did not. Camallanus cotti prevalence and mean abundance were higher in the Hawaiian summer (47.7%, 0.79) than in winter (25.8%, 0.36). A seasonal relationship of C. cotti levels is likely explained by extensive rains associated with the Hawaiian winter season, which may act to decrease parasite transmission by flushing infected poeciliid hosts, intermediate copepod hosts, and possibly free-living infective worm stages downstream. Bothriocephalus acheilognathi displayed low prevalence and mean abundance in both summer (4.0%, 0.06) and winter (6.5%, 0.07), and it may be difficult to detect seasonal changes due to these low levels. Camallanus cotti prevalence and mean abundance remained relatively constant from the summer of 1995 to the summer of 1999, indicating that levels of this roundworm are stable in Waianu Stream. Whereas B. acheilognathi prevalence and mean abundance were low during the summer of 1995 and the summer of 1997, a dramatic peak in prevalence and mean abundance was observed in the summer of 1998 (41.2%, 1.06), with levels decreasing sharply in the summer of 1999 (4.4%, 0.07). It appears that B. acheilognathi also is present in stable populations at low levels, even though levels rose sharply during a single year.

The development and morphogenesis of Camallanus cotti Fujita, 1927 (Nematoda: Camallanidae), with notes on its phylogeny and definitive host range.

The freshwater fish nematode Camallanus cotti Fujita, 1927 (Spirurida: Camallanidae) is naturally distributed in East, South and Southeast Asia. During the past three or four decades the species has been disseminated to Europe, North America, Australia and Hawaii, mainly due to the extensive ornamental fish trade. In the present study, its development and larval morphogenesis is described. Newborn first-stage larvae are ingested by copepods and within hours reach the haemocoel, where the worms moult twice. At 22 degrees C ambient temperature, fully-developed third-stage larvae appear on day 11 p.i. After transmission into the fish intestine two further moults occur before the adult stage is reached. At 23 degrees C water temperature, the final moult is initiated around day 33 p.i. in males and between 34 and 42 days p.i. in females. Morphologically the young and older adults are similar, but the buccal capsule of both young males and young females is not yet fully sclerotised. Based on the close similarity in the development and adult morphology of the buccal capsule in C. cotti and three congeners from Europe and North America, we suggest, in general agreement with earlier workers, that the Camallanus Railliet & Henry, 1915 originated in tropical Asia and subsequently radiated to species when adapting to new climatic and/or host-related conditions in the Old World and the New World. C. cotti may thus represent a lineage of speciation which did not apparently involve extensive changes in relation to their natural geographical distribution or definitive host range.

Selection pressure towards monoxeny in Camallanus cotti (Nematoda, Camallanidac) facing an intermediate host bottleneck situation.

This paper describes the ability of the Asian fish nematode Camallanus cotti to carry out both heteroxeny, i.e. an indirect life-cycle using copepods as intermediate host, and monoxeny, i.e. direct infection and development in the definitive fish host. C. cotti occurs naturally in various freshwater teleosts in Asia. During the past decades it has been disseminated into closed or semi-closed aquaculture systems and aquaria around the world, mainly due to the ornamental fish trade. Under such conditions the species may frequently face a bottleneck situation with regard to the availability of copepods. It is known that C. cotti may reproduce and persist in copepod-free aquaria for several months. In order to investigate whether C. cotti has selected towards monoxeny in water systems lacking copepods, in contrast to the opposite selection pressure when copepods are present, 2 separate infection trials were run. It was shown that the parasite can infect the fish host both indirectly via copepods, and directly. However, C. cotti has significantly higher fitness, expressed as survival to maturity, when transmitted indirectly compared to the direct transmission mode. We suggest that the ability of aquarium populations of C. cotti to carry out a direct life-cycle is favoured by selection in order to avoid extinction whenever copepods are absent. It still remains unknown, however, whether the parasite shows the same characteristics in the wild.

Camallanus carangis Olsen, 1954 (Nematoda: Camallanidae) reported from French Polynesia and Hawai'i with a redescription of the species.

The marine fish parasite Camallanus carangis Olsen, 1954 (Nematoda: Camallanidae) is redescribed from the honeycomb grouper Epinephelus merra (Serranidae) in the Tuamotu Islands of French Polynesia and the glasseye Heteropriacanthus cruentatus (Priacanthidae) in Hawai'i, both coral reef fishes. Camallanus carangis is reported for the first time from French Polynesia and Hawai'i. Camallanus marinus Schmidt and Kuntz, 1969 is designated a synonym of C. carangis and Camallanus paracarangis Velasquez, 1980 is regarded as a species inquirenda. Additionally, the genus Oncophora may need to be redefined to accommodate Camallanus aotea Slankis and Korotaeva, 1974, which bears more resemblance to Oncophora than Camallanus.

Pathobiology associated with the spiruroid nematodes Camallanus oxycephalus and Spinitectus carolini in the intestine of green sunfish, Lepomis cyanellus.

The histopathology induced by Camallanus oxycephalus and Spinitectus carolini in the intestine of green sunfish Lepomis cyanellus was examined. At what is assumed to be more recent attachment sites, penetration of C. oxycephalus was restricted to the mucosal layer, causing complete destruction of the columnar epithelium. At what appeared to be older attachment sites, the parasite penetrated deep into the intestinal wall, even to the circular muscle layer. At these sites of attachment, there were flask-shaped ulcers in the mucosal and submucosal layers. Surrounding the ulcers, there was granulomatous tissue, with extensive fibrosis at the surface; the fibrosis probably reduced the loss of body fluids and blood cells from the intestinal wall. The number of goblet cells in the posterior end of the intestine increased significantly, from 92.6 cells/mm in uninfected fish to 168.4 cells/mm in fish infected with more than 5 C. oxycephalus. Adult S. carolini also causes severe damage to the intestinal wall of green sunfish. Parasite penetration was not limited to the mucosal layer. Mature worms were observed invading both the muscular and serosal layers. Penetration by the parasite was usually associated with the local loss of columnar epithelium and infiltration by lymphocytes and granulocytic leukocytes. Fibrosis in the mucosal layer was commonly observed. There was goblet cell hyperplasia in the anterior portion of the intestine, with numbers of goblet cells increasing from 64.4 cells/mm in uninfected fish to 95.0 cells/mm in heavily infected fish.

The development of Procamallanus (Spirocamallanus) neocaballeroi (Nematoda: Camallanidae), a parasite of Astyanax fasciatus (Pisces) in Mexico.

The development of the nematode Procamallanus (Spirocamallanus) neocaballeroi (Caballero-Deloya, 1977), an intestinal parasite of the characid fish, Astyanax fasciatus (Cuvier) in Mexico, was studied in the experimental copepod intermediate host, Mesocyclops sp. After the copepod's ingestion of free first-stage larvae of the nematode, these penetrate into the haemocoel of the intermediate host; they moult twice (on the 3rd and 4-5th day p.i. at 21-22 degrees C) before they attain the third, infective stage. The third-stage larva already possesses the large buccal capsule subdivided into an anterior broad portion with eight spiral thickenings (as observed in lateral view) and a narrow posterior portion, and its tail tip bears three conical processes. The definitive host acquires infection by feeding on infected copepods; in the intestine of this fish, the nematode larvae undergo two more moults (on the 10th and 14-15th day p.i. at 25-32 degrees C) before attaining their maturity. The prepatent period is approximately two months.

Seasonal dynamics of Camallanus anabantis infections in the climbing perch, Anabas testudineus, from the freshwater swamps near Kalyani town, West Bengal, India.

The seasonal changes of the nematode Camallanus anabantis Pearse, 1933, in the climbing perch (Anabas testudineus) from the freshwater swamps near Kalyani town, West Bengal, India were studied during the period from February 1988 to August 1989. The nematode exhibited a one-year cycle. Larvated females occurred in the fishes from October to February at a water temperature of 12 degrees-29 degrees C. New infection of fishes occurred from February to May and occasionally in September. The fourth-stage larvae, the males, and the young females (without eggs), although irregularly, were found present throughout the year. Egg-laden females occurred in the fishes in August, October to February, and March.

Identification and characterization of the proteolytic enzymes in the developmental stages of the eel-pathogenic nematode Anguillicola crassus.

The proteolytic activities of homogenates prepared from the second larva (L2) and the third larva (L3) as well as the adult stage of the eel-pathogenic nematode Anguillicola crassus were examined using hemoglobin, azocoll, elastin-orcein, and keratin azure as substrates. Whole bodies of L2 larvae, the anterior third of the bodies of L3 larvae, and the anterior fifth of the bodies of adults were studied. Extracts of L2 contained a trypsin-like proteinase exhibiting a molecular weight of 38,000 Da on gelatin-substrate gel electrophoresis. The proteinase showed a pH optimum at 8 and activity against azocoll and keratin. An apparent molecular weight of 25,000 Da was determined for the trypsin-like proteinase of the L3. This enzyme possessed collagenolytic, keratinolytic and slight elastinolytic activity at an optimal pH of 8. Samples of adults contained an aspartyl proteinase with a molecular weight of 90,000 Da. When hemoglobin was used as the substrate, the enzyme displayed optimal activity at pH 5. It was concluded that the proteinases of the larval stages are penetration enzymes, whereas that of the adult stage is a digestive enzyme.