Dataset Information

Functional Ultrastructure of the Excretory Gland Cell in Zoonotic Anisakids (Anisakidae, Nematoda).

ABSTRACT: Excretory and secretory products are crucial for parasite infectivity and host immunomodulation, but the functioning and ultrastructure of the excretory gland cell (EC) that produces these products are still scarcely understood and described. In light of growing reports on anisakiasis cases in Europe, we aimed to characterise the EC of larval Anisakis pegreffii and adult Pseudoterranova azarasi. In the latter, EC starts 0.85 mm from the head tip, measuring 1.936 × 0.564 mm. Larval EC shows a long nucleus with thorn-like extravaginations toward the cytoplasm, numerous electron-dense and -lucent secretory granules spanning from the perinuclear to subplasmalemmal space, an elevated number of free ribosomes, small, spherical mitochondria with few cristae and a laminated matrix, small and few Golgi apparatuses, and few endoplasmic reticula, with wide cisternae complexes. Ultrastructure suggests that anaerobic glycolysis is the main metabolic pathway, obtained through nutrient endocytosis across the pseudocoelomic surface of the EC plasmalemma and its endocytic canaliculi. Thorn-like extravaginations of EC karyotheca likely mediate specific processes (Ca2+ signaling, gene expression, transport, nuclear lipid metabolism) into the extremely wide EC cytosol, enabling focal delivery of a signal to specific sites in a short time. These functional annotations of parasitic EC should help to clarify anisakiasis pathogenesis.

SUBMITTER: Mladineo I

PROVIDER: S-EPMC6912704 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Functional Ultrastructure of the Excretory Gland Cell in Zoonotic Anisakids (Anisakidae, Nematoda).

Mladineo Ivona I Hrabar Jerko J Smodlaka Hrvoje H Palmer Lauren L Sakamaki Kristen K Keklikoglou Kleoniki K Katharios Pantelis P

Cells 20191117 11

Excretory and secretory products are crucial for parasite infectivity and host immunomodulation, but the functioning and ultrastructure of the excretory gland cell (EC) that produces these products are still scarcely understood and described. In light of growing reports on anisakiasis cases in Europe, we aimed to characterise the EC of larval Anisakispegreffii and adult Pseudoterranovaazarasi. In the latter, EC starts 0.85 mm from the head tip, measuring 1.936 × 0.564 ...[more]

PMID: 31744245

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Project description:BackgroundAnisakiasis is a foodborne disease caused by the third-stage larvae (L3) of two species belonging to the genus Anisakis: Anisakis pegreffii and Anisakis simplex sensu stricto. Both species have been the subject of different -omics studies undertaken in the past decade, but a reliable in vitro culture protocol that would enable a more versatile approach to functional studies has never been devised. In nature, A. pegreffii shows a polyxenous life-cycle. It reproduces in toothed whales (final host) and disseminates embryonated eggs via cetacean faeces in the water column. In the environment, a first- (L1) and second-stage larva (L2) develops inside the egg, and subsequently hatched L2 is ingested by a planktonic crustacean or small fish (intermediate host). In the crustacean pseudocoelom, the larva moults to the third stage (L3) and grows until the host is eaten by a fish or cephalopod (paratenic host). Infective L3 migrates into the visceral cavity of its paratenic host and remains in the state of paratenesis until a final host preys on the former. Once in the final host's gastric chambers, L3 attaches to mucosa, moults in the fourth stage (L4) and closes its life-cycle by becoming reproductively mature.MethodsTesting two commercially available media (RPMI 1640, Schneider's Drosophila) in combination with each of the six different heat-inactivated sera, namely foetal bovine, rabbit, chicken, donkey, porcine and human serum, we have obtained the first reliable, fast and simple in vitro cultivation protocol for A. pegreffii.ResultsSchneider's Drosophila insect media supplemented with 10% chicken serum allowed high reproducibility and survival of adult A. pegreffii. The maturity was reached already at the beginning of the third week in culture. From collected eggs, hatched L2 were maintained in culture for 2 weeks. The protocol also enabled the description of undocumented morphological and ultrastructural features of the parasite developmental stages.ConclusionsClosing of the A. pegreffii life-cycle from L3 to reproducing adults is an important step from many research perspectives (e.g., vaccine and drug-target research, transgenesis, pathogenesis), but further effort is necessary to optimise the efficient moulting of L2 to infective L3.

Project description:L3 larvae of anisakid nematodes are an important problem for the fisheries industry and pose a potential risk for human health by acting as infectious agents causing allergies and as potential vectors of pathogens and microrganisms. In spite of the close bacteria-nematode relationship very little is known of the anisakids microbiota. Fresh fish could be contaminated by bacteria vectored in the cuticle or in the intestine of anisakids when the L3 larvae migrate through the muscles. As a consequence, the bacterial inoculum will be spread, with potential effects on the quality of the fish, and possible clinical effects cannot be discarded. A total of 2,689,113 16S rRNA gene sequences from a total of 113 L3 individuals obtained from fish captured along the FAO 27 fishing area were studied. Bacteria were taxonomically characterized through 1803 representative operational taxonomic units (OTUs) sequences. Fourteen phyla, 31 classes, 52 orders, 129 families and 187 genera were unambiguously identified. We have found as part of microbiome an average of 123 OTUs per L3 individual. Diversity indices (Shannon and Simpson) indicate an extraordinary diversity of bacteria at an OTU level. There are clusters of anisakids individuals (samples) defined by the associated bacteria which, however, are not significantly related to fish hosts or anisakid taxa. This suggests that association or relationship among bacteria in anisakids, exists without the influence of fishes or nematodes. The lack of relationships with hosts of anisakids taxa has to be expressed by the association among bacterial OTUs or other taxonomical levels which range from OTUs to the phylum level. There are significant biological structural associations of microbiota in anisakid nematodes which manifest in clusters of bacteria ranging from phylum to genus level, which could also be an indicator of fish contamination or the geographic zone of fish capture. Actinobacteria, Aquificae, Firmicutes, and Proteobacteria are the phyla whose abundance value discriminate for defining such structures.

Project description:Background: Anisakiasis is a zoonotic disease caused by accidental ingestion of live Anisakis spp. third-stage larvae present in raw or undercooked seafood. Symptoms of this emerging infectious disease include mild-to-severe abdominal pain, nausea, and diarrhea. Some patients experience significant allergic reactions. Aims: In order to better understand the onset of anisakiasis, we aimed to: (i) histopathologically describe severe inflammatory/hemorrhagic infection site lesions in Sprague-Dawley rats experimentally infected with Anisakis pegreffii larvae; and (ii) qualitatively and quantitatively characterize the transcriptomes of affected tissues using RNA-Seq. Methodology: The experiment was performed on 35 male rats, sacrificed at 5 time points (6, 10, 18, 24, and 32 h post-infection). Gastric intubation was performed with 10 A. pegreffii larvae (N = 5 infected rats per time point) or 1.5 ml of saline (external control N = 2 rats). 16 pools, seven for muscle tissues and nine for stomach tissues, were created to obtain robust samples for estimation of gene expression changes depicting common signatures of affected versus unaffected tissues. Illumina NextSeq 500 was used for paired-end sequencing, while edgeR was used for count data and differential expression analyses. Results: In total, there were 1372 (855 up and 517 down) differentially expressed (DE) genes in the Anisakis-infected rat stomach tissues, and 1633 (1230 up and 403 down) DE genes in the muscle tissues. Elicited strong local proinflammatory reaction seems to favor the activation of the interleukin 17 signaling pathway and the development of the T helper 17-type response. The number of DE ribosomal genes in the Anisakis-infected stomach tissue suggests that A. pegreffii larvae might induce ribosomal stress in the early infection stage. However, the downstream pathways and post-infection responses require further study. Histopathology revealed severe inflammatory/hemorrhagic lesions caused by Anisakis infection in the rat stomach and muscle tissues in the first 32 h. The lesion sites showed infiltration by polymorphonuclear leukocytes (predominantly neutrophils and occasional eosinophils), and to a lesser extent, macrophages. Conclusion: Understanding the cellular and molecular mechanisms underlying host responses to Anisakis infection is important to elucidate many aspects of the onset of anisakiasis, a disease of growing public health concern.

Project description:The genus Pseudoterranova includes parasite species of cetaceans and pinnipeds. The third stage larva (L3) of seal-infecting species occur in second intermediate or paratenic fish hosts mainly in neritic waters. This study firstly describes a Pseudoterranova L3 from meso/bathypelagic fishes off Macaronesia. L3s were morphologically and genetically studied by light microscopy and sequencing of the mtDNA cox2 and entire ITS rDNA genes. Bayesian inferences were performed with sequences from the larvae and selected sequences from GenBank. The nematode L3s were molecularly identified as Pseudoterranova ceticola, a parasite of kogiid whales. Such larvae were collected from Bolinichthys indicus, Chauliodus danae, Eupharynx pelecanoides, Diaphus rafinesquii, D. mollis, Diretmus argenteus and Maulisia argipalla. They mainly occurred in the viscera of these fishes. Pseudoterranova ceticola L3 were small (< 12 mm) and whitish, and a prominent characteristic is a circumoral ridge extending from the ventral boring tooth which differentiate them from Pseudoterranova spp. L3 maturing in pinnipeds and Terranova sensu lato larvae that mature in poikilotherms. The shape of the tail: conical, long, pointed, ventrally curved and lacking mucron also distinguish these larvae from those of the pinniped-infecting Pseudoterranova spp. Phylogenetic analyses based on mtDNA cox2 and ITS rDNA sequences suggest that P. ceticola is closely related to Skrjabinisakis spp., and not with Pseudoterranova spp. parasitizing pinnipeds. The related species Skrjabinisakis paggiae, S. brevispiculata and S. physeteris (until recently belonging to genus Anisakis), are as P. ceticola also parasites of physeteroid cetaceans. The morphology and morphological variation of the larvae of the cetacean parasite P. ceticola is thoroughly described for the first time. These L3 can readily be morphologically distinguished from those of the pinniped-infecting Pseudoterranova spp. The parasite likely completes its life cycle in the mesopelagic and bathypelagic realm, with meso/bathypelagic fish as 2nd intermediate or paratenic hosts and kogiids as final host. Thus, Pseudoterranova from cetaceans appear to be morphologically, genetically, and ecologically differentiated to those from pinnipeds, suggesting that they are not congeneric.

Dataset Information

Functional Ultrastructure of the Excretory Gland Cell in Zoonotic Anisakids (Anisakidae, Nematoda).

Publications

Functional Ultrastructure of the Excretory Gland Cell in Zoonotic Anisakids (Anisakidae, Nematoda).

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