Desmozoon lepeophtherii n. gen., n. sp., (Microsporidia: Enterocytozoonidae) infecting the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae)

Abstract

Background: A microsporidian was previously reported to infect the crustacean parasite, Lepeophtheirus salmonis (Krøyer, 1837) (Copepoda, Caligidae), on farmed Atlantic salmon (Salmo salar L.) in Scotland. The microsporidian was shown to be a novel species with a molecular phylogenetic relationship to Nucleospora (Enterocytozoonidae), but the original report did not assign it to a genus or species. Further studies examined the development of the microsporidian in L. salmonis using electron microscopy and re-evaluated the molecular findings using new sequence data available for the group. Here we report a full description for the microsporidian and assign it to a new genus and species.

Results: The microsporidian infects subcuticular cells that lie on the innermost region of the epidermal tissue layer beneath the cuticle and along the internal haemocoelic divisions. The mature spores are sub-spherical with a single nucleus and an isofilar polar filament with 5-8 turns in a double coil. The entire development is in direct contact with the host cell cytoplasm and is polysporous. During early merogony, a diplokaryotic nuclear arrangement exists which is absent throughout the rest of the developmental cycle. Large merogonial plasmodia form which divide to form single uninucleate sporonts. Sporogonial plasmodia were not observed; instead, binucleate sporonts divide to form two sporoblasts. Prior to final division, there is a precocious development of the polar filament extrusion apparatus which is associated with large electron lucent inclusions (ELIs). Analyses of DNA sequences reveal that the microsporidian is robustly supported in a clade with other members of the Enterocytozoonidae and confirms a close phylogenetic relationship with Nucleospora.

Conclusion: The ultrastructural findings of the precocious development of the polar filament and the presence of ELIs are consistent with those of the Enterocytozoonidae. However, the confirmed presence of an early diplokaryotic stage and a merogonial plasmodium that divides to yield uninucleate sporonts instead of transforming into a sporogonial syncitium, are features not currently associated with the family. Yet, analyses of DNA sequence data clearly place the microsporidian within the Enterocytozoonidae. Therefore, due to the novelty of the copepod host, the ultrastructural findings and the robust nature of the phylogenetic analyses, a new genus should be created within the Enterocytozoonide; Desmozoon lepeophtherii n. gen. n. sp. is proposed.

Figure 1

a-b - TEM of mature spores of Desmozoon lepeophtherii n. gen., n. sp. Sagittal section of spores detailing seven coils of an isofilar polar filament (white arrows) in a double coil arrangement. The polar filament has an electron dense core and is composed of concentric layers of varying electron densities (inset a). A thick electron lucent endospore wall (black asterisks) is surrounded by a thinner electron dense exospore layer (black arrows). The single nucleus (n), posterior vacuole (pv) and polaroplast (pp) are all prominent features of the mature spore. The polaroplast is located at the anterior end of the spore and accommodates the manubroid part of the polar filament (white asterisk) and the attachment disc. Scale bars 1 μm (200 nm inset a).

Figure 2

SEM of freeze-fractured L. salmonis and transverse abdominal semi-thin histological section. a) SEM of freeze-fractured louse showing a large xenoma (x) developing beneath the cuticle (cu). The epidermal tissue layer remains uninfected directly beneath the cuticle (white arrows) but has a different more coarse appearance (white asterisk) bordering the haemocoel (h). The xenoma has been fractured open revealing that it is packed with microsporidian spores (s). b) Transverse abdominal semi-thin section from an infected louse. Developing xenomas (x) contain both mature spores (s) and developing stages (d) and can originate from beneath the cuticle (cu) or from the haemocoelic divisions that separate the haemal sinuses (black arrows). The epidermal tissue layer beneath the cuticle remains intact (white arrows). Scale bars a 25 μm, b 50 μm.

Figure 3

TEM of microsporidian infection beneath the cuticle, xenoma structure and merogonial replication of Desmozoon lepeophtherii n. gen., n. sp. a) The microsporidian infection (mi) is found beneath the cuticle (cu) and originates from the innermost portion of the epidermal tissue layer (el). b) A small xenoma contains mature spores (s) and reveals that the host nucleus (white asterisks) is not infected by the microsporidian but is grossly hypertrophic and has numerous branches and folds. c) An early meront stage with a single nucleus in diplokaryotic formation (di). d) A divisional meront showing cytoplasmic constrictions (black arrows), that contains two nuclei in diplokaryotic arrangement (di). e) A large divisional merogonial plasmodium (pl) situated next to a host cell nucleus (hn), black arrows indicate nuclear activity associated with nuclear dissociation of the diplokaryon in neighbouring meronts. f) An enlarged view of the same plasmodium, with single unpaired nuclei (sn), undergoing division via plasmotomy (black arrows). White arrows indicate electron-dense laminate bodies lying on the nuclear membrane indicating recent nuclear activity due to nuclear dissociation of the diplokaryotic arrangement. Scale bars a-b 5 μm, c-f 2 μm.

Figure 4

TEM of sporogonial stages of Desmozoon lepeophtherii n. gen., n. sp. a) An early divisional sporont with tubules in the host cell cytoplasm has a modest thickening of the plasma membrane (white arrows) and precocious development of the pf (black arrows) associated with the nucleus and cytoplasmic cisternae (white asterisks) that start to form the pf bundles. b) Sporont with a thickening plasma membrane (white arrows), a diffuse nucleus showing signs of recent activity (black arrows), the developing ad (black asterisks) is associated with a nuclear invagination, from which the developing pf extends. Tubules are present in the host cell cytoplasm. c) Immature sporoblasts have a more defined nucleus and show signs of pf organisation in to bundles (black arrows), which are arranged in close proximity to large ELIs (black asterisks), tubules are still present in the host cell cytoplasm. d) Maturing sporoblasts have features of mature spores such as a pp and a more mature pf. ELIs (black asterisks) have dark granules associated with the membranes (white arrows). e) Some sporoblasts at an equivalent developmental stage to (d) have two sets of pf apparatus (white/black asterisks) both with associated ELIs. f) An early spore with a fully formed exospore layer (black arrows) and a developing endospore layer (white arrows). This late stage shows a typical internal arrangement seen in mature spores; ad and pp are located at the anterior of the spore, the single nucleus and rows of pf (white asterisks) are medially positioned, ELIs (black asterisks) are posteriorly located and will form part of the posterior vacuole. A secretion from the exospore forms a fragile interfacial envelope which creates a void surrounding the spore (open black arrows). All scale bars 1 μm. ad (anchoring disc); n (nucleus); pf (polar filament); pp (polaroplast); t (tubules).

Figure 5

Other ultrastructural observations associated with Desmozoon lepeophtherii n. gen., n. sp. a and b) Secretions from mature spores (s) are sometimes observed that form multiple and regular layers which have a fine electron dense core, and surround the spore (white arrows). The secretions originate from the exospore surface and, when first produced, do not have an electron dense core (black arrows). c) At the border of a xenoma (white arrows) concentric whorls of the secretions have formed adjacent to a sporoblast (sb), the individual layers appear more compact but still maintain an electron dense core (black arrows). d and inset) Host cell nuclei of microsporidian-infected cells were frequently observed with intranuclear inclusions, which were of a crystalline appearance with a regular pattern. Scale bars a, b and d (inset) 500 nm, c 1 μm, d 2 μm.

Figure 6

Maximum parsimony generated phylogenetic tree constructed using SSU rDNA sequence alignments from representative taxa of the Enterocytozoonidae. The tree utilises 328 parsimony informative characters and is rooted to the related Microsporidium sp. infecting Daphnia. The scale represents number of base changes and the numbers at the nodes indicate bootstrap support values from 1000 resamplings. AH = Atlantic halibut; AS = Atlantic salmon; CS = chinook salmon; DP = Daphnia; ES = English sole; RT = rainbow trout.