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Fig. 6 in Description, molecular identification and pathological lesions of Huffmanela persica sp. nov. (Nematoda: Trichosomoididae: Huffmanelinae) from the daggertooth pike conger Muraenesox cinereus

Fig. 6 Maximum likelihood (ML) phylogram reconstructed using the 18S rDNA dataset of the new species Huffmanela persica sp. nov. and other related species within the families Trichosomoididae,Trichinellidae, Trichuridae and Capillariidae. ML analysis was performed using the substitution model K2 + G with 1000 bootstrap replications

Fig. 5 in Description, molecular identification and pathological lesions of Huffmanela persica sp. nov. (Nematoda: Trichosomoididae: Huffmanelinae) from the daggertooth pike conger Muraenesox cinereus

Fig. 5 Photomicrographs from histological sections of infected tissues of a daggertooth pike conger eel. A, B Histological sections of the stomach infected by eggs of Huffmanela persica sp. nov. at various stages of development as well as degenerated encysted metazoans (⁎). C, D Sections of the tunica serosa of the stomach parasitized with completely developed eggs. E Sections of the ovarian lamellae infected by both clusters of immature (➔) and developing (➤) eggs, representing immature and previtellogenic oocytes embedded within a loose fibro-granulomatous infiltration containing histiocytes and eosinophilic granular leukocytes (⁎). F Magnified view of histological section of infected ovary showing a fibro-granulomatous infiltrate surrounding clusters of eggs at different stages of development (mainly highly developed eggs, ➤). G High magnification (100×) view of a fully developed egg of H. persica (cross-sectional view) showing larva in-folded within the eggshell and a protruding polar plug at either end. H Less developed eggs with a nearly central nucleus (⁎) and thin eggshell layer and I fully developed eggs containing twisted larvae of H. persica cut on varying planes of section

Fig. 4 in Description, molecular identification and pathological lesions of Huffmanela persica sp. nov. (Nematoda: Trichosomoididae: Huffmanelinae) from the daggertooth pike conger Muraenesox cinereus

Fig. 4 Scanning electron micrographs of poorly developed (A–D) and fully developed (E–I) eggs of Huffmanela persica sp. nov. (separated from infected ovary). Less developed eggs spherical shaped (white arrow shows a shrunken and wrinkled egg) and with no evidently developed polar plugs. Fully developed eggs oblong and containing two plugs at poles. Eggs completely surrounded by a UL bearing uniformly based mammiform mounds adorned with tendril-like vermiform appendage emerging from the apex, occasionally adjoined to that of a neighboring mound (green arrowheads). Note the illusory appearance of serrated-like ridges (occasionally in the form of illusory interconnecting ridges, black arrows) in side views which is caused by overlapping of the mound bases (well observed in less developed eggs, red arrowheads). Outer surface of eggshell with irregular protuberances (yellow arrowheads)

Fig. 1 in Description, molecular identification and pathological lesions of Huffmanela persica sp. nov. (Nematoda: Trichosomoididae: Huffmanelinae) from the daggertooth pike conger Muraenesox cinereus

Fig. 1 Representation of an advanced egg of Huffmanela persica sp. nov. with measurements considered in this study. Total length with protruding polar plug and UL (black line); total length without protruding polar plug and UL (red line); total width with UL (yellow line); total width without UL (light blue line); shell thickness with UL (green line); shell thickness without UL (white line); polar plug width (dark blue line)

Fig. 2 in Description, molecular identification and pathological lesions of Huffmanela persica sp. nov. (Nematoda: Trichosomoididae: Huffmanelinae) from the daggertooth pike conger Muraenesox cinereus

Fig. 2 Macroscopic and microscopic appearance of fully developed eggs of Huffmanela persica sp. nov. A Grossly visible lesions of eggs previously deposited by adult forms of H. persica sp. nov. in the form of dark spots of various size within the infected tissues (ovary and serosa of stomach) of Muraenesox cinereus. B Wet mount prepared from infected ovary illustrating variously oriented advanced eggs of H. persica sp. nov. within the egg clusters

Fig. 3 in Description, molecular identification and pathological lesions of Huffmanela persica sp. nov. (Nematoda: Trichosomoididae: Huffmanelinae) from the daggertooth pike conger Muraenesox cinereus

Fig. 3 General morphology and surface ornamentation pattern of eggs of Huffmanela persica sp. nov. in various stages of development. A–H Photomicrographs and a–h corresponding line drawings of individual eggs at different stages of development (scale bars: 20 µm). A, a Eggs in stage I at very early stage, probably meiosis I, with a spherical nucleus in granular cytoplasm and incompletely developed chitinous layer and polar plugs (note early appearance of superficial projections of UL already apparent). B, b Eggs in stage II at later stage of development (probably meiosis II); chitin deposition appears to be complete. C, c Two-celled mitotic stage of early embryonic development (embryonated). D, d Later multicellular stage of embryonic development; chitinous layer still uniformly translucent with no apparent division into outer and inner chitinous layers. E, e Eggs in stage III with bean-like embryo and chitinous layer appearing two-layered under bright-field (light) microscopy with darker inner layer. F, f Tadpole-like embryos with UL appearing to have been partially dislodged from chitinous layer. G, g Eggs in stage IV with darker-brown shell; embryo now vermiform (larvated) and in-folded three times (pretzel stage). H, h Later stage IV egg with chitinous layer very dark brown; larva nearing final development and folded 5–6 times. I–T Photomicrographs of less developed (I–L), moderately developed (M–P) and fully developed (Q–T) eggs, where the first set of images (I, M, Q) represents overview of these variously advanced eggs, and the second (J, N, R), third (K, O, S) and fourth (L, P, T) series of images focus on the pattern of their surface ornamentation by adjusting the focal plane. Black and yellow arrows represent illusions of superficial ridges (well demonstrated in less developed eggs; occasionally appearing as interconnecting ridges, blue arrowhead) and sculptures on the egg surface, respectively. Green arrowheads exhibit irregular protuberances on the eggshell surface. Red arrowheads indicate an illusory spinous appearance in fully developed eggs

Fig. 5 in Huffmanela lusitana sp. n. (Nematoda: Trichosomoididae) infecting pouting, Trisopterus luscus (Teleostei: Gadidae) off the Atlantic coast of Portugal

Fig. 5. Hypaxial muscle of pouting containing eggs of Huffmanela lusitana sp. n. Longitudinal sections displayed at same scale and showing the relationship between darkness rating of flesh and degree of egg infection. Muscle section from fish graded as normal had 0 eggs (A); muscle section from fish graded as slight to moderate darkening had about 19 eggs (B); muscle section from fish graded as intense darkening had about 89 eggs (C). H&E. Bar: 100 μm.

Fig. 6 in Huffmanela lusitana sp. n. (Nematoda: Trichosomoididae) infecting pouting, Trisopterus luscus (Teleostei: Gadidae) off the Atlantic coast of Portugal

Fig. 6. Hypaxial muscle of pouting infected with eggs of H. lusitana sp. n. (H&E stain). Longitudinal sections: (A) immature eggs (1) and advanced brownshelled eggs (2) with a linear distribution; (B) clearshelled (1) and brown-shelled eggs (2). Cross sections: (C) eggs in different stages of development and intense inflammatory lesions and muscle destruction (note the eosinophilic color of polar plugs at arrows); (D) eggs and nematodes in cross section [(1, 4, 5) eggs with intercellular location; (2) worm with intracellular location, probably 3rd or 4th stage larva; (3) apical view of a polar plug]. H&E. Bar: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 in Huffmanela lusitana sp. n. (Nematoda: Trichosomoididae) infecting pouting, Trisopterus luscus (Teleostei: Gadidae) off the Atlantic coast of Portugal

Fig. 2. Presumptive developmental stages of eggs and larvae of Huffmanela lusitana sp. n. from pouting (a, a') Stage 1: apparent unfertilized egg surrounded only by vitelline membrane with no evidence of polar plugs. (b, b') Stage 2: clear-shelled egg with polar plugs consisting of an outer and inner layer. (c, c') Stage 3: amber-shelled egg with bilayer eggshell observable; outer layer translucent and innermost typically dark (circle). (d, d') Stage 4: brown-shelled egg. (e, e', f, f') Stage 5: advanced brown-shelled egg with larva and outer layer of polar plugs. (g, g') Stage 6: fully developed brown-shelled egg; outer layer of polar "plugs" missing; egg apparently ready to hatch. (h, h') Freshly hatched larva expressed from egg under coverslip pressure. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 3 in Description, molecular identification and pathological lesions of Huffmanela persica sp. nov. (Nematoda: Trichosomoididae: Huffmanelinae) from the daggertooth pike conger Muraenesox cinereus

Fig. 3 (See legend on previous page.)

FIGURE 1 in Huffmanela spp. (Nematoda, Trichosomoididae) parasites in coral reef fishes off New Caledonia, with descriptions of H. balista n. sp. and H. longa n. sp.

FIGURE 1. Outer surface of the swimbladder of Abalistes stellatus (fish JNC2163). Dark zones are accumulations of eggs of Huffmanela balista n. sp.

FIGURE 5 in Huffmanela spp. (Nematoda, Trichosomoididae) parasites in coral reef fishes off New Caledonia, with descriptions of H. balista n. sp. and H. longa n. sp.

FIGURE 5. Histogram of egg length of the two Huffmanela species parasitic in Gymnocranius grandoculis. JNC892: eggs from the gills, syntypes of H. filamentosa Justine, 2004. JNC1726, JNC 2166, JNC2174, eggs of H. lata n. sp. JNC1726: eggs from the mesentery. JNC2166 and JNC2174: eggs from the swimbladder.

FIGURE 7. X in Huffmanela spp. (Nematoda, Trichosomoididae) parasites in coral reef fishes off New Caledonia, with descriptions of H. balista n. sp. and H. longa n. sp.

FIGURE 7. X-ray photographs of the spinal cord in two Bodianus perditio. A. Specimen not parasitized by Huffmanela ossicola. B. Specimen heavily parasitized by Huffmanela ossicola. Negative prints.

FIGURE 3 in Huffmanela spp. (Nematoda, Trichosomoididae) parasites in coral reef fishes off New Caledonia, with descriptions of H. balista n. sp. and H. longa n. sp.

FIGURE 3. Eggs and larvae of Huffmanela balista n. sp. from Abalistes stellatus. A–J. Eggs. A–B, C–D. Optical section and surface views of same eggs. F, G. Eggs with hatching larvae (note that hatching occurs in both clear and dark eggs). H. Dark-brown egg. I. Black egg, larva present but not visible. J. Black egg, larva present but not visible, envelope visible. K. L1 larva, spontaneously hatched.

FIGURE 6. A in Huffmanela spp. (Nematoda, Trichosomoididae) parasites in coral reef fishes off New Caledonia, with descriptions of H. balista n. sp. and H. longa n. sp.

FIGURE 6. A. Egg of Huffmanela mexicana Moravec et Fajer-Avila, 2000, for comparison with other species. B–D. Hatching eggs and larvae of Huffmanela ossicola Justine, 2004 from the gills of Bodianus perditio.

Fig. 4 in Huffmanela lusitana sp. n. (Nematoda: Trichosomoididae) infecting pouting, Trisopterus luscus (Teleostei: Gadidae) off the Atlantic coast of Portugal

Fig. 4. Wet mount of H. lusitana sp. n. eggs hypaxial muscle of pouting. The eggs are arranged in rows along muscle fibres.

Fig. 1 in Huffmanela lusitana sp. n. (Nematoda: Trichosomoididae) infecting pouting, Trisopterus luscus (Teleostei: Gadidae) off the Atlantic coast of Portugal

Fig. 1. Pouting, Trisopterus luscus after removing the skin to show intense darkening of flesh caused by millions of very dark eggs of Huffmanela lusitana sp. n.

Fig. 3 in Huffmanela lusitana sp. n. (Nematoda: Trichosomoididae) infecting pouting, Trisopterus luscus (Teleostei: Gadidae) off the Atlantic coast of Portugal

Fig. 3. Scanning electron microscopy of Huffmanela lusitana sp. n. eggs in muscle of pouting. Eggs arranged in rows among muscle fibers (150x) (A); Huffmanela egg on the surface of the muscle fibers (m) with eggshell (sh) surrounded by a smooth vitelline membrane (*) (1 500x) (B), except at the polar plugs (arrow) (1 500x) (C); Egg partially crushed. The cuticle of the larva (l) shows regularly spaced, transverse cuticular ridges (3 500x) (D).

Fig. 7 in Huffmanela lusitana sp. n. (Nematoda: Trichosomoididae) infecting pouting, Trisopterus luscus (Teleostei: Gadidae) off the Atlantic coast of Portugal

Fig. 7. Hypaxial muscle of pouting infected with eggs of H. lusitana sp. n. (H&E stain). Cross sections: (A) diffuse inflammatory reaction involving eggs at different stages of development; (B) granuloma with six eggs. (C) calcification of muscle tissue (arrow); (D) degenerative changes with regular cystic structures [of presumed parasitic nature (arrows)]. H&E. Bars: A, B, D, 100 μm; C, 50 μm.

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Fig. 8 in Huffmanela lusitana sp. n. (Nematoda: Trichosomoididae) infecting pouting, Trisopterus luscus (Teleostei: Gadidae) off the Atlantic coast of Portugal

Fig. 8. Darkened flesh of pouting rejected during preparation by end consumer.