MORPHOLOGICAL REDESCRIPTION OF DIROFILARIA IMMITIS

J. Parasitol., 96(3), 2010, pp. 499 504 F American Society of Parasitologists 2010 MORPHOLOGICAL REDESCRIPTION OF DIROFILARIA IMMITIS Adriano P. Furtado, Francisco T. V. Melo, Elane G. Giese, and Jeannie N. dos Santos* Laboratório de Biologia Celular e Helmintologia Profa. Dra. Reinalda Marisa Lanfredi, Instituto de Ciências Biológicas, Universidade Federal do Pará, Guamá, Belém, Pará, Brazil, 66075-110. e-mail: jeannie@ufpa.br ABSTRACT: Morphological descriptions of Dirofilaria immitis are scarce. For this reason, we carried out morphological studies using both light and scanning electron microscopy for this filaroid species. Morphometric and morphological data were compatible with previous descriptions of D. immitis, but several anatomical structures are described by scanning electron microscopy for the first time, such as details of the cuticular striations, positioning of amphids, visualization of anal and vulvar opening, descriptions of deirids, lateral line, the pair of phasmids in the posterior end in females, and visualization of a small pair of latero-terminal papillae in the posterior end in males. Dirofilaria immitis is the most common filaroid found parasitizing dogs, cats, and other carnivores throughout the world. It is transmitted by various species of mosquitoes (Insecta: Diptera: Culicidae) (McCall et al., 2008). This parasite is the causative agent for heartworm disease and occurs primarily in the right ventricle and adjacent pulmonary artery. Using light microscopy, several authors (López-Neyra, 1947; Yamaguti, 1962; Yorke and Maplestone, 1962) have described D. immitis. However, scanning electron microscopy (SEM) has become an important tool that can add new taxonomic characters to previous species descriptions made by light microscopy (Chagas-Moutinho et al., 2007; Torres et al., 2007), as well as contributing to descriptions of new species (Santos et al., 2008; Giese et al., 2009). Few reports have described D. immitis using this technique, although Wong and Brummer (1978) conducted a comparative study of filarial longitudinal ridges, and included D. immitis, while Rodrigues-Silva et al. (1999) described the rugosae area and the posterior end of male D. immitis. The present report is a redescription of D. immitis by light microscopy, and SEM. MATERIALS AND METHODS Filaroids were collected from the right ventricle and pulmonary artery of domestic pet dogs in the cities of Salvaterra (00u459120S, 48u319000W) and São Sebastião da Boa Vista (01u439050S, 49u319450W), Marajo Island, Para State, Brazil. This area is endemic to canine dirofilariasis according to Furtado et al. (2009). Routinely, domestic dogs with visceral leishmaniasis are killed by public health agencies (SESPA and SESMA) in these cities. These agencies authorized 30 necropsies of dogs (20 in Salvaterra and 10 in São Sebastião da Boa Vista) so that we could undertake the present investigation. The collected worms were washed immediately in phosphate buffered saline, ph 7.4, and fixed in hot AFA (2% acetic acid, 3% formaldehyde, and 95% ethanol 70 degrees GL) at 60 C. For light microscopy, 2-cm sections of the anterior and posterior ends of 18 females and 7 male D. immitis were observed using a light microscope, equipped with a camera lucida, after dehydration in ethanol series (70 100%), then cleared in Aman s lactophenol for 72 hr. Measurements are given in millimeters, followed by ranges in parentheses. A male and female filaroid from each municipality were deposited in the Helminthological Collection of the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil (CHIOC no. 35658A/B and CHIOC no. 35659A/B). For SEM, 2-cm sections of the anterior and posterior ends of 10 males and females were processed according the methods of Mafra and Lanfredi (1998) and observed using a JEOL JSM 6390 LV scanning microscope. Received 28 May 2009; revised 17 August 2009, 19 October 2009, 23 October 2009, 21 January 2010; accepted 22 January 2010. * To whom correspondence should be addressed. DOI: 10.1645/GE-2178.1 REDESCRIPTION Dirofilaria immitis (Figs. 1 19; Table I) General diagnosis: Elongated worm, filiform, with cephalic extremity slightly thin and rounded (Figs. 1, 4). Terminal oral opening, circular, without lips, surrounded by 4 pairs of small cephalic papillae and 2 lateral amphids (Fig. 5). Amphids presenting basal opening directed to posterior region of body (Fig. 6). Esophagus divided in muscular and glandular regions, but without clear demarcation between these regions (Fig. 1). Small lateral deirids situated between nerve ring and esophageal intestine junction (Fig. 1), in center of cuticular depression, next to lateral line, slightly dislocated for ventral region. Deirids with a filamentous structure, base wider than apex, and longitudinal concavity. Apex trifurcated (Figs. 7, 8). Body surface with delicate transversal striation (Fig. 9). Cuticular striations random in cephalic extremity (around cephalic papillae, amphids, and oral opening) (Fig. 5) and in posterior extremity of females (near phasmids) (Fig. 14). Absent cuticular transversal striations surrounding deirids (Figs. 7, 8), vulvar opening (Fig. 12), and anus (Fig. 13). Inconspicuous lateral lines constituted by discrete discontinuous ridges in anterior and posterior portion of filarids (Fig. 10), and by 2 or 3 parallel continuous lines throughout body (Fig. 11). Male: Body: 137.6 (119.4 162.3) length, 0.41 (0.3 0.5) width. Esophagus 1.21 (1.08 1.46) length, 0.11 (0.09 0.13) width. Nerve ring to anterior end 0.40 (0.35 0.47), in first third of esophagus. Deirids to anterior end 0.57 (0.29 0.77) (Figs. 7, 8). Ventral rugosae area in spiraled posterior end of males with cuticular parallel ridges of varied length. Ridges follow longitudinal axis of body in larger spirals (Fig. 15), gradually dislocating from transversal position in smaller spirals (Fig. 16). In region of larger spirals, number of ridges varies from 7 to 11. Each ridge composed of small units formed of prolongated cuneiform cuticular elevations, with base alleviated contour, rectilinear apex of varied length, shorter when perpendicular to common fine cuticular transversal striations throughout body (Fig. 17). Numerous rounded cuticular elevations in a line at end of each ridge and in spaces between these ridges (Fig. 17). Similar structures also observed laterally from rugosae area to dorsal region of worm, but more random. Paired and unpaired pre-cloacal papillae, varying from 1 to 6 in number (Figs. 2, 3, 18). Small pair of ad-cloacal papillae located lateral to cloacal opening (Figs. 3, 18). Five pairs of post-cloacal papillae: 1 pair ventral, 1 large pair ventro-lateral, 2 small pairs ventro-lateral, 1 pair terminal (Figs. 2, 3, 18 [and insert]). Pair of ventral phasmids between last 2 pair post-cloacal papillae (Figs. 2, 3, 18). Large spicule 0.34 (0.31 0.39), lying in channel formed by small spicule 0.17 (0.15 0.19) (Fig. 19). Gubernaculum absent. Tail: 0.10 (0.09 0.11). Female: Body: 218.3 (177.0 272.0) length, 0.49 (0.40 0.61) width. Esophagus length 1.29 (1.05 1.57), 0.12 (0.09 0.17) width (Fig. 1). Nerve ring to anterior end 0.41 (0.33 0.52), in first third of esophagus (Fig. 1). Deirids to anterior end 0.70 (0.56 0.97). Vulva next to anterior end, with an elliptical and transverse vulvar opening, delimited by fine, but not prominent, lips, without papillae or fringes (Fig. 12) located at 2.68 (2.23 3.26) to anterior end, just behind esophageal intestine junction (Fig. 1). Rounded terminal end. Anus sub-terminal with incurved opening (Fig. 13), toward posterior extremity, situated 0.17 (0.15 0.27) to posterior end. Pair of latero-ventral phasmids with circular openings (Fig. 14). Viviparous, didelphic, and opisthodelphic ovaries. 499

500 THE JOURNAL OF PARASITOLOGY, VOL. 96, NO. 3, JUNE 2010 FIGURES 1 3. Dirofilaria immitis by light microscopy. (1) Anterior end of female, showing the nerve ring, esophagus, deirids, first portion of intestine, and the vulvar opening. Bar 5 200 mm. (2) Lateral view of male posterior end, showing the large and small spicules, pre-cloacal papillae, large ventrolateral post-cloacal papillae, and the phasmids. Bar 5 100 mm. (3) Ventral view of male posterior end, with the pre-cloacal papillae, a small pair of adcloacal papillae, the cloacal opening, 4 pairs of post-cloacal papillae (1 pair ventral, 1 large pair ventro-lateral, and 2 small pairs ventro-lateral), a pair of ventral phasmids, and large and small spicules. Bar 5 100 mm.

FURTADO ET AL. REDESCRIPTION OF D. IMMITIS 501 FIGURES 4 8. Dirofilaria immitis by scanning electron microscopy. (4) Anterior end, showing the vulva (arrow). Bar 5 500 mm. (5) Detail of the anterior end, with oral opening (arrow), 4 pairs of cephalic papillae (arrow heads), and amphids (asterisks). Bar 5 20 mm. (6) Detail of amphid (arrow), with the anfidial opening (arrow head). Bar 5 5 mm. (7) Detail of a totally everted deirid (arrow), located in the center of cuticular depression. Bar 5 2 mm. (8) Detail of a partially everted deirid (arrow). Bar 5 5 mm.

502 THE JOURNAL OF PARASITOLOGY, VOL. 96, NO. 3, JUNE 2010 FIGURES 9 16. Dirofilaria immitis by scanning electron microscopy. (9) Detail of fine transversal cuticular striation (arrow heads). Bar 5 5 mm. (10) Lateral line in anterior portion of the body. Bar 5 5 mm. (11) Inconspicuous lateral line throughout length of body (arrow heads). Bar 5 50 mm. (12) Vulva opening (arrow), without cuticular striations surrounding the structure (arrow head). Bar 5 10 mm. (13) Anal aperture (arrow), without cuticular striations surrounding the structure (arrow head). Bar 5 10 mm. (14) Posterior extremity of female, showing the phasmids (arrow head). Bar 5 5 mm. (15) Rugosae area of males, with cuticular ridges (arrow heads) longitudinally disposed in the large spirals. Bar 5 20 mm. (16) Detail of the rugosae area of males, showing the cuticular ridges (arrow heads) transversally disposed in the smaller spirals. Bar 5 20 mm. Taxonomic summary Host: Canis familiaris (Linnaeus, 1758) (Carnivora: Canidae). Site of infection: Pulmonary artery and right ventricle. Locality: Salvaterra (00u459120S, 48u319000W) and São Sebastião da Boa Vista (01u439050S, 49u319450W), Marajo Island, Para State, Brazil. Deposition of specimens: Four voucher specimens in Helminthological Collection of the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil (CHIOC no. 35658A/B and CHIOC no. 35659A/B). Remarks Several new anatomical variations have been described for D. immitis. For example, Murata et al. (2003) and Sano et al. (2005) examined D. immitis from leopards and penguins in a Japanese zoo. They reported the presence of delicate longitudinal cuticular ridges along the entire length of the body using ordinary light microscopy. Nonetheless, both reports showed that these filarids also presented molecular characteristics similar to the D. immitis from canids. These data disagree with those of Wong and

FURTADO ET AL. REDESCRIPTION OF D. IMMITIS 503 FIGURES 17 19. Dirofilaria immitis by scanning electron microscopy. (17) Rugosae area, with segmented cuticular ridges, consisting of elevated cuneiform cuticle (arrows), elevated round cuticle (arrow heads). Bar 5 10 mm. (18) Ventral view of male posterior end, revealing paired pre-cloacal papillae (larger arrows), a pair of small ad-cloacal papillae (smaller arrows), a pair of post-cloacal ventral papillae (p), large post-cloacal ventro-lateral papillae (P), and ventral phasmids (arrow heads). Bar 5 30 mm. Detail (insert) of 1 terminal papilla in male. Bar 5 5 mm. (19) Details of large (arrow) and small spicules (arrow head). Bar 5 30 mm. TABLE I. Comparison of Dirofilaria immitis morphometric parameters. Taxonomic parameters Data for this work Leidy (1856) apud Lent and Teixeira-de-Freitas (1937) López-Neyra (1947) Female Male Female Male Female Male Body length 218.3 (177 272) 137.6 (119 162) 150 300 120 180 210 310 120 200 Body width 0.49 (0.40 0.61) 0.41 (0.34 0.5) 1.0 1.3 0.6 0.9 1.0 1.3 0.6 0.7 Esophagus length 1.29 (1.05 1.57) 1.21 (1.08 1.46).. 1.2 1.5. Esophagus width 0.12 (0.09 0.17) 0.11 (0.09 0.13).... Nerve ring 0.41 (0.33 0.52) 0.40 (0.35 0.47).. 0.4. Deirids 0.70 (0.56 0.97) 0.57 (0.29 0.77).... Vulvae 2.68 (2.23 3.26). 2.3 3.4. 2.1 3.4. Tail 0.17 (0.15 0.27) 0.10 (0.09 0.11).. 0.18 0.21 0.09 0.10 Large spicule. 0.34 (0.31 0.39). 0.3. 0.3 Small spicule. 0.17 (0.15 0.19). 0.17 0.22. 0.17 0.26 Male cloacal papillae distribution Unpaired and paired pre-cloacal (1 6); 1 pair ad-cloacal; 5 pairs post-cloacal (1 pair ventral, 1 large pair ventro-lateral, 2 small pairs ventro-lateral, 1 pair latero-terminal). Unpaired pre-cloacal (4 5); 3 5 pairs postcloacal (first and fourth laterally dislocated papillae) + frequently an unpaired papillae + unique terminal papillae

504 THE JOURNAL OF PARASITOLOGY, VOL. 96, NO. 3, JUNE 2010 Brummer (1978), who described D. immitis without longitudinal cuticular ridges using SEM. Because of these discrepancies, we carried out a detailed morphological study in D. immitis using light microscopy and SEM. Several characteristics have been previously described for the cephalic end of D. immitis (Wong and Brummer, 1978). These include the circular and terminal oral opening without lips, the number and distribution of cephalic papillae and amphids, and transverse cuticular striations. In the present work, pre-cloacal papillae were found in varying numbers (1 6), and may be both unpaired and paired. A pair of ad-cloacal papillae, i.e., the first and second pair of post-cloacal papillae, were observed in all specimens by light microscopy and SEM. The third and fourth pair of post-cloacal papillae were best observed by light microscopy. In contrast, terminal post-cloacal papillae were best seen by SEM. The pre-cloacal papillae distribution is similar to the descriptions of Wong and Brummer (1978) and Rodrigues-Silva et al. (1999), although López-Neyra (1947) described a different distribution of post-cloacal papillae for D. immitis. Based on this variability, Rodrigues-Silva et al. (1999) concluded that the cloacal papillae distribution cannot be considered as a reliable taxonomic character for D. immitis. The general characteristic of spiraled posterior end in males, including the rugosae area (arrangement of longitudinal and rounded cuticular striations), are similar to Wong and Brummer (1978) descriptions. Our observations, however, differ from those of Sano et al. (2005) in positioning of male phasmids. Using light microscopy, the latter authors observed phasmids situated near the tail tip. However, with light microscopy and SEM, we found these structures to be positioned ventrally. These differences could be artifactual or erroneous interpretations of penguin filarid morphology observed by light microscopy. Data presented in the present study show that D. immitis found in the thoracic organs (pulmonary arteries and right ventricle) of domestic dogs have morphological characteristics compatible with the descriptions and measures reported previously by others authors (López-Neyra, 1947; Yamaguti, 1962; Yorke and Maplestone, 1962; Anderson and Bain, 1976). However, we have also added several new features for D. immitis using SEM, thereby providing a much more robust characterization of the species. ACKNOWLEDGMENTS The authors thank the Para State Secretary of Public Health (Secretaria Estadual de Saúde Pública SESPA, 7 a Regional de Proteção Social) and the Municipal Secretaries of Health (Secretaria Municipal de Saúde) in Salvaterra and São Sebastião da Boa Vista for permission to undertake this study; Dra. 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