doi:10.1111/jfd.12083 Journal of Fish Diseases 2013, 36, 853–859 Morphological aspects and histological effects of the attachment organ of Parabrachiella sp. (Copepoda: Lernaeopodidae) on the grey mullet, Mugil liza Valenciennes S E Plaul1, M M Montes2, C G Barbeito1 and S R Martorelli2 1 Facultad de Ciencias Veterinarias, C
atedra de Histolog
ıa y Embriolog
ıa, UNLP. La Plata, Bs. As., Argentina 2 CEPAVE (Centro de Estudios Parasitol
ogicos y de Vectores), UNLP. La Plata, Bs. As., Argentina Abstract The genus Parabrachiella Wilson, 1915 (Lernaeopodidae) is represented by copepods that are highly adapted to a parasitic way of life. In Argentina, only P. insidiosa var. lageniformis Heller, 1865, P. chevreuxii Van Beneden, 1891 and P. spinicephala Ringuelet, 1945 have been cited, but none of these have been reported on mugilids. Recently, other species of this genus were found attached to the nasal cavities of juvenile grey mullets, Mugil liza Valenciennes, from Samboromb
on bay, Buenos Aires province. In this study, the prevalence and mean intensity of the Parabrachiella sp. on grey mullet is investigated. In addition, the damage the parasite imposes on its hosts is examined through evaluation of histological sections and immunostaining for proliferative cell nuclear antigen (PCNA). The morphology of the parasite’s bulla is described from light and scanning electron micrographs. Keywords: bulla, Mugil liza, nasal cavity, Parabrachiella sp., proliferative cell nuclear antigen (PCNA). Introduction Copepods of the family Lernaeopodidae are a successful group of parasites that are found mainly on marine fish (Benkirane, Coste & Raibaut Correspondence S E Plaul, Facultad de Ciencias Veterinarias,
tedra de Histolog
ıa y Embriolog
ıa, UNLP. La Plata, Buenos Ca Aires, Argentina (e-mail: splaul@museo.fcnym.unlp.edu.ar) Ó 2013 John Wiley & Sons Ltd 853 € 1999; Oktener & Trilles 2009) but have also been reported from certain freshwater species (Kabata 1992; Piasecki, Mlynarczyk & Hayward 2010). Only the adult females are parasitic (Sch€aperclaus 1991) and can be easily recognised by their morphology and mode of attachment. This involves the second maxillae which are modified and fused to a mushroom-shaped-anchoring structure known as the bulla (Benkirane et al. 1999; Piasecki et al. 2004). The morphology of the bulla shows great variability within the lernopodids. It is produced by the frontal gland of the cephalothorax during the larval stage and, once inserted into the tissue of the host, it fuses with the second maxillae and becomes a permanent attachment organ which allows the lernaeopodid to obtain its food (Kabata 1992; Castro & Gonz
alez 2005; Piasecki, Sez kowska-Jakubowska & Sobecka 2006). The genus Parabrachiella Wilson, 1915 includes 67 species (Piasecki et al. 2010). In Argentina, only three members of the genus Parabrachiella have been recorded as parasites of marine and brackish water species; Neobrachiella chevreuxii Van Beneden, 1891 (= P. chevreuxii) in Micropogonias furnieri, Scianidae (see Sardella, Etchegoin & Martorelli 1995; Alarcos & Etchegoin 2010), N. insidiosa var. lageniformis Heller, 1865 (= P. insidiosa var. lageniformis) in Merluccius hubbsi, Merlucidae (see Sardella & Timi, 1996) and N. spinicephala Ringuelet, 1945 (= P. spinicephala) in Pinguipes brasilianus, Pinguipidae (see Etchegoin, Timi & Lanfranchi 2006). At present, there are few reports of histopathology associated with Parabrachiella attachment and feeding, and the aim of this study was to analyse Journal of Fish Diseases 2013, 36, 853–859 the pathogenicity of these parasites and the morphological characteristics of the attachment organ. To investigate possible parasite induced damage to the host, histological sections through tissues at the site of parasite attachment were taken and immunohistochemistry was performed to evaluate proliferative cells. The study looks at the number of proliferating cells seen in the immediate vicinity of a parasite attaching and compares them with the number observed away from the point of parasite attachment and in uninfected co-specifics (Dezfuli et al. 2012). Furthermore, morphological characteristics of the bulla were observed under light microscopy and scanning electron microscopy (SEM). Materials and methods A total of one hundred and sixty-one juvenile specimens of juvenile grey mullet, Mugil liza Valenciennes (Mugilidae), were captured from two areas S E Plaul et al. Parabrachiella sp. in nasal cavity of mugilids in the Samboromb
on Bay, Buenos Aires province, Argentina during 2009. Of these, sixty-five specimens were collected in the Salado river (35º50′S, 57º25′W) and ninety-six specimens in the Aj
o river (36º 20′S, 56º 54′W) (Fig. 1). Limnological parameters such as water temperature, salinity, dissolved oxygen and pH were taken (Fig. 2). In both cases, fish were collected using a modified Garlito/Bituron fixed net (Colautti 1998) and a haul net (10 m length with 5 mm stretched mesh in the wings and 2.5 mm stretched mesh in the cod ends). Fish were weighed, and the total length of each fish was recorded. Nasal, oral-branchial cavities and the external body surface of each mullet were examined for parasitic copepods. Parasites were counted, dissected from the host tissue with needles, fixed in a 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.0) for 4 h at 4 °C, postfixed for 1 h in 1% osmium tetroxide in 0.1 M cacodylate buffer and then dehydrated in a graded series of ethanol. Thereafter, specimens for the Figure 1 Position of the Salado and Aj
o rivers within Samboromb
on Bay, Buenos Aires province. Ó 2013 John Wiley & Sons Ltd 854 S E Plaul et al. Parabrachiella sp. in nasal cavity of mugilids Journal of Fish Diseases 2013, 36, 853–859 Figure 2 Environmental parameters of water in the Salado and Aj
o rivers during 2009. SEM study were critical point-dried and sputtercoated with gold. Specimens were viewed and photographed using Philips SEM 505 and Soft Imaging System ADDA II (SIS) software. Taxonomic identification of the collected specimens to the generic level was made using the keys of Kabata (1979) and Piasecki et al. (2010). Additional infected specimens of grey mullet were fixed in 10% buffered formalin, and representative material was selected and processed for histological examination. Copepods collected from the nasal cavity were prepared for wax embedding and histology following standard methods. Histological sections were stained with haematoxylin and eosin and PAS technique. Cell proliferation within the sections was evaluated by immunohistochemistry using a monoclonal PCNA antibody (19F4, Boehringer) following the procedure of Ortego et al. (1994). Afterwards, the sections were counter-stained with haematoxylin. The prevalence and intensity of infection (Margolis et al. 1981) were calculated for each parasite species in relation to the number of fish infected *100/total number of fish and the total number of parasites/total number of infected fish, respectively (Table 1). Results A total of 228 adult females of the family Lernaeopodidae were found attached to the nasal cavity wall of the grey mullet, M. liza. According to the morphology of the second maxilla and the presence of posterior processes, these specimens were assigned to the genus Parabrachiella. Similarities between species make it extremely difficult to distinguish one species from another, but a high-resolution analysis of the posterior margin of the trunk revealed significant differences in the anal region. All parasitized fish were found in March, prevalence and intensity in both rivers are shown in Table 1. The limnological parameters (Fig. 2) indicated that during the summer, both rivers have high temperature and low salinity levels. Unlike the Salado river, the Aj
o river has a low concentration of dissolved oxygen and a slightly acidic pH, which may explain the higher observed prevalence of the parasite (49%). Table 1 Prevalence and intensity of Parabrachiella sp. from Mugil liza Salado river
river Ajo Ó 2013 John Wiley & Sons Ltd 855 Number of mullet examined Size range, cm Weight, g Prevalence Intensity 65 96 2.13–19.17 3.64–23.40 0.22–67.53 1.05–398.61 1.53% 49.00% 6.00 2.31 Journal of Fish Diseases 2013, 36, 853–859 Pathology Pathogenicity of the parasite was low; the infected fish showed no macroscopic signs of damage at the attachment sites, where no haemorrhage was observed. Evaluation of histological sections by light microscopy showed that alterations in the tissue were consistent with a chronic process. Histological examination revealed local hyperplasia due to irritation caused by the parasite in the attachment site. The bulla of the parasite crosses the epidermis and fixes to the surface of the dermis. Opposed to this site, where the parasite is supported, atrophy in the epithelium of the nasal cavity wall was observed (Fig. 3). Near the point of attachment, cellular infiltration of lymphocytes, eosinophilic granule cells and rodlet cells were detected as part of the host’s inflammatory response (Fig. 4). Although mucous cells were present in the epidermis, their number and size were not affected. The immunohistochemical technique showed that there were numerous PCNA-positive nuclei in both the basal/germinal and middle stratum of the epidermis as compared to the levels seen in uninfected (control) hosts (Fig. 5). Structure of the parasite’s attachment organ SEM microphotographs show that the elongated second maxillae remain separate except for the distal end where they fuse forming a ring around the Figure 3 Position of Parabrachiella sp. within the nasal cavity of its host Mugil liza. Areas of hyperplasia (black arrow) are evident close to the point of parasite attachment (AS). The area where the parasite is attached shows signs of atrophy in the epithelium (white arrows). AS, attachment site; OE, olfactory epithelium; ON, olfactory nerve; OS, olfactory sac; P, parasite. Ó 2013 John Wiley & Sons Ltd 856 S E Plaul et al. Parabrachiella sp. in nasal cavity of mugilids bulla, in which marks of the maxillary suture can be seen (Fig. 6). Light microscopy showed that the bulla, the attachment organ, penetrates the nasal cavity tissue where it is deeply embedded (Fig. 7a). This organ is composed mainly of two parts, a manubrium and an anchor. The manubrium, long and cylindrical, is connected posteriorly to the tips of the second maxillae and, anteriorly, it is expanded forming the anchor which has the shape of a thin cup excavated in the centre and flattened along its whole surface (Fig. 7b). The matrix of the bulla, in longitudinal section, showed the presence of canals crossing the manubrium that opens at the anchor in several pores displayed in a regular circle (Fig. 7 inset). The presence of fibres that stain intensely with eosin and PAS was also detected. Discussion In mugilids, only two species of Parabrachiella have been reported: P. mugilis Kabata, Raibaut & Ben Hassine, 1971 from Liza aurata and L. saliens (see Kabata, Raibaut & Ben Hassine 1971; Benkirane et al. 1999) in the Palearctic region and N. exilis Shiino, 1956 (= P. exilis) from M. platanus (= M. liza) (see Knoff, Luque & Takemoto 1994; Knoff, Luque & Amato 1997) and M. cephalus (Castro Romero & Baeza Kuroki 1986) from Brazil and Chile, respectively, in the Neotropical region. Parabrachiella sp. from the grey mullet is the first species of the genus reported parasitizing the nasal cavities. This microhabitat is remarkable because P. mugilis and P. exilis were reported parasitizing the gills and fins. Members of the genus have, however, been recorded as parasites of the branchial and oral cavities in some marine fish such as Paralichthys californicus (see Piasecki 1993), M. furnieri (see Sardella et al. 1995; Alarcos & Etchegoin 2010), and Trigla lucerna (see Benkirane et al. 1999).The implications of this parasitosis for farmed, ornamental and wild fish in Argentina are unknown. The morphology of the bulla varies markedly within lernaeopodids, but this morphology is quite stable among the species of a genus. Kabata & Cousens (1972) concluded that the morphology is linked with the type of host and distinguished three types of bullae which correspond to three types of hosts: freshwater teleosts, marine teleosts and Elasmobranchii. The bulla of Parabrachiella sp. is included within type II found in S E Plaul et al. Parabrachiella sp. in nasal cavity of mugilids Journal of Fish Diseases 2013, 36, 853–859 (a) (b) Figure 4 Cellular infiltration found in the epithelium as part of the inflammatory response of the host. (a) Rodlet cells (black arrow). (b) Eosinophilic granule cells (white arrows). In both images, an infiltration of lymphocytes can be seen. OE, olfactory epithelium; OS, olfactory sac; P, parasite. (a) (b) Figure 5 Immunohistochemical characterization of PCNA labelled nuclei in the nasal cavity of Mugil liza. (a) In the control group, proliferating nuclei (PCNA-positive) can be seen in a few cells in the basal area of the olfactory sac epithelium (arrows). Inset: detail of epithelium at higher magnification. (b) In the treated group, PCNA-positive cells can be seen in both the basal and middle stratum of the epidermis (arrows). Inset: detail of epithelium at higher magnification. OS, olfactory sac; P, parasite. marine fish. These bullae are consistent with species attached to the tegument and are characterized by large-diameter cups flattened on the host tissues (Benkirane et al. 1999). The pathogenicity of the parasite is low, but lesions that are consistent with a chronic process can be seen. This group is characterized by their relatively large size, and their attachment mechanism, however, its species seem to inflict less damage than other parasitic copepods like Lernaea or Ergasilus. Disturbances in the environment, as well as differentiation and degeneration, are the main factors affecting the cellular components of the epidermis (Kang et al. 1998; Nolan et al. 2000). The Ó 2013 John Wiley & Sons Ltd 857 skin is the first barrier of protection of the body against pathogens; however, few studies have been performed in this organ associated with cell proliferation. The light-microscopical lesions described in the current study resulted in local hyperplasia due to irritation caused by the parasite in the attachment site. Coinciding with these results, the immunohistochemical technique marking PCNA was intense mainly in the middle stratum of the mullet’s nasal cavity epithelium. Changes in the expression of PCNA can provide an early indication of deviations to normal functioning. Dezfuli et al. (2012) have observed that the number of PCNA-positive cells in regions close to the point Journal of Fish Diseases 2013, 36, 853–859 (b) (a) Figure 6 (a) A scanning electron micrograph of a female Parabrachiella sp. 9130. (b) Extremity of second maxillae with bulla, 9250. Note that the elongated second maxillae (M) remain separated and unite only at their distal end around the bulla (Bu). An, antenna; Bu, bulla; C, cephalosome; M, maxillae; PP, posterior processes; T, trunk. (a) S E Plaul et al. Parabrachiella sp. in nasal cavity of mugilids The final effects depend, however, on more than one factor and are influenced by the intensity of infection, the site affected and often by environmental parameters such as temperature, oxygen and salinity level (Kabata 1992). According to Johnson et al. (2004), temperature is the most important environmental factor controlling the developmental time of parasitic copepods and the rate at which their population size increases in the absence of treatments. We observed that these three factors together with the pH are related to prevalence of the parasite. In the case of the Ajo river, low concentrations of dissolved oxygen together with high temperatures and low salinity seem to make fish more prone to parasitic infections. The prevalence and intensity of infection in the current study significantly declined in late autumn, which coincides with the decreasing water temperature and the observations made in other similar studies (Plaul, Garc
ıa Romero & Barbeito 2010). The present study is the first report of the effects of the Parabrachiella sp. penetration in the catadromous fish M. liza. The results may contribute to the implementation of appropriate treatments for different types of physiological changes and the development of preventive techniques in fish farming. This is very important due to the development of aquaculture in many South American countries (Poquet 1979). (b) Figure 7 (a) Insertion of Parabrachiella sp. into the tegument of the olfactory sac of its host Mugil liza. (b) Bulla composed of two parts, manubrium and anchor. Inset: detail of the anchor, the latter is crossed by several canals displayed in a regular circle. A, anchor; Bu, bulla; M, maxillae; Ma, manubrium; OS, olfactory sac; P, parasite. of parasite attachment was significantly higher than the number observed in uninfected individuals. The present work as well as the previous study mentioned demonstrated that increased cell proliferation is a general reaction produced by parasite attachment. The general condition of the fish sampled in this study was not evaluated but because other lernaeopodids have been documented to reduce the egg production of hatchery rainbow trout (Gall, McClendon & Schafer 1972), it is desirable to assess the impact of this parasite on the growth, maturation, and physiology of the host (Nagasawa & Urawa 2002). Ó 2013 John Wiley & Sons Ltd 858 Acknowledgements We would to thank to Lic. Agustin Solari for providing samples of juveniles M. liza and Felicia Cardillo for helping in Parabrachiella collection. We also thank Drs Federico Lozano and Pablo Marino for reading the MS. References Alarcos A.A. & Etchegoin J.A. (2010) Parasite assemblages of estuarine-dependent marine fishes from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). Parasitology Research 107, 1083–1091. Benkirane O., Coste F. & Raibaut A. (1999) On the morphological variability of the attachment organ of Lernaeopodidae (Copepoda: Siphonostomatoida). Folia Parasitologica 46, 67–75. Castro R. & Gonz
alez M.T. (2005) Clavellotis sebastidis sp. nov. (Copepoda, Lernaeopodidae) parasitic on Sebastes oculatus Valenciennes, 1833 from Argentina. Acta Parasitologica 50, 74–79. Journal of Fish Diseases 2013, 36, 853–859 Castro Romero R. & Baeza Kuroki H. (1986) Some species of Neobrachiella Kabata, 1979 (Copepoda: Lernaopodidae) parasitic on Chilean fishes, with description of Neobrachiella paralichthyos sp. nov. from Paralichthys adspersus (Syteindachner). Crustaceana 51, 245–253. Colautti D. (1998) Sobre la utilizaci
on de trampas para peces en las lagunas pamp
asicas. Revista de Ictiolog
ı a 6, 17–23. Dezfuli B.S., Giari L., Lui A., Squerzanti S., Castaldelli G., Shinn A.P., Manera M. & Lorenzoni M. (2012) Proliferative cell nuclear antigen (PCNA) expression in the intestine of Salmo trutta trutta naturally infected with an acanthocephalan. Parasites & Vectors 5, 198. doi: 10.1186/ 1756-3305-5-198 Etchegoin J.A., Timi J.T. & Lanfranchi A.L. (2006) Redescription of Neobrachiella spinicephala (Ringuelet, 1945) parasitic on Pinguipes brasilianus Cuvier. Acta Parasitologica 51, 290–293. Gall G.A.E., McClendon E.L. & Schafer W.E. (1972) Evidence on the influence of the copepod (Salmincola californiensis) on the reproductive performance of a domestical strain of rainbow trout (Salmo gairdneri). Transactions of the American Fisheries Society 101, 345–346. Johnson S.C., Treasurer J.W., Bravo S., Nagasawa K. & Kabata Z. (2004) A review of the impact of parasitic copepods on marine aquaculture. Zoological Studies 43, 229–243. Kabata Z. (1979) Parasitic Copepoda of British Fishes. pp. 468, The Ray Society, The British Museum, London. Kabata Z. (1992) Copepods parasitic on fishes: keys and notes for identification of the species. In: Synopses of the British Fauna (New Series) N° 47. (ed. by D.M. Kermack, R.S.K. Barnes & J.H. Crothers Editors), pp.264. The Linnean Society of London and the Estuarine and Coastal Sciences Association, Oegstgeest, Netherlands. Kabata Z. & Cousens B. (1972) The structure of the attachment organ of Lernaeopodidae (Crustacea: Copepoda). Journal of the Fisheries Research Board of Canada 29, 1015–1023. Kabata Z., Raibaut A. & Ben Hassine O.K. (1971) Eubrachiella mugilis n. sp., un copepode parasite de muges de Tunisie. Bulletin de l’Institut National Scientifique et Technique d’Oceanographie et de Peche de Salammbo 2, 87–93. Kang W.S., Moon Y.W., Han J.W., Park N.G. & Kim H.H. (1998) Induced epidermal cell turnover in the seawateradapted guppy, Poecilia reticulata. Korean Journal of Biological Sciences 2, 521–527. Knoff M., Luque J.L. & Takemoto R.M. (1994) Parasitic copepods on Mugil platanus G€unther (Osteichthyes: Mugilidae) from the coast of the State of Rio de Janeiro, Brazil. Revista Brasileira de Parasitologia Veterin
a ria 3, 45– 56. Knoff M., Luque J.L. & Amato J.F.R. (1997) Community ecology of the metazoan parasites of grey mullets, Mugil platanus (Osteichthyes: Mugilidae) from the littoral of the State of Rio de Janeiro, Brazil. Revista Brasileira de Biologia 57, 441–454. Margolis L., Esch G.W., Colmes J.C., Curie A.M. & Schad G.A. (1981) The use of ecological terms in parasitology. The Journal of Parasitology 68, 133. Ó 2013 John Wiley & Sons Ltd 859 S E Plaul et al. Parabrachiella sp. in nasal cavity of mugilids Nagasawa K. & Urawa S. (2002) Infection of Salmincola californiensis (Copepoda: Lernaeopodidae) on juvenile masu salmon (Oncorhynchus masou) from a stream in Hokkaido. Bulletin of the National Salmon Resources Center 5, 7–12. Nolan D.T., Hadderingh R.H., Spanings F.A.T., Jenner H.A. & Wendelaar Bonga S.E. (2000) Effects of short-term acute temperature elevation on sea trout smolts (Salmo trutta L.) in tap water and in Rhine water: effects on skin and gill epithelia, hydromineral balance and gill specific Na+K+ATPase activity. Canadian Journal of Fisheries and Aquatic Sciences 57, 708–718. € Oktener A. & Trilles J.P. (2009) Four parasitic copepods on marine fish (Teleostei and Condrichthyes) from Turkey. Acta Adriatica 50, 121–128. Ortego L.S., Hawkins W.E., Walker W.W., Krol R.M. & Benson W.H. (1994) Detection of proliferating cell nuclear antigen in tissue of three small fish species. Biotechnic and Histochemistry 69, 317–323. Piasecki W. (1993) Description of Neobrachiella sp. (Copepoda, Siphonostomatoida, Lernaeopodidae) parasitic in the buccal cavity of California halibut (Paralichthys californicus). Wiadomos’ci Parazytologiczne 39, 149–153. Piasecki W., Goddwin A.E., Eiras J.C. & Nowak B.F. (2004) Importance of Copepoda in freshwater aquaculture. Zoological Studies 43, 193–205. Piasecki W., Sezkowska-Jakubowska A. & Sobecka E. (2006) Comparative description of males of two species of Achtheres von Nordmann, 1832 (Copepoda: Siphonostomatoida: Lernaeopodidae) infecting zander and European perch. Folia Parasitologica 53, 211–216. Piasecki W., Mlynarczyk M. & Hayward C.J. (2010) Parabrachiella jarai sp. nov. (Crustacea: Copepoda: Siphonostomatida) parasitic on Sillago sihama (Actinopterygii: Persiformes: Sillaginidae). Experimental Parasitology 125, 55–62. Plaul S.E., Garc
ıa Romero N. & Barbeito C.G. (2010) Distribution of the exotic parasite, Lernaea cyprinacea (Copepoda, Lernaeidae) in Argentina. Bulletin of the European Association of Fish Pathologists 30, 65–73. Poquet M. (1979) Aportaciones al estudio morfol
ogico de algunas especies de cop
epodos de peces del litoral mediterr
aneo. Miscela nea Zoolo gica 5, 161–171. Sardella N.H. & Timi J.T. (1996) Parasite communities of Merluccius hubbsi from the Argentinian-Uruguayan common fishing zone. Fisheries Research 27, 381–388. Sardella N.H., Etchegoin J.A. & Martorelli S.R. (1995) Parasite community of Micropogonias furnieri (whitemouth croaker) in Argentina. Boletim del Instituto Oceanografico de Venezuela 34, 41–47. Sch€aperclaus W.(1991) Diseases caused by copepods. In: Fish Diseases Vol.2 (ed. by W. Sch€aperclaus, H. Kulow & K. Schreckenbach), pp. 908–913. Published for the U.S. Department of the Interior and the National Science Foundation, Washington, DC. Amerind, New Delhi. Received: 27 September 2012 Revision received: 7 December 2012 Accepted: 7 December 2012