Levinseniella (Levinseniella) gavilanicola sp. nov. (Trematoda: Microphallidae) en el gavilan cangrejero Buteogallus anthracinus de Venezuela y nota sobre su ciclo de vida.
The trematodes of the family Microphallidae are small parasites present in the digestive tract of most vertebrates. The genus Levinseniella Stiles and Hassall, 1901, contained more than 40 species described mainly from birds around the world, and metacercariae of these species use mainly crustaceans as second intermediate hosts. Of these, only three especies have been described from South America: Levinseniella (M.) cruzi  in Anas bahamensis in Brasil; L. anenteron Szidat, 1964  in Larus marinus dominicanus in Argentina, and L. (L.) venezuelensis  in Ereunetes pusillus in Venezuela. A few studies on life cycles of Levinseniella have been previously reported on L (L.) brachysoma (Creplin, 1837) Stiles and Hassall, 1901; L (L.) propinqua Jaegerskiold, 1907; L (M.)cruzi ; L (M.) amnicolae ; L (M.) ophidea ; and L. deblocki . To date, only L (L.) venezuelensis has been described in Venezuela. A new microphallid, L (L.) gavilanicola sp. nov. is described in Venezuela from both naturally and experimentally infected vertebrate hosts. The metacercariae of this species were found in the decapods Uca rapax, which served as second intermediate hosts. However, the first intermediate host still needs to be elucidated.
MATERIALS AND METHODS
The study was carried out at Cajigal Municipality, Sucre State, between 1993 and 1999. In 1993, one Buteogallus anthracinus (Deppe, 1830; Falconiformes: Accipitridae) was caught using Japanese nets from El Paujil, Cajigal Municipality, Sucre State, Venezuela. Ten digeneans were recovered from the small intestine, intestinal caecum, and rectum of the bird, and the worms were fixed under cover slip pressure in 5% hot formalin and stored in 70% ethanol. In addition, during 1999, 27 specimens of Uca rapax (Smith, 1870; Brachyura: Ocypodidae) were collected during the period of low tide from La Ceiba and El Paujil. The carapace of the crab was removed by cutting with scissors around the margin of the dorsal surface, its internal tissues were removed, and examined under dissecting microscope. Metacercarial cysts were collected from the abdominal tissues that cover the insides of the dorsal carapace and hepatopancreas. Cysts were preserved in 0,75% saline for further studies and to be used in experimental infections. Twenty metacercarial cysts were force-fed with pipette to two chicks (Gallus gallus domesticus Linnaeus, 1758) and one duck (Cairina moschata Linnaeus, 1758). In addition, two rats (Rattus norvegicus Berkenhout, 1758) and three mice (Mus musculus Linnaeus, 1758) were fed with infected crabs. The experimental animals were examined 4-10 days postinfection and 120 adult trematodes were recovered from small intestine, of which 13 from chicks, 11 from the duck, 28 from rats and 68 from mice. Due to difficulties in studying live metacercariae, these were allowed to excyst naturally in 0.50% solution of trypsin in phosphate buffer at pH 7. 2 and at 26[grados]C. Metacercariae were excysted after four hours in the solution. Some excysted metacercariae and adults were studied live, and other were used for permanent preparations which were killed in hot 0.75% saline, fixed in AFA (alcohol-formalin-acetic acid), and stained in Semichon's acetocarmine after dehydration in a series of ascending alcohols, cleared in oil cloves and mounted in Canada balsam. The drawings were made with a drawing tube on a Carl Zeiss Axioscope microscope attachment, modelo A1, Germany.
All measurements are given in micrometers ([micro]m). The trematodes were identified using the keys of some investigators [3, 7, 8, 14].
RESULTS AND DISCUSSION
Levinseniella (Levinseniella) gavilanicola sp. nov. (FIGS. 1-8).
Description of adult fluke (FIGS. 1-4, 8). Fifty specimens were studied and the measurents are given on TABLE I. Body with linguiform shape, tegument with spines, extending up to the end of intestinal caeca. Preoral lobe weakly developed. Subterminal oral sucker, with two non glandular lateral auricular papillae and a muscular ring at posterior end. Prepharynx, well developed, slightly thick, followed by a muscular pharynx. A long and wide oesophagus bifurcates to two intestinal caeca over anterior to seminal vesicle, extending posteriorly to the lateral margins at the level of acetabulum. Round acetabulum, smaller than oral sucker. Testes symmetrical, post acetabular. Efferent tubes leave towards anterior part of body at the middle of ventral part of each testis, joining at equatorial level of acetabulum forming deferent duct, entering posterior to seminal vesicle. Elongate seminal vesicle, located anterior to the acetabulum, filled with sperms, covered with a fine non muscular membrane, which continues over the prostatic complex. Pars prosistica relatively short, surrounded by prostatic cells. Short ejaculatory duct, ending in a muscular ovoid male genital papilla, projecting into the genital atrium. Genital pore located on the left of acetabulum, opening to the genital atrium, above or sideways of the male genital papilla. Genital atrium located on the left side and close to acetabulum, containing nine accessory atrial sacs (also called atrial fingerlings), with two rows of sclerotized bars, one with four on the base of atrium and the other with five over it.Well developed female pouch without sclerotic structures, connected with genital atrium by a muscular layer at the base of the male genital papilla. Ovary with smooth or slightly wavy edges, located to the right of acetabulum, immediately anterior to right testis, the oviduct emerges on mid lateral side of ovary, it enlarges immediately to form ootype, surrounded by Mehlis glands, which later forms a small seminal receptacle and Laurer's canal. The oviduct extends posteriorly to unite with the uterus and the main vitellogenic duct. Uterus postacetabular, lateral branches sometimes reach to anterior margin of testes. Lightly muscular metraterm reaches genital atrium close to the male genital papilla. Small eggs, operculate, yellow in color. Vitelline cells localized at postero-lateral to testes, forming two groups of 6-8 follicles on each side. V-shaped excretory bladder, excretory pore subterminal surrounded by glandular cells.
Type host: Natural definitive host: Buteogallus anthracinus.
Experimental Hosts: Chick (Gallus gallus domesticus), duck (Cairina moschata), mouse (Mus musculus), rat (Rattus norvegicus).
Site of infection: Adults in small intestine, intestinal caecum and rectum.
Second intermediate host: Fiddler crab (Uca rapax).
Site of infection: Metacercaria: Tissues that covers the insides of the dorsal carapace and hepatopancreas.
Location: La Ceiba, El Paujil, Cajigal Municipality, Sucre State, Venezuela.
Holotype: It is deposited in the Parasitological Collection of the Institute for Research in Biomedicine and Applied Sciences "Dr. Susan Tai", Universidad de Oriente, Cumana, Sucre State, Venezuela, IIBCA No. 1226.
Paratype: IIBCA No. 1227, at the above address.
Etymology: The specific name is derived from the common name of the natural host, gavilan.
Metacercariae (FIGS. 5-7).
Description. (Based on 23 specimens from naturally infected Uca rapax, 13 from La Ceiba and 10 from El Paujil, and measurements based on 46 worms and given in micrometers (pm)). Cyst almost spherical, 536-593 X 512-570, double walled, thick outer membrane, whitish, and internally hyaline. Body of excysted metacercariae elongate, 948-1,871 x 256-589, slightly reduced in the preacetabular region. Integument covered with spines that reach acetabular level. Preoral lobe, 5 x 20, not well developed. Conspiscuous nervous system, with a nerve commissure located above the pharynx, two short antero-lateral nerves reach the oral sucker and two long posterior nerves reach the level of seminal vesicle. Oral sucker well developed, 95-181 x 101-191, with a pair of non glandular lateral auricular papillae and a muscular post oral ring. Preacetabular distance: 436-1,096. Short prepharynx: 10-30 x 25-166; Muscular pharynx: 50-166 x 10-65. Oesophagus, 151-414 x 10-30, long and wide; short intestinal caeca, extending to acetabular level. Acetabulum almost spherical, 95-151 x 95-136, postequatorial, spineless. Postacetabular distance: 223-558. Symmetrical testes, right testis: 30-70 x 75-131, and left testis: 35-70 x 35-70. Posttesticular distance: 203-456. Arched seminal vesicle, 50343 x 50-277, full of sperms, between intestinal caeca and acetabulum. Bulbous pars prostatica, 34-129 x 53-161, well developed with many prostate cells, surrounded by a thin membrane with a short ejaculatory duct ending in a male genital papilla, located over the genital atrium. Genital pore opens to genital atrium. Genital atrium lateral to the acetabulum, containing nine accessory atrial sacs, with sclerotized structures in the form of bars, arranged in two rows, one anterior with five and one posterior with four. Female pouch lies adjacent to the genital atrium. Oval ovary, 40-161 x 75-181, anterior to right testis, oviduct short, ootype cells surrounded by Mehlis glands. Small seminal receptacle. Uterus lies posttesticular, some metacercariae (from La Ceiba) contained some developing eggs, 16-21 x 10-16. Muscular metratermo.Vitelline glands well developed, located in two lateral groups of 6 and 8 follicles each . Excretory bladder "V" shaped, the primary excretory ducts are divided at the junction of the oesophageal bifurcation, excretory duct divided into anterior and posterior, each with four flame cells. Flame cells formula: [(2 + 2) + (2 + 2)] = 16. Sub-terminal excretory pore, surrounded by glandular cells.
According to Deblock , this new species must be included in the family Microphallidae Ward, 1901, subfamily Levinseniellinae Stiles and Hassall, 1901, and in the genus Levinseniella (Levinseniella) Stiles and Hassall, 1901, on the basis of the morphological features of the worms. Members of the genus Levinseniella are very similar morphologically, the features of some organs, i.e., the accessory atrial sacs, presence or absence of female pouch are used to differentiate the species. Heard  revised the genus Levinseniella and divided it into four groups based on the number of accessory atrial sacs and the presence or absence of female pouch. Deblock and Pearson  reviewed the genus. Levinseniella and created two new subgenus, Levinseniella and Monarrhenos to include those species with or without female pouch.Yamaguti  created a new genus, Heardlevinseniella, based on the absence of female pouch and having a large oral sucker with lateral auricular papillae and a muscular ring on its posterior part. Overstreet and Perry  considered that the diagnostic characters that Yamaguti used to differentiate these two subgenera are not sufficiently viable and synonimised the subgenus Heardlevinseniella to Levinseniella. According to the classification of Heard , Levinseniella (L.) gavilanicola sp. nov., clearly belongs to Group III (numerous accessory atrial sacs (7 or more) and presence of female pouch), and according to Deblock , the new species falls into genus Levinseniella (Levinseniella) Stiles and Hassall, 1901.
Levinseniella (L.) carteretensis  is the only species included by Heard  into the group III, which is very similar to the new species described herein, but differs due to the absence of a pair of lateral auricular papillae in the oral sucker and a postoral muscular ring; in addition, it bears a weakly developed male papilla and 7-10 accessory atrial sacs. L (M.)hunteri  is the only species of the genus Levinseniella that shares with L (L.) gavilanicola sp. nov., the same number of accessory atrial sacs (9) and their arrangement in the wall of the genital atrium. However, the new species can be distinguished from L (M.) hunteri by the presence of sclerotized parts in its accessory atrial sacs, a pair of lateral auricular papillae on the oral sucker and female pouch. Levinseniella (L.) gavilanicola sp. nov., differs from all the members of the genus Levinseniella described in South America, i.e., L (M.) cruzi  from Brazil. L. anenteron (Szidat, 1964)  from Argentina and L (L.) venezuelensis  from Venezuela. L (M.) cruzi lacks a pair of lateral auricular papillae, post-oral muscular ring, female pouch and its genital atrium contains 6-10 accesssory atrial sacs without sclerotized structures and its vitellaria contains 10-12 folicles on each size of the body. Further, L. (M) cruzi used the birds, Rollandia. rolland chilensis and Himantopus melanurus as natural hosts and the freshwater shrimp Palaemonetes argentinus as natural intermediate secondary host. The absence of a digestive system and the presence of four accessory atrial sacs in L. anenteron, serves to separate it from the new species. L (L.) venezuelensis shared with the present species in possessing a pair of lateral auricular papillae on its oral sucker, a postoral muscular ring and the presence of female pouch. However, the more distinct difference between both species are the number of accessory atrial sacs (six in L (L.) venezuelensis and nine in L (L.) gavilanicola sp. nov.), without sclerotized structures in the former and at least 2-3 row of spines on the acetabulum. According to Fischthal and Nasir  the ring of acetabular spines appears unique for the species.
A few studies on the life cycles of Levinseniella have been reported on: L (L.) brachysoma, L (L.) propinqua, L (M) cruzi, L (M.) amnicolae, L (M.) ophidea, and L. deblocki. The first two species can be distinguished from L (L.) gavilanicola by having three and four accessory atrial sacs and by the absence of lateral auricular papillae and postoral ring (reduced in L (L.) propinqua). In addition by their use on of Isopods and Amphipods (only in L (L.) propinqua) as second intermediate hosts and Charadriformes and Anseriformes birds, as definitive hosts. L (L.) cruzi has already been differentiated from L (L.) gavilanicola (see above). L (M.) amnicolae is distinguished from the new species by its smaller digestive system, the number of accessory atrial sacs (4), without sclerotized structures, and the absence of female pouch, and an oral sucker without lateral auricular papillae and postoral ring. Additionally, it uses Asellus communis as second intermediate host. L (M.) ophidea can be separated from L (L.) gavilanicola sp. nov. by the use of Thamnophis elegans (snake) and Rana catesbeiana (bullfrog) as natural definitive hosts and several species of leeches: Glossiphoniaea complanata, Helobdella stagnalis, Erpodella punctate and Dina parva as second intermediate hosts. Also, by the absence of lateral auricular papillae, postoral ring and female pouch. In addition, L (M.) ophidea possesses 9-15 accesory atrial sacs and its vitellaria has two groups of 5-10 follicles each. Levinseniella (M.) deblocki, can be distinguished from the present species by having more than 9 accesory atrial sacs and by lacking a female pouch. Considering the differences described earlier of L (L.) gavilanicola sp. nov. from its closely related species and for being the only species reported from the hawk, Buteogallus anthracinus, as its natural host, and the crab Uca rapax as second natural intermediate host, it appears reasonable to consider the species of the present study as a new species of the genus Levinseniella Stiles and Hassall, 1901.
A new species, Levinseniella (Levinseniella) gavilanicola (Digenea: Microphallidae) and its life cycle is described for the first time in Venezuela from naturally infected crab hawk, Buteogallus anthracinus (type host). The new species differs from other species of this group mainly in the number of atrial accessory sac (9 with sclerotized structures) and its arrangement in the wall of the genital atrium. The adults worms were recovered experimentally from chicken, duck, rat and mice after infecting them with metacercariae from the crab Uca rapax.
Recibido: 14/10/2014. Aceptado: 30/04/2015.
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Marcos Julio Diaz (1), Erika Gomez (1), Abul Kashem Bashirullah (2) and Del Valle Guillarte (1)
(1) 1nstituto de Investigaciones en Biomedicina y Ciencias Aplicadas "Dra. Susan Tai". (2) Instituto Oceanografico de Venezuela, Universidad de Oriente, Cumana, estado Sucre, Venezuela. firstname.lastname@example.org
TABLE I MEASUREMENTS OF Levinseniella (Levinseniella) gavilanicola sp. nov. Host Buteogallus Gallus(Gallus) Rattus anthracinus domesticus norvegicus Number 8 7 28 Examined Body Length 1,045-1,776 1,051-1,358 820-1,666 Breadth 476-629 410-43 435-512 Oral sucker 147-174 126-156 121-182 107-161 131-161 101-192 Prepharynx 40-120 40-50 50-101 20-40 20-30 20-40 Pharynx 60-90 70-80 55-85 50-80 50-60 50-75 Oesophagus 160-410 136-166 101-303 10-20 20-25 15-25 Seminal vesicle 60-121 55-90 65-111 202-262 227-292 181-282 Acetabulum 100-153 116-121 101-142 112-139 106-111 101-142 Right testis 30-50 45-50 40-111 80-112 60-126 80-126 Left testis 30-60 65-85 45-116 80-120 50-90 101-151 Pars prostatica 80-107 69-75 43-91 90-134 75-134 59-134 Ovary 80-150 75-111 55-126 140-180 126-151 75-176 Eggs 18-24 21-24 18-26 10-13 10-13 10-16 Host Mus Cairina musculus moschata Number 18 5 Examined Body Length 487-1,333 1,051-1,615 Breadth 282-461 384-512 Oral sucker 136-161 126-166 60-161 131-161 Prepharynx 20-75 75-101 15-45 20-25 Pharynx 30-90 65-95 25-70 50-65 Oesophagus 126-181 126-328 15-20 15-30 Seminal vesicle 50-85 60-116 80-287 151-252 Acetabulum 55-126 111-136 55-121 101-141 Right testis 40-50 45-55 40-106 80-176 Left testis 40-50 45-70 60-80 75-116 Pars prostatica 40-72 67-99 59-107 80-134 Ovary 60-29 60-146 65-146 121-181 Eggs 21-26 18-24 10-13 10-13