New host record and geographical distribution of Nematoda parasitizing Hypophthalmus marginatus Valenciennes (Siluriformes) from the Tocantins River, Brazil

Hypophthalmus marginatus is among the most commercialized fish from the Amazon region, with fish fillets exported to Southeastern Brazil and other countries. In the present study, the H. marginatusanalyzed were parasitized by third-stage larvae of Hysterothylaciumsp. and Anisakissp. These nematodes are being reported for the first time in H. marginatus collected from the Tocantins River, representing a new host and geographical records, and expanding knowledge of the hosts of these nematodes in the Neotropical Region. A brief description with original measurements is presented.

Anisakid Larvae from Anchovies in the South Coast of Korea

Anisakiasis (anisakidosis) refers to a foodborne zoonosis caused by ingesting raw or undercooked marine fish or cephalopods infected with anisakid larvae. The present study was performed to investigate the prevalence of anisakid larvae in anchovies (Engraulis japonica) purchased from 2 local markets in Gyeongsangnam-do, the Republic of Korea (=Korea), during 2018–2019. Anchovies were transported to our laboratory and examined by pepsin-HCl artificial digestion technique followed by microscopic observations and molecular analyses. The overall prevalence of anisakid larvae was 19.5% (39/200), from which a total of 51 larvae (av. 1.3 larvae/infected anchovy) were recovered. Sequencing of the larvae targeting the ITS region, including ITS1, 5.8S rRNA, and ITS2 genes confirmed the species of larvae as Anisakis pegreffii (54.9%; 28/51), Hysterothylacium sinense (23.5%; 12/51), and Hysterothylacium aduncum (21.5%; 11/51). The results suggested that anchovies could be a potential source of human anisakiasis in Korea.

Molecular Identification of Anisakis Larvae Extracted by Gastrointestinal Endoscopy from Health Check-up Patients in Korea

Anisakiasis is a zoonotic disease induced by anisakid nematodes, and endoscopic inspection is used for a diagnosis or remedy for it. Anisakis simplex, Anisakis physeteris, and Pseudoterranova decipiens had been reported to be the major species causing human infections, particularly, in Japan. However, in Korea, recent studies strongly suggested that Anisakis pegreffii is the major species of human infections. To support this suggestion, we collected anisakid larvae (n=20) from 20 human patients who were undergone gastrointestinal endoscopy at a health check-up center in Korea, and molecular identification was performed on the larvae using PCR-RFLP analysis and gene sequencing of rDNA ITS regions and mtDNA cox2. In addition, anisakid larvae (n=53) collected from the sea eel (Astroconger myriaster) were also examined for comparison with those extracted from humans. The results showed that all human samples (100%) were identified as A. pegreffii, whereas 90.7% of the samples from the sea eel were A. pegreffii with the remaining 9.3% being Hysterothylacium aduncum. Our study confirmed that A. pegreffii is the predominant species causing human anisakiasis in Korea, and this seems to be due to the predominance of this larval type in the fish (sea eels) popularly consumed by the Korean people. The possibility of human infection with H. aduncum in Korea is also suggested.

Histopathological changes induced by Hysterothylacium deardorffoverstreetorum larvae (Nematoda: Raphidascarididae) in Priacanthus arenatus Cuvier, 1829 (Actinopterygii) / Alterações histopatologicas induzidas por larvas de Hysterothylacium deardorffoverstreetorum (Nematoda: Raphidascarididae) em Priacanthus arenatus Cuvier, 1829 (Actinopterygii)

Abstract Priacanthus arenatus is a fish that occurs in the waters of the western Atlantic Ocean and has few records of parasitism. This study aimed to report histological changes caused by the larvae of Hysterothylacium deardorffoverstreetorum. Between July and December, 2013, 30 samples of P. arenatus were obtained from fish markets in the cities of Niterói and Rio de Janeiro, state of Rio de Janeiro, Brazil. Fragments of spleen, liver, stomach wall and mesentery tissues containing nodules and free parasites were extracted to histopathological study. Through histological analysis it was verified that these nodules consisted of connective tissue capsules, in which the parasites were located, with granulomatous inflammation mainly comprised of macrophages and lymphocytes. These nodules also consisted of a central portion of acellular amorphous material, formed by concentric layers, which was slightly basophilic and contained tunnels and, sometimes, parasite debris. The presence of tunnels in acellular amorphous material suggests that the larvae are initially located at the center of the nodules and then they migrate to the surface.

Resumo Priacanthus arenatus é um peixe que ocorre nas águas do Atlântico ocidental com poucos relatos de parasitimo. Este estudo objetivou relatar as características histológicas causadas pelas larvas de H. deardorffoverstreetorum em P. arenatus. Entre julho e dezembro de 2013, 30 espécimes de P. arenatus foram obtidas de mercados de peixes das cidades de Niterói e Rio de Janeiro, Estado do Rio de Janeiro, Brasil. Fragmentos de baço, fígado, parede do estômago e mesentério contendo nódulos foram extraídos para estudo histopatológico. Foi observado através de análise histológica que esses nódulos eram constituídos por uma cápsula de tecido conjuntivo, onde se localizavam os parasitas viáveis, com infiltrado inflamatório granulomatoso, constituído, predominantemente, por macrófagos e linfócitos e por porção central de material amorfo acelular, formado por lâminas concêntricas, ligeiramente basofílico, contendo túneis e, por vezes, restos parasitários. A presença de túneis no material amorfo acelular dos nódulos analisados histologicamente sugerem que as larvas se localizam inicialmente no centro do granuloma e depois migram para a superfície do mesmo.

Resposta imunológica a antígenos de Hysterothylacium deardorffoverstreetorum de peixes teleósteos / Imunne response against Hysterothylacium deardorffoverestreetorum from teleost fish

Anisaquidose é uma doença provocada por parasitos da família Anisakidae e se caracteriza por manifestações gastrointestinais e alérgicas. O Anisakis simplex é o parasito mais patogênico ao homem e altamente alergênico. Porém, outros anisaquídeos também são danosos aos humanos, mas é desconhecida a imunogenicidade dessas larvas. O objetivo deste trabalho foi avaliar o potencial imunogênico do parasito Hysterothylacium deardorffoverestreetorum (HD) em modelo murino. Camundongos da linhagem BALB/c foram divididos em três grupos experimentais e receberam as preparações antigênicas obtidas de larvas de HD: extrato bruto de larvas (EBH), extrato secretado/ excretado de larvas (ESH) e extrato bruto de larvas após excreção/secreção (EEH). Amostras séricas foram obtidas em diferentes dias após imunização para determinação dos níveis de anticorpos específicos pelo ensaio imunoenzimático (ELISA). Os resultados demonstram aumento na produção de imunoglobulina (Ig) G após a segunda imunização, com aumento progressivo após a terceira imunização. Já em relação à IgE, a reatividade foi mais tardia, demonstrando aumento progressivo após a terceira imunização. Foi avaliada a imunidade celular por meio da intradermorreação, como resultado estatisticamente significativo em relação ao controle utilizado. Este experimento é a primeira descrição da potencialidade patogênica desse parasito em mamíferos e representa um avanço no diagnóstico da anisaquidose humana.(AU)

Anisaquidosis is a disease caused by parasites of Anisakidae family and is characterized by gastrointestinal and allergic reactions. The Anisakis simplex is a more pathogenic Anisakidae to humans and is highly allergenic. However, other species of this family also have characteristics that are harmful to humans, but little is known about the immunogenicity this parasites. The objective of this study was to experimentally assess the immunogenic potential of the parasite Hysterothylacium deardorffoverestreetorum (H.D) in mice. Mice of inbred BALB/c strain were divided into three groups and received three immunizations of the following antigenic preparations obtained from L3 larvae H.D: Crude larval extract of H.D (CEH) Extract secreted / excreted larvae H.D. (ESH) and crude extract of larvae after excretion / secretion (EEH). Serum samples were obtained on different days after immunization to determine the levels of circulating specific antibodies by enzyme-linked immunosorbent assay (ELISA). The results show increased production of immunoglobulin (Ig) G after the second immunization with a gradual increase after the third immunization. Regarding IgE reactivity, this occurred later, demonstrating a progressive increase only after the third immunization. Cellular immunity was evaluated by intradermal, and showed statistically significant result compared to the control used. This experiment is the first description of the pathogenic potential of this parasite in mammals and represents a breakthrough in the diagnosis of human Anisakidosis.(AU)

Anisakidae and Raphidascarididae larvae parasitizing Selene setapinnis (Mitchill, 1815) in the State of Rio de Janeiro, Brazil / Larvas Anisakidae e Raphidascarididae parasitos de Selene setapinnis (Mitchill, 1815) no Estado do Rio de Janeiro, Brasil

Between February and August, 2012, thirty specimens of Atlantic moonfish, Selene setapinnis, were purchased in local markets in Niterói, State of Rio de Janeiro, Brazil, with the aim of analyzing the presence of anisakid nematodes, establishing their rates of parasitism and infection sites, due to importance in the sanitary inspection. A total of sixty nematode larvae, belonging to at least two species were found: nine larvae of Terranova sp., Anisakidae, with prevalence (P) of 13.3%, mean intensity (MI) of 2.25, mean abundance (MA) of 0.30 and range of infection intensity (RI) from 1 to 6; and 51 larvae of Hysterothylacium fortalezae, Raphidascarididae, with P = 26.7%, MI = 6.40, MA = 1.70, and RI = 1-17. The infection sites for Terranova sp. were the mesentery and liver serosa; and for H. fortalezae, the infection sites were the mesentery, abdominal cavity and liver serosa. New morphological data from scanning electron microscopy, on the external structures of H. fortalezae (mainly at the posterior end), are presented. This is the first report of H. fortalezae parasitizing S. setapinnis.

De Fevereiro a Agosto de 2012, trinta espécimes de peixe galo, Selene setapinnis, foram adquiridos de mercados locais em Niterói, Estado do Rio de Janeiro, Brasil, com o objetivo de analisá-los quanto à presença de nematoides anisaquídeos, estabelecendo seus índices parasitários e sítios de infecção, devido a sua importância na inspeção sanitária. Do total de sessenta larvas de nematoides, pelo menos duas espécies foram encontradas: nove larvas de Terranova sp., Anisakidae, com prevalência (P) de 13,3%, intensidade média (IM) de 2,25, abundância média (AM) de 0,30, e amplitude de variação da intensidade de infecção (AI) de 1 a 6; e 51 larvas de Hysterothylacium fortalezae, Raphidascarididae, com P = 26,7%, IM = 6,40, AM = 1,70, e AI = 1 a 17. Os sítios de infecção para Terranova sp. foram o mesentério e a serosa do fígado; e para H. fortalezae, os sítios de infecção foram o mesentério, cavidade abdominal e serosa do fígado. Novos dados morfológicos das estruturas externas, principalmente da extremidade posterior de H. fortalezae, são evidenciados por microscopia eletrônica de varredura. Este é o primeiro relato de H. fortalezae parasitando S. setapinnis.

Parasites of the Mutton Snapper Lutjanus analis (Perciformes: Lutjanidae) in Alagoas, Brazil / Parasitas de Cioba Lutjanus analis (Perciformes: Lutjanidae) em Alagoas, Brasil

A parasitological survey was carried out on a sample of sixty mutton snappers (Lutjanus analis) that were caught on the coast of Alagoas, northeastern Brazil. The parasite diversity and infection levels were low. The ectoparasite Rocinela signata Schioedte & Meinert, 1879 (Isopoda: Aegidae), and larvae of two endoparasites, Trypanorhyncha gen. sp. and Hysterothylacium sp. (Nematoda: Anisakidae), were detected. The significance of these parasites is discussed in the context of their transmission pathways and potential impact.

Um estudo parasitológico foi realizado em 60 exemplares de cioba, Lutjanus analis, capturados ao largo de Alagoas, no Nordeste do Brasil. A diversidade e níveis de infecção parasitária foram baixos. Foram detectados o ectoparasito Rocinela signata Schioedte & Meinert, 1879 (Isopoda: Aegidae) e dois endoparasitos, larvas de Trypanorhyncha gen. sp. e larvas de Hysterothylacium sp. (Nematoda: Anisakidae). O significado desses parasitos é discutido no âmbito das suas vias de transmissão e potencial impacto.

Endohelminths in Cichla piquiti (Perciformes, Cichlidae) from the Paraná river, São Paulo state, Brazil / Endohelmintos de Cichla piquiti (Perciformes, Cichlidae) procedentes do rio Paraná, estado de São Paulo, Brasil

Fifty specimens of Cichla piquiti were collected from the Paraná River downstream of the Ilha Solteira Hydroelectric Power Station in Brazil and surveyed for endohelminth parasites. All fish were parasitised by at least one helminth species (overall prevalence [P] = 100%). Eight parasite taxa were present: the nematode Procamallanus (Procamallanus) peraccuratus in the intestines; third-stage larvae of the anisakids Contracaecum sp. and Hysterothylacium sp. in the visceral cavity, mesentery and serosa of the stomach and intestines and on the liver and spleen; the trematodes Austrodiplostomum compactum in the eye (metacercariae) and Genarchella genarchella in the stomach; and the cestodes Proteocephalus macrophallus, Proteocephalus microscopicus, and Sciadocephalus megalodiscus in the intestines. Hysterothylacium sp. larvae (P = 86%) and P. microscopicus (P = 74%) were the most prevalent parasites. Anisakids were more prevalent and abundant in the dry season. A negative correlation between the abundances of Hysterothylacium sp. and P. microscopicus was observed, suggesting a competitive/antagonistic relationship between these parasites. Cichla piquiti represents a new host for four parasite species. These new records significantly increase the list of parasites of C. piquiti, contributing to the knowledge of the host-parasite relationship and the geographical distribution of these helminths.

Cinquenta espécimes de Cichla piquiti foram colhidos do Rio Paraná, a jusante da Usina Hidrelétrica de Ilha Solteira, Brasil, na pesquisa de endohelmintos. Todos os peixes estavam parasitados por, pelo menos, uma espécie de helminto (prevalência total [P] = 100%). Foram encontrados oito taxa de parasitas: os nematóides Procamallanus (Procamallanus) peraccuratus no intestino; larvas de terceiro estádio dos anisaquídeos Contracaecum sp. e Hysterothylacium sp. na cavidade abdominal, mesentério e serosa do estômago e do intestino, e sobre o fígado e baço; metacercárias do trematódeo Austrodiplostomum compactum no olho e um indivíduo adulto de Genarchella genarchella no estômago; os cestóides Proteocephalus macrophallus, Proteocephalus microscopicus e Sciadocephalus megalodiscus no intestino. Larvas de Hysterothylacium sp. (P = 86%) e P. microscopicus (P = 74%) foram os parasitas mais prevalentes. Anisaquídeos apresentaram maior prevalência e abundância no período seco. Foi observada uma correlação negativa entre as abundâncias de Hysterothylacium sp. e P. microscopicus, sugerindo uma relação de antagonismo/competição entre esses parasitas. Cichla piquiti é descrito como novo hospedeiro para quatro espécies de parasitas. Estes novos registros aumentam significativamente a lista de parasitas de C. piquiti, contribuindo para o conhecimento da relação parasito-hospedeiro e distribuição geográfica desses helmintos.

Nematodes of zoonotic importance in Cynoscion guatucupa (Pisces) in the state of Rio de Janeiro / Nematóides de importância zoonótica em Cynoscion guatucupa (Pisces) no estado do Rio de Janeiro

Between January and August 2012, thirty specimens of Cynoscion guatucupa (Cuvier, 1830) caught off coast of the municipality of Itajaí, state of Santa Catarina, Brazil (26° 54' 28″ S and 48° 39' 43″ W) and commercialized in the state of Rio de Janeiro were investigated for the presence of zoonotic nematodes. In total 853 larvae of four nematode parasite species were found. There were three species of Anisakidae: three larvae of Anisakis sp., with prevalence (P) of 10%, mean intensity (MI) of 1.0, mean abundance (MA) of 0.1 and infection site (IS) in the mesentery; five of Terranova sp. with P = 13.3%, MI = 1.25, MA = 0.17, range of infection (RI) from 1 to 2 and IS = mesentery; and seven of Contracaecum sp. with P = 6.6%, MI = 3.5, MA = 0.23, RI = 1 to 4 and IS = mesentery and abdominal cavity. There was one species of Raphidascarididae: 838 larvae of Hysterothylacium deardorffoverstreetorum with P = 83.3%, MI = 33.52, MA = 27.93, RI = 1 to 219 and IS = mesentery, liver serosa and abdominal cavity. This is the first report of larvae of Anisakis sp. and Contracaecum sp in C. guatucupa in Brazil.

Entre janeiro e agosto de 2012, 30 espécimes de Cynoscion guatucupa (Cuvier, 1830) pescados no litoral do município de Itajaí, estado de Santa Catarina, Brasil (26° 54' 28″ S e 48° 39' 43″ W) e comercializados no estado do Rio de Janeiro, foram investigados quanto á presença de nematóides zoonóticos. Foram encontradas 853 larvas de quatro espécies de nematóides parasitos. Três espécies pertencem á Anisakidae, sendo três larvas de Anisakis sp., com prevalência (P) de 10%, intensidade média (IM) de 1, abundância média (AM) de 0,1 e sítio de infecção (SI) o mesentério; cinco de Terranova sp., com P = 13,3%, IM = 1,25, AM = 0,17, amplitude de variação da intensidade de infecção (AI) de 1 a 2 e SI = mesentério; e sete de Contracaecum sp., com P = 6,6%, IM = 3,5, AM = 0,23, AI = 1 a 4 e SI = mesentério e cavidade abdominal. Uma espécie pertence á Raphidascarididae, sendo 838 larvas de Hysterothylacium deardorffoverstreetorum com P = 83,3%, IM = 33,52, AM = 27,93, AI = 1 a 219 e SI = mesentério, serosa do fígado e cavidade abdominal. Este é o primeiro registro de larvas de Anisakis sp. e Contracaecum sp. em C. guatucupa no Brasil.

Anisakidae nematodes in the blackfin goosefish, Lophius gastrophysus Miranda-Ribeiro, 1915 purchased in the State of Rio de Janeiro, Brazil / Nematoides Anisakidae no peixe sapo-pescador, Lophius gastrophysus Miranda-Ribeiro, 1915 comercializado no Estado do Rio de Janeiro, Brasil

The blackfin goosefish is included in a prized fish category, representing valuable fishery resource worldwide. The aim of this study was to identify the Anisakidae larvae parasitizing this fish species, considering the hygienic-sanitary and public health importance of these parasites infecting specimens of the blackfin goosefish, Lophius gastrophysus Miranda-Ribeiro, 1915 (Lophiidae), purchased from markets in the municipalities of Cabo Frio, Niterói, Duque de Caxias and Rio de Janeiro; parasitological indices and sites of infection are presented. From March 2000 to December 2005, 87 specimens of Lophius gastrophysus were investigated for helminths. Seventeen fish (19.5%) were parasitized with larvae of anisakid nematodes. Anisakis simplex , Hysterothylacium sp. and Raphidascaris sp. appeared with prevalence of 1.14, 12.6 and 12.6%, intensity and mean intensity of infection of 1, 2.81, 10.5 and mean abundance of 0.01, 0.35 and 1.33 respectively. The sites of infection were stomach, stomach and intestine serosas and abdominal cavity. This is the first record of Anisakis simplex, Hysterothylacium sp. and Raphidascaris sp. for this species of fish.

Nematoides Anisakidae no peixe sapo-pescador, Lophius gastrophysus Miranda-Ribeiro, 1915 comercializados no Estado do Rio de Janeiro, Brasil. O peixe sapo-pescador é muito apreciado e representa um valioso recurso pesqueiro em nível mundial. O objetivo deste estudo foi o de identificar larvas de Anisakidae pela sua importância higiênico-sanitária e na saúde pública, cujos parasitos infectam espécimes do peixe sapo-pescador, Lophius gastrophysus Miranda-Ribeiro, 1915 (Lophiidae), obtidos em mercados dos municípios de Cabo Frio, Niterói, Duque de Caxias e Rio de Janeiro; são apresentados os índices parasitológicos e sítios de infecção dos helmintos encontrados. Foram investigados 87 espécimes de Lophius gastrophysus no período de março de 2000 a dezembro de 2005. Desses, 17 (19,5%) estavam parasitados por larvas de anisaquídeos. Anisakis simplex, Hysterothylacium sp. e Raphidascaris sp. apareceram com prevalências de 1,14, 12,6 e 12,6%, intensidade e intensidade média de infecção de 1, 2,81 e 10,5, abundância média de 0,01, 0,35 e 1,33 respectivamente. Os sítios de infecção foram o estômago, serosas do estômago e intestino e cavidade abdominal. Este é o primeiro registro de Anisakis simplex, Hysterothylacium sp. e Raphidascaris sp. nesta espécie de peixe.

Genetic and morphological characterisation of a new species of the genus Hysterothylacium (Nematoda) from Paralichthys isosceles Jordan, 1890 (Pisces: Teleostei) of the Neotropical Region, state of Rio de Janeiro, Brazil

Taking into account the difficulties of taxonomic identification of larval anisakid nematodes based on morphological characters, genetic analyses were performed, together with those usually applied, in order to identify anisakid larvae found in the flounder Paralichthys isosceles from the littoral of the state of Rio de Janeiro, Brazil. The analysis of 1,820 larvae revealed a new species, similar to Hysterothylacium MD, Hysterothylacium 2, Hysterothylacium KB and Hysterothylacium sp regarding the absence of the larval tooth, an excretory pore situated below the nerve ring level, and slender lateral alae. Moreover, the new species differs from Hysterothylacium fortalezae and Hysterothylacium reliquens with regard to the number and size of spines present on the tail end and from Hysterothylacium patagonicus by the absence of interlabia. The maximum parsimony and neighbour joining tree topologies based on the 18S ribosomal DNA gene, complete internal transcribed spacer region and cytochrome oxidase 2 (COII) gene demonstrated that the Brazilian larvae belong to Raphidascarididae and represent a unique genetic entity, confirmed as a new Hysterothylacium species. Furthermore, the new species presents COII genetic signatures and shares polymorphisms with Raphidascarididae members. This is the first description of a new anisakid species from Brazil through the integration of morphological and molecular taxonomy data.

A new nematode Hysterothylacium aetobatum from ray fish, Aetobatus narinari (Euphrasen)

The present paper reports a new species of a nematode of Aetobatus narinari caught off the Bay of Bengal at Visakhapatnam. It could be distinguished by body measurements, arrangement of papillae on the lips, length of intestinal caecum, position of vulva, number and arrangement of caudal papillae, position and size of spicules and shape of tail. The new species is named as Hysterothylacium aetobatum.

Investigation on the Nematode of Hysterothylacium aduncum (Anisakidae) from Bohai Sea and Yellow Sea in China / 中国寄生虫学与寄生虫病杂志

Objective To investigate Hysterothylacium aduncum (Anisakidae) infection in marine fishes from Bohai Sea and Yellow Sea. Methods Nematodes were collected from the digestive tract of fishes, fixed with hot 4% formalin and preserved in 70% ethanol for study. The specimens were cleared in lactophenol for light microscopical examination, and properly treated for scanning electron microscopy (SEM). Results Among the fishes examined, 14 out of 93 species (15.1%) were found infected by H.aduncum, with a higher prevalence in the fish of Lophius litulon(66.7%), Scomber-omorus niphonius(47.5%), and Gadus macrocephalus(33.3%). H.aduncum infection was first recorded in elasmobranch-Raja smirnovi. Morphological differences of H.aduncum were observed, including the width of lateral alae and the length of intestinal caecum. Conclusion H.aduncum in fishes of Bohai Sea and Yellow Sea in China may be a complex species, and its high prevalence in some fishes reminds the risk of anisakiasis by eating raw fishes.

MORPHOLOGICAL AND TAXONOMICAL STUDIES ON ANISAKIDAE LARVAE FOUND IN MARINE FISHES OF CHINA II. GULF OF TONG KING / 中国寄生虫学与寄生虫病杂志

A survey on Anisakidae larvae in 29 species(134 specimens) of marine fishes in the Gulf of Tong King has been carried out.Anisakidae larvae were detected in 15 out of 29 species.The detected specimens were identified as larvae of Anisakis simplex,Hysterothylacium and Pseudoterranova.The parasitization rate of Anisakis simplex larvae,the main pathogen of anisakiasis,in fishes was 30.6% (41/134),while the parasitization rates of Hysterothylacium and Pseudoterranova larvae were comparatively low.Hysterothylacium larvae China type I detected from Muraenesox clnereus and Trichiurus Tiaumela was a new record.Their morphological characteristics were summarized as follows: 1.Length 10.78-14.18mm,Width 0.25-0.38mm,the length of the esophagus is 1.14-1.73mm,intestinal cecum 0.77-1.24mm and ventricular appendage 6.27-8.40mm,extending parallelly with the intestine to the last quarter of the larva; 2.Boring tooth was present,but mucron was absent; 3.No genital anlage was observed.