ITS-2 rDNA sequencing of Gnathostoma species (Nematoda) and elucidation of the species causing human gnathostomiasis in the Americas

From several gnathostome species the complete internal transcribed spacer ITS-2 ribosomal DNA (rDNA) repeat sequence and a fragment of the 5.8S rDNA were obtained by direct polymerase chain reaction cycle-sequencing and silver-staining methods. The size of the complete ITS-1 sequence in agarose gel electrophoresis was also obtained. The ITS-2 enabled the differentiation of Gnathostoma spinigerum from Thailand and Gnathostoma binucleatum from Mexico and Ecuador and confirmed the validity of the latter. Gnathostoma turgidum, Gnathostoma sp. I (=Gnathostoma procyonis sensu Almeyda-Artigas et al., 1994), and Gnathostoma sp. II (=G. turgidum sensu Foster, 1939 pro parte), all from Mexico, proved to be independent species, but Gnathostoma sp. III, also from Mexico, could not be differentiated from G. turgidum. In Mexico and Ecuador, gnathostomes involved in human infection and that had been classified as G. spinigerum belong to G. binucleatum. The 5.8S rDNA sequences of the 6 Gnathostoma species studied were identical. The results of the ITS-1 agreed with those results of ITS-2.     

Gnathostoma binucleatum (Spirurida: Gnathostomatidae) from the freshwater fish in Tabasco, Mexico

Human gnathostomiasis is a food-born parasitic disease of relative importance in many countries in Southeast Asia. It is caused by several species of nematodes of the genus Gnathostoma. In Mexico is an emerging public health problem since 1970, when first cases were reported. Until today, larval morphometric characters that have been proposed to differentiate between the three species of Gnathostoma present in this country, are not satisfactory. Recently, the presence of advanced third-stage larvae AdvL3 (infective form for humans) in freshwater fishes from Pantanos de Centla, Tabasco. was recorded but their specific identity was not clarified . Examination of four species of freshwater fishes from the same locality revealed that three of them: Petenia splendida (n=58), Cichlasoma managuense (n=35) and Gobiomorus dormitor (n=9) were infected by 15 AdvL3 of Gnathostoma binucleatum. Specific identity was obtained comparing the internal transcribed spacer 2 (ITS2) of the ribosomal DNA with sequences reported in Genbank. This is the first record of G. binucleatum in P. splendida and G. dormitor from Tabasco and the first specific determination of the parasite in the locality.     

Study of the ethiological agent of gnathostomosis in Nayarit,Mexico

In order to clarify the specific identity of the etiological agent of human gnathostomosis in Nayarit State, Mexico, morphological and molecular studies were conducted on advanced third stage larvae obtained from human and fish tissue. Cathorops fuerthii from Agua Brava lagoons complex, was the only fish species found to be infected among four species surveyed. Morphological variability does not allow specific identification of the larvae. Internal transcribed spacer 2 of the ribosomal DNA was sequenced for six larvae (five from fish, one from human tissue). Low divergence in the sequences of Nayarit larvae and Gnathostoma binucleatum (0.24% or less) indicate that the larvae examined belong to this species.     

Larval Gnathostoma hispidum detected in the red banded odd-tooth snake, Dinodon rufozonatum rufozonatum, from China

A total of 205 larval gnathostomes were collected from 18 (22.5%) of 80 red banded odd-tooth snakes, Dinodon rufozonatum rufozonatum, which had been smuggled from China and confiscated at Customs in Busan, Republic of Korea. In order to identify the species, some of the larvae were observed by a light microscope and a scanning electron microscope (SEM). The larvae were 2.18 x 0.29 mm in average size, and had a pair of lips at the anterior end, a muscular esophagus, 2 pairs of cervical sacs, and brownish intestines. The head bulb was characteristically equipped with 4 rows of hooklets; the average number of hooklets in each respective row was 38.6, 40.5, 41.5, and 43.7. In SEM views, the mouth evidenced a pair of lateral lips of equal size in a half-moon shape. Each lip featured a couple of labial papillae and a small amphid located between the 2 papillae. The hooklets on the head bulb had single-pointed, posteriorly-curved tips. The cuticular spines were larger and more densely distributed on the anterior part of the body, and decreased gradually in size and number toward the posterior body. On the basis of these morphological characteristics, the larvae were identified as the third stage larvae of Gnathostoma hispidum.     

Discovery of an endemic area of Gnathostoma turgidum infection among opossums, Didelphis virginiana, in Mexico

Gnathostomosis, caused by Gnathostoma binucleatum, is a serious public health issue in Mexico. Although 2 other Gnathostoma spp., G. turgidum and G. lamothei, have been found in wild animals, their natural life cycle or their relation to human disease remains unclear. While we were conducting an epidemiological survey on Gnathostoma spp. in Sinaloa State, Mexico, we found an endemic area for G. turgidum in common opossums, Didelphis virginiana, located in Tecualilla, Sinaloa. The species identification was carried out by morphological and molecular biological methods. This is the first record of an endemic area for G. turgidum infection in opossums, D. virginiana, in the Americas.     

A new species of Gnathostoma (Nematoda: Gnathostomatidae) in Procyon lotor hernandezii from Mexico

Gnathostoma lamothei n. sp., inhabiting the stomach of Procyon lotor hernandezii Wagler, 1831, in Tlacotalpan, Veracruz State, and Rio Sapo, Oaxaca, Mexico, is described. This new species differs from all other congeners by having the posterior half of the body surface covered by rows of tiny round bosses instead of spines, or lacking ornamentations. Sequences of the ITS2 of the ribosomal DNA of G. lamothei n. sp. are compared with sequences of other species of the genus recorded in Mexico; they show a wide divergence (<50%) with Gnathostoma binucleatum Almeyda-Artigas, 1991, and Gnathostoma turgidum Stossich, 1902, and high similarity with Gnathostoma sp. I sequence (99.2%). On the basis of morphometric traits and sequences, previous records of Gnathostoma sp. I (=Gnathostoma procyonis of Almeyda-Artigas et al., 1994, not Chandler, 1942, and Gnathostoma neoprocyonis nomen nudem) in Mexico are referred to as the new species.     

Detection of Gnathostoma spinigerum Third-Stage Larvae in Snakeheads Purchased from a Central Part of Myanmar

To examine the infection status of freshwater fish with Gnathostoma spp. larvae in Myanmar, we purchased 15 snakeheads, Channa striatus, from a local market in a suburban area of Naypyidaw, the new capital city. Two larval gnathostomes were collected using an artificial digestion technique, and observed by a light microscope and a scanning electron microscope. The size of an intact larva was 2.65 mm long and 0.32 mm wide. The characteristic morphology of the larvae included the presence of a long esophagus (0.80 mm long), 2 pairs of cervical sacs (0.43 mm long), and a characteristic head bulb with 4 rows of hooklets. The number of hooklets in the 1st, 2nd, 3rd, and 4th row was 45, 48, 50, and 52, respectively. Based on these morphological characters, the larvae were identified as the advanced 3rd-stage larvae of Gnathostoma spinigerum. This is the first report of detection of G. spinigerum 3rd-stage larvae in the central part of Myanmar. Our study suggests that intake of raw meat of snakehead fish in Myanmar may result in human gnathostomiasis.     

Infection Status of Gnathostoma spinigerum Larvae in Asian Swamp Eels,Monopterus albus,Purchased from Local Markets in Cambodia

Present study was performed to know the infection status of Gnathostoma sp. larvae in swamp eels from Cambodia. We purchased total 30 Asian swamp eels, Monopterus albus, from local markets in Pursat and Takeo Provinces and Phnom Penh on May and November 2017 and May 2018. All collected eels were transferred to our laboratory with ice and each of them was examined by artificial digestion method. A total of 15 larval gnathostomes (1–5 larvae) were detected from 55.6% (5/9) swamp eels in Pursat Province. No larval gnathostomes were found in 21 swamp eels in Takeo Province and Phnom Penh. The advanced third-stage larvae (AdL₃) detected were 2.575–3.825 (3.250) mm in length and 0.375–0.425 (0.386) mm in width. They had the characteristic head bulb (av. 0.104×0.218 mm) with 4 rows of hooklets, long muscular esophagus (1.048 mm), and 2 pairs of cervical sacs (0.615 mm). The number of hooklets in 4 rows on the head bulb was 41, 44, 47, and 50. In scanning electron microscopy, characteristic features were 4 rows of hooklets on the head bulb, cervical papillae, tegumental spines regularly arranged in transverse striations, and anus. The larval gnathostomes were identified as AdL₃ of Gnathostoma spinigerum based on the morphological characters. By the present study, it has been confirmed that G. spinigerum larvae are infected in Asian swamp eels, M. albus, in Pursat Province, Cambodia.     

Larval Gnathostoma spinigerum Detected in Asian Swamp Eels,Monopterus albus,Purchased from a Local Market in Yangon,Myanmar

The present study was performed to determine the infection status of swamp eels with Gnathostoma sp. larvae in Myanmar. We purchased total 37 Asian swamp eels, Monopterus albus, from a local market in Yangon in June and December 2013 and 2014. All collected eels were transferred with ice to our laboratory and each of them was examined by the artificial digestion technique. A total of 401 larval gnathostomes (1-96 larvae/eel) were detected in 33 (89.2%) swamp eels. Most of the larvae (n=383; 95.5%) were found in the muscle. The remaining 18 larvae were detected in the viscera. The advanced third-stage larvae (AdL₃) were 2.3-4.4 mm long and 0.25-0.425 mm wide. The characteristic head bulb (0.093 × 0.221 mm in average size) with 4 rows of hooklets, muscular long esophagus (1.025 mm), and 2 pairs of cervical sacs (0.574 mm) were observed by light microscopy. The average number of hooklets in the 1st, 2nd, 3rd, and 4th rows was 41, 45, 48, and 51, respectively. As scanning electron microscopic findings, the characteristic 4-5 rows of hooklets on the head bulb, a cervical papilla, tegumental spines regularly arranged in the transverse striations, and an anus were well observed. Based on these morphological characters, they were identified as the AdL₃ of Gnathostoma spinigerum. By the present study, it has been confirmed for the first time that Asian swamp eels, M. albus, from Yangon, Myanmar are heavily infected with G. spinigerum larvae.     

Discovery of larval Gnathostoma nipponicum in frogs and snakes from Jeju-do (Province), Republic of Korea

A survey was performed to find out the intermediate hosts of Gnathostoma nipponicum in Jeju-do (Province), the Republic of Korea. In August 2009 and 2010, a total of 82 tadpoles, 23 black-spotted pond frogs (Rana nigromaculata), 7 tiger keelback snakes (Rhabdophis tigrinus tigrinus), 6 red-tongue viper snakes (Agkistrodon ussuriensis), and 2 cat snakes (Elaphe dione) were collected in Jeju-do and examined by the pepsin-HCl digestion method. Total 5 gnathostome larvae were detected in 3 (50%) of 6 A. ussuriensis, 70 larvae in 3 of 7 (42.9%) R. tigrinus tigrinus, and 2 larvae in 2 of 82 (8.7%) frogs. No gnathostome larvae were detected in tadpoles and cat snakes. The larvae detected were a single species, and 2.17 × 0.22 mm in average size. They had characteristic head bulbs, muscular esophagus, and 4 cervical sacs. Three rows of hooklets were arranged in the head bulbs, and the number of hooklets in each row was 29, 33, and 36 posteriorly. All these characters were consistent with the advanced third-stage larvae of G. nipponicum. It has been first confirmed in Jeju-do that R. nigromaculata, A. ussuriensis, and R. tigrinus tigrinus play a role for intermediate and/or paratenic hosts for G. nipponicum.     

The first record of a confirmed human case of Gnathostoma doloresi infection

The first case of Gnathostoma doloresi infection in a human was found in Miyazaki Prefecture, Japan. A whole length of the parasite was dissected out from biopsied skin and was identified as the third-stage larva of G. doloresi based on the morphological characteristics of the hooklets of the head bulb and also on the number of nuclei in the intestinal epithelial cells of the parasite.     

Phenotypic variability in the Caribbean Orange Icing sponge Mycale laevis (Demospongiae: Poecilosclerida)

Sponge species may present several morphotypes, but sponges that are morphologically similar can be separate species. We investigated morphological variation in Mycale laevis, a common Caribbean reef sponge. Four morphotypes of M. laevis have been observed (1) orange, semi-cryptic, (2) orange, massive, (3) white, semi-cryptic, and (4) white, massive. Samples of M. laevis were collected from Key Largo, Florida, the Bahamas Islands, and Bocas del Toro, Panama. Fragments of the 18S and 28S rRNA ribosomal genes were sequenced and subjected to phylogentic analyses together with sequences obtained for 11 other Mycale species and additional sequences retrieved from GenBank. Phylogenetic analyses confirmed that the genus Mycale is monophyletic within the Order Poecilosclerida, although the subgenus Aegogropila is polyphyletic and the subgenus Mycale is paraphyletic. All 4 morphotypes formed a monophyletic group within Mycale, and no genetic differences were observed among them. Spicule lengths did not differ among the 4 morphotypes, but the dominant megasclere in samples collected from Florida and the Bahamas was the strongyle, while those from Panama had subtylostyles. Our data suggest that the 4 morphotypes constitute a single species, but further studies would be necessary to determine whether skeletal variability is due to phentotypic or genotypic plasticity.     

Geographic differentiation and speciation in Cerion–a preliminary discussion of patterns and processes

Cerion's exuberant morphological diversity (600 described 'species') combined with the extreme rarity of reproductive isolation among morphotypes (only one unambiguous case of sympatry in the Bahamas) has long made this genus an object of fascination for evolutionists and of frustration for taxonomists. We have pursued an integrated approach of genetic and morphometric study based on field investigations of ecology and biogeography in the Bahamas. Cerion's morphotypes are not distributed haphazardly, but show definite patterns of correlation with habitat and geography. Although all morphotypes interbreed, hybrid zones tend to be narrow and characterized by highly local genetic anomalies–unique alleles present in neither parental population. Different patterns of covariance in ontogeny, and habitat preferences, also indicate mat the morphotypes are distinctive, non-amalgamating entities (despite little difference in the frequencies of structural genes among them) that may be called species once a definition based on strict reproductive isolation is abandoned. Variation in structural genes, anatomy and morphology is non-concordant, but orderly for each criterion. Similar morphologies are often polyphyletic and evolved repeatedly as one possible ontogenetic route within a developmental program common to all Cerion. Although we cannot always distinguish among competing causes for observed patterns, we can establish genetic, morphological, anatomical, and biogeographic criteria for decisions when adequate evidence is available. The species of Cerion will be reduced by more than an order of magnitude from a list currently described.     

Phylogenomic and morphological data reveal hidden patterns of diversity in the national tree of Brazil,Paubrasilia echinata

Premise

Paubrasilia echinata (common names, pau brasil, brazilwood) is the national tree of Brazil and an endangered species endemic to the Brazilian Atlantic Forest. Over its wide distribution of 2000 km, its leaflets morphology exhibits extensive plasticity. Three morphotypes are commonly identified based on leaf size, but it is unclear if they represent distinct taxa or a single polymorphic species. This study aims to clarify the taxonomic position of the three morphotypes to inform conservation decisions.

Methods

A morphometric study of leaf characters of herbarium specimens was coupled with genetic analyses using genotype-by-sequencing data. We used maximum-likelihood and coalescent methods to evaluate the phylogenetic and population structure of the species. We compared these with a morphological dendrogram built from hierarchical clustering.

Results

Two of the three morphotypes formed separately evolving lineages, the third morphotype formed two geographically separate lineages, and northern trees with intermediate leaf morphology formed a separate fifth lineage. Leaflet size varied by over 35-fold, and although morphological clustering generally matched the genetic patterns, there were some overlaps, highlighting the cryptic diversity within this group.

Conclusions

Our genetic and morphological results provide some evidence that cultivated trees from different states in Brazil seem to have a limited genetic origin and do not reflect the broader genetic and geographical diversity of the species. As a result, more care is likely needed to preserve the overall genomic diversity of this endangered and iconic species.     

Morphological and genetic differences in ecologically distinct populations of Petrosia (Porifera, Demospongiae)

The taxonomic status of two sponge tnorphotypes living sympatrically in Mediterranean caves and usually ascribed to Petrosia ficiformis was elucidated on the basis of morphological, morphometric and genetic features. The two morphotypes, spherical and cylindrical, showed differences in the shape and size of spicules and in the morphology of the aquiferous system. Electrophoretic analyses demonstrated that the two morphotypes are reproductively independent and so they should be considered as distinct biological species. The spicular features allow attribution of the cylindrical morphotype to P.ficiformis and the spherical one to P. clavata. For these two species parasympatric speciation is proposed.     

Cryptic diversity in the pen shell Atrina pectinata (Bivalvia: Pinnidae): high divergence and hybridization revealed by molecular and morphological data

Cryptic species have been increasingly revealed in the marine realm through an analytical approach incorporating multiple lines of evidence (e.g., mtDNA, nuclear genes and morphology). Illustrations of cryptic taxa improve our understanding of species diversity and evolutionary histories within marine animals. The pen shell Atrina pectinata is known to exhibit extensive morphological variations that may harbour cryptic diversity. In this study, we investigated A. pectinata populations along the coast of China and one from Japan to explore possible cryptic diversity and hybridization using a combination of mitochondrial (cytochrome c oxidase subunit I, mtCOI) and nuclear (ribosomal internal transcribed spacer, nrITS) genes as well as morphology. Phylogenetic analyses of mtCOI ‘DNA barcoding gene’ sequences resolved six divergent lineages with intralineage divergences between 0.4% and 0.8%. Interlineage sequence differences ranged from 4.3% to 22.0%, suggesting that six candidate cryptic species are present. The nrITS gene revealed five deep lineages with Kimura 2‐parameter distances of 3.7–30.3%. The five nuclear lineages generally corresponded to mtCOI lineages 1–4 and (5 + 6), suggestive of five distinct evolutionary lineages. Multiple nrITS sequences of significant variance were found within an individual, clearly implying recent hybridization events between/among the evolutionary lineages, which contributed to cytonuclear discordance. Morphologically, five morphotypes matched the five genetic lineages, although the intermediates may well blur the boundaries of different morphotypes. This study demonstrates the importance of combining multiple lines of evidence to explore species cryptic diversity and past evolutionary histories.     

Five confirmed human cases of gnathostomiasis nipponica recently found in northern Japan.

Five confirmed human cases of gnathostomiasis nipponica exhibiting creeping eruption and itching were found sporadically from the autumn of 1991 to the winter of 1992 in the northern region of the mainland of Japan. In all cases, a causative gnathostome with 3 transverse rows of hooklets on the head bulb was detected in biopsied skin. The morphological characteristics agreed with the advanced third-stage larvae of Gnathostoma nipponicum. Within a few weeks before development of symptoms, all patients had histories of eating raw freshwater fishes, kokanee (Salmo nerka nerka), carp (Cyprinus carpio), crucian carp (Carassius gibelio langsdorfi), or common ice-fish (Salangichthys microdon). However, they had never eaten raw loach, which is known as a source of human infections with G. nipponicum.     

Morphological variation in the tropical anole, Anolis casildae (Squamata: Polychrotidae)

We describe morphological variation (scalation and coloration) observed among eight individuals of the Panamanian lizard species Anolis casildae. This variation was not observed in the holotype and aids in identification of this recently described species (originally described on the basis of a single, male specimen). This species occurs only in the Reserva Forestal Fortuna (Chiriquí Province) and the adjacent Bosque Protector Palo Seco (Bocas del Toro Province) in western Panama. Anolis casildae can be distinguished from all other Panamanian anole species via six features: (1) two enlarged superciliary scales (the first larger than the second); (2) an anterior nasal scale in contact with the rostral scale or separated from the rostral by one scale; (3) 6-8 sublabial scales to the center of the eye; (4) 3-4 scales between the supraobital semicircles; (5) unique coloration (4-6 oblique brown bands interspersed by blue-outlined yellow patches; dewlap is a dirty cream color with broad yellow scale rows irregularly interspersed with smaller emerald green scales) and (6) A. casildae occurs from 1,050 to 1,400 m in the Cordillera Central. We also compare our natural history observations of A. casildae to a similar large anole, A. frenatus, a species which we believe A. casildae to be closely related.     

Gnathostoma binucleatum: excretion-secretion antigen analysis obtained from advanced third-stage larvae in in vitro culture

The paper describes an introductory characterization of antigenic stimulation of excretion-secretion products (ESP) of Gnathostoma binucleatum advanced third-stage larvae cultured in vitro and proteinases present in this products. Excretory and secretory proteins were obtained after 10 larvae were maintained in 5% CO(2) RPMI medium. The supernatant was collected each week for two months. The proteins were dialyzed, concentrated, and separated in 10% SDS-PAGE gels under reducing conditions and transferred to nitrocellulose paper for immunoblot analyses. G. binucleatum immunized mice serum was used to determine protein antigenicity. Four proteins of 40, 80, 120, and 208 kDa persisted for two months and three proteins, 80, 120, and 208 kDa were recognized for antibodies of mice. In SDS-PAGE gelatin substrate gels ESP resolved as two proteins with molecular weight of 80 and 208 kDa that were sensitive to a metalloproteinase inhibitor, and thus it may be inferred that they might be used for diagnosis of gnathostomiasis.     

中国马鞭草科植物的花粉形态

本文对我国18属91种3变种马鞭草科(Verbenaceae)植物的花粉形态进行了详细地观察研究。其中对15属27种进行了扫描电镜观察。本科为花粉多类型的科之一。根据萌发孔的数目、特征和纹饰特点,本文将马鞭草科分为9个花粉类型:1.具疣3沟花粉类型;2.具刺3沟花粉类型;3.具细网8沟花粉类型;4.具模糊颗粒-细网3沟花粉类型;5.具网3沟花粉类型;6.具散沟(6-8条)花粉类型;7.具3孔花粉类型;3.具细网3沟花粉类型,4.具模糊颗粒一细网3沟花粉类型,5.具网3沟花粉类型,6.具散沟(6-8条)花粉类型,7.具3孔花粉类型,8.具3孔沟花粉类型,9.具3(4)孔沟花粉类型。