Gnathostoma spinigerum Infection in the Upper Lip of a Korean Woman: An Autochthonous Case in Korea

Autochthonous human gnathostomiasis had never been reported in the Republic of Korea. We report here a case of Gnathostoma spinigerum infection in a 32-year-old Korean woman, presumed to have been infected via an indigenous route. The patient had experienced a painful migratory swelling near the left nasolabial fold area of the face for a year, with movement of the swelling to the mucosal area of the upper lip 2 weeks before surgical removal of the lesion. Histopathological examinations of the extracted tissue revealed inflammation with heavy eosinophilic infiltrations and sections of a nematode suggestive of a Gnathostoma sp. larva. The larva characteristically revealed about 25 intestinal cells with multiple (3-6) nuclei in each intestinal cell consistent with the 3rd-stage larva of G. spinigerum. The patient did not have any special history of travel abroad except a recent trip, 4 months before surgery, to China where she ate only cooked food. The patient is the first recorded autochthonous case of G. spinigerum infection in Korea.     

A Case of Anisakidosis Caused by Pseudoterranova decipiens Larva

Pseudoterranova decipiens larva is a rare cause of anisakiasis. Indeed, prior to the present study, there had been only 12 reports of larval P. decipiens infection in the Republic of Korea. In June 2011, an anisakid larva, 32.1 mm in length and 0.88 mm in width, and finally identified as the third stage larva of P. decipiens owing to the presence of an intestinal cecum but lacking ventricular appendage, was discovered in a 61-year-old woman during the course of endoscopy executed as a part of routine physical examinations. The patient had eaten raw a rockfish 13 hr prior to the endoscopy, but showed no symptoms of anisakiasis. This paper is the 13th report of P. decipiens infection in Korea.     

Biocontrol Potential of Steinernema thermophilum and Its Symbiont Xenorhabdus indica Against Lepidopteran Pests: Virulence to Egg and Larval Stages

Under laboratory conditions, the biocontrol potential of Steinernema thermophilum was tested against eggs and larval stages of two important lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura (polyphagous pests), as well as Galleria mellonella (used as a model host). In terms of host susceptibility of lepidopteran larvae to S. thermophilum, based on the LC₅₀ 36 hr after treatment, G. mellonella (LC₅₀ = 16.28 IJ/larva) was found to be more susceptible than S. litura (LC₅₀ = 85 IJ/larva), whereas neither host was found to be significantly different from H. armigera (LC₅₀ = 54.68 IJ/larva). In addition to virulence to the larval stages, ovicidal activity up to 84% was observed at 200 IJ/50 and 100 eggs of H. armigera and S. litura, respectively. To our knowledge this is the first report of entomopathogenic nematode pathogenicity to lepidopteran eggs. Production of infective juvenile (IJ) nematodes/insect larva was also measured and found to be positively correlated with rate of IJ for H. armigera (r = 0.990), S. litura (r = 0.892), as well as G. mellonella (r = 0.834). Both Phase I and Phase II of symbiotic bacteria Xenorhabdus indica were tested separately against neonates of H. armigera and S. litura by feeding assays and found to be virulent to the target pests; phase variation did not affect the level of virulence. Thus S. thermophilum as well as the nematode’s symbiotic bacteria applied separately have the potential to be developed as biocontrol agents for key lepidopteran pests.     

An update to the taxonomy of the genus Gastroserica Brenske (Coleoptera,Scarabaeidae, Sericini)

Based on the examination of newly collected material and additional specimens housed in Chinese collections, our knowledge of Gastroserica Brenske, 1897, is expanded. Here, seven new species are described, including habitus photographs and illustrations of the male genitalia: Gastroserica haoyui sp. n. (China: Zhejiang Prov.), G. fengduana sp. n. (China: Sichuan Prov.), G. wenzhui sp. n. (China: Guangxi Prov.), G. damingshanica sp. n. (from China: Guangxi Prov.), G. jinxiuensis sp. n. (China: Guangxi Prov.), G. liboensis sp. n. (China: Yunnan Prov.) and G. carolusi sp. n. (Laos). Additionally, we provide a distribution map of the new taxa and new distribution records of the known taxa.     

Sequence Analysis of cytb Gene in Echinococcus granulosus from Western China

Echinococcus granulosus is the causative agent of cystic echinococcosis with medical and veterinary importance in China. Our main objective was to discuss the genotypes and genetic diversity of E. granulosus present in domestic animals and humans in western China. A total of 45 hydatid cyst samples were collected from sheep, humans, and a yak and subjected to an analysis of the sequences of mitochondrial cytochrome b (cytb) gene. The amplified PCR product for all samples was a 1,068 bp band. The phylogenetic analysis showed that all 45 samples were identified as E. granulosus (genotype G1). Ten haplotypes were detected among the samples, with the main haplotype being H1. The haplotype diversity was 0.626, while the nucleotide diversity was 0.001. These results suggested that genetic diversity was low among our samples collected from the west of China based on cytb gene analysis. These findings may provide more information on molecular characteristics of E. granulosus from this Chinese region.     

Target Host Finding by Steinernema feltiae and Heterorhabditis bacteriophora in the Presence of a Non-Target Insect Host

The ability of Steinernema feltiae or Heterorhabditis bacteriophora infective juveniles (IJ), when applied to the soil surface, to infect a Galleria mellonella larva at the base of a soil-filled cup (276 cm³) was evaluated in the presence and absence of 100 larvae of a non-target insect, the aphid midge Aphidoletes aphidimyza, near the soil surface. In all four trials with either S. feltiae or H. bacteriophora, A. aphidimyza presence did not affect the number of IJ finding and infecting a G. mellonella larva. Steinernema feltiae and H. bacteriophora IJ movement (as measured by the percentage of IJ aggregating on either side of an experimental arena) in the presence of one or many A. aphidimyza larvae was evaluated in agar- and soil-filled petri dishes, respectively. Infective juvenile movement in the presence of A. aphidimyza did not differ from random, indicating that IJ were not attracted to A. aphidimyza. It is suggested, therefore, that A. aphidimyza does not reduce IJ efficacy when these two forms of biological control agent are present together in a field situation even though it is known that A. aphidimyza is susceptible to IJ of these species.     

A description of preimaginal stages of Pseudaspidapion botanicum Alonso-Zarazaga & Wang, 2011 (Apionidae,?Curculionoidea)

The preimaginal stages including egg, mature larva and pupa of Pseudaspidapion botanicum Alonso-Zarazaga & Wang, 2011 were described and figured, diagnostic characters of larva and pupa were discussed, and corresponding biological information was supplied. The nomenclature of frontal setae in the larva compared with curculionid weevils, the absence of the hypopharyngeal bracon in the larva, and the metafemoral setae in the pupa were discussed. Common and different characters among the larvae of Pseudaspidapion botanicum, Aspidapion radiolus (Marsham, 1802) and Aspidapion aeneum (Fabricius, 1775) were also provided.     

Intramuscular Sparganosis in the Gastrocnemius Muscle: A Case Report

Sparganosis is a parasitic infection caused by the plerocercoid tapeworm larva of the genus Spirometra. Although the destination of the larva is often a tissue or muscle in the chest, abdominal wall, extremities, eyes, brain, urinary tract, spinal canal, and scrotum, intramuscular sparganosis is uncommon and therefore is difficult to distinguish from a soft tissue tumor. We report a case of intramuscular sparganosis involving the gastrocnemius muscle in an elderly patient who was diagnosed using ultrasonography and MRI and treated by surgical excision. At approximately 1 cm near the schwannoma at the right distal sciatic nerve, several spargana worms were detected and removed.     

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.     

Review of the Parasa undulata (Cai, 1983) species group with the first conifer-feeding larva for Limacodidae and descriptions of two new species from China and Taiwan (Lepidoptera,Limacodidae)

Although the caterpillars are well-known for the stings and magnificent coloration, the systematics of Limacodidae is historically neglected and chaotic due to the difficulty in matching the larval with adult stages as well as the very conservative and convergent adult morphology. One of the biggest taxonomic problems surrounds a collective group from Southeastern Asia, termed the “green limacodid moths”, which harbours at least 90 species placed in the genus Parasa Walker, 1859 and 14 “subunits”. The P. undulata group was previously composed of 3 species from China and Taiwan, and characterized only by wing pattern. This species group is extensively studied herein with two new species described, i.e. P. viridiflamma sp. n. (Taiwan) and P. minwangi sp. n. (S. China), and discovery of female genitalia of three species, presenting new phylogenetic insights in this potentially paraphyletic genus. In addition, one limacodid larva was found to be feeding exclusively on Picea (Pinaceae) in Taiwan. Its identity, Parasa pygmy Solovyev, 2010 in P. undulata group, is confirmed through matching its COI sequence to the adult. This discovery is also biologically significant because the previous known host breadth of Parasa was of polyphagy on various angiosperm plant families. This case, therefore, represents the first record of conifer-feeding behavior in this family as well as the first of specialized herbivory in the genus. Meanwhile, the background match between Picea leaves and larval coloration is shared with other Picea-feeding insects. This phenomenon is worth of further investigation in the aspect of convergent evolution of crypsis associated with a particular plant.     

A novel spliceosome-mediated trans-splicing can change our view on genome complexity of the divergent eukaryote Giardia intestinalis

Although spliceosomal introns are an abundant landmark in eukaryotic genomes, the nuclear genome of the divergent eukaryote Giardia intestinalis, the causative agent of giardiasis, has been considered as “intron-poor” with only five canonical (cis-spliced) introns. However, three research groups (including ours) have independently reported a novel class of spliceosomal introns in the G. intestinalis genome. Three protein-coding genes are split into pieces in the G. intestinalis genome, and each of the partial coding regions was independently transcribed into polyadenylated premature mRNAs (pre-mRNAs). The two pre-mRNAs directly interact with each other by an intermolecular-stem structure formed between their non-coding portions, and are then processed into mature mRNAs by spliceosome-mediated trans-splicing. Here, we summarize the recently published works on split introns (“splintrons”) in the G. intestinalis genome, and then provide our speculation on the functional property of the Giardia spliceosomes based on the putative ratio of splintrons to canonical introns. Finally, we discuss a scenario for the transition from typical GT-AG boundaries to non-typical AT-AC boundaries in a particular splintron of Giardia.     

Identification of an immunogenic protein of Giardia lamblia using monoclonal antibodies generated from infected mice

The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs) specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6) by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein) and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90%) of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.     

An improved method to obtain antigen-excreting Toxocara canis larvae

Toxocara canis is a dog helminth which causes visceral larva migrans (VLM) when infecting humans as a larva. The infection is demonstrated by detecting IgG antibodies against excretory-secretory larval antigens (ESLA) in serum by ELISA. The production of ESLA involves the collection of adult worms from dog puppy stools, the separation of eggs from dissected uteri, and the in vitro growing of egg-derived larvae, following the time-consuming and laborious protocol described by De Savigny [De Savigny, D.H., 1975. In vitro maintenance of T. canis larvae and a simple method for the production of Toxocara ES antigen for the uses in serodiagnostic tests for visceral larva migrans. Journal of Parasitology 61, 781-782]. In this work, an improved protocol for obtaining T. canis larvae is described. The modifications proposed improved the efficiency of the original De Savigny method in three ways: (i) increasing the parasite yield up to five fold, (ii) improving the larval purity, and (iii) markedly reducing the execution time of the protocol.     

Revision of three camaenid and one bradybaenid species (Gastropoda,Stylommatophora) from China based on morphological and molecular data,with description of a new bradybaenid subspecies from Inner Mongolia,China

We have revised the taxonomy of three camaenid and one bradybaenid species from China and described one new subspecies of the genus Bradybaena (Family Bradybaenidae) from Inner Mongolia, China. The genitalia of three Satsuma (Family Camaenidae) species S. mellea stenozona (Moellendorff, 1884), S. meridionalis (Moellendorff, 1884), comb. n. and S. uncopila (Heude, 1882), comb. n. assigned to the genus Bradybaena previously,lack a dart sac and mucous glands. Moreover, the molecular phylogeny has revealed close relationships between the three species and the genus Satsuma. Two species, S. stenozona (Moellendorff, 1884) from Fuzhou and Ganesella citrina Zilch, 1940 from Wuyi Mountain, are considered as synonymous and should be a subspecies of S. mellea mellea (Pfeiffer, 1866) because of the morphological and molecular similarities. Meanwhile, the other two are placed in the genus Satsuma: S. meridionalis (Moellendorff, 1884), comb. n. and S. uncopila (Heude, 1882), comb. n. G. virgo Pilsbry, 1927 differs from species of the genera Ganesella and Satsuma not only in its shell, but also in anatomical characters, such as having a dart sac and mucous gland, and lacking a flagellum. Additionally, phylogenetic analyses highly support the sister relationship with other Bradybaena species. Thus, placement of G. virgo Pilsbry, 1927 in the genus Bradybaena issuggested.     

Phylogenetic Relationships of a Distinct Species of Globodera from Portugal and Two Punctodera Species

Evolutionary relationships based on ribosomal DNA (rDNA) sequence data for a previously unknown species of Globodera from Portugal, Punctodera chalcoensis from Mexico, and P. punctata from Estonia, plus previously published sequences, support the following relationships: (((Cactodera weissi, G. artemisiae, C. milleri), ((G. sp. Bouro, G. sp. Canha, G. sp. Ladoeiro), ((G. pallida, G. rostochiensis), (P. chalcoensis, P. punctata)))), Heterodera avenae). Globodera sp. from Portugal, which can be confused with potato cyst nematodes by phytosanitary services when the identification is based only on morphological characters, is clearly different based on our molecular data. In addition, the rDNA data show the Globodera sp. to be only distantly related to other European Globodera species that parasitize Asteraceae. Punctodera chalcoensis and P. punctata form a sister clade to the G. pallida + G. rostochiensis clade.     

Ancyronyx Erichson, 1847 (Coleoptera,Elmidae) from Mindoro,Philippines, with description of the larvae and two new species using DNA sequences for the assignment of the developmental stages

Ancyronyx buhid sp. n. and Ancyronyx tamaraw sp. n. are described based on adults and larvae, matched using their cox1 or cob DNA sequence data. Additional records of Ancyronyx schillhammeri Jäch, 1994 and Ancyronyx minerva Freitag & Jäch, 2007 from Mindoro are listed. The previously unknown larva of Ancyronyx schillhammeri is also described here, aided by cox1 data. The new species and larval stages are described in detail and illustrated by SEM and stacked microscopic images. Keys to the adult and larval Ancyronyx species of Mindoro and an updated checklist of Philippine Ancyronyx species are provided. The usefulness as bioindicators, the phylogenetic relationships and biogeographic aspects affecting the distribution patterns are briefly discussed.     

Molecular and Biochemical Characterization of a Novel ��-N-Acetyl-D-Hexosaminidase with Broad Substrate-Spectrum from the Aisan Corn Borer, Ostrinia Furnacalis

Insect β-N-acetyl-D-hexosaminidases with broad substrate-spectrum (IBS-Hex) are the homologues of human β-N-acetyl-D-hexosaminidase A/B (HsHex A/ B). These enzymes are distributed in most insect species and vary in physiological roles. In this study, the gene encoding an IBS-Hex, OfHEX2, was cloned from the Asian corn borer, Ostrinia furnacalis. Recombinant OfHex2 was expressed in Pichia pastoris and purified to homogeneity. By structure-based sequence alignment, three sequence segments with high diversity among IBS-Hexs were firstly concluded. Furthermore, the residue pair N423-R424/ D452-L453 important for the specificity of human β-N-acetyl-D-hexosaminidase subunits α/β toward charged/ non-charged substrates was not conserved in OfHex2 and other IBS-Hexs. Unlike HsHex A, OfHex2 could not degrade charged substrates such as 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide, ganglioside GM2 and peptidoglycan. OfHex2 showed a broad substrate-spectrum by hydrolyzing β1-2 linked N-acetyl-D-glucosamines from both α3 and α6 branches of biantennary N-glycan and β1-4 linked GlcNAc from chitooligosaccharides as well as β1-3 linked or β1-4 linked N-acetyl-D-galactosamine from oligosaccharides of glycolipids. Real-time PCR analysis demonstrated that the expression of OfHEX2 was up-regulated in the intermolt stages (both larva and pupa), and mainly occurred in the carcass rather than in the midgut during the feeding stage of fifth (final) instar larva. This study reported a novel IBS-Hex with specific biochemical properties, suggesting biodiversity of this class of enzymes.     

Molecular Detection of Giardia intestinalis from Stray Dogs in Animal Shelters of Gyeongsangbuk-do (Province) and Daejeon,Korea

Giardia is a major public health concern and considered as reemerging in industrialized countries. The present study investigated the prevalence of giardiosis in 202 sheltered dogs using PCR. The infection rate was 33.2% (67/202); Gyeongsangbuk-do and Daejeon showed 25.7% (39/152, P<0.0001) and 56% (28/50), respectively. The prevalence of infected female dogs (46.7%, P<0.001) was higher than in male dogs (21.8%). A higher prevalence (43.5%, P<0.0001) was observed in mixed breed dogs than purebred (14.1%). Although most of the fecal samples collected were from dogs of ≥1 year of age which showed only 27.4% positive rate, 61.8% (P<0.001) of the total samples collected from young animals (<1 year of age) were positive for G. intestinalis. A significantly higher prevalence in symptomatic dogs (60.8%, P<0.0001) was observed than in asymptomatic dogs (23.8%). Furthermore, the analysis of nucleotide sequences of the samples revealed that G. intestinalis Assemblages A and C were found in the feces of dogs from Gyeongsangbuk-do and Daejeon. Since G. intestinalis Assemblage A has been known to infect humans, our results suggest that dogs can act as an important reservoir of giardiosis in Korea. Hence, hygienic management should be given to prevent possible transmission to humans.     

Distant Neighbor Base Sequence Context Effects in Human Nucleotide Excision Repair of a Benzo[a]pyrene-derived DNA Lesion

The effects of non-nearest base sequences, beyond the nucleotides flanking a DNA lesion on either side, on nucleotide excision repair (NER) in extracts from human cells were investigated. We constructed two duplexes containing the same minor groove-aligned 10S (+)-trans-anti-B[a]P-N²-dG (G?) DNA adduct, derived from the environmental carcinogen benzo[a]pyrene (B[a]P): 5′-C-C-A-T-C-G?-C-T-A-C-C-3′ (CG?C-I), and 5′-C-A-C3-A4-C5-G?-C-A-C-A-C-3′ (CG?C-II). We used polyacrylamide gel electrophoresis to compare the extent of DNA bending, and molecular dynamics simulations to analyze the structural characteristics of these two DNA duplexes. The NER efficiencies are 1.6(± 0.2)-fold greater in the case of the CG?C-II than the CG?C-I sequence context in 135-mer duplexes. Gel electrophoresis and self-ligation circularization experiments revealed that the CG?C-II duplex is more bent than the CG?C-I duplex, while molecular dynamics simulations showed that the unique -C3-A4-C5- segment in the CG?C-II duplex plays a key role. The presence of a minor groove-positioned guanine amino group, the Watson-Crick partner to C3, acts as a wedge; facilitated by a highly deformable local -C3-A4- base step, this amino group allows the B[a]P ring system to produce a more enlarged minor groove in CG?C-II than in CG?C-I, as well as a local untwisting and enlarged and flexible Roll only in the CG?C-II sequence. These structural properties fit well with our earlier findings that in the case of the family of minor groove 10S (+)-trans-anti-B[a]P-N²-dG lesions, flexible bends and enlarged minor groove widths constitute NER recognition signals, and extend our understanding of sequence context effects on NER to the neighbors that are distant to the lesion.     

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage. HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.