Morphological Comparison and Taxonomic Utility of Copulatory Structures of Selected Nematode Species

Spicules of 9 Meloidogyne, 2 Heterodera, 3 Globodera, and 12 other plant-parasitic, insect-parasitic, and free-living nematodes were excised and examined using scanning electron microscopy (SEM). Gubernacula of some of the species were also excised, and their structure was determined. The two spicules of all species examined were symmetrically identical in morphology. The spicule typically consisted of three parts: head, shaft, and blade with dorsal and ventral vela. The spicular nerve entered through the cytoplasmic core opening on the lateral outer surface of the spicule head and generally communicated with the exterior through one or two pores at the spicule tip. Spicules of Xiphinema sp. and Aporcelaimellus sp. were not composed of three typical parts, were less sclerotized, and lacked a cytoplasmic core opening and distal pores. Spicules of Aphelenchoides spp. had heads expanded into apex and rostrum and had very arcuate blades with thick dorsal and ventral edges (limbs). Gubernaculum shapes were stable within a species, but differed among species examined. The accessory structures of Hoplolaimus galeatus consisted of a tongue-shaped gubernaculum with two titillae at its distal end and a plate-like capitulum terminating distally in two flat, wing-like structures. A comparison of spicules of several species of Meloidogyne by SEM and light microscopy revealed no striking morphological differences.     

New species of Rhabdochona Railliet, 1916 (Nemata: Rhabdochonidae) from Rainbow Trout in California Streams

Three new species of Rhabdochona Railliet, 1916 are described and illustrated from Salmo gairdneri Richardson (rainbow trout) in freshwater streams in California: Rhabdochona californiemis n. sp., R. paxmani n. sp., and R. satmonis n. sp. Rhabdochona californiensis n. sp. is characterized by 14 anteriorly directed teeth in the prostome, egg devoid of filaments or floats, male and female tail terminus with a single mucro, left (long) spicule slender with a moderate distended podoid terminal end, spicular ratio 1:3.8. Rhabdochona paxmani n. sp. is characterized by 10 teeth in the prostome, eggs with polar floats, left (long) spicule slender with podoid terminus distended and having a minute subterminal spine; right spicule with prominent gorgeret (barb), spicular ratio 1:4.3, male and female tail terminus with a cuticular conical rounded short projection. Rhabdochona salmoni, n. sp. is characterized by 10 teeth anteriorly directed in the prostome, eggs with polar floats, left spicule slender with a distended podoid terminus; right spicule with a sharply indented gorgeret, spicular ratio 1:4.3, male and female tail terminus with a conical or rounded tip.     

Intestinal Nematodes from Small Mammals Captured near the Demilitarized Zone,Gyeonggi Province,Republic of Korea

A total of 1,708 small mammals (1,617 rodents and 91 soricomorphs), including Apodemus agrarius (n = 1,400), Microtus fortis (167), Crocidura lasiura (91), Mus musculus (32), Myodes (= Eothenomys) regulus (9), Micromys minutus (6), and Tscherskia (= Cricetulus) triton (3), were live-trapped at US/Republic of Korea (ROK) military training sites near the demilitarized zone (DMZ) of Paju, Pocheon, and Yeoncheon, Gyeonggi Province from December 2004 to December 2009. Small mammals were examined for their intestinal nematodes by necropsy. A total of 1,617 rodents (100%) and 91 (100%) soricomorphs were infected with at least 1 nematode species, including Nippostrongylus brasiliensis, Heligmosomoides polygyrus, Syphacia obvelata, Heterakis spumosa, Protospirura muris, Capillaria spp., Trichuris muris, Rictularia affinis, and an unidentified species. N. brasiliensis was the most common species infecting small mammals (1,060; 62.1%) followed by H. polygyrus (617; 36.1%), S. obvelata (370; 21.7%), H. spumosa (314; 18.4%), P. muris (123; 7.2%), and Capillaria spp. (59; 3.5%). Low infection rates (0.1-0.8%) were observed for T. muris, R. affinis, and an unidentified species. The number of recovered worms was highest for N. brasiliensis (21,623 worms; mean 20.4 worms/infected specimen) followed by S. obvelata (9,235; 25.0 worms), H. polygyrus (4,122; 6.7 worms), and H. spumosa (1,160; 3.7 worms). A. agrarius demonstrated the highest prevalence for N. brasiliensis (70.9%), followed by M. minutus (50.0%), T. triton (33.3%), M. fortis (28.1%), M. musculus (15.6%), C. lasiura (13.2%), and M. regulus (0%). This is the first report of nematode infections in small mammals captured near the DMZ in ROK.     

Bursaphelenchus anatolius n. sp. (Nematoda: Parasitaphelenchidae), an Associate of Bees in the Genus Halictus

Bursaphelenchus anatolius n. sp., a phoretic associate of Halictus bees from Ankara, Turkey, is described and illustrated. Bursaphelenchus anatolius n. sp. is closest to B. kevini, which is phoretically associated with Halictus bees from the Pacific Northwest. Bursaphelenchus anatolius n. sp. and B. kevini appear to be sister taxa based upon several shared morphological features, similar life histories involving phoresy with soil-dwelling Halictus bees, and molecular analysis of the near-full-length small subunit rDNA, D2D3 expansion segments of the large subunit rDNA, and partial mitochondrial DNA COI. Bursaphelenchus anatolius n. sp. can be differentiated from all other species of Bursaphelenchus based upon spicule morphology. The paired spicules are uniquely shaped and ventrally recurved, and both B. anatolius n. sp. and B. kevini possess extending flaps that open when the spicules are protracted beyond the cloaca. Population growth of B. anatolius n. sp. was measured at 23 °C in the laboratory on cultures of the fungus Monilinia fructicola grown on lactic acid-treated, 5% glycerol-supplemented potato dextrose agar. Nematode population densities rapidly increased from 110 to about 110,000/9-cm-diam. dish within 21 days.     

Steinernema neocurtillis n. sp. (Rhabditida: Steinernematiclae) and a Key to Species of the Genus Steinernema

Steinernema neocurtillis n. sp. isolated from the mole cricket Neocurtilla hexadactyla Perty can be distinguished from other members of the genus by characteristics of the first-generation male and the third-stage infective juvenile (IJ). In the male, the distance from the anterior end to the excretory pore (DAE) is less than the body width at the excretory pore; D% (DAE divided by length of esophagus x 100) is low at 19. The gubernaculum legth is greater than three-fourths the spicule length. Range of the ratio gubernaculum length divided by spicule length is 0.82-0.93 in the first-generation male and 0.92-1.00 in the second-generation male. In the IJ, the distance from the anterior end to the excretory pore is extremely short (18 μm), causing the D% and E% (DAE divided by tail length x 100) to be low (D% = 23 and E% = 12). Average body length of the IJ is 885 μm.     

Description and SEM Observations of Meloidogyne sasseri n. sp. (Nematoda: Meloidogynidae), Parasitizing Beachgrasses

Meloidogyne sasseri n. sp. is described and illustrated from American beachgrass (Ammophila breviliffulata) originally collected from Henlopen State Park and Fenwick Island near the Maryland state line in Delaware, United States (6). Its relationship to M. graminis, M. spartinae, and M. californiensis is discussed. Primary distinctive characters of the female perineal pattern were a high to rounded arch with shoulders, widely spaced lateral lines interrupting transverse striations, a sunken vulva and anus, and coarse broken striae around the anal area. Second-stage juvenile body length was 554 μm (470-550), stylet length 14 μm (13-14.5), tail length 93 μm (83-115), tapering to a finely rounded terminus. Male stylet length 20 μm (19-21.5), spicule length 33 μm (30-36). Scanning electron microscope observations provided additional details of perineal patterns and face views of the female, male, and J2 head. Wheat, rice, oat, Ammophila sp., Panicum sp., bermudagrass, zoysiagrass and St. Augustinegrass were tested as hosts. Distribution of the species was the coasts of Delaware and Maryland. The common name "beachgrass root-knot" is proposed for M. sasseri n. sp.     

Two New Species of Aphelenchoididae (Nemata) from Declining Red Pines in Maryland

Aphelenchoides resinosi n. sp. and Ektaphelenchus joyceae n. sp. are described and illustrated from red pines of the Allegheny plateau of Maryland, USA. The new species were found in trees infested with Bursaphelenchus xylophilus. Primary diagnostic characters of A. resinosi females are constriction of the head, basal stylet knobs, tong postuterine sac, two incisures in the lateral field, and conical tail four to five anal body widths long with a simple terminal mucro. Diagnostic characters of the males are two pairs of subventral caudal papillae and spicule shape: Primary diagnostic characters of E. joyceae females are a slight constriction of the head, six similar lips, conical tail, and short postuterine sac. Diagnostic characters of the males are spicule size and shape, a single row of spermatocytes, and one pair of caudal papillae. Within-tree distributions of A. resinosi and E. joyceae are presented. A total of 70% of both red-needled and chlorotic-needled trees in the study were positive for A. resinosi and E. joyceae. Branch hierarchy was related to the percentage of samples positive for A. resinosi.     

Anchitrema sanguineum (Digenea: Anchitrematidae) Accidentally Found during Colonoscopy of a Patient with Chronic Abdominal Pain: A Case Report

In November 2007, a 46-year-old male Thai patient presented with chronic abdominal pain for over 3 years. Colonoscopy revealed a small parasite of about 2 × 1 mm in size attached to the cecum mucosa. The worm was removed endoscopically, fixed, and stained for morphological observations. The specimen was identified as Anchitrema sanguineum (Digenea: Anchitrematidae), a trematode first reported in a reptile, Chamaeleo vulgaris, from Egypt, and then sporadically found in the intestines of insectivorous bats and other mammals. The patient was treated with praziquantel but no more worms were found in his stool. His symptoms improved slightly but not cured completely. It remains unclear whether the chronic abdominal pain of the patient was caused by this trematode infection. Whatever is the pathogenicity of this trematode, this is the first human case of A. sanguineum infection in the literature.     

Morphology and Description of Bursaphelenchus platzeri n. sp. (Nematoda: Parasitaphelenchidae), an Associate of Nitidulid Beetles

Bursaphelenchus platzeri n. sp., an associate of nitidulid beetles in southern California, is described and illustrated. Adult males and females of B. platzeri n. sp. were examined by scanning electron microscopy for ultrastructural comparisons with other members of the genus. Bursaphelenchus cocophilus (red ring nematode) appears to be the closest related taxon to B. platzeri n. sp. based upon shared morphological features of the fused spicules, female tail shape, phoresy with non-scolytid beetles, and molecular analysis of the near full-length small subunit (SSU) rDNA. Unfortunately, sequence data from the D2D3 expansion segments of the large subunit (LSU) rDNA and partial mitochondrial DNA COI did not help resolve the relationship of nearest relative. In addition to significant molecular sequence differences in SSU, LSU, and COI, B. platzeri n. sp., which is an obligate fungal feeder, can be differentiated from B. cocophilus because it is an obligate parasite of palms. Bursaphelenchus platzeri n. sp. can be differentiated from all other species of Bursaphelenchus by the length and shape of the female tail and spicule morphology. The spicules are fused along the ventral midline and possess unfused cucullae; the fused unit appears to function as a conduit for sperm. Population growth of B. platzeri n. sp. was measured in a time-course experiment at 25°C in the laboratory on cultures of the fungus Monilinia fructicola grown on 5% glycerol-supplemented potato dextrose agar (GPDA). Nematode population densities rapidly increased from 25 to approximately 200,000/culture within 14 d and then plateaued for up to 28 d.     

Schistogram changes after administration of antischistosomal drugs in mice at the early phase of Schistosoma mansoni infection

Mice infected with Schistosoma mansoni were treated with oxamniquine, praziquantel, artesunate at the pre-patent phase, aiming at observing schistogram alterations. Half of the animals were perfused five days post-treatment for counting and classification of immature worms, based on pre-established morphological criteria (schistogram); the remaining animals were evaluated 42 or 100 days after infection and perfusion of the portal-system was performed for collection and counting of adult worms and oogram. It was observed that oxamniquine and artesunate treatment administered at the pre-postural phase causes significant reduction in the number of immature and adult worms. However, there was little reduction with praziquantel when used at the dose of 400 mg/kg for treatments administered 14, 15, 21 or 23 days post-infection. Artesunate was responsible for significant alterations in development of young worms, as well as for a higher number of worms presenting intestinal damages. Immature adult worms were detected in mice treated with artesunate or oxamniquine at the pre-patent phase of infection and recovered by perfusion 100 days after infection. Schistogram proved to be a very useful tool for experimental evaluation of the activity of antischistosomal drugs and a good model to identify the most sensitive stages to drugs.     

Steinernema diaprepesi n. sp. (Rhabditida: Steinernematidae), a Parasite of the Citrus Root Weevil Diaprepes abbreviatus (L) (Coleoptera: Curculionidae)

A nematode collected from Diaprepes abbreviatus is identified and described as a new species, Steinernema diaprepesi n. sp. The new species is closely related to S. feltiae, S. glaseri, and S. oregonense and can be distinguished from these species by the following characteristics: Males: Spicule averaging 79 (71-90) µm and spicule shape; D% (distance from anterior end to excretory pore/ esophagus length × 100) about 80; the ratio SW (spicule length/anal body width) about 1.8. Females: Vulva with short, double- flapped epiptygma; tail terminus usually with 5 papillae-like structures. Infective juveniles: Body averaging 1,002 (880-1,133) µm, EP (distance from anterior end to excretory pore) = 74 (66-83) µm; tail length = 83 (65-91) µm, and E% (EP/tail length × 100) = 89.6 (78-114). Lateral field pattern variable, the formula for the arrangement of ridges from head to tail is: 2, 6, 7, 8, 4, 2. The portion with eight ridges is the longest. This new species can be differentiated further from three closest species (S. feltiae, S. glaseri, and S. oregonense) by characteristic sequences of their ITS regions, including sequence lengths, ratios of similarity, composition, and differences in base characters in sequence alignment.     

Tylenchulus palustris Parasitizing Peach Trees in the of nited States

Most morphological characteristics of three populations of a Tylenchulus sp. from peach roots in Alabama, Arkansas, and Georgia did not differ from those of T. palustris paratypes. However, some mature females differed slightly from those of T. palustris paratypes from Florida. These mature females were more swollen in the posterior portion of their bodies, and they possessed digitate postvulval body sections with round rather than conoid termini. These morphological variants had a wide postvulval section core (PVSC), as do T. palustris paratypes; they did not differ from the paratypes in other characteristics. Second-stage juveniles and males were less morphologically variable and were not different from the paratypes. No males were found in populations from Alabama and Georgia. The Tylenchulus sp. from three peach sites was determined to be T. palustris. This is the first report of T. palustris on an economically important crop.     

Paraiotonchium muscadomesticae n. sp. (Tylenchida: lotonchiidae) a Parasite of the House Fly (Musca domestica) in Brazil and a Key to Species of the Genus Paraiotonchium

Paraiotonchium muscadomesticae n. sp., a parasite of the house fly, Musca domestica L., is described and illustrated from material collected in Brazil. The life cycle of P. muscadomesticae is similar to that of P. autumnale (Nickle), consisting of alternating gamogenetic and parthenogenetic generations. Paraiotonchium muscadomesticae n. sp. can be distinguished from P. nicholasi Slobodyanyuk, P. autumnale (Nickle) Slobodyanyuk, and P. crassirostris (Yatham &Rao) Siddiqi by the shorter body length of young heterosexual females, 652 g.m (530-709) for P. muscadomesticae compared to 750 μm or more (801-1,050) for the others. Paraiotonchium muscadomesticae is close to P. nicholosi but differs from it by V ratio and spicule length (V = 80-84; spicule = 16-21 p.m in P. muscadomesticae compared to V = 73-78; spicule = 25-35 μm in P. nicholasi). Paraiotonchium muscadomesticae and P. nicholasi differ from all species of this genus by the absence of a bursa on males of these two species.     

Ochoterenella esslingeri n. sp. (Nematoda: Onchocercidae: Waltonellinae) from Bokermannohyla luctuosa (Anura: Hylidae) in Minas Gerais,Brazil, with notes on Paraochoterenella Purnomo & Bangs, 1999

The waltonelline Ochoterenella esslingeri n. sp., a filarial parasite of the anuran Bokermannohyla luctuosa in Minas Gerais, Brazil is described. Several characters distinguish this new species from the 15 species presently included in the genus: the cuticular ornamentation of the female that is restricted to the posterior region of the body, the irregular arrangement of the small, rounded bosses, the postoesophageal vulva, the short glandular oesophagus, the size and shape of the microfilariae, the long left spicule and high spicular ratio. Irregularly arranged, tiny, rounded bosses are common in the monotypic genus Paraochoterenella from an Indonesian ranid, which is not well defined but likely valid. In the Neotropical Realm, the type hosts of the species of Ochoterenella are Hylidae (O. esslingeri n. sp.), Leptodactylidae (two species) and the remaining 13 species were described from the giant toad Rhinella marina (Bufonidae).     

Caudal Papillae in Romanomermis culicivorax

Distribution of caudal papillae in adult Romanomermis culicivorax was determined by scanning electron microscopy. Ninety eight caudal papillae, each containing one pore, were present in males but absent in females. Papillae were arranged in three longitudinal rows, one ventral, two ventrolateral; the middle ventral row bifurcated anterior to the spicule. The appearance of the papillae was different anterior and posterior to the spicule. The role of the caudal papillae in mediating copulatory behavior was discussed.     

A Horsehair Worm,Gordius sp. (Nematomorpha: Gordiida), Passed in a Canine Feces

Nematomorpha, horsehair or Gordian worms, include about 300 freshwater species in 22 genera (Gordiida) and 5 marine species in 1 marine genus (Nectonema). They are parasitic in arthropods during their juvenile stage. In the present study, the used gordian worm was found in the feces of a dog (5-month old, male) in July 2014. Following the worm analysis using light and scanning electron microscopes, the morphological classification was re-evaluated with molecular analysis. The worm was determined to be a male worm having a bi-lobed tail and had male gonads in cross sections. It was identified as Gordius sp. (Nematomorpha: Gordiidae) based on the characteristic morphologies of cross sections and areole on the cuticle. DNA analysis on 18S rRNA partial sequence arrangements was also carried out, and the gordiid worm was assumed to be close to the genus Gordius based on a phylogenic tree analysis.     

Teratorhabditis palmarum n. sp. (Nemata: Rhabditidae): An Associate of Rhynchophorus palmarum and R. cruentatus

Teratorhabditis palmarum n. sp., an associate of the palm weevils Rhynchophorus palmarum and R. cruentatus is described. Teratorhabditis palmarum was isolated from newly emerged adults and cocoons of R. palmarum from red-ring diseased coconut palms, Cocos nucifera, in Trinidad and Ecuador, and from red-ring diseased oil palms, Elaeis guineensis, in Colombia. Teratorhabditis palmarum was also associated internally with newly emerged adults of R. cruentatus from mature transplanted cabbage palmettos, Sabal palmetto, in Florida. Dauer juveniles of T. palmarum infested the genital capsule and body cavity of newly emerged adult female and male palm weevils. Adult nematodes isolated from cocoons and dauer juveniles from newly emerged palm weevils were cuhurable on bacterial lawns on several solid media. Females of T. palmarum have a single anteriorly directed ovary; vulva at 93-96% of the body length; short, hemispherical spicate tail; three or four teeth in the metastom; cuticle with distinct transverse punctations that change abruptly at the level of the procorpus to indentations of alternating size and arrangement; and eggs with cuticular sculpturing. Males have a crenate, peloderan bursa with a 2 + 5 + 3 pattern of bursal rays (7 extend to the margin of bursa); spicules linear, completely fused at the distal tip and dorsally for 50% of the total spicule length.     

Description of Hemicycliophora biosphaera n. sp. from Arizona (Nemata: Criconematidae)

Hemicycliophora biosphaera n. sp. (Nemata: Criconematidae) was found in soil from a fallow field plot within the Biosphere 2 Center, Oracle, Arizona. The nematode species is characterized by continuous and irregular breaks in transverse striae in the lateral field, smooth annules, a rounded-truncate lip region with rounded anterior margins, three lip annules, first labial annule elevated and widened laterally, dome-shaped and elevated labial disc, stylet length (76-97 (μm), VA%T value (30-59), 234-273 body annules, and tail with a terminus offset, cylindrical to slightly conoid digit. Hemicycliophora biosphaera n. sp. most closely resembles H. armandae but differs from it in body width (30-39 vs. 38-54 μm), stylet length (76-97 vs. 95-119 μm), greater number of annules between the excretory pore and esophagus base (4-16 vs. 2), length of the tail terminal spike (16-28 vs. 32 μm), lower Rvan value (9-15 vs. 16), and indistinct spetanatheca vs. distinct spermatheca.     

Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.     

Susceptibility of Laboratory Rodents to Trichinella papuae

Members of the genus Trichinella are small nematodes that can infect a wide range of animal hosts. However, their infectivity varies depending on the parasite and host species combination. In this study, we examined the susceptibility of 4 species of laboratory rodents, i.e., mice, rats, hamsters, and gerbils to Trichinella papuae, an emerging non-encapsulated Trichinella species. Trichinella spiralis and Trichinella pseudospiralis were also included in this study for comparison. Fifteen animals of each rodent species were infected orally with 100 muscle larvae of each Trichinella species. Intestinal worm burden was determined at day 6 and 10 post-inoculation (PI). The numbers of muscle larvae were examined at day 45 PI. The reproductive capacity index (RCI) of the 3 Trichinella species in different rodent hosts was determined. By day 6 PI, 33.2-69.6% of the inoculated larvae of the 3 Trichinella species became adult worms in the small intestines of the host animals. However, in rats, more than 96% of adult worms of all 3 Trichinella species were expelled from the gut by day 10 PI. In gerbils, only 4.8-18.1% of adult worms were expelled by day 10 PI. In accordance with the intestinal worm burden and the persistence of adults, the RCI was the highest in gerbils with values of 241.5±41.0 for T. papuae, 432.6±48 for T. pseudospiralis, and 528.6±20.6 for T. spiralis. Hamsters ranked second and mice ranked third in susceptibility in terms of the RCI, Rats yielded the lowest parasite RCI for all 3 Trichinella species. Gerbils may be an alternative laboratory animal for isolation and maintenance of Trichinella spp.