Identification of Entomopathogenic Nematodes in the Steinernematidae and Heterorhabditidae (Nemata: Rhabditida)

This paper contains taxonomic keys for the identification of species of the genera Steinernema and Heterorhabditis. Morphometrics of certain life stages are presented in data tables so that the morphometrics of species identified using the keys can be checked in the tables. Additionally, SEM photographs and diagnoses of the families and genera of Steinernematidae and Heterorhabditidae are presented.     

Effect of Entomopathogenic Nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) on Meloidogyne mayaguensis Rammah and Hirschmann (Tylenchida: Meloidoginidae) Infection in Tomato Plants

Some studies suggest that entomopathogenic nematodes (EPN) affect plant-parasitic nematode populations. Here, the effects of live and dead IJ of Heterorhabditis bacteriophora JPM4, H. baujardi LPP7, Steinernema feltiae SN and S. carpocapsae All were evaluated against eggs and J2 of the plant-parasitic nematode Meloidogyne mayaguensis. According to treatment, 100 IJ were applied with 350 eggs, 350 J2 or 175 eggs + 175 J2 to tomato plants. Bioassays were conducted in March to May and repeated in September to November 2005. Both experiments lasted 9 weeks, and the variable evaluated was number of galls per plant. When eggs were used for infections in the first trial, plants exhibited lower gall number compared to control when live and dead H. baujardi IJ and live S. feltiae IJ were added (9.7, 4.5, 7.3 and 85.7 galls, respectively). In the second trial, live S. feltiae and S. carpocapasae IJ influenced gall formation compared to control (14.33, 14.57 and 168.02 galls, respectively). When J2 were used for infections, plants with live H. baujardi IJ presented less galls when compared to control in both trials (38.3 and 355.7 galls in the first trial and 145.2 and 326.2 in the second one, respectively). Infection with a mixture of J2 and eggs resulted in fewer galls than when live S. feltiae IJ were present in both trials, compared to control (38.3 and 44.2 galls vs. 275.3 and 192.2 galls, respectively). We conclude that H. baujardi and S. feltiae apparently may be inhibiting egg hatching and J2 infection.     

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae,Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.     

Mortality of Immature Houseflies (Musca domestica L.) in Artificial Diet and Chicken Manure after Exposure to Encapsulated Entomopathogenic Nematodes (Rhabditida: Steinernematidae, Heterorhabditidae)

The entomopathogenic nematodes, Steinernema felitae (=bibionis) and Heterorhabditis megidis, were encapsulated in calcium alginate and their efficacy was tested against immature houseflies. Aliquots of capsules (15 ml) containing either 1 000 000, 500 000, 250 000 or 125 000 nematodes were added to 70-ml portions of grassmeal diet containing either eggs of first, second or third instar larvae. After 2 days, the treatment with 1 000 000 encapsulated S. feltiae (=bibionis) had killed 94% of housefly eggs and 90% of first instar larvae. By day 6, both of these mortalities had increased significantly (P 0.005) to 100%. By day 2, in the same medium, 1 000 000 encapsulated H. megidis had killed 71.4% of eggs and 90% of first instar larvae. This increased significantly (P 0.01) by day 6, to 99.2% and 100% respectively. Another experiment was carried out where immature houseflies were placed in chicken manure. The emergence of houseflies as adults was used to measure the effect of the encapsulated nematodes. Depending on the numbers of nematodes and the original stage of the housefly, the treatment with encapsulated S. feltiae resulted in 55-96% reduction in adult housefly emergence, whereas treatment with encapsulated H. megidis resulted in 35-98% reduction in emergence. Finally, when encapsulated nematodes were presented as a bait to adult houseflies, little infectivity was observed.     

Structure,development, and evolutive patterns of spermatozoa in rhabditid nematodes (Nematoda: Rhabditida)

Spermatogenesis of five rhabditid nematodes was studied using transmission electron microscopy and is described herein. Structure and development of nematode sperm in all available representatives of the extensive order Rhabditida have been analysed and the main characteristics of each infraorder are discussed. The ancestral sperm of the order Rhabditida was reconstructed using maximum likelihood and Bayesian methods based on 44 ultrastructural sperm characters. The hypothetical ancestral spermatogenesis of the order Rhabditida agrees with the previously suggested “rhabditid” pattern and appears to be conserved throughout the order Rhabditida. Despite the enormous variation of rhabditid nematodes, few groups deviate from the ancestral pattern. This conserved pattern can be informative within the phylum Nematoda at order level, but poses limitations when used in taxonomic and phylogenetic analysis within Rhabditida.     

Susceptibility of Sap Beetles (Coleoptera: Nitidulidae) to Entomopathogenic Nematodes

Nitidulid beetles (Coleoptera) are considered to be serious pests of date palms throughout the world. They attack ripe fruit, causing it to rot, and damage is reflected in both reduced yield and lower fruit quality. The present study was aimed at an evaluation of the susceptibility to different sap beetles to entomopathogenic nematodes. We further tested nematode efficacy in pots filled with soil infested by third instar larvae of the two beetle species. In Petri dish assay, mortality levels of Carpophilus humeralis and C. hemipterus exposed to Heterorhabditis sp. IS-5 strain indicated that the latter is less susceptible to nematode infection. Exposure of both sap beetle species to different nematode strains gave moderate levels of mortality (35-65%) with the heterorhabditid strains HP88, IS-5 and IS-25. The IS-12 strain of Heterorhabditis sp. showed poor virulence (<35% mortality) against larvae of C. humeralis as well as larvae and pupae of C. humipterus. The nematode species S. riobrave showed moderate virulence (35-65%) mortality to larvae and pupae of S. humeralis as well as to larvae of C. hemipterus . Exposure of C. hemeralis to different concentrations of Heterorhabditis sp. IS-5 in pots containing soil resulted in high mortality (>65%). In contrast, the lower concentrations (500 and 1000 nematodes/pot) caused low mortality (35%) of C. hemipterus . Other heterorhabditid strains caused 95-100% mortality of C. humeralis in pot assay. The HP88 strain of H. bacteriophora and the Tx strain of Steinernema riobrave showed poor effectiveness. Incubation of different nematode strains with the C. humeralis larvae at high temperature (32 C) resulted in an increase in insect mortality with the IS-12 and IS-21 strains. Reduced mortality was recorded with the HP88 strain treatment at the higher temperature. The IS-5 and IS-12 strains were equally effective in all three soil types tested, whereas the IS-19 strain was more effective in the Almog type soil than in the others.     

Survey of Heterorhabditidae and Steinernematidae (Rhabditida,Nematoda) in Western Canada

A survey was done in the summer months along the Alaska Highway, in other parts of British Columbia, in northern Alberta, and in the Yukon Territory for steinernematid and heterorhabditid nematodes occurring in the top 10 cm of soil. Steinernema feltiae and Steinernema spp. were found at 18 and Heterorhabditis megidis at 7 sites of 125 sampled. Most nematodes were found where visible insect infestation occurred and where human influence on the habitat was substantial (e.g., agricultural, forested and bush-hedgerow habitats); none was found in grassland or virgin forests. Heterorhabditis megidis occurred in only the southern, warmer, drier region of British Columbia. In the laboratory some steinernematid isolates and H. megidis killed Galleria mellonella larvae at 13 and 22 C, whereas some isolates of Steinernema killed the larvae at only 13 C. Steinernema spp. from three high altitude sites with low, average July temperatures (13-14 C) are cold-active in that they produced infective juveniles at 13 C and killed G. mellonella at 6 C.     

Population and infection dynamics of Daubaylia potomaca (Nematoda: Rhabditida) in Helisoma anceps

Daubaylia potomaca is an unusual parasite for several reasons. Specifically, it has a direct life cycle in which it uses a planorbid snail, Helisoma anceps , as the definitive host. In addition, adult females have been shown to be both the infective stage and the only stage documented to be shed from a live, infected host. Finally, adults, juveniles, and eggs have been observed in all tissues and blood spaces of the host, suggesting the parasite consumes and actively migrates through host tissue. The present study examined the population and infection dynamics of D. potomaca in Mallard Lake, a 4.9-ha public access pond in the Piedmont region of North Carolina. In particular, the study examined the role of seasonality on the prevalence and mean intensity of infection of D. potomaca in the snail host. Data collected from August 2008 to October 2009 suggest that prevalence and mean intensity were inversely related in the spring and fall. Prevalence in fall 2008 was 10.3% but increased to 47.3% in spring 2009. Conversely, intensity was high in fall 2008 at 52.4 ± 8.9 worms/infected host but dropped to 3.1 ± 0.3 worms/infected host in spring 2009. During the same time, the parasites within the snails went from highly aggregated populations in the fall to a less aggregated distribution in the spring. It is hypothesized that D. potomaca induces mortality of the snail hosts during the winter, followed by a rapid recruitment event of the nematodes by the snail population after torpor.     

Evaluation of Entomopathogenic Nematodes for the Control of Mediterranean Fruit Fly (Diptera: Tephritidae)

The virulence of various entomopathogenic nematode (EPN) strains was evaluated against the Mediterranean fruit fly, C. capitata . The selected nematodes were assessed for their infectivity for the final larval stage of the insect host and under varying environmental conditions. Among 12 EPN strains tested, Steinernema riobrave Texas ( Sr TX) and Heterorhabditis sp. IS-5 (H IS-5), showed high activity and induced >80% mortality. Six EPN strains showed limited activity (>30% mortality), and four strains had no effect (<20% mortality). Sr TX was more effective than H IS-5. Mature C. capitata larvae were most susceptible to nematode infection during the first 4h after they began to emerge from their diet to pupate. Activity of the two nematode strains at a constant inoculation rate was dependent on insect larval density. The highest activity was recorded at 1.88 larvae cm -2 and decreased at higher larval densities. EPN activity was also directly related to nematode density. Maximal activity was shown at a density of 150 infective juveniles cm -2 . A similar activity pattern was also recorded with Sr TX in four different soil types. The persistence of this EPN in the soil extended over 5 days but there was no activity after 14 days. Except for a lower activity under cool conditions (17°C), temperatures ranging between 22 and 41°C, or moisture levels in the treated soil ranging between 3 and 20%, had no significant effect on nematode activity. Our results suggest that application of Sr TX against C. capitata may have potential for controlling C. capitata .     

Pathogenicity of Two Species of Entomopathogenic Nematodes Against the Greenhouse Whitefly,Trialeurodes vaporariorum (Hemiptera: Aleyrodidae), in Laboratory and Greenhouse Experiments

The greenhouse whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) is a polyphagous pest in greenhouse crops. The efficacy of two entomopathogenic nematodes (EPN), Steinernema feltiae and Heterorhabditis bacteriophora, as biological control agents against T. vaporariorum was evaluated using two model crops typical of vegetable greenhouse productions: cucumber and pepper. Laboratory tests evaluated adults and second nymphal instars for pest susceptibility to different EPN species at different concentrations of infective juveniles (IJ; 0, 25, 50, 100, 150, 200, and 250 IJ per cm²); subsequent greenhouse trials against second nymphal instars on cucumber and pepper plants evaluated more natural conditions. Concentrations were applied in combination with Triton X-100 (0.1% v/v), an adjuvant for increasing nematode activity. In laboratory studies, both life stages were susceptible to infection by the two nematode species, but S. feltiae recorded a lower LC₅₀ than H. bacteriophora for both insect stages. Similarly, in greenhouse experiments, S. feltiae required lower concentrations of IJ than H. bacteriophora to reach the same mortality in nymphs. In greenhouse trials, a significant difference was observed in the triple interaction among nematode species × concentration × plant. Furthermore, the highest mortality rate of the second nymphal instars of the T. vaporariorum was obtained from the application of S. feltiae concentrated to 250 IJ/cm² on cucumber (49 ± 1.23%). The general mortality caused by nematodes was significantly higher in cucumber than in pepper. These promising results support further investigation for the optimization of the best EPN species/concentration in combination with insecticides or adjuvants to reach a profitable control of this greenhouse pest.     

Molecular phylogeny of the Tylenchina and evolution of the female gonoduct (Nematoda: Rhabditida)

Tylenchina are a morphologically and functionally diverse group of nematode species that range from free-living bacteriovores, over transitory grazing root-hair feeders to highly specialized plant-parasites with complex host associations. We performed phylogenetic analyses of small subunit rDNA sequences from 97 species including an analysis that account for the RNA secondary structure in the models of evolution. The present study confirms the sister relationship of the bacteriovore Cephalobidae with the predominantly plant-parasitic Tylenchomorpha. All analyses appoint the fungal-feeding Aphelenchidae and Aphelenchoididae as being polyphyletic but the morphology based hypothesis of their monophyly could not be significantly rejected. Within the Tylenchomorpha, the families that exclusively parasitize higher plants are joined in a single clade. However, only the monophyletic position of the (super)families Hoplolaimidae and Criconematoidea were supported; Anguinidae, Tylenchidae, Belonolaimidae and Pratylenchidae appeared to be paraphyletic or polyphyletic. Parsimony and likelihood ancestral state reconstruction revealed that burrowing endoparasitism and sedentary endoparasitism each evolved, respectively, at least six and at least three times independently, mostly from migratory ectoparasitic ancestors. Only root-knot nematodes have evolved from burrowing endoparasitic nematodes. Traditional classifications are partially misled by this convergent evolution of feeding type and associated morphology. Contrastingly, mapping attributes of the gonoduct cellular architecture, including newly obtained data of 18 species belonging to the Aphelenchoidea, Criconematoidea, Anguinidae and Panagrolaimidae, revealed a broad congruence of the gonoduct characters and the molecular phylogenetic hypothesis. Yet, the presence of an offset spermatheca and proliferation of uterus cells has evolved multiple times, the latter associated with derived endoparasitic feeding specialization and resulting reproduction mode. Ancestral state reconstruction further revealed that the gonoduct of the morphologically and ecologically dissimilar tylenchid and cephalobid nematodes evolved from a common ancestor.     

Pathogenicity of Microtetrameres centuri Barus, 1966 (Nematoda: Tetrameridae) in Meadowlark

Pathogenicity of adult and third-stage Microtetrameres centuri Barus, 1966 in meadowlarks (Sturnella spp.) is reported for the first time. Adults do not encyst; their effects are mainly mechanical. Some glandular atrophy of its avian host is detected but no marked inflammatory response is exhibited. Juveniles also encyst experimentally in the perivisceral sinus of the grasshopper (alternate host) hemocoel.     

The action of the anterior feeding apparatus of Caenorhabditis elegans (Nematoda: Rhabditida)

From analysis of ciné film, combined with ultrastructural observations, the anterior feeding structures and their behaviour in the free-living microbivorous nematode Caenorhabditis elegans during ingestion in dense and sparse suspensions of Escherichia coli are described In dense suspensions bacteria accumulate in the buccal cavity against the three metastomal flaps that nearly close it, and are then swallowed down the three tubular apical expansions of the triradiate oesophageal lumen when the flaps open. Excess medium is then expelled, as the oesophageal lumen closes and traps the swallowed bacteria. The flaps and oesophagus operate by contractions in the seven most anterior oesophageal muscle cells, probably coordinated via proprioceptive nerve endings between the cells. About one million nematode hours are needed to extract 1 g of bacteria from 30 ml of medium. With few or no bacteria, the head moves more, the metastomal flaps do not close and medium seems to pass in and out of the buccal cavity, probably as part of the widespread exploration phase of food-finding behaviour. Abnormal feeding behaviour, control and functions of the metastomal flaps and associated structures, and control of food intake volume are discussed.     

Entomopathogenic Nematodes: Biodiversity, Geographical Distribution and the Convention on Biological Diversity

This paper addresses three major issues. Firstly, molecular taxonomy and its application to elucidate the biodiversity and biogeography of entomopathogenic nematodes is considered. Accurate identification is fundamental for understanding biodiversity, and because these nematodes are morphologically conservative, molecular techniques will provide the insights necessary to develop a robust, morphologically based taxonomy. Secondly, a review of the knowledge on their biogeography and habitat specificity, including a consideration of the limitations to the available data is given. Much of the information is presented in two tables which summarize the distributions of recognized species at continental and national levels. Thirdly, this paper provides a brief consideration of the Convention on Biological Diversity and its implications for future work with entomopathogenic nematodes and biological control.     

Laboratory infection of terrestrial isopods (Crustacea: Isopoda) with neoaplectanid and heterorhabditid nematodes (Rhabditida: Nematoda)

Under laboratory conditions, the nematodes Neoaplectana carpocapsae and Heterorhabditis heliothidis were able to infect and develop inside the hemocoel of terrestrial isopods belonging to the genus Porcellio. Armidillidium vulgare was also infected by N. carpocapsae, but this host was less susceptible than Porcellio. Results show that, under suitable ecological conditions, it would be feasible to utilize N. carpocapsae as a biological control agent of sowbugs (Porcellio spp.). This is the first report showing the ability of neoaplectanids and heterorhabditids to invade representatives of the Crustacea.     

Influence of Salinity on Survival and Infectivity of Entomopathogenic: Nematodes

Exposure to NaC1, KCI, and CaCl₂ affected the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema glaseri differently. Survival, virulence, and penetration efficiency of S. glaseri were not affected by these salts. At high concentrations, however, all three salts inhibited its ability to move through a soil column and locate and infect a susceptible host. Calcium chloride and KCl had no effect on H. bacteriophora survival, penetration efficiency, or movement through a soil column, but moderate concentrations of these salts enhanced H. bacteriophora virulence. NaCl, however, adversely affected each of these parameters at high salinities (>16 dS/m). Salt effects on S. glaseri are attributed solely to interference with nematode host-finding ability, whereas the NaCl effects on H. bacteriophora are attributed to its toxicity and possibly to interference with host-finding behavior.     

Structure of the pharynx in the adult nematode Anguillicoloides crassus (Nematoda: Rhabditida)

The structure of the pharynx of the adult female nematode Anguillicoloides crassus (Spirurina) has been studied for the first time using light and transmission electron microscopy. The cylindrical pharynx consists of a short anterior muscular corpus and an enlarged posterior glandular and muscular postcorpus. The main cellular components of the pharynx of A. crassus include the muscle cells, the marginal cells, the nerve cells, and 1 dorsal and 2 subventral glands. New observations for nematodes include: (1) the non-contractile regions of pharyngeal musculature in the corpus have specific appearance; (2) the ventrosublateral longitudinal nerve in the pharynx has an enlarged, enucleated anterior part, with a pronounced palmate projections; and (3) abundant lysosomelike membranous bodies consisting of myelinlike figures of varied size present in marginal cells and pharyngointestinal valve. The 2 subventral glands and, apparently, the single dorsal gland, have their openings at the same level, i.e., at the border between the corpus and postcorpus. The pharyngeal-intestinal valve joins the pharynx to the intestine. Knowledge of the ultrastructure of these complex characters may be useful in understanding of functional features, and for comparative morphology as well as evolutionary considerations within the Chromadorea.     

A Brief Report on the Chromosome Number of Neodiplogaster pinicola and Panagrellus redivivoides (Nematoda: Rhabditida)

Both Neodiplogaster pinicola and Panagrellus redivivoides reproduce amphimictically, with XO type of sex determination. In N. pinicola, primary spermatocytes have six bivalent chromosomes and one univalent; after two meiotic divisions, sperm are produced with either six or seven chromosomes. In primary oocytes, with seven bivalents, meiosis is initiated by entrance of a sperm. After two meiotic divisions, three polar nuclei are produced, and egg and sperm pronuclei fuse. Cleavage begins after the egg is laid. Males have a 2n number of 13 chromosomes; females, 14. In P. redivivoides, primary spermatocytes have four bivalents and one univalent. After two meiotic divisions, spermatids are produced with either four or five well separated chromosomes. In primary oocytes, the first maturation division is initiated after penetration of a sperm; after two meiotic divisions, each egg has five chromosomes. Cleavage begins immediately after fusion of egg and sperm pronuclei, and embryonic development and hatching occur within the uterus. Males have a 2n chromosome number of 9; females, 10.     

Neoaplectana Steiner, 1929 a junior synonym of Steinernema Travassos, 1927 (Nematoda; Rhabditida)

Summary A type specimen of Steinernema kraussei and a population of this nematode from the type host were compared with three species of Neoaplectana. No characters were found to separate the two genera and so Neoplectana Steiner, 1929 is considered to be a junior synonym of Steinernema Travassos, 1927. Valid species now included within the genus Steinernema are: S. kraussei (Steiner, 1923) Travassos, 1927 (type species); S. glaseri (Steiner, 1929) n.comb.; S. feltiae (Filipjev, 1934) n.comb. and S. bibionis (Bovien, 1937) n.comb. A key is given to these four species and their junior synonyms are listed.