Review of Pasteuria penetrans: Biology,Ecology, and Biological Control Potential

Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite that has shown great potential as a biological control agent of root-knot nematodes. Considerable progress has been made during the last 10 years in understanding its biology and importance as an agent capable of effectively suppressing root-knot nematodes in field soil. The objective of this review is to summarize the current knowledge of the biology, ecology, and biological control potential of P. penetrans and other Pasteuria members. Pasteuria spp. are distributed worldwide and have been reported from 323 nematode species belonging to 116 genera of free-living, predatory, plant-parasitic, and entomopathogenic nematodes. Artificial cultivation of P. penetrans has met with limited success; large-scale production of endospores depends on in vivo cultivation. Temperature affects endospore attachment, germination, pathogenesis, and completion of the life cycle in the nematode pseudocoelom. The biological control potential of Pasteuria spp. have been demonstrated on 20 crops; host nematodes include Belonolaimus longicaudatus, Heterodera spp., Meloidogyne spp., and Xiphinema diversicaudatum. Pasteuria penetrans plays an important role in some suppressive soils. The efficacy of the bacterium as a biological control agent has been examined. Approximately 100,000 endospores/g of soil provided immediate control of the peanut root-knot nematode, whereas 1,000 and 5,000 endospores/g of soil each amplified in the host nematode and became suppressive after 3 years.     

Commercial Development and Future Prospects for Entomogenous Nematodes

Although entomogenous nenmtodes generally have many of the attributes of the ideal biocontrol agent, many of these attributes make the nematodes less than desirable for commercial production. Environmental limitations, lack of patent protection, "shelf life," shipping problems, and the need for users to receive specialized training are factors that have discouraged the involvement of larger companies. The future of these nematodes as commercially available biocontrol products appears to lie with the smaller "cottage industries" or with government-subsidized production. Problems encountered with attempts to produce commercially the mosquito parasite Romanomermis culicivorax are discussed.     

Salts and the Infectivity of Romanomermis culicivorax

The effects of common inorganic ions found in freshwater on the infectivity of Romanomermis culicivorax and the survival of its host, Culex pipiens, were tested. In general, the median lethal concentrations found for R. culicivorax were greater than the reported median concentrations of these ions in freshwater but less than the reported maximum natural concentrations. The ion toxicity for R. culicivorax (on a molar basis) increased in the following order: sodium < potassium < calcium; aml chloride < carbonate = sulfate < nitrate < nitrite < phosphate. The larvae of C. pipiens were generally 20 to 75 times as tolerant of higher ion concentrations as were the preparasitic stages of R. culicivorax.     

Cellular pattern formation, establishment of polarity and segregation of colored cytoplasm in embryos of the nematode Romanomermis culicivorax

We have begun to analyze the early embryogenesis of Romanomermis culicivorax, an insect-parasitic nematode phylogenetically distant to Caenorhabditis elegans. Development of R. culicivorax differs from C. elegans in many aspects including establishment of polarity, formation of embryonic axes and the pattern of asymmetric cleavages. Here, a polarity reversal in the germline takes place already in P₁ rather than P₂, the dorsal-ventral axis appears to be inverted and gut fate is derived from the AB rather than from the EMS blastomere. So far unique for nematodes is the presence of colored cytoplasm and its segregation into one specific founder cell. Normal development observed after experimentally induced abnormal partitioning of pigment indicates that it is not involved in cell specification. Another typical feature is prominent midbodies (MB). We investigated the role of the MB region in the establishment of asymmetry. After its irradiation the potential for unequal cleavage in somatic and germline cells as well as differential distribution of pigment are lost. This indicates a crucial involvement of this region for spindle orientation, positioning, and cytoplasmic segregation. A scenario is sketched suggesting why and how during evolution the observed differences between R. culicivorax and C. elegans may have evolved.     

Growth and Development of Romanomermis culicivorax In Vitro

Various combinations of vertebrate and invertebrate tissue culture and microbiological media were utilized in an attempt to culture Romanomermis culicivorax (Mermithidae: Nematoda) in vitro. Most media were unsuitable and caused nematodes to become lumpy, vacuolated, and granular. Slow and limited growth and development of internal structures of the nematodes were obtained with variously supplemented Grace''s tissue culture and Schneider''s Drosophila media. In an enriched Grace''s medium, development attained by the nematodes after 3-4 wk was comparable to 4-5-day-old parasites grown in vivo in the mosquito host, Culex pipiens. Two molts were observed in vitro. Maximum dimensions in vitro were 7.0-mm length and 87-μm width at the widest point. The stichosome, stichocytes, and trophosome developed prominently. A filiform tail and highly cuticularized tube persisted throughout the culture period in vitro.     

Polyamine Synthesis by the Mermithid Nematode Romanomermis culicivorax

The polyamine and amino acid composition of the mermithid nematode, Romanomermis culicivorax, and its host, Aedes aegypti, was determined. Putrescine, spermidine, spermine, cadaverine and two acetylated spermidine derivatives were present in parasitic juveniles, newly-emerged post-parasites, and eggs of R. culicivorax. Whole insect homogenates of fourth-instar A. aegypti contained the same array of polyamines, except that the putrescine:spermidine ratio was the inverse of that in parasitic R. culicivorax. Polyamines and amino acids were in greater concentrations in the nematode eggs than in other developmental stages investigated. Both the host and nematode possess the biosynthetic capacity for polyamine biosynthesis, as evidenced by measurable activities of ornithine decarboxylase in the host''s tissues and the nematode''s free-living stages.     

Uptake of Lipids by the Entomophilic Nematode Romanomermis culicivorax

Romanomermis culicivorax juveniles were dissected out of Aedes aegypti larvae 7 days after infection and incubated under controlled conditions in isotonic saline containing a ¹⁴C-labeled fatty acid (palmitic acid), monoacylglycerol (glycerol monoolein), or triacylglycerol (glycerol tripalmate) nutrient source. The mermithid absorbed each of these lipids from the incubation medium, the rate of uptake being greatest for glycerol monoolein. No lipase activity was detected in whole nematode homogenates or in the media in which the nematodes were incubated. It is suggested that the nematode transports complex lipid molecules across its outer cuticle intact.     

Field Evaluation of Steinernema feltiae Against the Web-spinning Larch Sawfly Cephalcia lariciphila

Field trials were conducted in Rheola Forest, Wales, Great Britain, to determine the effectiveness of Steinernema feltiae UK strain in controlling the web-spinning larch sawfly Cephalcia lariciphila. Foliar sprays at the rate of 5,000-20,000 nematodes/100 cm branch resulted in 3.4-29.4% infection of sawfly larvae. Soil application of 200 nematodes/cm² resulted in 61% infection of sawfly prepupae and 17.3% of pupae. Prepupal infection ranged from 4.8 to 14.7% 1 year after nematode application. Soil applications of this nematode show that it has potential for biological control of sawfly prepupae.     

Soil Applications of Steinernematid and Heterorhabditid Nematodes for Control of Colorado Potato Beetles,Leptinotarsa decemlineata (Say)

Three strains of Steinernema feltiae Filipjev (All, Mexican, and Breton strains) and one of Heterorhabditis heliothidis (Khan, Brooks, and Hirschmann) were evaluated for their potential to control Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), larvae and pupae in the soil. In laboratory studies, H. heliothidis and S. feltiae (Mexican strain) produced the highest mortality (6 days posttreatment) of CPB when applied to the surface of a soil column containing mature CPB larvae 5 cm below. Mortality ranged from 80 to 90% at rates of 79-158 nematodes/cm². Similar results were seen in a field microplot study with all four nematodes; S. feltiae (Mexican strain) and H. heliothidis were most effective. Adult CPB emergence was reduced 86.5-100% after application of 31-93 H. heliothidis/cm² and 88.4-100% with 93-155 S. feltiae (Mexican strain)/cm². The All strain of S. feltiae was moderately effective (ca. 80% reduction at 93-155 nematodes/cm²), while the Breton strain was ineffective (< 40% reduction at 155 nematodes/cm²). In small plots of potatoes enclosed in field cages, application of H. heliothidis and S. feltiae (Mexican strain) at rates of 93-155 nematodes/cm² before larval CPB burial in the soil resulted in 66-77% reduction in adult CPB emergence. Soil applications of these nematodes show potential for biological control of CPB.     

Induced Resistance to Meloidogyne hapla by other Meloidogyne species on Tomato and Pyrethrum Plants

Advance inoculation of the tomato cv. Celebrity or the pyrethrum clone 223 with host-incompatible Meloidogyne incognita or M. javanica elicited induced resistance to host-compatible M. hapla in pot and field experiments. Induced resistance increased with the length of the time between inoculations and with the population density of the induction inoculum. Optimum interval before challenge inoculation, or population density of inoculum for inducing resistance, was 10 days, or 5,000 infective nematodes per 500-cm³ pot. The induced resistance suppressed population increase of M. hapla by 84% on potted tomato, 72% on potted pyrethrum, and 55% on field-grown pyrethrum seedlings, relative to unprotected treatments. Pyrethrum seedlings inoculated with M. javanica 10 days before infection with M. hapla were not stunted, whereas those that did not receive the advance inoculum were stunted 33% in pots and 36% in field plots. The results indicated that advance infection of plants with incompatible or mildly virulent nematode species induced resistance to normally compatible nematodes and that the induced resistance response may have potential as a biological control method for plant nematodes.     

The Effects of Temperature on the Infectivity of Romanomermis culicivorax

The survival time (ST) of the preparasitic larvae of Rornanomermis culicivorax was determined by measuring motility at 1, 6, 12, 18, 21, 27, 30, and 37 C; the ST₅₀ at each of these temperatures was 2.3, 2.2, 2.0, 2.0, 1.7, 1.6, 0.9, and 0.7 days, respectively. About one-third of the preparasites infected first-instar larvae of Culex pipiens within 24 h at 27 C. The preparasites were infective at 12 to 33 C with the optimum infectivity at 21-33 C. Lower temperatures decreased the percent infectivity but increased the time that the nematodes remained infective. The time required for host infection increased as the preparasitic larvae aged at 15, 21, and 27 C.     

Compatibility of Soil Amendments with Entomopathogenic Nematodes

The impact of inorganic and organic fertilizers on the infectivity, reproduction, and population dynamics of entomopathogenic nematodes was investigated. Prolonged (10- to 20-day) laboratory exposure to high inorganic fertilizer concentrations inhibited nematode infectivity and reproduction, whereas short (1-day) exposures increased infectivity. Heterorhabditis bacteriophora was more sensitive to adverse effects than were two species of Steinernema. In field studies, organic manure resulted in increased densities of a native population of Steinernema feltiae, whereas NPK fertilizer suppressed nematode densities regardless of manure applications. Inorganic fertilizers are likely to be compatible with nematodes in tank mixes and should not reduce the effectiveness of nematodes used for short-term control as biological insecticides, but may interfere with attempts to use nematodes as inoculative agents for long-term control. Organic manure used as fertilizer may encourage nematode establishment and recycling.     

Parasitism of Heterodera schachtii and Meloidogyne javanica by Hirsutella rhossiliensis in Microplots over Two Growing Seasons

Numbers of cyst and root-knot nematodes and percentage parasitism by the nematophagous fungus Hirsutella rhossiliensis were quantified in microplots over 2 years. The microplots contained either sugarbeets in loam infested with Heterodera schachtii or tomatoes in sand infested with Meloidogyne javanica. The fungus was added to half of the microplots for each crop. Although H. rhossiliensis established in both microplot soils, the percentage of nematodes parasitized did not increase with nematode density and nematode numbers were not affected by the fungus. The results indicate that long-term interactions between populations of the fungus and cyst or root-knot nematodes will not result in biological control.     

Nutrient Composition of Romanomermis culicivorax in Relation to Egg Production and Metabolism

The nutrient composition of postparasitic females (newly emerged juveniles, newly molted adults, and spent adults) and eggs of Romanomermis culicivorax was studied. Throughout post-parasitic development, proteins increased and lipids decreased progressively as a proportion of the dry weight; the proportion of glycogen within the nematodes remained stable. The greatest decrease in the lipid moiety occurred during egg production. Eggs contained relatively low levels of lipids (12% dry weight), and ca. 20% of the dry weight of the eggs was unaccounted for by lipid, protein, and glycogen determinations. Chitin, mucoproteins, and peptides were present in the eggs. The fatty acid composition of nematodes remained constant during postparasitic development; eggs contained a similar profile of fatty acids as postparasites, with marginally higher content of unsaturated fatty acids. Radiotracer studies showed that the eggs could oxidize glucose and palmitic acid.     

In Vivo Growth of Romanomermis culicivorax: Biochemical Changes During Parasitism

Biochemical analyses of total protein, lipid, carbohydrate, DNA, amino acid, and length, width, and dry weight measurements are reported for different stages of Romanomermis culicivorax cultured in the mosquito, Culex pipiens. The Bradford technique for assaying total protein was the most sensitive and reliable biochemical technique tested for assaying in vivo growth of R. culicivorax. Increases in total protein, lipid, carbohydrate, and dry weight during growth from preparasite to postparasite were greater than 6,900-fold for females and 2,300-fold for males. DNA increased 650-fold and 233-fold during development to female and male postparasites, respectively. The proportions of amino acids for preparasites were significantly different (P ≤ 0.01) from female and male postparasites for all amino acids tested, except methionine and tyrosine. Female and male postparasites were similar in protein, lipid, carbohydrate, DNA, and most amino acid proportions, but were significantly different in relative concentrations of serine, glycine, and alanine (P ≤ 0.01). Preliminary results suggest that the use of amino acid ratios from female postparasites improves the in vitro culture performance of R. culicivorax.     

Operons Are a Conserved Feature of Nematode Genomes

The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and “spliced leader” (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.     

Susceptibility of Psorophora columbiae Larvae over Time to Parasitism by Romanomermis culicivorax

The ability of Romanomermis culicivorax preparasites to penetrate and infect Psorophora columbiae decreased substantially after ca. 28 hours. Parasitism at temperatures typical of Louisiana rice fields (i.e., 26, 29, and 32 ± 0.5 C) showed a significant linear decrease (P < 0.01) as the percentage of older larval instars increased at the times of exposure. These data emphasize the need for a synchronous field application of preparasites to challenge the rapid development of early instars of Ps. columbiae. Applications of postparasites rather than insecticide treatments to potential mosquito breeding habitats may offer greater flexibility in larval mosquito control programs.     

Teratological Development in the Cephalic Anatomy of the Nematode Romanomermis culicivorax

Adults of Romanomermis culicivorax obtained from mass cultures were examined by scanning and transmission electron microscopy to determine the organization of their anterior sense organs. The normal pattern apparently consists of two lateral amphids plus six cephalic papillae. Lateral cephalic papillae contain two sense organs, each with a cuticular pore, while subdorsal and subventral papillae have three sense organs, each with a cuticular pore. About 30% of females and 80% of males examined showed aberrant developments in these sense organs. Both cuticle and underlying cells (hypodermis and neurons) are affected; some sense organs are absent while others are incompletely formed. Few aberrant worms were found in a smaller collection of worms reared in lower population densities. Perhaps aberrant forms are examples of teratological development resulting from, or promoted by, conditions used for mass rearing of biological control agents.     

Entomopathogenic Nematodes as a Model System for Advancing the Frontiers of Ecology

Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae have a mutualistic–symbiotic association with enteric γ-Proteobacteria (Steinernema–Xenorhabdus and Heterorhabditis–Photorhabdus), which confer high virulence against insects. EPNs have been studied intensively because of their role as a natural mortality factor for soil-dwelling arthropods and their potential as biological control agents for belowground insect pests. For many decades, research on EPNs focused on the taxonomy, phylogeny, biogeography, genetics, physiology, biochemistry and ecology, as well as commercial production and application technologies. More recently, EPNs and their bacterial symbionts are being viewed as a model system for advancing research in other disciplines such as soil ecology, symbiosis and evolutionary biology. Integration of existing information, particularly the accumulating information on their biology, into increasingly detailed population models is critical to improving our ability to exploit and manage EPNs as a biological control agent and to understand ecological processes in a changing world. Here, we summarize some recent advances in phylogeny, systematics, biogeography, community ecology and population dynamics models of EPNs, and describe how this research is advancing frontiers in ecology.     

Potential of Foliar,Dip, and Injection Applications of Avermectins for Control of Plant-Parasitic Nematodes

Studies were conducted to determine the potential of two avermectin compounds, abamectin and emamectin benzoate, for controlling plant-parasitic nematodes when applied by three methods: foliar spray, root dip, and pseudostem injection. Experiments were conducted against Meloidogyne incognita on tomato, M. javanica on banana, and Radopholus similis on banana. Foliar applications of both avermectins to banana and tomato were not effective for controlling any of the nematodes evaluated. Root dips of banana and tomato were moderately effective for controlling M. incognita on tomato and R. similis on banana. Injections (1 ml) of avermectins into banana pseudostems were effective for controlling M. javanica and R similis, and were comparable to control achieved with a conventional chemical nematicide, fenamiphos. Injections of 125 to 2,000 μg/plant effectively controlled one or both nematodes on banana; abamectin was more effective than emamectin benzoate for controlling nematodes.