The life cycle of Anguina agrostis: Development in host plant

Bird A.F. and Stynes B.A. 1981. The life cycle of Anguina agrostis: Development in the host plant. Internationaljournal for Parasitology11: 431–440. The growth and development of the infective second stage “dauer” larvae (DL₂) of Anguina agrostis into adults have been followed under field conditions in rye grass (Lolium rigidum). Three moults were observed to occur during the parasitic phase of development. From the third (second parasitic) moult onwards, there was much more variability in the size of the female nematodes than in the males and sexual dimorphism became very pronounced. The transition from the DL₂ to the second stage parasitic larva (PL₂) is marked by the disappearance of the numerous lipid storage granules which are characteristic of the DL₂, and the development in the PL₂ of an intestine which becomes more pronounced in each succeeding stage, particularly in the adult female. Anguina agrostis is unusual among parasitic nematodes in that the DL₂ has the thickest cuticle of all stages, including adults. The L₄ and adult males have thicker cuticles than the females at the same stages of development. Moulting appears to involve resorption of the innermost basal zone of the shed cuticle as well as morphological and chemical changes to the epicuticle.     

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.     

Genetic mapping of variation in dauer larvae development in growing populations of Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the appropriate induction of dauer larvae development within growing populations is likely to be a primary determinant of genotypic fitness. The underlying genetic architecture of natural genetic variation in dauer formation has, however, not been thoroughly investigated. Here, we report extensive natural genetic variation in dauer larvae development within growing populations across multiple wild isolates. Moreover, bin mapping of introgression lines (ILs) derived from the genetically divergent isolates N2 and CB4856 reveals 10 quantitative trait loci (QTLs) affecting dauer formation. Comparison of individual ILs to N2 identifies an additional eight QTLs, and sequential IL analysis reveals six more QTLs. Our results also show that a behavioural, laboratory-derived, mutation controlled by the neuropeptide Y receptor homolog npr-1 can affect dauer larvae development in growing populations. These findings illustrate the complex genetic architecture of variation in dauer larvae formation in C. elegans and may help to understand how the control of variation in dauer larvae development has evolved.     

Alteration in cellular acetylcholine influences dauer formation in Caenorhabditis elegans

Altered acetylcholine (Ach) homeostasis is associated with loss of viability in flies, developmental defects in mice, and cognitive deficits in human. Here, we assessed the importance of Ach in Caenorhabditis elegans development, focusing on the role of Ach during dauer formation. We found that dauer formation was disturbed in choline acetyltransferase (cha-1) and acetylcholinesterase (ace) mutants defective in Ach biosynthesis and degradation, respectively. When examined the potential role of G-proteins in dauer formation, goa-1 and egl-30 mutant worms, expressing mutated versions of mammalian G_o and G_q homolog, respectively, showed some abnormalities in dauer formation. Using quantitative mass spectrometry, we also found that dauer larvae had lower Ach content than did reproductively grown larvae. In addition, a proteomic analysis of acetylcholinesterase mutant worms, which have excessive levels of Ach, showed differential expression of metabolic genes. Collectively, these results indicate that alterations in Ach release may influence dauer formation in C. elegans. [BMB Reports 2014; 47(2): 80-85]     

For the Insect Pathogen Photorhabdus luminescens, Which End of a Nematode Is Out?

The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae. The dauer juvenile (DJ) stage nematode selectively retains P. luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host. We report the results of studying the transmission of the bacteria by its nematode vector. Cells of P. luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus. Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria. Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth. This regurgitation reaction was induced by a low-molecular-weight, heat- and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures. Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph. The ability to visualize P. luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae. This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study.     

Nemtaode-Vector Relationships in the Pine Wilt Disease System

Pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease in North America and Japan. Dispersal stage dauer larvae are transported to new host trees on the body surface and within the tracheal system of several beetle species. Worldwide, 21 species of Cerambycidae, 1 genus of Buprestidae, and 2 species of Curculionidae are known to carry pinewood nematode dauer larvae upon emerging from nematode-infested trees. Five species of cerambycids in the genus Monochamus are known to transmit dauer larvae to new host trees, four North American species and one Japanese species. Primary transmission to healthy trees occurs through beetle feeding wounds on young branches. Secondary transmission to stressed trees or recently cut logs occurs through Monochamus oviposition sites.     

Adhesion of conidia of the fungus Dilophospora alopecuri to the cuticle of the nematode Anguina agrostis,the vector in annual ryegrass toxicity

The adhesion of conidia of the fungus Dilophospora alopecuri to the surface of the second stage dauer larva (DL₂) of the nematode Anguina agrostis (syn. A. funesta) was examined using both light and electron optics. The process of attachment does not lead to any apparent damage to the epicuticle of the nematode. Photographs of sections cut tangentially through the setulose appendages of the conidia show that a mucilagenous fibrillar material appears to be exuded from the highly convoluted surface of these appendages. This material adheres to the surface of the nematode cuticle and is deposited in the transverse annulations. The adhesion of these spores to DL₂ of A. agrostis was examined in 4822 nematodes from four galls. The mean percentage of DL₂ with spores adhering was 64% and ranged from 43 to 85%. This adhesion was compared with that of Corynebacterium rathayi from bacterial galls and was found to coincide. Thus, bacteria adhere to nematodes with D. alopecuri conidia attached and these conidia adhere to nematodes with C rathayi attached. Furthermore, DL₂ that are free from conidial adhesion appear to be free from bacterial adhesion and, in most instances, DL₂ that remain from from bacterial adhesion remain free from conidial adhesion. These observations draw attention to the potential of D. alopecuri as an agent for the biological control of annual ryegrass toxicity. Conidial adhesion to A. agrostis differs from bacterial adhesion to this nematode in that no visible damage to the cuticle takes place. The concept that adhesion of microorganisms to nematodes occurs in two phases, one involving site recognition and the other, if it occurs, involving physiological interaction and morphological change is discussed.     

Temporal Pattern of Pinewood Nematode Exit from the Insect Vector Monochamus carolinensis

Laboratory-reared Monochamus carolinensis (Olivier) were used to study the temporal pattern of pinewood nematode dauer larval exit from this beetle vector. Exit rates of dauer larvae were determined by comparing the mean number of dauer larvae carried by adult beetles 0, 7, 14, or 21 days after emergence from the log in which they developed. Density of dauer larvae was highest in beetles on the day of their emergence and dropped slowly through the subsequent age classes. The rate of nematode exit was low during the first week (4.5%) and higher during weeks 2 (20.5%) and 3 (13.1%). A total of 38.1% of the initial dauer larvae exited the beetles during the 3-week observation period.     

The Influence of Environmental Factors on the Respiration of Plant-Parasitic Nematodes

Respiration of selected nematode species was measured relative to CO₂ level, temperature, osmotic pressure, humidity, glucose utilization and high ionic concentrations of sodium and potassium.In general, respiration was stimulated most by the dominant environmental factors at levels near those expected in the nematode''s "natural" habitat. Soil-inhabiting nematodes utilized O₂, most rapidly with high (1-2%) CO₂ whereas a foliar nematode (Aphelenchoides ritzemabosi) did so with 0.03% CO₂, the concentration typically found in air. Temperature optima for respiration corresponded closely to those for other activities. Ditylenchus dipsaci and Pratylenchus penetrans adults and Anguina tritici and A. agrostis second-stage larvae respired within the range of osmotic pressures from 0 to 44.8 arm and respiration of their drought-resistant stages was stimulated by increasing osmotic pressure which accompanies the onset of drought. Rehydration of A. tritici and A. agrostis larvae with RH as low as 5% stimulated measurable respiration. Glucose utilization from liquid medium by A. tritici larvae or A. ritzembosi was not detectable. Supplemental Na⁺ stimulated respiration of Anguina tritici, K⁺ did not.     

Effect of Monochamus carolinensis on the Life History of the Pinewood Nematode,Bursaphelenchus xylophilus

The development of Bursaphelenchus xylophilus in pine wood infested with and free of Monochamus carolinensis was investigated. Formation of third-stage dispersal juveniles occurred in the presence and absence of pine sawyer beetles. The proportion of third-stage dispersal juveniles in the total nematode population was negatively correlated with moisture content of the wood. Formation of nematode dauer juveniles was dependent on the presence of the pine sawyer beetle. Dauer juveniles were present in 3 of 315 wood samples taken from non-beetle-infested Scots pine bolts and 81 of 311 samples taken from beetle-infested bolts. Nematode densities were greater in wood samples taken adjacent to insect larvae, pupae, and teneral adults compared with samples taken from areas void of insect activity. Nematodes recovered from beetle larvae, pupae, and teneral adults were mostly fourth-stage dauer juveniles, although some third-stage dispersal juveniles were also recovered. Dauer juvenile density was highest on teneral adult beetles.     

Liquid culture mass production of biocontrol nematodes,<Emphasis Type="Italic"> Heterorhabditis bacteriophora</Emphasis> (Nematoda: Rhabditida): improved timing of dauer juvenile inoculation

Heterorhabditis bacteriophora is used in biological control of soil-borne insect pests in horticulture and turf. Mass production is carried out in monoxenic liquid cultures pre-incubated with the symbiont of the nematodes, the bacterium Photorhabdus luminescens, before nematode dauer juveniles (DJ) are inoculated. As a response to bacterial food signals, the DJ recover from the developmentally arrested dauer stage, grow to adults and produce DJ offspring. Variable DJ recovery after inoculation into cultures of P. luminescens often causes process failure due to low numbers of adult nematodes in the medium. In order to enhance DJ recovery, improve nematode population management and increase yields, the optimal timing for DJ inoculation was sought. The process parameter pH and respiration quotient (RQ) were recorded in order to test whether changes can be used to identify the best moment for DJ inoculation. When DJ were inoculated during the lag and early logarithmic growth phases of P. luminescens cultures, DJ recovery was low and almost no nematode reproduction was obtained. High populations of P. luminescens phase variants were recorded. Recovery and yields increased when DJ were inoculated during the latter log phase during which the RQ dropped to values <0.8 and the pH reached a maximum. The highest DJ recovery and yields were observed in cultures that were inoculated during the late stationary growth phase. This period started with the increase of the pH after its distinct minimum at pH <8.0. Thus optimal timing for DJ inoculation can be defined through monitoring of the pH in the P. luminescens culture.     

Rhabditis (Oscheius) pheropsophi n. sp. (Rhabditida: Rhabditidae)

Rhabditis (Oscheius) pheropsophi n. sp., associated with cadavers of the bombardier beetle, Pheropsophus aequinoctialis, is described from material collected in Brazil. Mean body length of the female is 1,217 μm, of the male 872 μm, and of the dauer juvenile 568 μm. The female has six lips with one papilla on each lateral lip and two on each sublateral lip; stoma wall thickened dorsally, metarhabdions with warts, excretory pore near base of esophagus, tail long (c = 8), and phasmids prominent, protruding on scanning electron microscope preparations. The male has 10 pairs of bursal ribs, with the terminal pair considerably smaller than the others; spicules fused distally two-thirds of their length. The new species can be distinguished from other members of the Dolichura-group by its fused spicules.     

Heritability of the Liquid Culture Mass Production Potential of the Entomopathogenic Nematode Heterorhabditis bacteriophora

The infective stage of entomopathogenic nematodes ( Heterorhabditis spp.) is the mobile, but developmentally arrested dauer juvenile (DJ). For commercial application, nematodes are produced in liquid culture. Prior to the inoculation of the DJ, their symbiotic bacterium Photorhabdus luminescens is cultured. The DJ exit from the arrested stage (recovery) and develop to reproductive adults. Recovery is a response to bacterial food signals. In liquid culture the percentage of DJs recovering from the DJ stage is highly variable, which significantly influences the number of reproducing hermaphrodites and the final DJ yields. The liquid culture yield is defined by the number of DJ mL -1 harvested from the medium. The heritability of the disposition to recover from the DJ stage and of the final DJ yield in liquid culture has been evaluated. From a hybrid strain of H. bacteriophora 30 homozygous inbred lines were established by inbreeding over seven generations. These inbred lines were propagated in liquid culture and DJ recovery and yields were recorded. The calculated heritability for the DJ recovery was low ( h 2 = 0.38). No significant genetic variability could be detected for this trait. In contrast, a high heritability ( h 2 = 0.90) was found for the total number of DJs produced in the liquid medium.     

Commensal Nematodes in the Glands,Genitalia, and Brood Cells of Bees (Apoidea)

Seven species of bees from the eastern United States, representing four families in the Apoidea, were dissected and examined for nematode associates. Dufour''s glands in females of Halictus ligatus, Augochlora pura mosieri, and Augochlorella gratiosa (Halictidae) from Florida were infested with dauer juveniles of Aduncospiculum halicti (Diplogasteridae). The Dufour''s glands of Colletes thoracicus (Colletidae) females from Maryland were infested with dauer juveniles of a new species of Koerneria sp. (Diplogasteridae), and abdominal glands of females of Andrena alleghaniensis (Andrenidae) from New York were infested with dauer juveniles of another new species of Koerneria. The lateral and median oviducts, Dufour''s glands, and poison sacs in females of Anthophora abrupta (Anthophoridae) from Maryland and Alabama were infested with dauer juveniles of a new species of Bursaphelenchus sp. (Aphelenchoididae). Cross sections of the nematode-infested poison sacs of A. abrupta revealed two types of humoral host defense reactions.     

Effect of attachment of Corynebacterium rathayi on movement of Anguina agrostis larvae

Bird A. F. and Riddle D. L. 1984. Effect of attachment of Corynebacterium rathayi on movement of Anguina agrostis larvae. International Journal for Parasitology 14: 503–511. The movement of freshly hatched larvae (FHL₂) and dauer larvae (DL₂) of Anguina agrostis was compared on ‘agarose’ plates. The DL₂S moved faster and over greater distances. They were not attracted to Corynebacterium rathayi on agar plates, but contact with this bacterium, in most instances, markedly reduced their speed of movement. This reduction was found to be approximately proportional to the concentration of the bacteria (from 8 × 10⁵ to 8 × 10⁸ per ml) to which the DL₂ were exposed prior to observation of their movement, as was the number of bacteria observed to be adhering when viewed under the light microscope. This type of bacterial attachment appeared to be largely stage specific as it was much more pronounced and characteristically different in the DL₂ from that in the FHL₂. This interaction between the DL₂ and the bacterium was similar in material from fresh and from rehydrated nematode galls so that it was apparently not dependent on any surface cuticular changes associated with anhydrobiosis. Furthermore, pretreatment of the nematodes with the surfactant SDS did not influence attachment. An electron microscope study of sections cut through DL₂ exposed to bacteria showed that this interaction was indeed not a surface phenomenon but that the bacteria exerted a pathological effect on the nematode. The bacterium's capsular material actually penetrated and broke down the nematode epicuticle and part of the cortical zone. These observations make it easier to understand the dramatic physiological responses of this nematode to these bacteria.     

Facultative Vivipary is a Life-History Trait in Caenorhabditis elegans

Organisms partition their resources among growth, maintenance, and reproduction and, when resources become limiting, the allocation to one process necessitates reduced allocation to others. When starved, Caenorhabditis elegans adults retain progeny internally which then consume the parent body contents, and some of those larvae use the resources to reach the resistant, long-lived dauer stage. If starved under similarly extreme conditions, larvae from eggs laid outside of the body are unable to develop into dauers. We interpret this switch from ovipary, or laying eggs, to bearing live young as facultative vivipary. This switch is induced by starvation of late fourth-stage larvae, young adults, or gravid adults. In C. elegans, vivipary is the altruistic allocation of all available parental energy and nutrients to progeny, with the associated costs to adult hermaphrodites of truncated life span and fecundity. As a life-history trait, facultative vivipary is a survival-enhancing response to stress that may provide insights into the evolution of reproduction and longevity.     

Specificity of extrafloral nectar induction by herbivores differs among native and invasive populations of tallow tree

Background and Aims

Invasive plants can be released from specialist herbivores and encounter novel generalists in their introduced ranges, leading to variation in defence among native and invasive populations. However, few studies have examined how constitutive and induced indirect defences change during plant invasion, especially during the juvenile stage.

Methods

Constitutive extrafloral nectar (EFN) production of native and invasive populations of juvenile tallow tree (Triadica sebifera) were compared, and leaf clipping, and damage by a native specialist (Noctuid) and two native generalist caterpillars (Noctuid and Limacodid) were used to examine inducible EFN production.

Key results

Plants from introduced populations had more leaves producing constitutive EFN than did native populations, but the content of soluble solids of EFN did not differ. Herbivores induced EFN production more than simulated herbivory. The specialist (Noctuid) induced more EFN than either generalist for native populations. The content of soluble solids in EFN was higher (2·1 times), with the specialist vs. the generalists causing the stronger response for native populations, but the specialist response was always comparable with the generalist responses for invasive populations.

Conclusions

These results suggest that constitutive and induced indirect defences are retained in juvenile plants of invasive populations even during plant establishment, perhaps due to generalist herbivory in the introduced range. However, responses specific to a specialist herbivore may be reduced in the introduced range where specialists are absent. This decreased defence may benefit specialist insects that are introduced for classical biological control of invasive plants.     

Epidemiology of Anguina agrostis on Highland Colonial Bentgrass

The epidemiology of Anguina agrostis was investigated in field plots of Colonial bentgrass (cv. Highland), Agrostis tenuis, near Corvallis, Oregon. Each October from 1990-92, nylon mesh pouches, each containing 10 galls, were buried in the field or placed on the soil surface in microplots. Pouches were collected monthly or bimonthly between December and June and nematodes per gall counted. Nematode egression from galls began in late March and was completed by mid-May, corresponding to the period of floral initiation in bentgrass. In 1991 and 1992, 0.09-m² plots were inoculated with 0, 1, 5, 15, 50, 120, or 200 galls/plot. The disease severity (number of galls) and disease incidence (% seed heads with galls) increased linearly at inoculum densities below 50 galls/ plot. At higher inoculum densities, disease increase approached an asymptote. In 1991, plots were established to determine the characteristics of disease spread. Disease foci were established by placing 0, 5, 50, or 500 galls along 30-cm sections of row in the fall. In July 1992, seed heads were harvested at 30 and 60 cm from each focus within and across plant rows. Most infestations were found within 30 cm of foci at all inoculum levels. At high inoculum densities, the distribution of galls was aggregated with the majority of galls located on less than 10% of the seed heads. These disease spread and incidence data suggest populations of A. agrostis increase slowly in bentgrass in Oregon.     

Interaction Between Neoaplectana carpocapsae and a Granulosis Virus of the Armyworm Pseudaletia unipuncta

Neoaplectaua carpocapsae developed and reproduced in armyworm hosts infected with a granulosis virus (GV). Macerated tissues of dauer juveniles from GV-infecled hosts had sufficient GV to infect 1st and 2nd instar armyworms. Electron-microscope examination of dauer juveniles and adult female nematodes confirmed the presence of GV in the lumen of the intestine. No GV was observed in other tissues of the nematode.