Stimuli for cuticle formation and ecdysis in vitro of the infective larva of Anisakis sp. (Nematoda: Ascaridoidea)

Third-stage larvae of the genus Anisakis from the fish Leionura atun (Trichiuroidei: Perciformes) form a new cuticle and moult in vitro in about 72 h. If the culture medium is Krebs-Ringer under 5% carbon dioxide in air at 37°C, relatively few moult and survival is poor. But more moult and survival is enhanced if worms are incubated in tissue culture medium 199, even if the gas phase is air, although they moult more quickly if it contains 5% carbon dioxide. In both Krebs-Ringer and 199 the benefits of high concentrations of carbon dioxide only accrue if the gas is present during the first 40 h of incubation. Worms do not feed in these media until they have moulted.     

Reversal of order of ecdysis in Haemonchus contortus (Nematoda)

Exposure of Haemonchus contortus infective larvae to carbon dioxide under specific conditions is believed to activate a succession of events that ultimately control both exsheathment and subsequent development to the fourth stage. Rarely at pH 6, but always at pH 2, worms fail to exsheath yet develop to the fourth stage surrounded by cuticles two and three. Failure to exsheath is probably a consequence of inactive enzymes in exsheathing fluid, because exposure to EDTA, which inactivates exsheathing fluid, reduced exsheathment and yielded sheathed fourth-stage worms. Although exsheathment is blocked at pH 2, CO2 can activate the receptor for exsheathment as readily at pH 2 as at pH 6. Therefore the hypothesis that an early common pathway controls both exsheathment and development is not invalidated.     

Comparison of the methods applicable for the pathogenicity assessment of entomopathogenic nematodes

Single infective juveniles of Heterorhabditis bacteriophora, H. megidis (Nematoda: Heterorhabditidae), Steinernema arenarium, S. carpocapsae and S. feltiae (Nematoda: Steinernematidae) were used to infect single Galleria mellonella (Lepidoptera: Pyralidae) larvae. Four parameters of entomopathogenic nematodes pathogenicity were assessed: the mortality of insects, infectivity of nematodes, number of nematodes established per single G. mellonella, and degree of infective juveniles colonization (percent of infective juveniles which intestine was colonized by symbiotic bacteria). The accuracy, repeatability, and versatility for different species of EPNs in bioassay arenas were compared. Our modifications of the original methods yielded ~ 50% higher efficiency of infective juveniles in cell culture plates and > 20% higher efficiency in centrifuge test tubes. The efficiency of nematodes in cell culture plates (39–77%) was relatively low, especially in the case of Heterorhabditis spp. In the bioassay arena, infective juveniles migrated between cells. The results of our studies indicate that the pathogenicity of EPNs should be assessed in centrifuge test tubes. In these arenas, the infectivity of single IJs was ~ 90% for Heterorhabditis spp. and ~ 95% for Steinernema spp. The degree of colonization of the EPN isolates by symbiotic bacteria was in the range of 96–98%.     

Organic and inorganic acids as the stimulus for exsheathment of infective juveniles of nematodes

Petronijevic T., Rogers W. P. and Sommerville R. I. 1986. Organic and inorganic acids as the stimulus for exsheathment of infective juveniles of nematodes. International Journal for Parasitology 16: 163–168. A variety of organic and inorganic acids stimulated exsheathment of infective juveniles of Nematospiroides dubius over the range pH 2–7. Activity, in relation to pK values, suggests that the undissociated form of the acid was the active agent. Under normal conditions of exposure, exsheathment of infective juveniles of Haemonchus contortus was not obtained below pH 6, and at higher pH values activity was low except in the presence of CO₂ at pressures >9-5 kPa in the gas phase. Brief exposure to HCl at concentrations up to 2N did not induce exsheathment but in the presence of CO₂ in the gas phase, 95.3 kPa, activity was obtained in 0-O1N HCl when the times of exposure were 3–25 min.The differences in the responses of N. dubius and H. contortus to a stimulus at low pH values may be attributed to the stability of the exsheathing enzyme of N. dubius in acid media as well as to the relative sensitivity of the two species to stimuli generally.     

Carbon dioxide assimilation from air and water by duckweed Spirodela polyrrhiza (L.) Schleid

Carbon dioxide assimilation by duckweed, S. polyrrhiza, was measured using a glass assimilation box and ¹⁴C-NaHCO₃, under different pH conditions of water. S. polyrrhiza assimilates carbon dioxide from both air and water. The carbon assimilation from air is comparable to the assimilation from water under normal pH conditions.     

Potential negative effects on biological control by Sancassania polyphyllae (Acari: Acaridae) on an entomopathogenic nematode species

Sancassania polyphyllae (Acari: Acaridae) is associated with larvae of the white grub, Polyphylla fullo (Coleoptera: Scarabaeidae), and will feed on the infective juveniles of entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae which are important biological control agents of soil insect pests. We conducted laboratory studies to determine the potential negative effects this mite species might have on biological control of soil insect pests. Our objectives in this study were to (1) determine the response of S. polyphyllae adult mites to a nematode-killed insects on agar, (2) evaluate the predation by mites on Steinernema feltiae infective juveniles from nematode-killed insects on agar and in soil, and (3) assess predation efficiency of the mite on the infective juveniles in the soil. On agar, we found (1) significantly more adult female mites near or on a nematode-killed Ceratitis capitata (Diptera: Tephritidae) larva than near or on the freeze-killed larva or a bamboo mimic suggesting that a chemical or an odor from the nematode-killed larva attracted the mites, and (2) 10 mites consumed 96% of infective juveniles that emerged from an insect cadaver. In soil with a nematode-killed insect, the average number of infective juveniles recovered was <30 when mites were present, whereas the average number of infective juveniles recovered was >375 when the mites were absent. When the infective juveniles alone were placed in different depths in relation to the mites in the soil column for 4 and 10 days, S. polyphyllae was not as efficient at finding the infective juveniles when they were separated from each other in the soil lending support to the idea that the mites were cueing in on the cadaver as a food resource. Our data suggest that emerging infective juveniles from an insect cadaver in the soil in the presence of S. polyphyllae can adversely affect biological control because of nematode consumption by the mites.     

Assessing the influence of the entomopathogenic nematode Heterorhabditis baujardi LPP7 (Rhabiditina) on embryogenesis and hatching of the plant-parasitic nematode Meloidogyne mayaguensis (Tylenchina)

Two assays were conducted to assess the influence of infective juveniles (IJs) of Heterorhabditis baujardi LPP7 on the embryogenesis and hatching of Meloidogyne mayaguensis. In the first assay, eggs were incubated in water alone or in the presence of infective juveniles, and completion of embryogenesis was evaluated 14 days later. In the second assay, unhatched second-stage juveniles were incubated in distilled water alone or in the presence of infective juveniles. Cumulative hatching was compared at various time intervals. Embryogenesis was not affected, whereas second-stage juveniles hatching was delayed probably because of the eggs permeability to noxious metabolites released by Photorhabdus luminescens, which is the bacterial symbiont of H. baujardi.     

Effect of Entomopathogenic Nematode Concentration on Survival during Cryopreservation in Liquid Nitrogen

Entomopathogenic nematodes are used for biological control of insect pests. A method for improved cryopreservation of infective juvenile stage nematodes has been developed using Steinernema carpocapsae and Heterorhabditis bacteriophora. Optimum survival for both species was achieved with 12,000 infective juveniles/ml in glycerol and 7,500/ml in Ringer''s solution. For S. carpocapsae, maximum survival also was observed with 60,000 infective juveniles/ml in glycerol and 25,000/ml in Ringer''s solution. These concentrations resulted in 100% post-cryopreservation survival of S. carpocapsae and 100% retention of original virulence to Galleria mellonella larvae. This is the first report of achieving 100% survival of an entomopathogenic nematode after preservation in liquid nitrogen. Maximum survival of H. bacteriophora following cryopreservation was 87%.     

Response of Steinernema carpocapsae Infective Juveniles to the Plasma of Three Insect Species

Exsheathed infective juveniles of Steinernema carpocapsae All strain were attracted to the plasma of three species of insects in agar plate bioassays. Plasma of Pieris rapae crucivora, Spodoptera litura, and Agrotis segetum attracted 88.6%, 80.4%, and 64.4%, respectively, of Steinernema carpocapsae juveniles added to plates. Autoclaved plasma of S. litura larvae attracted more juveniles than saline controls, but less than nonautoclaved plasma. The active agent passed through a 14,000 MW dialysis membrane.     

Exsheathment of the infective larva of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island wallaby Macropus eugenii.

The infective larva of L. eugenii is enveloped in two cuticles which are discarded when the larva exsheaths in the sacculated portion of the wallaby's stomach. In vitro larvae exsheathed in a 0·85% solution of sodium chloride at 37°C, buffered to pH 7 with bicarbonate ion and 40% carbon dioxide. Survival was enhanced if the liquid phase contained medium 199 and serum, and exsheathment was quicker if exposure to carbon dioxide was 1 h rather than 1 day or 7 days. As larvae exsheathed, contractions of the pharynx commenced, and medium was ingested, even when larvae were enveloped in both cuticles. The stimuli for exsheathment and the subsequent pattern of events are like those already recognised in some trichostrongyles.     

Trichinella spiralis: Effect of environmental temperature on mice

Effects of environmental temperature on Trichinella spiralis-infected mice were studied. Groups of mice were acclimated to temperatures of 8–10 C, 20–25 C, and 36 C for a period of 5 days and then were exposed to 100 infective juveniles of T. spiralis via stomach intubation. After being maintained at the above temperatures for 5 days, they were examined for adult worms. Results indicate that mice maintained at 36 C tend to harbor significantly higher numbers of adults and that there is a direct relationship between temperature and worm number. Effects of mouse strain differences on these results were also examined. Of those strains tested, inbred Swiss albino mice proved to be superior hosts for T. spiralis.     

Undissociated bases as the stimulus for the development of early parasitic stages of nematodes

Petronijevic T. and Rogers W. P. 1987. Undissociated bases as the stimulus for the development of early parasitic stages of nematodes. International Journal for Parasitology 17 :911–915. The effects of NH₄Cl and NH₂CH₃ on infective stages of Haemonchus contortus, Nematospiroides dubius and Ascaris suum have been compared with the action of H₂CO₃. Detailed experiments were carried out with H. contortus. NH₄Cl at pH 8 under air was less toxic to infective stages than it was to free-living juveniles of Panagrellus redivivus. The induction of exsheathment or hatching by bases was slow (20–30 h) though the time of development of 4th stage H. contortus was not proportionally increased. Activity at pH 6 was less than that at pH 8. In contrast to the action of H₂CO₃ as the stimulus, NH₄Cl was not Ca²⁺-dependent. Prolonged exposure to anoxia at pH 8 was toxic, but in the presence of NH₄Cl or H₂CO₃ toxicity was decreased. The inhibition of exsheathment due to Dnp at pH 7 was greater when NH₄Cl was the stimulus.The process whereby host signals induce development of parasitic stages is discussed.     

Emergence and Dispersal Patterns of Two Isolates of the Entomopathogenic Nematode Steinernema feltiae

Few studies have addressed the role of the sexes in the emergence and dispersal of entomopathogenic nematodes from host insects. Individuals of two isolates of Steinernema feltiae, UK76 and SBIl, emerging from Galleria mellonella cadavers were classed as Non-Dispersed (remaining on the cadaver for up to nine days) and Dispersed (actively moving away from the cadaver). Sex ratios within both classes were examined in infective (individuals that successfully invaded bait G. mellonella) and entire (infective and noninfective individuals that matured in hanging drops of G. mellonella haemolymph) populations. Sex ratios differed significantly from 1:1 only in the SBIl Non-Dispersed entire population (female bias) and SBIl Non-Dispersed infective population (male bias). For each isolate, Dispersed individuals were significantly more infective than Non-Dispersed. However, only 11% of SBIl and 22% of UK76 Non-Dispersed individuals were found to be mature infective juveniles (IJ) compared with 78% of SBIl and 82% of UK76 Dispersed individuals (based on survival in SDS). Infective juveniles dispersing towards distant radial bait G. mellonella tended to migrate from the head region of the natal cadaver. For each isolate, a higher proportion of males than females arrived early at distant baits. SBIl males survived alone in G. mellonella cadavers for longer periods than did females, which supports the “male colonization” hypothesis.     

Attraction of entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora to the red palm weevil (Rhynchophorus ferrugineus)

The red palm weevil (RPW), Rhynchophorus ferrugineus, is a serious pest of date palms. Its larvae bore deep into the trunk disrupt the vascular tissues and kill the infested trees. Behavioral features of entomopathogenic nematodes (EPNs), reflected by attraction and distribution patterns, are fundamental aspect in determining their parasitic ability and potential management of RPW. We studied the attraction behavior of the EPNs Steinernema carpocapsae and Heterorhabditis bacteriophora to the RPW under simulated natural conditions in tubes to evaluate their infective potential. In all experiments, a certain proportion of infective juveniles (IJs) (16–20%) stayed near the inoculated site and a major proportion (38–48%) was attracted to the host end. Both H. bacteriophora and S. carpocapsae were efficient crawlers, climbing up and descending when locating their insect host. They were efficiently attracted to the various larval sizes and stages of the RPW life cycle. Host localization by ascending movement was more prominent in S. carpocapsae than in H. bacteriophora. In general, H. bacteriophora is classified as a cruiser forager and S. carpocapsae as an ambusher. However, in this study, we discovered a higher percentage of cruiser foragers among S. carpocapsae IJs. They dispersed much faster and their cruising behavior was prominent characteristic in controlling the cryptic RPW concealed in organic habitats.     

Infection of Anastrepha ludens following soil applications of Heterorhabditis bacteriophora in a mango orchard

To determine the efficacy of Heterorhabditis bacteriophora Poinar (Nematoda: Heterorhabditidae) for control of Anastrepha ludens (Loew) (Diptera: Tephritidae), field experiments were performed in a mango orchard with soil temperatures of 24–29 °C. The density of third‐instar A. ludens (50–500 larvae per plot) released into 0.25 m² wood‐framed experimental plots containing soil (16% wt/wt moisture) previously treated with 125 infective juveniles per square centimetre soil surface did not significantly influence the prevalence of infection by H. bacteriophora. In subsequent experiments, the percentages of infection of fly pupae were positively correlated with the concentration of infective stages applied to soil plots. The highest average percentage of infection (74% at 250 infective juveniles per square centimetre) was observed when fly larvae were released simultaneously onto soil, compared to larvae that emerged from laboratory‐infested mangoes over a period of 8 days (52% infection at 500 infective juveniles per square centimetre). Double applications of infective juveniles at an interval of 4 days did not greatly improve the prevalence of infection (～10% higher) compared to single applications. Between 9 and 15% of larvae that remained within infested mangoes became infected by nematodes, irrespective of the concentration of nematodes applied to each experimental plot. We conclude that effective control of A. ludens requires very high densities of H. bacteriophora. The successful use of this nematode for biocontrol of A. ludens will depend on identifying ways of overcoming the fly's ability to avoid infection.     

Granulosis virus in the intestinal lumen of Neoaplectana carpocapsae: Retention of infectivity after treatment with formaldehyde or high pH

High pH values (>11.0) cause the dissolution of occlusion bodies of the granulosis virus (GV) of Pseudaletia unipuncta and subsequent inactivation of the virus within 24 hr. The GV is also inactivated within 48 hr by 0.04% formaldehyde. The GV is found in the intestinal lumen of infective third stage nematodes (dauer juveniles) of Neoaplectana carpocapsae when development occurs in GV-infected hosts. The GV in these dauer juveniles retains its infectivity even when the nematodes are placed into an alkaline solution with pH values of 11.1 or 12.1 or in 0.04% formaldehyde up to 336 hr. However, significant loss of infectivity of GV occurs when the nematodes are in formaldehyde but not at high pH values. The dauer juveniles are ensheathed by the second stage cuticle. This cuticle probably protects the GV in the intestinal lumen of the nematode from the high pH and formaldehyde.     

Releases of ingested phytoplankton carbon by Daphnia magna

1. The carbon budgets and assimilation efficiencies (AEs) of adults and juveniles of Daphnia magna were quantified using ¹⁴C as a tracer. Animals were fed pure Chlamydomonas reinhardtii or Scenedesmus obliquus at different food concentrations. Carbon AEs (46–70%) were comparable at food concentrations of 0.03–0.30 mg C L^?1 for both algal species, but decreased to 34–49% when the food concentration further increased by 10‐fold. The carbon AEs were significantly and negatively correlated with the food level. 2. During the postdigestive period, partitioning of ingested carbon into different compartments including excretion, respiration and egestion was not influenced by the food species and life stage. There was a negative correlation between respiration (as % of total loss) and food concentration and a positive correlation between egestion (as % of total loss) and food concentration. Dissolved organic carbon (DOC) and CO₂ accounted for 55–72% and 9–37%, respectively, of the total carbon loss from juveniles fed both algal diets. For adults, DOC and CO₂ contributed to 44–64% and 20–47% of the total carbon loss, respectively. Particulate organic carbon (POC) was a minor pathway for the overall carbon loss. 3. The turnover and release budget of structural carbon (as moults and neonate reproduction) were further evaluated in long‐term experiments at different algal concentrations. Food concentration did not affect the carbon efflux or the carbon allocation into different physiological compartments except for respiration. Juveniles had twofold lower carbon turnover rate (0.12–0.16 day^?1) than those of the adults (0.32–0.35 day^?1). In adults, comparable carbon was allocated into DOC (35–42%) and reproduction (27–35%), which were the dominant routes for carbon loss. For the juveniles, DOC accounted for 42–64% of the total carbon loss. 4. About 21–38% of the total DOC released by adults and juveniles was associated with the high molecular weight organic carbon fraction (>5 kDa). Our results show that carbon was mainly lost by D. magna in the form of DOC during assimilation process as well as from the structural materials. Reproduction or maternal transfer was another major drain of body carbon for adult D. magna.     

Carbon dioxide exchange in the needles of the common spruce in southern taiga spruce forests

Dynamics of carbon dioxide exchange in the Common Spruce (Picea abies L.) in relation to environmental factors was monitored during several seasons. Direct linear dependence of photosynthesis rate from the levels of air temperature and illumination was found, and correlation coefficients were 0.860 (p < 0.001) and 0.704 (p < 0.001). It was found that seasonal maximum of net photosynthesis production was attained at temperatures of 23–25°C. A decrease in temperature optimum was associated with reduction of the CO₂ assimilation intensity level. The impact of environmental factors on photosynthesis intensity is discussed in terms of the developed model. Using this model, we demonstrated that temperature and illumination dynamics in toto accounts for 82% of changes in photosynthesis rate. It is the air temperature that exerts the strongest influence on the process of photosynthesis. According to our calculations, the net photosynthesis level was three times higher than the level of respiration. This is indicative of a positive carbon dioxide balance in the needles of the Common Spruce.     

Heamolysis during in vitro CO2 removal from human blood using a membrane lung

Haemolysis of human blood has been examined in vitro as a function of pH in the range 7.2–8.0. The hydrogen ion concentration of freshly donated blood from 11 donors was manipulated in 42 experiments, entirely by altering the carbon dioxide fraction of air with which the blood was equilibrated using a membrane lung. In contrast to the known alkalaemic haemolysis which occurs in canine blood, we observed no correlation between plasma haemoglobin concentrations and blood pH. We conclude that alkalaemic haemolysis is unlikely to complicate the clinical application of extracorporeal carbon dioxide removal in the management of acute respiratory failure.     

Mass Spectrometry–Driven Discovery of Neuropeptides Mediating Nictation Behavior of Nematodes

Neuropeptides regulate animal physiology and behavior, making them widely studied targets of functional genetics research. While the field often relies on differential -omics approaches to build hypotheses, no such method exists for neuropeptidomics. It would nonetheless be valuable for studying behaviors suspected to be regulated by neuropeptides, especially when little information is otherwise available. This includes nictation, a phoretic strategy of Caenorhabditis elegans dauers that parallels host-finding strategies of infective juveniles of many pathogenic nematodes. We here developed a targeted peptidomics method for the model organism C. elegans and show that 161 quantified neuropeptides are more abundant in its dauer stage compared with L3 juveniles. Many of these have orthologs in the commercially relevant pathogenic nematode Steinernema carpocapsae, in whose infective juveniles, we identified 126 neuropeptides in total. Through further behavioral genetics experiments, we identify flp-7 and flp-11 as novel regulators of nictation. Our work advances knowledge on the genetics of nictation behavior and adds comparative neuropeptidomics as a tool to functional genetics workflows.