Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) are lethal parasites of soil dwelling insects. We collected 168 soil samples from five provinces, all located in southern Thailand. Eight strains of EPNs were isolated and identified to species using restriction profiles and sequence analysis. Five of the isolates were identified as Heterorhabditis indica, and one as Heterorhabditis baujardi. Two undescribed Steinernema spp. were also discovered which matched no published sequences and grouped separately from the other DNA restriction profiles. Behavioral tests showed that all Heterorhabditis spp. were cruise foragers, based on their attraction to volatile cues and lack of body-waving and standing behaviors, while the Steinernema isolates were more intermediate in foraging behavior. The infectivity of Thai EPN strains against Galleria mellonella larvae was investigated using sand column bioassays and the LC50 was calculated based on exposures to nematodes in 24-well plates. The LC50 results ranged from 1.99-6.95 IJs/insect. Nine centimeter columns of either sandy loam or sandy clay loam were used to determine the nematodes’ ability to locate and infect subterranean insects in different soil types. The undescribed Steinernema sp. had the greatest infection rate in both soil types compared to the other Thai isolates and three commercial EPNs (Heterorhabditis bacteriophora, Steinernema glaseri and Steinernema riobrave).
Entomopathogenic Nematodes; Foraging Behavior; Galleria mellonella; Heterorhabditis; Steinernema
Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.
For malaria transmission to occur, a mosquito must ingest and harbor the parasites for approximately two weeks while the parasites complete multiple developmental stages. Until development is complete and the malaria parasites invade the mosquito salivary glands, transmission to another host cannot occur. Upon completion of parasite development, transmission is possible with every subsequent bite. In this study we demonstrate that tissue-specific overexpression of a single activated protein kinase that is essential to insulin signaling in the mosquito can dramatically reduce parasite development. This kinase – Akt – has been described as a critical cell signaling node that regulates a range of physiological processes. In addition to the impact on parasite development, increased Akt signaling also reduced the average mosquito lifespan relative to controls, thereby limiting the window of opportunity for successful parasite transmission. Thus, we demonstrate that genetic manipulation of one key signaling protein directly reduces parasite development in the insect vector as well as the duration of mosquito infectivity.
A dynamic decision-making system that includes a mass of indistinguishable agents could manifest impressive heterogeneity. This kind of non-homogeneity is postulated to result from macroscopic behavioral tactics employed by almost all involved agents. A State-Space Based (SSB) mass event-history model is developed here to explore the potential existence of such macroscopic behaviors. By imposing an unobserved internal state-space variable into the system, each individual’s event-history is made into a composition of a common state duration and an individual specific time to action. With the common state modeling of the macroscopic behavior, parametric statistical inferences are derived under the current-status data structure and conditional independence assumptions. Identifiability and computation related problems are also addressed. From the dynamic perspectives of system-wise heterogeneity, this SSB mass event-history model is shown to be very distinct from a random effect model via the Principle Component Analysis (PCA) in a numerical experiment. Real data showing the mass invasion by two species of parasitic nematode into two species of host larvae are also analyzed. The analysis results not only are found coherent in the context of the biology of the nematode as a parasite, but also include new quantitative interpretations.
Extremists; Heterogeneity; Interval censoring; Logistic regression; Maximum likelihood estimation; Nematode; Parasite infection; Weibull distribution
Observations from nematodes to mammals indicate that insulin/insulin-like growth factor signaling (IIS) regulates lifespan. As in other organisms, IIS is conserved in mosquitoes and signaling occurs in multiple tissues. During bloodfeeding, mosquitoes ingest human insulin. This simple observation suggested that exogenous insulin could mimic the endogenous hormonal control of aging in mosquitoes, providing a new model to examine this phenomenon at the organismal and cellular levels. To this end, female Anopheles stephensi mosquitoes were maintained on diets containing human insulin provided daily in sucrose or three times weekly by artificial bloodmeal. Regardless of delivery route, mosquitoes provided with insulin at 1.7×10−4 and 1.7×10−3 μmol l−1, doses 0.3-fold and 3.0-fold higher than non-fasting blood levels, died at a faster rate than controls. In mammals, IIS induces the synthesis of reactive oxygen species and downregulates antioxidants, events that increase oxidative stress and that have been associated with reduced lifespan. Insulin treatment of mosquito cells in vitro induced hydrogen peroxide synthesis while dietary supplementation reduced total superoxide dismutase (SOD) activity and manganese SOD activity relative to controls. The effects of insulin on mortality were reversed when diets were supplemented with manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable SOD mimetic agent, suggesting that insulin-induced mortality was due to oxidative stress. In addition, dietary insulin activated Akt/protein kinase B and extracellular signal-regulated kinase (ERK) in the mosquito midgut, suggesting that, as observed in Caenorhabditis elegans, the midgut may act as a ‘signaling center’ for mosquito aging.
malaria; mosquito; Plasmodium; Anopheles; aging; insulin; oxidative stress; antioxidant
Directly viewing soil-dwelling entomopathogenic and plant-parasitic nematodes in situ is difficult, if not impossible. As a result, researchers have developed a diverse array of bioassays which assess nematode behavioral traits within arenas designed to simulate various aspects of the natural habitat. However, reliably rendering what we can see in the laboratory into accurate predictions of how nematodes achieve their objectives in the field is challenging. In the current review, we systemically assessed the goals and attributes of several of the assays most commonly used to investigate nematode host finding and host invasion behavior. By illuminating the relative strengths and limitations of each assay, we hope to improve our ability to develop meaningful predictions for the field.
behavior; chemotaxis; ecology; electrophysiology; olfactometer; soil column
Entomopathogenic nematodes, Heterorhabditis indica and Steinernema riobrave, were tested for virulence and reproductive yield in Tenebrio molitor that were fed wheat bran diets with varying lipid- and protein-based supplements. Lipid supplements were based on 20% canola oil, peanut, pork or salmon, or a low lipid control (5% canola). Protein treatments consisted of basic supplement ingredients plus 0, 10, or 20% egg white; a bran-only control was also included. Some diet supplements had positive effects on nematode quality, whereas others had negative or neutral effects. All supplements with 20% lipids except canola oil caused increased T. molitor susceptibility to H. indica, whereas susceptibility to S. riobrave was not affected. Protein supplements did not affect host susceptibility, and neither lipid nor protein diet supplements affected reproductive capacity of either nematode species. Subsequently, we determined the pest control efficacy of progeny of nematodes that had been reared through T. molitor from different diets against Diaprepes abbreviatus and Otiorhynchus sulcatus. All nematode treatments reduced insect survival relative to the control (water only). Nematodes originating from T. molitor diets with the 0% or 20% protein exhibited lower efficacy versus D. abbreviatus than the intermediate level of protein (10%) or bran-only treatments. Nematodes originating from T. molitor lipid or control diets did not differ in virulence. Our research indicates that nutritional content of an insect host diet can affect host susceptibility to entomopathogenic nematodes and nematode fitness; therefore, host media could conceivably be optimized to increase in vivo nematode production efficiency.
diet; entomopathogenic nematode; Heterorhabditis; in vivo, mass production; Steinernema
Meloidogyne partityla is a parasite of pecan and walnut. Our objective was to determine interactions between the entomopathogenic nematode-bacterium complex and M. partityla. Specifically, we investigated suppressive effects of Steinernema feltiae (strain SN) and S. riobrave (strain 7–12) applied as infective juveniles and in infected host insects, as well as application of S. feltiae's bacterial symbiont Xenorhabdus bovienii on M. partityla. In two separate greenhouse trials, the treatments were applied to pecan seedlings that were simultaneously infested with M. partityla eggs; controls received only water and M. partityla eggs. Additionally, all treatment applications were re-applied (without M. partityla eggs) two months later. Four months after initial treatment, plants were assessed for number of galls per root system, number of egg masses per root system, number of eggs per root system, number of eggs per egg mass, number of eggs per gram dry root weight, dry shoot weight, and final population density of M. partityla second-stage juveniles (J2). In the first trial, the number of egg masses per plant was lower in the S. riobrave-infected host treatment than in the control (by approximately 18%). In the second trial, dry root weight was higher in the S. feltiae-infected host treatment than in the control (approximately 80% increase). No other treatment effects were detected. The marginal and inconsistent effects observed in our experiments indicate that the treatments we applied are not sufficient for controlling M. partityla.
Biological control; entomopathogenic nematode; Meloidogyne partityla; pecan; Steinernema; Xenorhabdus
Entomopathogenic nematodes are lethal insect parasites that reproduce exclusively inside their hosts in nature. Infection decisions made by the free-living infective-stage juveniles have an impact on reproductive success, but it is likely that mating decisions are made by adults while inside their host. We investigated sexual communication between male and female adult stages of Steinernema carpocapsae (Rhabditida: Steinernematidae) to assess whether mating is chemically mediated during the adult stage or results from incidental encounters between adults inside the insect host. To assess chemical communication, we measured the behavioral response of adult male S. carpocapsae to several different potential sources of chemical information. Male S. carpocapsae responded to virgin females only and were not influenced by mated conspecific females, conspecific males, or heterospecific females. These results show that species-specific communication takes place between adult entomopathogenic nematodes within the host cadaver just prior to mating.
behavior; entomopathogenic nematode; insect; mating; parasite; pheromone; reproductive isolation; Steinernema
Entomopathogenic nematodes are potent biopesticides that can be mass-produced by in vitro or in vivo methods. For in vivo production, consistently high infection rates are critical to efficiency of the process. Our objective was to optimize in vivo inoculation of Steinernema carpocapsae and Heterorhabditis bacteriophora in Galleria mellonella and Tenebrio molitor by determining effects of inoculation method, nematode concentration, and host density. We found immersing hosts in a nematode suspension to be approximately four times more efficient in time than pipeting inoculum onto the hosts. The number of hosts exhibiting signs of nematode infection increased with nematode concentration and decreased with host density per unit area. This is the first report indicating an effect of host density on inoculation efficiency. We did not detect an effect of nematode inoculum concentration on nematode yield per host or per gram of host. Yield was affected by host density in one of the four nematode-host combinations (S. carpocapsae and T. molitor). We conclude that optimization of inoculation parameters is a necessary component of developing an in vivo production system for entomopathogenic nematodes.
culture; Heterorhabditis bacteriophora; in vivo; production; Steinernema carpocapsae
Rearing conditions have been shown to affect several aspects of entomopathogenic nematode biology, including dispersal behavior and infectivity. The present study explores the differences in development rate of Heterorhabditis bacteriophora and Steinernema carpocapsae when infective juveniles (IJ) were collected in water using the standard White trap method vs. natural emergence from cadavers into sand. We exposed Galleria mellonella to IJ entompopathogenic nematodes treated in one of three ways: collected in a White trap, allowed to emerge directly into sand, or collected in a White trap and treated with a cadaver homogenate. When S. carpocapsae IJ were allowed to emerge from cadavers directly into sand and then allowed to infect new hosts, they developed into adults at a faster rate than IJ that were collected with White traps. The difference in development was not due to differential infection rates. No difference in development stages was detected amount the same H. bacteriophora treatments.
entomopathogenic nematodes; Heterorhabditis bacteriophora; host; rearing conditions; Steinernema carpocapsae
Tomato seedlings in a growth chamber were inoculated with 150 Meloidogyne incognita eggs and 25 infective juveniles (IJ)/cm² of Steinernema feltiae, S. riobrave, or Heterorhabditis bacteriophora. With the exception of seedling roots treated with H. bacteriophora, all seedlings treated with entomopathogenic nematodes had fewer M. incognita juveniles inside roots and produced fewer eggs than the control seedlings. Tomato plants in the greenhouse were infested with 4,000 M. incognita eggs and treated 2 weeks before, 1 week before, at the same time, 1 week after, or 2 weeks after with 25 or 125 IJ/cm² of S. feltiae, S. riobrave, or H. bacteriophora. Plants with pre- and post-infestation applications of S. feltiae or S. riobrave suppressed M. incognita. Plants treated with H. bacteriophora 1 week before and at the time of infestation suppressed M. incognita. Increasing the rate of H. bacteriophora and S. feltiae from 25 to 125 IJ/cm² improved M. incognita suppression.
biological control; entomopathogenic nematodes; Heterorhabditis bacteriophora; Lycopersicum esculentum; Meloidogyne incognita; Steinernema feltiae; S. riobrave; suppression; tomato