Parasitism of Heterotylenchus autumnalis Nickle (Nematoda: Sphaerulariidae) to the Face Fly,Musca autumnalis De Geer (Diptera: Muscidae)

New information is reported on the parasitism of Heterotylenchus autumnalis upon its principal known host, Musca autumnalis. Black to brown spots are produced on the cuticle of all infected host larvae where the nematode penetrated. The principal damage to the host is castration of the female. In laboratory tests nematode larvae were not infective and did not leave the hosts before the female fly was 1 1 days old. Nematode larvae removed from infected male flies infected other hosts, but it is believed that in nature these larvae are unable to leave the host.     

Nematodes, bacteria, and flies: a tripartite model for nematode parasitism

More than a quarter of the world's population is infected with nematode parasites, and more than a hundred species of nematodes are parasites of humans [1-3]. Despite extensive morbidity and mortality caused by nematode parasites, the biological mechanisms of host-parasite interactions are poorly understood, largely because of the lack of genetically tractable model systems. We have demonstrated that the insect parasitic nematode Heterorhabditis bacteriophora, its bacterial symbiont Photorhabdus luminescens, and the fruit fly Drosophila melanogaster constitute a tripartite model for nematode parasitism and parasitic infection. We find that infective juveniles (IJs) of Heterorhabditis, which contain Photorhabdus in their gut, can infect and kill Drosophila larvae. We show that infection activates an immune response in Drosophila that results in the temporally dynamic expression of a subset of antimicrobial peptide (AMP) genes, and that this immune response is induced specifically by Photorhabdus. We also investigated the cellular and molecular mechanisms underlying IJ recovery, the developmental process that occurs in parasitic nematodes upon host invasion and that is necessary for successful parasitism. We find that the chemosensory neurons and signaling pathways that control dauer recovery in Caenorhabditis elegans also control IJ recovery in Heterorhabditis, suggesting conservation of these developmental processes across free-living and parasitic nematodes.     

The effects of host age and superparasitism by the parasitoid, Microplitis rufiventris on the cellular and humoral immune response of Spodoptera littoralis larvae

To study the dynamics of stage-dependent immune responses in Spodoptera littoralis (Boisd.) larvae (Lepidoptera: Noctuidae), single and superparasitism experiments were carried out using the parasitoid Microplitis rufiventris Kok. (Braconidae: Hymenoptera). Compared to younger (preferred) host larvae, the older (non-preferred) host larvae displayed a vigorous humoral response that often damaged and destroyed the single wasp egg or larva. Superparasitism and host age altered both the cellular and humoral immune responses. Younger host larvae showed a stronger encapsulation response compared to older host larvae. Moreover encapsulation rates in younger hosts (e.g., second instar) decreased with increasing numbers of parasitoid eggs deposited/larvae. In older larvae, the encapsulation rate was low in fourth, less in fifth and absent in sixth instar hosts. Conversely, the order and magnitude of the cellular immune response in S. littoralis hosts were highest in second instar larvae with the first instar larvae being a little lower. The immune response steadily decreased from the third through to the fifth instar and was least obvious in the sixth instar. In contrast, the general humoral immune response was most pronounced in sixth instar larvae and diminished towards younger stages. The results suggest that both cellular and humoral responses are stage-dependent. Wasp offspring in younger superparasitized host larvae fought for host supremacy with only one wasp surviving, while supernumerary wasp larvae generally survived in older superparasitized larvae, but were unable to complete development. Older instars seem to have a method for immobilizing/killing wasp larvae that is not operating in the younger instars.     

Litomosoides sigmodontis: vaccine-induced immune responses against Wolbachia surface protein can enhance the survival of filarial nematodes during primary infection

Wolbachia are bacteria present within the tissues of most filarial nematodes. Filarial nematode survival is known to be affected by immune responses generated during filarial nematode infection and immune responses to Wolbachia can be found in different species harbouring filarial nematode infections, including humans. Using the rodent filarial model Litomosoides sigmodontis, we show that pre-exposure to wolbachia surface protein in a Th1 context (but not in a Th2-context) enhances worm survival on subsequent challenge. This study suggests that despite abundant evidence that pro-inflammatory reactions to the endosymbiont have detrimental effects on the both the nematode and mammalian host, they may under some circumstances be beneficial to the nematode.     

Laboratory Culture and Life History of Heleidomermis magnapapula in Its Host, Culicoides variipennis (Diptera: Ceratopogonidae)

The mermithid parasite Heleidomermis magnapapula was maintained in larvae of the midge Culicoides variipennis for 20 months in enamel pans containing nutrient-rich water and polyester pads as a substrate. Inseminated female mermithids were introduced to the pad surface when the host was in the late second or early third-instar. Host larvae were harvested from the pans 9 days after exposure and held in tap water for nematode emergence. Preparasite yield was positively correlated with female nematode size and averaged 1,267 preparasites/female. Male and female nematodes emerged an average of 12.2 and 13.4 days after host exposure, respectively. Supplemental host food (Panagrellus) during the final days of parasitism did not alter time of emergence. Parasites emerging singly were 64% females, whereas superparasitized hosts yielded males (up to nine/host). Nematode carryover into the adult midge normally occurred at a level of 0.5-2.5%. Parasite load (nematodes/ parasitized individual) in midge adults was lower than that of larvae from the same cohort, and adult midges were more likely to harbor female parasites. Exposure of fourth-instar host larvae resulted in higher levels of adult parasitism (up to 17%).     

Factor Affecting Sex Ratios of a Mermithid Parasite of Mosquitoes

The ratio of male to female Reesimermis nielseni Tsai and Grundmann, a nematode parasite of mosquito larvae, increased as the number of parasites per host increased. Hosts with a single nematode produced 9% males compared with essentially 100% males in hosts with more than 7 parasites; hosts with 3 nematodes produced about equal numbers of males and females. Males of R. nielseni generally emerged before females because of the earlier death of multiple-infected mosquitoes. The species of the host mosquito influenced the sex ratio, but the size of a specific host at the time of invasion did not. Host diet also had a noticeable influence on the sex ratio of the nematode: singly infected hosts from a starved population produced 92% males compared with 13% in the normally fed group. The importance of these factors in the mass rearing of R. nielseni is discussed.     

Seasonal dynamics of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis as a response to abiotic factors and abundance of insect hosts

The seasonal dynamics of entomopathogenic nematodes (EPNs) of the genus Steinernema and Heterorhabditis were studied during one season in meadow and oak wood habitats, in the vicinity of Ceské Budejovice, Czech Republic. The influences of soil temperature, moisture, and abundance of suitable hosts on EPN dynamics were investigated. The host range of these nematodes, in both habitats was also observed. A total of four EPN species were found in both habitats. Steinernema affine was the dominant species both in oak wood and in meadow. Additionally, the oak wood habitat was inhabited by S. kraussei and S. weiseri; the meadow habitat by Heterorhabditis bacteriophora. The mean abundance of total EPN community was 28,000ind./m(2) in oak wood and 11,000ind./m(2) in meadow. The seasonal dynamics of entomopathogenic nematodes in both habitats were characterized by high nematode densities in the beginning of the season, followed by a rapid decrease, and then stabilization. EPN abundances did not show any apparent correlation with soil temperature and moisture, but they were negatively correlated with the abundance of suitable insect hosts. Inter- and intraspecific competition for limited nutrients (hosts) probably played a major role in EPN seasonal dynamics. Broad host range of entomopathogenic nematodes in both habitats was predominantly represented by dipteran and coleopteran larvae. Most common hosts belonged to the families Asilidae, Bibionidae, and Empididae (Diptera), as well as Carabidae and Curculionidae (Coleoptera).     

Does scavenging extend the host range of entomopathogenic nematodes (Nematoda: Steinernematidae)?

Living and freeze-killed natural and laboratory hosts, with different susceptibility to entomopathogenic nematodes, were exposed to the larvae of Steinernema affine and Steinernema kraussei in two different experimental arenas (Eppendorf tubes, Petri dishes), and the success of the colonisation and eventual progeny production were observed. Both nematodes were able to colonise both living and dead larvae of Galleria mellonella (Lepidoptera) and adult Blatella germanica (Blattodea) even though the progeny production in dead hosts was lower on average. Living carabid beetles, Poecilus cupreus, and elaterid larvae (Coleoptera) were resistant to the infection, however, both nematodes were able to colonise and multiply in several dead P. cupreus and in a majority of dead elaterid larvae. By scavenging, EPNs can utilise cadavers of insects that are naturally resistant to EPN infection, and so broaden their host range.     

Quantification of Invasion of Two Strains of Steinernema carpocapsae (Weiser) into Three Lepidopteran Larvae

Studies with last instar larvae of the fall armyworm, Spodoptera frugiperda (J. E. Smith), the black cutworm, Agrotis ipsilon (Hufnagel), and the greater wax moth, Galleria mellonella (L.) were used to quantify the invasive ability of two strains (All and Mexican) of Steinernema carpocapsae and to determine how factors in the bioassay procedure affect both nematode invasion and host mortality. Nematode invasive ability was variable, with 10-50% of nematodes successfully infecting the host. The percentage of infectives invading the host (invasion efficiency) was positively related to increases in length of host exposure time and number of hosts per arena, negatively related to increases in substrate surface area per host, and not affected by nematode concentration. There was a direct relationship between concentration applied and the number of nematodes invading the host. Mortality was less affected than invasion efficiency by bioassay conditions and appears to be a much less sensitive index of nematode activity than invasive ability.     

Exploration of host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster

The facultative intracellular bacterial pathogen Listeria monocytogenes is capable of replicating within a broad range of host cell types and host species. We report here the establishment of the fruit fly Drosophila melanogaster as a new model host for the exploration of L. monocytogenes pathogenesis and host response to infection. Listeria monocytogenes was capable of establishing lethal infections in adult fruit flies and larvae with extensive bacterial replication occurring before host death. Bacteria were found in the cytosol of insect phagocytic cells, and were capable of directing host cell actin polymerization. Bacterial gene products necessary for intracellular replication and cell-to-cell spread within mammalian cells were similarly found to be required within insect cells, and although previous work has suggested that L. monocytogenes virulence gene expression requires temperatures above 30 degrees C, bacteria within insect cells were found to express virulence determinants at 25 degrees C. Mutant strains of Drosophila that were compromised for innate immune responses demonstrated increased susceptibility to L. monocytogenes infection. These data indicate L. monocytogenes infection of fruit flies shares numerous features of mammalian infection, and thus that Drosophila has the potential to serve as a genetically tractable host system that will facilitate the analysis of host cellular responses to L. monocytogenes infection.     

Influence of Host Nutrients on the Parasitic Development of Mermis nigrescens (Mermithidae)

Schistocerca gregaria nymphs and adults of both sexes were infected with eggs of Mermis nigrescens. Mermithid larvae grew more slowly in nymphal hosts, and emerging larvae were smaller than those from adult hosts. The longer the larvae remained in the host, the greater their size. Those developing in adult female hosts were longest. Single mermithid larvae that were transferred to a second host continued to grow and were significantly longer at emergence than larvae that developed solely in one host. In adult hosts that were infected with 40-300 M. nigrescens eggs, the percentage of mermithids that became males was strongly dependent on host weight at infective doses of 90 eggs or more. Results are discussed in relation to nutrient stress on the larvae and its importance in developing in vitro culture techniques.     

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.     

Optimization of Inoculation for In Vivo Production of Entomopathogenic Nematodes

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.     

The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads

Actively growing cultures of Photorhabdus luminescens were encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads, containing approximately 2.5 x 10(7)Photorhabdus cells per bead, when mixed with sterilized soil and exposed to Spodoptera litura larvae resulted in 100% mortality in 48 h, while the use of alginate encapsulated Heterorhabditis nematode resulted in 40% mortality after 72 h. The bacteria were reisolated from the dead insect thus proving Koch's postulates and demonstrating the ability of P. luminescens to kill the insect host on their own, independent of the symbiont nematode. The LC(50) dose of Photorhabdus cells was estimated at 1010 cells per larva for killing S. litura 6th instar larvae in 48 h.     

Protein-poor diet reduces host-specific immune gene expression in Bombus terrestris

Parasites infect hosts non-randomly as genotypes of hosts vary in susceptibility to the same genotypes of parasites, but this specificity may be modulated by environmental factors such as nutrition. Nutrition plays an important role for any physiological investment. As immune responses are costly, resource limitation should negatively affect immunity through trade-offs with other physiological requirements. Consequently, nutritional limitation should diminish immune capacity in general, but does it also dampen differences among hosts? We investigated the effect of short-term pollen deprivation on the immune responses of our model host Bombus terrestris when infected with the highly prevalent natural parasite Crithidia bombi. Bumblebees deprived of pollen, their protein source, show reduced immune responses to infection. They failed to upregulate a number of genes, including antimicrobial peptides, in response to infection. In particular, they also showed less specific immune expression patterns across individuals and colonies. These findings provide evidence for how immune responses on the individual-level vary with important elements of the environment and illustrate how nutrition can functionally alter not only general resistance, but also alter the pattern of specific host–parasite interactions.     

Entomopathogenic Nematodes for Control of Insect Pests Above and Below Ground with Comments on Commercial Production

Entomopathogenic nematodes (EPNs) have been utilized in classical, conservation, and augmentative biological control programs. The vast majority of applied research has focused on their potential as inundatively applied augmentative biological control agents. Extensive research over the past three decades has demonstrated both their successes and failures for control of insect pests of crops, ornamental plants, trees and lawn and turf. In this paper we present highlights of their development for control of insect pests above and below ground. The target insects include those from foliar, soil surface, cryptic and subterranean habitats. Advances in mass-production and formulation technology of EPNs, the discovery of numerous efficacious isolates/strains, and the desirability of reducing pesticide usage have resulted in a surge of commercial use and development of EPNs. Commercially produced EPNs are currently in use for control of scarab larvae in lawns and turf, fungus gnats in mushroom production, invasive mole crickets in lawn and turf, black vine weevil in nursery plants, and Diaprepes root weevil in citrus in addition to other pest insects. However, demonstrated successful control of several other insects, often has not lead to capture of a significant share of the pesticide market for these pests.     

Effects of Meloidogyne incognita on Nitrogen Fixation in Soybean

The effects of a North Carolina population of Meloidogyne incognita on N₂ fixation on root-knot-susceptible ''Lee 68'' and moderately resistant ''Forrest'' soybean were evaluated 50, 75, I00, and 135 days after inoculation with nematodes. Nematodes stimulated N₂ fixation in Lee 68 by 50 days and in Forrest by 75 days. At all other intervals, N₂ fixation was either depressed or unaffected by nematodes. Additional observations indicate that the susceptibility of Lee 68 is associated with greater rates of penetration by larvae and more favorable responses of host tissues to nematodes than occur in Forrest. With time, however, the histological reactions of both hosts became less favorable for nematode development. Resistant or hypersensitive responses became common in Forrest by 75 days but not in Lee 68 until 90 days after inoculation. This population of M. incognita may stimulate N₂ fixation at a specific time interval and depress it at others; therefore, disease of susceptible soybeans caused by this nematode is probably not primarily due to a net loss of fixed nitrogen but to pathogenicity similar to that which occurs on nonlegume hosts.