Instar Galleria Mellonella Research Articles

Nematicidal Effects of Four Terpenes Differ among Entomopathogenic Nematode Species

Entomopathogenic nematodes (EPNs) have been studied for more than half a century, and employed for insect pest management using augmentation, conservation, and classical biological control approaches. As obligate lethal parasitoids of insect larvae, EPN navigate a chemically complex soil environment and interact with their insect hosts, plants, and each other. EPN responses to various terpenes, such as herbivore-induced plant volatiles, have the potential to enhance EPN efficacy through their attraction. However, several of the terpenes are currently being formulated as biological fungicides, insecticides, and acaricides for above- or below-ground applications. We conducted laboratory experiments to investigate the possible nematicidal effect of four terpenes, carvacrol, geraniol, eugenol, and thymol, to two heterorhabditids and two steinernematid species. Each terpene showed nematicidal activity against at least two of the four EPN species, with carvacrol showing the strongest activity and Heterorhabditis bacteriophora the highest sensitivity. Despite the high sensitivity of both heterorhabditids and near-zero sensitivity of the steinernematids to thymol, carvacrol, and eugenol, an increasing effect was observed when steinermatid nematodes were exposed to geraniol, and a decreasing effect for heterorhabditids, with H. bacteriophora exhibiting higher mortality than H. indica. The virulence of the nematodes towards fourth instar Galleria mellonella was also tested after exposure to the median lethal doses of each terpene. No significant difference in virulence was observed between nematodes that were exposed or not exposed to sublethal doses. The experiments suggest that the tested terpenes have a strong effect on EPN viability, which should be considered when combining the two approaches in IPM. The terpenes did not have a universal effect on all nematode species, which merits further investigation, while virulence tests suggest that sublethal doses of these terpenes have no effect on the host-killing performance of EPNs.

The Effects of Magnetic Iron Oxide Nanoparticles (Fe3O4) on Some Biological Aspects of Galleria mellonella L. (Lepidoptera: Pyralidae)

In this study, 18-38 nm-sized and spherical-shaped nanopowder Fe3O4 NPs concentrations (0.4, 2, 10, 50, 250 µg/10 µl) was force-fed to sixth instar (180 ± 20 mg) Galleria mellonella (Lepidoptera: Pyralidae) larvae under laboratory conditions. The effects of magnetic iron oxide nanoparticles (Fe3O4) on the pupal and adult developmental times, pupal and adult weights and adult longevity of G. mellonella were recorded. Results showed that treating G. mellonella with 250 μg/10 µl Fe3O4 NPs significantly increased pupal weights. Additionally, while adult developmental time increased post 250 μg/10 µl Fe3O4 NPs treatment, it was observed that pupal developmental time, pupal and adult weights, and adult longevity were not statistically significantly different when compared to the control

Effects of Zinc oxide Nanoparticles (ZnO NPs) on Hemocyte Types of Galleria mellonella

In this study, 70 nm-sized and nanorod-shaped Zinc oxide nanoparticle (ZnO NPs) concentrations (0.5, 1, 2.5, 5, and 10 µg/10 µl) was force-fed to fourth instar (110 ± 20 mg) Galleria mellonella (Lepidoptera: Pyralidae) larvae. The effects of ZnO NPs on plasmatocyte, granulocyte, spherulocyte, prohemocyte, oenocytoid, and coagulocyte numbers in hemolymph of G. mellonella larvae was determined. Results showed that treating G. mellonella with 10 μg/10 µl ZnO NPs significantly decreased spherulocytes numbers, whereas numbers of plasmatocyte, granulocyte, prohemocyte, oenocytoid, and coagulocyte numbers did not differ significantly when compared to the control group after 24 h force feeding treatment. There was a statistically significant difference between the experimental groups in the prohemocyte numbers of larvae that exposed to 1 and 5 μg/10 µl ZnO NPs.

Fenoxycarb Effects 7th instar Galleria mellonella Larvae by Triggering Extra Larval Stages

Insect growth regulators are specific chemicals with hormonal effects on insects and these types of chemicals target to reduce the nonspecific effects of pesticides on nature and living things. In this study, we investigated the effects of one of the insect growth regulators, fenoxycarb which mimics the juvenile hormone action, on the growth of a harmful lepidopteran species Galleria mellonella, and it’s some developmental parameters. For this purpose, fenoxycarb was applied on day 0 of 7th instar larvae of the greater wax moth, Galleria mellonella. The treatment with 100 ng fenoxycarb had no developmental effect on Galleria mellonella larvae. The treatment with relatively high doses of fenoxycarb on day 0 induced the supernumerary larvae formation of 99% and larvae which molted extra larval instar formed healthy pupae in the ratio of 98-99%. Consequently, the effects of fenoxycarb on Galleria mellonella larvae occurred in a dose-dependent manner. Although fenoxycarb is a potent insect growth regulator, late Galleria mellonella larvae are not susceptible to this chemical, unlike some other Lepidopter insect species.

Effects of the copper oxide nanoparticles (CuO NPs) on Galleria mellonella hemocytes

In this study, 38 nm-sized and flake-like-shaped CuO NPs (10, 50, 100, 150 μg/10 µl/larva) were force-fed to fourth instar (100 ± 20 mg) Galleria mellonella (Lepidoptera: Pyralidae) larvae under the laboratory conditions. The effects of CuO NPs on total hemocyte counts (THCs) and the frequency of viable, mitotic, apoptotic, necrotic, and micronucleated hemocyte indices were detected with the double-staining protocol by hematoxylin and eosin (H&E) stains. The total hemocyte counts (THCs) did not change significantly in G. mellonella larvae at all concentrations for 24 h and 72 h post-force-feeding treatment. The ratio of viable hemocytes decreased at 50, 100, 150 μg/10 µl concentrations in 24 h and 72 h when compared with untreated larvae. The increases in the percentage of mitotic and micronucleated hemocytes were statistically significant at 150 μg/10 µl in 24 h. The results showed that high concentrations (>10 μg/10 µl) of CuO NPs increased the percentage of apoptotic hemocytes in 24 h. 100 and 150 μg/10 µl of CuO NPs caused a significant increase in the percentage of necrotic hemocytes in 24 h. The decrease in the percentage of mitotic hemocytes at 10, 100 and 150 μg/10 µl in 72 h was statistically significant. Apoptotic hemocytes increased and were found to be higher at 100 and 150 μg/10 µl of CuO NPs in 72 h in comparison with the untreated larvae. Finally, we observed an increase in the percentage of necrotic hemocytes at 150 μg/10 µl in 72 h.

A Bacillus thuringiensis Chitin-Binding Protein is Involved in Insect Peritrophic Matrix Adhesion and Takes Part in the Infection Process.

Bacillus thuringiensis (Bt) is used for insect pest control, and its larvicidal activity is primarily attributed to Cry toxins. Other factors participate in infection, and limited information is available regarding factors acting on the peritrophic matrix (PM). This study aimed to investigate the role of a Bt chitin-binding protein (CBPA) that had been previously shown to be expressed at pH 9 in vitro and could therefore be expressed in the alkaline gut of lepidopteron larvae. A ∆cbpA mutant was generated that was 10-fold less virulent than wild-type Bt HD73 towards Ostrinia furnacalis neonate larvae, indicating its important role in infection. Purified recombinant Escherichia coli CBPA was shown to have a chitin affinity, thus indicating a possible interaction with the chitin-rich PM. A translational GFP–CBPA fusion elucidated the localization of CBPA on the bacterial surface, and the transcriptional activity of the promoter PcbpA was immediately induced and confirmed at pH 9. Next, in order to connect surface expression and possible in vivo gut activity, last instar Galleria mellonella (Gm) larvae (not susceptible to Bt HD-73) were used as a model to follow CBPA in gut expression, bacterial transit, and PM adhesion. CBPA-GFP was quickly expressed in the Gm gut lumen, and more Bt HD73 strain bacteria adhered to the PM than those of the ∆cbpA mutant strain. Therefore, CBPA may help to retain the bacteria, via the PM binding, close to the gut surface and thus takes part in the early steps of Bt gut interactions.

Activity of Steinernema colombiense in plant-based oils.

Entomopathogenic nematodes (EPNs) are excellent biological control agents. Although traditionally EPN application targeted belowground insects, their aboveground use can be supported if combined with adjuvants. We hypothesized that EPN infective juveniles (IJs) could be combined with plant-based oils as adjuvants, without decreasing their efficacy against insect larvae under various scenarios. Specifically, our objectives were to evaluate the activity of Steinernema colombiense (Nematoda: Steinernematidae) when mixed with two plant-based oils (coconut and olive oils) and maintained at different temperatures and times, or combined with entomopathogenic fungi. First, we evaluated how these oils affected IJ survival and virulence against last instar Galleria mellonella (Lepidoptera: Pyralidae) larvae when maintained at five different temperatures (4, 8, 14, 20, and 24°C) and five incubation times (1, 3, 7, 14, and 21 days), using water as control treatment. Second, we evaluated virulence when combined with these two oils as well as with water (control) and combined with the entomopathogenic fungi (EPF), Beauveria bassiana (Hypocreales: Clavicipitaceae). Infective juvenile survival was higher in coconut than olive oil and water mixtures up to 7 days at 4°C. Conversely, olive oil supported higher larval mortality than coconut oil at 4 to 20°C and 14 days. Similarly, the number of days needed to kill insect larvae increased at extreme temperatures (4 and 24°C) after 14 days. Finally, the EPN + EPF combination showed an additive effect compared to EPN and EPF single treatments. Our findings indicate that our plant-based oil mixtures maintain viable IJs at moderate temperatures and up to 7 to 14 days, and can be used in single EPN mixtures or combined with EPF.

DESCRIPTION OF NEW SPECIES OF METARHABDITIS LONGICAUDATUS (NEMATODA: RHABDITIDAE) WITH THREE NEW RECORDS FROM SINDH, PAKISTAN

Many rhabditid nematodes like Metarhabditis belonging to the bacteriophage group are considered to be necromenic associates of insects and used to be facultative entomopathogenic nematodes or as biocontrol agents. These nematodes can be safely used against insect pests of different crops and vegetables. In the present study, nematode specimens of new and known species were collected from different areas of Sindh and isolated from soil samples by baiting technique using last instar Galleria mellonella. One new nematode species Metarhabditis longicaudata n. sp. and three new records of M. adenobia, M. amasactae, and M. rainai (Nematoda: Rhabditidae) were found from Sindh. M. longicaudata n. sp. differed from the species of the genus Metarhabditis by having long un-covered tail spike. It was also characterized by a large sized male 1286 (1154-1325) µm; longer male tail 69.5 (62-76) µm; longer spicule 43 (40-46) µm; leptoderan bursa; longer gubernaculum 27 (20-34) µm. The female was also of large size of 1507 (1366-1684) µm with larger tail 102 (94-112) µm and longer pharynx 231 (206-265) µm. The genus Metarhabditis is reported for the first time from Sindh, Pakistan.

Biological characterization of the entomopathogenic nematode, Steinernema innovationi: a South African isolate.

Abstract Entomopathogenic nematode species perform differently under different environmental conditions; therefore, the authors investigated the biological and environmental characteristics that could optimize performance of Steinernema innovationi. The authors studied the effect of temperature on infectivity and reproduction, the foraging behavior and host range. Thermal activity was optimal between 22 and 25°C. Highest infective juvenile (IJ) yields in last instar Galleria mellonella were observed at 22°C (333,014 IJs/g) and 25°C (354,165 IJs/g). An average of 26% of the IJs infected G. mellonella larvae at depths of 15 cm within 24 hr. Steinernema innovationi IJs raised greater than 95% of their body off the substrate and moved in a circular pattern, but did not jump. The hosts, Acheta domesticus, Chilo partellus, and Plutella xylostella showed the least susceptibility. All other hosts, [Eldana saccharina, Sesamia calamistis, Tenebrio molitor, G. mellonella, Cydia pomonella] suffered 100% larval mortality. Pupal mortality ranged from 47 to 68%. An LC50 and LC70 of 3 and 31 IJs/larva, respectively, were calculated for Agrotis ipsilon. The results indicate that S. innovationi is adapted to moderate temperatures. Furthermore, to the author’s knowledge this is the first report of a nematode (particularly from ‘glaseri group’) that shows cruiser-type behavior in movement, but also nictates.

Enhanced insecticidal activity of Chilo iridescent virus expressing an insect specific neurotoxin

Previously we have generated a recombinant Chilo iridescent virus (CIV) by inserting the green fluorescent protein gene (gfp) into the CIV 157L open reading frame (ORF) locus and showed that this recombinant (rCIV-Δ157L-gfp) was fully infectious both in cell culture as well as in insect larvae. This study opened up a new avenue for increasing the speed of kill of CIV and other iridoviruses by inserting virulence or toxin genes into the viral genome. In the current study we constructed a recombinant CIV (rCIV-Δ157L/gfp-AaIT) where the 157L ORF was replaced with both the AaIT neurotoxin gene from the scorpion Androctonus australis and the gfp gene, each under control of the viral major capsid protein (mcp) gene promoter. Recombinant virus was purified by successive rounds of plaque purification using Spodoptera frugiperda (Sf-9) cells. One-step growth curves for the recombinant viruses, rCIV-Δ157L/gfp-AaIT and rCIV-Δ157L-gfp, and wild-type CIVs in Sf-9 cells showed similar profiles. AaIT toxin expression in infected third instar Galleria mellonella larvae was confirmed by western blot analysis using an antibody against the AaIT protein. rCIV-Δ157L/gfp-AaIT infection at a concentration that kills 100% of the larvae caused paralysis in infected third instar G. mellonella larvae from two days after injection, whereas infection with non-AaIT containing viruses showed mortality starting much later (>10days). Bioassays on these larvae demonstrated that the speed of kill of CIV carrying AaIT was strikingly enhanced as compared to wild-type CIV. These results suggest that insertion of a toxin gene into CIV provides further opportunities to control a wide range of pest insects, such as weevils, using an iridovirus.

Virulence comparisons of high-temperature-adapted Heterorhabditis bacteriophora, Steinernema feltiae and S. carpocapsae

Summary Entomopathogenic nematodes (EPNs) are environmentally safe alternative control agents. Nematodes in the Heterorhabditidae and Steinernematidae families are widely used in biological control frameworks, especially for soil-inhabiting insect pests. In this experiment, Heterorhabditis bacteriophora (Poinar, 1976), Steinernema feltiae (Filipjev, 1934) and S. carpocapsae (Weiser, 1955) adapted at high temperature were assessed in order to detect differences in virulence between adapted and non-adapted populations. All species were exposed to 38 °C for 2 h. After this treatment, live infective juveniles (IJs) were used to infect to last instar Galleria mellonella (Linnaeus, 1758). larvae at the following doses: 1, 2, 3, 4 and 5 IJs/larva. The LD50 and LD90 were obtained for these species. Non-adapted populations of the nematode species were used as controls for this experiment. The results indicated that differences in S. feltiae and S. carpocapsae virulence between the adapted and non-adapted populations were significant; no significant difference was observed between the adapted and non-adapted H. bacteriophora populations.

Heterorhabditis georgiana n. sp. (Rhabditida: Heterorhabditidae) from Georgia, USA

AbstractIn a survey of entomopathogenic nematodes in Georgia, a nematode isolate of the genus Heterorhabditis was collected from soil by the insect-baiting technique and maintained in the laboratory on last instar Galleria mellonella (L.) larvae. Morphological and molecular studies of the isolate showed that the nematode is a new species. Light and scanning electron microscopy, DNA characterisation and phylogeny were used for this description. Heterorhabditis georgiana n. sp. is morphologically similar to H. bacteriophora and can be distinguished from this species mainly by male and female characters. Sixty percent of males have three pairs of papillae in the terminal group; 40% with three papillae on one side and two papillae on the other. The tail is conoid and slightly curved ventrally. The percentage of the gubernaculum to spicule length (GS%) is similar to that of H. mexicana (56) but higher than that of H. bacteriophora and species in the indica-group and the length of the spicule relative to anal body diam. (SW%) is lower than that of H. baujardi (172 vs 182), H. indica (172 vs 187) and H. bacteriophora (172 vs 174), but higher than that of H. amazonensis (172 vs 152), H. floridensis (172 vs 157) and H. mexicana (172 vs 167). The female can be differentiated from related species by its unique vulva pattern. In phylogenetic trees of ITS and D2D3 regions, the new species and H. bacteriophora form a monophyletic group. The new species has evolved five autapomorphic nucleotide character states based on analysis of ITS, differing from its sister taxon H. bacteriophora at 29 and three aligned positions of ITS and D2D3 regions, respectively.

Efficacy of Heterorhabditis baujardi LPP7 (Nematoda: Rhabditida) applied in Galleria mellonella (Lepidoptera: Pyralidae) insect cadavers to Conotrachelus psidii, (Coleoptera: Curculionidae) larvae

The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil causing severe reduction in fruit quality. We assessed its susceptibility to Heterhorhabditis baujardi LPP7 infective juveniles (IJs) in the greenhouse and under field conditions applying the nematodes in cadavers of seventh instar Galleria mellonella larvae. Field persistence of these nematodes in the soil was evaluated through G. mellonella-baiting. Insect cadaver concentrations of 2, 4 and 6 applied in pots in the greenhouse experiment caused significant mortality compared to the control. Significance differences were observed in the field between control and treatments only when six cadavers per 0.25 m2 were applied. Infective juveniles from the cadavers persisted 6 weeks after application in the field, but decreased greatly thereafter. Our work demonstrates that H. baujardi LPP7 IJs emerging from G. mellonella cadavers can be efficacious against guava weevil fourth instar larvae. Also, we demonstrated the long-term persistence of IJs in the soil.

Heterorhabditis amazonensis n. sp. (Rhabditida: Heterorhabditidae) from Amazonas, Brazil

AbstractIn a survey of entomopathogenic nematodes in Brazil, a nematode isolate of the genus Heterorhabditis was found. The nematode was collected from soil by the insect-baiting technique and maintained in the laboratory on last instar Galleria mellonella (L.) larvae. Morphological and molecular studies of the isolate showed that the nematode is a new species. Light and scanning electron microscopy, DNA characterisation and phylogeny were used for this description. Heterorhabditis amazonensis n. sp. is morphologically similar to H. baujardi, H. floridensis, H. mexicana and H. indica, and can be distinguished from these species mainly by male and female characters. Fifty percent of Heterorhabditis amazonensis n. sp. males have two pairs of bursal papillae in the terminal group; 25% with two papillae on one side and one papilla on the other side and 25% with one pair of papillae. Twenty percent of the population has a curved gubernaculum. The percentage of the gubernaculum to spicule length (GS%) is lower than that of H. mexicana (50 vs 56), and the length of the spicule relative to anal body diam. (SW%) is lower than that of H. mexicana (152 vs 167) and H. baujardi (152 vs 182). The female can be differentiated from related species by its unique vulva pattern. In a phylogenetic tree, the new species, H. floridensis, H. baujardi and H. mexicana form a monophyletic group, a sister group to H. indica. The new species has evolved three autapomorphic nucleotide character states, differing from its sister taxa H. mexicana at 22, and H. baujardi at 15, aligned positions.

Effects of diflubenzuron on the integument of fifth instarGalleria mellonella larvae

Effects of the chitin synthesis inhibitor diflubenzuron (DFB) on the integument of 5th instarGalleria mellonella L. larvae were investigated. When larvae were fed with semi-artificial diets containing 250, 500 and 1000 ppm of the compound, DFB affected the integument. The affected larvae failed to ecdysis, their cuticle was ruptured, lost haemolymph and blackened. In treated larvae, cuticle deposition was disrupted and the cuticle thickness was decreased by ∼50% compared with the untreated control, particularly at day 3 1/2. However, statistically there was no significant difference among the three concentrations (P>0.05). This may indicate that all three concentrations are equally effective in decreasing level. DFB at 500 and 1000 ppm also affected the epidermal cells and caused the occurrence of vacuoles.

Mattesia oryzaephili (Neogregarinorida: Lipotrophidae), a Pathogen of Stored-Grain Insects: Virulence, Host Range and Comparison with Mattesia dispora

The neogregarine, Mattesia oryzaephili (Neogregarinorida: Lipotrophidae) has only been reported from the sawtoothed grain beetle, Oryzaephilus surinamensis. The pathogen's presence in cadavers of the rusty grain beetle, Cryptolestes ferrugineus, in collapsed colonies prompted studies of its potential to control stored-product insects. Respective mortality rates in fourth instar C. ferrugineus and C. pusillus were 15.3 and 17.7% at 102 oocysts/g of diet and 89.4 and 80.5% at 105 oocysts/g. The mortality of fourth instar O. surinamensis exposed to 105 oocysts/g was only 12%. For C. ferrugineus larvae, there were no significant differences in mortality and infection between exposure to Mattesia dispora and exposure to M. oryzaephili (P>0.05), but for C. pusillus larvae, both responses were significantly higher for M. oryzaephili than M. dispora. Adult C. ferrugineus and O. surinamensis were similar in their responses to M. oryzaephili, with mortality not exceeding 20%, but differed in their responses to M. dispora, with O. surinamensis being more susceptible. The median lethal doses for larval Mediterranean flour moths, Ephestia kuehniella, were 7.9×107 M. oryzaephili oocysts/g of diet and 2.7×103 M. dispora oocysts/g of diet. In single dose assays of M. oryzaephili physiological host range, greater than 75% infection was achieved for Rhyzopertha dominica and Plodia interpunctella. More than half of oocysts germinated during passage through the guts of susceptible and resistant insects. Second and third instar Galleria mellonella were highly susceptible to M. oryzaephili infection, but fifth instars were not. Infection percentages in fifth instars exposed to 106 oocysts/g were significant only when boric acid or the stilbene, Blankophor®RHK were incorporated into the diet. Host range and general morphology confirm the identity of Mattesia oryzaephili.

Entomopathogenic nematodes and their symbiotic bacteria for the biological control of Hoplia philanthus (Coleoptera: Scarabaeidae)

Abstract Larvae of scarabaeid beetle Hoplia philanthus cause severe problems on sports turf, lawns, and pastures. The efficacy of three species of entomopathogenic nematodes, Heterorhabditis megidis, Steinernema feltiae, and Steinernema glaseri was tested against third instar of H. philanthus. In a dose response experiment, 0, 1, 10, 100, 1000, and 10,000 infective juveniles (IJs) were inoculated in wells of 24-well plates containing sand and a single H. philanthus larva. S. glaseri was most effective (LC50=4.6 and LC90=79.3 after 7 days exposure of IJs/larva) compared to H. megidis (LC50=9.7 and LC 90 =511.8 IJs / larva ). The lethal concentration of S. feltiae could not be determined because of the strain’s low virulence against third instar H. philanthus. In a second experiment, the pathogenicity of the symbiotic bacteria Photorhabdus luminescens, Xenorhabdus bovienii, and Xenorhabdus poinarii was studied. Three microliters of a bacterial suspension (containing 0, 25, 250, 2500, or 25,000 cells) were injected into the hemocoel of third instar of H. philanthus and last instar Galleria mellonella. P. luminescens and X. bovienii killed 100% of the larvae of both species after 72 h; X. poinarii caused lowest mortality for both insect species. In a third experiment, 3 μl of a cell-free filtrate of P. luminescens, X. bovienii, and X. poinarii were injected into the hemocoel of third instar of H. philanthus and last instar G. mellonella. The filtrates of P. luminescens and X. bovienii caused 100% mortality after 24 h to H. philanthus and G. mellonella. The X. poinarii filtrate was least toxic to both insect species. In pot trials, H. megidis and S. glaseri caused more than 80% mortality of H. philanthus larvae infesting potted perennial ryegrass 42 days after application of 2.5–7.5 billion nematodes/ha. The mortality was greater than the grub mortality caused by either S. feltiae (16%) or the control (10%).

Expression of larval hemolymph proteins ( Lhp) genes and protein synthesis in the fat body of greater wax moth ( Galleria mellonella) larvae during diapause

When one-day-old, last instar Galleria mellonella larvae are exposed to 18°C they enter diapause and cease further development for several months. During diapause a group of proteins (72–84 kDa) synthesized in the fat body and secreted into the hemolymph is markedly elevated. Partial sequencing of the N-terminus of two proteins from this group confirmed their identity with larval hemolymph proteins (LHP) belonging to the family of hexameric storage proteins. The expression of two Lhp genes of known sequence ( Lhp76 and Lhp82) were monitored in both diapausing and non-diapausing individuals. The expression of both genes and subsequent synthesis of the proteins (LHP76 and LHP82) is maintained until at least 90–100 days of diapause.

Apolipophorin-III and the Interactions of Lipoteichoic Acids with the Immediate Immune Responses of Galleria mellonella

We investigated the effects of lipoteichoic acids, surface components of Gram-positive bacteria, on the hemocytes and phenoloxidase activity in last instar Galleria mellonella larvae, as well as the binding of apolipophorin-III, an insect lipid-binding protein, to lipoteichoic acids. Binding of apolipophorin-III to lipoteichoic acid was studied using an assay based on 1,9-dimethylmethylene blue. Apolipophorin-III bound the lipoteichoic acids from Bacillus subtilis, Enterococcus hirae, and Streptococcus pyogenes and to intact cells of E. hirae. E. hirae lipoteichoic acid promoted the binding of apolipophorin-III to the cells of this species. All lipoteichoic acids tested caused a dose- and time-dependent drop in the total counts of hemocytes and, depending on the species of lipoteichoic acid, partial or complete depletion of plasmatocytes. Granulocyte counts were not affected. Apolipophorin-III prevented partially the loss of plasmatocytes due to B. subtilis lipoteichoic acid. All three lipoteichoic acids studied activated phenoloxidase in vitro; injections of B. subtilis lipoteichoic acid into the larvae elevated the phenoloxidase activity, whereas injections of E. hirae or S. pyogenes lipoteichoic acid, or apolipophorin-III alone, suppressed it. Apolipophorin-III decreased the activation of phenoloxidase by B. subtilis lipoteichoic acid.

Dispersal and Host-Finding Ability of Entomopathogenic Nematodes At Low Temperatures

To enhance the control of sedentary, subterranean insect pest species like Otiorhynchus sulcatus, 19 new alpine isolates of entomopathogenic nematodes were screened for infectivity at 9°C. For this purpose, nematode dispersal was studied in the presence of late instar Galleria mellonella and O. sulcatus, using a T-tube choice system filled with moist sand. For each isolate five characteristics were recorded: migration rate, average distance travelled, host-finding ability, parasitisation rate, and invasion rate. As expected, most isolates accumulated in the host compartments. The rank sum of the isolates, calculated according to the ranks attained for each of the five characteristics, classified seven Steinernema kraussei before the first S. feltiae. However, no single isolate was clearly the most infective. Steinernema sp. 1, Heterorhabditis CH-H-FLU91, and the commercially available S. feltiae OBS III yielded lowest scores. They are considered unsuitable control agents at low temperatures. The locomotory activity of the nematodes in dispersal assays with G. mellonella was significantly correlated with their efficacy at 8.5± 1°C against O. sulcatus in pot experiments. This indicates that T-tube choice experiments as performed in this study represent a meaningful tool for selecting cold-active nematodes. In supplementary dispersal experiments with larvae of Melolontha melolontha, biological control-performance of the four nematode species tested was meagre.