Biological control of Phlebotomus papatasi larvae by using entomopathogenic nematodes and its symbiotic bacterial toxins.

The sand fly Phlebotomus papatasi is an important disease-bearing vector. Five entomopathogenic nematodes (EPNs) - Steinernema carpocapsae DD136, Steinernema sp. (SII), S. carpocapsae all, S. abbasi, and Heterorhabditis bacteriophora HP88 - were applied as biocontrol agents against the late third instar larvae of P. papatasi. In addition, the effect of toxin complexes (TCs) of Xenorhabdus nematophila and Photorhabdus luminescens laumondii bacteria was evaluated. Results revealed that S. carpocapsae DD136 was the most virulent species followed by Steinernema sp. (SII) and S. carpocapsae all where LC50 were 472, 565, 962 IJs/ml, respectively. Also, the crude TCs were slightly more active and toxic than their fractionated protein. Histopathological examination of infected larvae with H. bacteriophora HP88 showed negative effect on their midgut cells. In conclusion, EPNs with their symbiotic bacteria are more effective as biocontrol agents than the crude or fractionated TCs against sand fly larvae.

Biological control of Phlebotomus papatasi larvae by using entomopathogenic nematodes and its symbiotic bacterial toxins

@# The sand fly Phlebotomus papatasi is an important disease-bearing vector. Five entomopathogenic nematodes (EPNs) – Steinernema carpocapsae DD136, Steinernema sp. (SII), S. carpocapsae all, S. abbasi, and Heterorhabditis bacteriophora HP88 – were applied as biocontrol agents against the late third instar larvae of P. papatasi. In addition, the effect of toxin complexes (TCs) of Xenorhabdus nematophila and Photorhabdus luminescens laumondii bacteria was evaluated. Results revealed that S. carpocapsae DD136 was the most virulent species followed by Steinernema sp. (SII) and S. carpocapsae all where LC50 were 472, 565, 962 IJs/ml, respectively. Also, the crude TCs were slightly more active and toxic than their fractionated protein. Histopathological examination of infected larvae with H. bacteriophora HP88 showed negative effect on their midgut cells. In conclusion, EPNs with their symbiotic bacteria are more effective as biocontrol agents than the crude or fractionated TCs against sand fly larvae.

Silver nanoparticles enhance the larvicidal toxicity of Photorhabdus and Xenorhabdus bacterial toxins: an approach to control the filarial vector, Culex pipiens.

Mosquito-control is still based mostly on chemical insecticides which are toxic and cause environmental deprivation. This study investigates synthesizing silver bio-nanoparticles (AgNPs) from nematode-symbiotic bacterial toxin complexes as an alternative larvicidal bioinsecticide agent against Culex pipiens larvae. Five species/strains of nematode-symbiotic bacteria, Xenorhabdus indica, Xenorhabdus spp., Photorhabdus luminescens laumondii HP88, Photorhabdus luminescens akhurstii HRM1 and Photorhabdus luminescens akhurstii HS1 were used. AgNPs were characterized by scanning electron microscopy and x-ray diffraction analysis. Larvae were initially exposed to descending concentrations (300, 150, 75, 37.5 and 18.75 µg/ml) of each of the five bacterial toxins (as positive controls) or to the bio-AgNPs synthesized from the same bacterial toxins (200, 100, 50, 25, 12.5, 6.25, 3.12 and 1.5 µg/ml) for 48 hours. Results of toxicity bioassays showed that mortality of treated larvae was concentration-dependent, toxins from X. indica, P. luminescens laumondii HP88 and P. luminescens akhurstii HS1 showed LC50 of 29, 28 and 2002 µg/ml, respectively. While, toxins from P. luminescens akhurstii HRM1 and Xenorhabdus sp. showed LC50 of 199, 318 µg/ml, respectively. Bio-AgNPs synthesized from, X. indica or Xenorhabdus sp. toxins have significantly increased their larvicidal activities (LC50 of 1.6, 3.7 µg/ml ) at 48h post-treatment. Moreover, bio-AgNPs synthesized from P. luminescens laumondii HP88, P. luminescens akhurstii HRM1 or P. luminescens akhurstii HS1 toxins significantly increased their larvicidal activities (LC50 of 2.1, 1.5, 13.9 µg/ml, respectively) at 48h post treatment. In conclusion, the highest larval toxicity was observed when larvae were treated with bio-AgNPs synthesized from P. luminescens akhurstii HRM1 and X. indica, followed by P. luminescens laumondii HP88 and Xenorhabdus sp. Subsequently, data of the present study suggest these bio-AgNPs toxin complexes as potentially effective bio-control candidates in the battle against mosquito. However, testing other types of bio-synthesized nanomaterials, and their synergistic combinations against different mosquito species still under investigation.

Effects of the entomopathogenic nematode, Heterorhabditis bacteriophora HP 88 and azadirachtin on the immune defence response and prophenoloxidase of Parasarcophaga surcoufi larvae (Diptera: Sarcophagidae).

Each third larval instar (155-175 mg) of the fleshfly, Parasarcophaga surcoufi was injected with 20 and 40 infective juveniles (IJs) of entomopathogenic nematode, Heterorhabditis bacteriophora, and with 0.5 and 1 microg of azadirachtin. Injection with 20 IJs decreased the total haemocyte count (THC) up to 40 h post-injection, except for its increase at 20 h. On the contrary, injection with 40 IJs increased the THC during the hours post-injection, except for its decrease at 40 h. The injection of nematode (40 IJs) decreased, the percentage of differential haemocyte count of P. surcoufi larvae, specially the granulocytes and plasmatocytes at 40 h. Whereas, prohaemocytes and oenocytoids were increased at 40 h post-infection. The granulocytes and plasmatocytes were engaged with encapsulation of the nematode, H. bacteriophora. The released symbiotic bacterium, Xenorhabdus luminescens was poorly phagocytosed by the granulocytes. After injection of the larvae with azadirachtin, THC and the percentage of the number of oenocytoids were particularly increased after 40 h of injection with azadirachtin; plasmatocytes and granulocytes were decreased at 10 h post-injection with 0.5 microg and at 40 h post-injection with 1 microg of azadirachtin. The most prominent haemocyte deformities in P. surcoufi larvae treated with azadirachtin were the release of cytoplasmic components from granulocytes together with bulging or lysis of plasmatocytes. Maximal haemolymph phenoloxidase (PO) activity was obtained at pH 6.2 and 6.6. This activity increased with increasing the pre-incubation time using laminarin, alpha-chymotrypsin and trypsin as activators. Injection of both nematode and azadirachtin significantly suppressed the haemolymph PO activity even in the presence of the activators laminarin, alpha-chymotrypsin and methanol. This suppression was dose-dependent and reached maximum at 30 h post-injection. The electrophoretic haemolymph protein profile was time-dependent as indicated by appearance and disappearance of protein bands. After 40 h post-injection with the nematode all the protein bands were replaced by new ones (probably containing immune protein). However, at this time injection with azadirachtin (1 microg) lead to appearance of 10 new protein bands & disappearance of an equal number of the present band.