Toxicological and ultrastructural effects of chitin synthesis inhibitors (lufenuron and chlorfluazuron) on third larval instars integument of Chrysomya albiceps (Diptera: Calliphoridae)

The impact of two chitin synthesis inhibitors, chlorfluazuron and triflumuron against the black cutworm, Agrotis ipsilon (Hufn.) was studied. Feeding technique was adopted. Different effects of these two chitin synthesis inhibitors on the 4 th instar larvae were investigated. Effect on biological aspects, larval duration, larval weight, percentage of pupation, pupal duration, pupal weight, percentage of adult emergence, adult longivity, total oviposition period, number of eggs per female, egg hatchability and sterility percentage were studied. Effect of both compounds on total proteins and glycogen levels was also investigated.

Management of khapra beetle, Trogoderma granarium Everts, using binary combinations of chitin synthesis inhibitors and inert dusts

The salmon louse (Lepeophtheirus salmonis) is an ectoparasite infecting Atlantic salmon (Salmo salar), which causes substantial problems to the salmon aquaculture and threatens wild salmon. Chitin synthesis inhibitors (CSIs) are used to control L. salmonis in aquaculture. CSIs act by interfering with chitin formation and molting. In the present study, we investigated the action of four CSIs: diflubenzuron (DFB), hexaflumuron (HX), lufenuron (LF), and teflubenzuron (TFB) on larval molt. As the mode of action of CSIs remains unknown, we selected key enzymes in chitin metabolism and investigated if CSI treatment influenced the transcriptional level of these genes. All four CSIs interfered with the nauplius II molt to copepodids in a dose-dependent manner. The EC50 values were 93.2 nM for diflubenzuron, 1.2 nM for hexaflumuron, 22.4 nM for lufenuron, and 11.7 nM for teflubenzuron. Of the investigated genes, only the transcriptional level of L. salmonis chitin synthase 1 decreased significantly in hexaflumuron and diflubenzuron-treated larvae. All the tested CSIs affected the molt of nauplius II L. salmonis larvae but at different concentrations. The larvae were most sensitive to hexaflumuron and less sensitive to diflubenzuron. None of the CSIs applied had a strong impact on the transcriptional level of chitin synthesis or chitinases genes in L. salmonis. Further research is necessary to get more knowledge of the nature of the inhibition of CSI and may require methods such as studies of protein structure and enzymological studies.

BackgroundThe larvicidal and biochemical effects of chitin synthesis inhibitors (CSIs), namely lufenuron, flufenoxuron and hexaflumuron against the newly molted penultimate instar larvae of the house fly Musca domestica, were investigated.MethodsDifferent concentrations from each tested compound were applied on forty individuals of M. domestica 2nd instar larvae. Four replicates were used for each concentration.ResultsThe recorded LC25 and LC75 values were (166.11, 68.33 and 56.43 ppm) and (732.33, 283.02 and 248.45 ppm) for lufenuron, flufenoxuron and hexaflumuron, respectively. The results showed significant (P < 0.05) increase of mortality in larvae treated with different tested CSIs compounds. Mortality was greater in larvae treated by hexaflumuron than lufenuron and flufenoxuron. The main metabolites were tested in the larval whole-body tissue homogenate and findings could be summarized as follows: tested concentrations of CSIs (a) predominantly reduced the total carbohydrate, protein, lipid and cholesterol content at certain ages tested. (b) Disturbed the total carbohydrate content particularly for larvae treated with LC75 concentration of hexaflumuron. (c) Exerted the protein and lipid profiles and this effect was much more pronounced in larvae treated with hexaflumuron. (d) Reduced the quantitative cholesterol content and this reduction was found to be increased with development.ConclusionsTested CSIs in particular hexaflumuron showed remarkable larval toxicity and reduced the main metabolites content in the larval whole-body tissue homogenate of the house fly, M. domestica.

Laboratory studies were carried out to investigate the efficacy of the chitin synthesis inhibitor (cyromazine) against all larval stages of Culex pipiens. The compound was tested its efficacy on larval mortality, larval duration, pupation, pupal duration and adult emergence. Also, the histological effects of cyromazine on the midgut, the integument, the fatbodies and the muscles of 4th larval instar treated as 1st larval instar were studied. The tested compound increased the larval mortality, decreased the percent pupation and adult emergence and prolonged the pupal duration. Also, this compound elicited histological effects on the midgut, the integument, the fat bodies and the muscles of 4th larval instar of C. pipiens treated as 1st larval instar with 0.01 and 0.1 ppm.

The objective of the current study was to determine the fumigant toxicity of Thuja essential oil against 3rd and 5th instar larvae of the greater wax moth, Galleria mellonella. Thuja essential oil was extracted from unripe fruits of Thuja orientalis plant by water distillation. The mortality of larvae at the end of larval period, pupation and adult emergence were tested at different concentrations of Thuja oil after a 24h exposure period ranging from 5 to 25µL for 3rd instar larvae and 80µL for 5th instar. The combined effect of Thuja essential oil and larvae resulting from parents irradiated as pupae of G.mellonella with 80Gy was investigated. The results demonstrated that the mortality increased by increasing oil concentration. The percent mortality of 3rd instar larvae reached 100% when exposed to a higher concentration of Thuja oil (25µL) after 24h, while in the case of 5th instar larvae the mortality was 73.3% at (80µL). Larvae of both instars resulting from parents of G. mellonella irradiated as pupae with 80Gy and exposed to the same concentrations of Thuja oil vapor became more susceptible to the oil as a result of the combined effect of the two agents. The LC50 and LC90 values for each experiment were calculated. The tested plant oil prolonged the larval - pupal periods and reduced both pupation percentage of adult emergence.

Artemisia herba alba and Allium sativum grow in Sinai peninsula and, Northern area of Egyptian Delta respectively. Both extracts plants were evaluated as anti–insect products in an effort to develop alternative insecticides considering the environment. The effect of Artemisia and Allium extracts on some Biological aspects as well as some digestive enzymes changes after treatment with those extracts have been studied. The results indicated that treatment the 3rd instar larvae of Spodoptera littoralis for 24hours revealed satisfactory effect in larval mortality (50%) and significant prolongation for both larval and pupal durations. This prolongation was accompanied with a decrease in the percentage of pupation and adult emergence. Digestive enzymatic activities were pronounced on estimation of Trehalase, Invertase and Amylase manifested obvious changes in the treated larvae of Spodoptera littoralis.

Purpose Combining gamma irradiation and nanotechnology has become one of the most promising new approaches for area-wide (AW) pest management in recent years. The laboratory trials were conducted to determine the combining effects of BT-AgNPs and gamma irradiation for controlling P. gossypiella. Radio-sensitivity of male pupae at different doses of gamma radiation and the effectiveness of biosynthesized silver nanoparticles using Bacillus thuringiensis on larval instar were assayed. Additionally, the ultrastructure changes on the alimentary canal of 4th instar larvae were studied to evaluate the impact of the combined approach at a cellular level. Materials and methods Laboratory- rearing technique was used for rearing Pectinophora gossypiella. The irradiation process was achieved at Co60 - Gamma Chamber (4000 A). Alanine dosimeters were used for measuring the average absorbed dose and dose mapping. Preparation of Silver nanoparticles (AgNPs) using Bacillus thuringiensis (Bt) and their characterization has been investigated. The treated 4th instar larvae by gamma irradiation or ∕and BT-AgNPs were dissected under the stereo microscope. The alimentary canal was obtained anatomically and Transmission Electron Microscope) was used in examining the stained sections. Results Based on the nonhatching eggs produced by irradiated males’ pupae, the values of effective doses were calculated. The effective doses ranged from 16 to 291 Gy for the ED25 - ED75. The sterility index reached 74.1% when irradiated with males by 291 Gy crossed with nonirradiated females and the adult emergence decreased to be 35.3%. The insecticidal potential of Bt-AgNPs on the 2nd and 4th larval instars was dose-dependent and its LC50 toxicity value was 0. 3 and 0. 4 mg/ml, respectively. The lethal concentration LC50 of the 2nd instar larvae increased the larval and pupal mortality to 55% and 44.4%, respectively, and reduced the adult emergence to be 55.6%. The combining effects of Bt-AgNPs with 291 Gy induced 100% pupae mortality and there was no adult emergence in F1 generation. Such effects also severed the ultrastructure deformity of the midgut of the 4th instar larvae after the two-day post-treatment. Conclusions The combining effects are recommended as an effective IPM program to control P. gossypiella by releasing sterile males (derived from pupae irradiated with 291 Gy) crossing with the normal females in the field, and reducing the fertility of the population to 31.2%. Subsequently, the resulted larvae treated with LC50 of Bt-AgNPs prevented the adult emergence and stopped the life cycle of P. gossypiella.

The study evaluated the efficacy of methanolic extracts of Ruta chalepensis (Rutaceae), Withania somnifera (Solanaceae), Cleome paradoxa (Capparaceae) and Heliotropium longiflorum (Boraginaceae) aerial parts against Culex pipiens larvae. Different concentrations (100-500 ppm) of the methanolic extracts of the plants were tested towards larval mortality and development of C. pipiens separately. Larval mortalities were counted at 2, 4 & 10 days after treatment. Egg hatchability was determined at 4 & 7 days after treatment. Successful pupation and adult emergence percentage were recorded daily till all control adults emerged. All plants extracts exhibited variable activities. The greatest effect was with R. chalepensis which showed acute (2 days) and chronic (10 days) LC50 of 132.6 & 96.56 ppm, respectively. Larval mortality up to 84.47% &85.53%, were observed with C. paradoxa and R. Chalepensis respectively. Egg hatch was significantly reduced about equal with R. chalepensis and W. somnifera extracts. Concentration levels of C. paradoxa (≥200 ppm) and H. longiflorum (>400 ppm) showed significant hindrance to the larval development and reduction to resulting pupae and adults. Drastic development retardation was shown with extract of C. paradoxa leaves (300 ppm), but only 15.3% & 5.6% of larvae reach pupal and adult stages respectively. The larvicidal activity of methanolic extracts of R. chalepensis, W somnifera, C. paradoxa, H. longiflorum proved to be effective against C. pipiens larvae without any human or animal risk.

BackgroundThe cotton leafworm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), is an economic pest on various crops worldwide. Farmers generally used to apply chemical pesticides to control the pest. The bio-control potential of the entomopathogenic nematodes (EPNs) as alternatives to harmful synthetic pesticides was examined in this study. The study aimed to determine the efficacy of EPNs isolates against the different stages of larvae of the cotton leafworm S. littoralis and the effect of time on mortality rate under laboratory conditions.ResultsEPNs isolates were tested at 4 different concentrations (0, 250, 500 and 1000 IJs/ml) in 150 ml plastic containers on last instar larvae (sixth instar) of S. litorallis. Experiments for third, fourth and fifth instar larvae of S. litorallis were carried out in petri dishes at different concentrations (0, 50, 100 and 200 IJs/ml) at 25 °C temperature. Mortality rates of larvae were calculated at 3 different times (48, 72, 96 h) after inoculation. The high mortality rate of last instar larvae (98.81%) of S. littoralis was caused by Steinernema feltiae (Tokat-Emir), followed by S. carpocapsae (Tokat-Bakışlı05) (95.24%) and then H. bacteriophora (11 KG) with (90.47%) at the highest concentration (1000 IJ/ml). The highest mortality rate of fifth instar larvae was caused by S. feltiae (Tokat-Bakışlı05) and S. carpocapsae (Tokat-Emir) with (100%) and (92.12%). In addition, the highest mortality rate of the 4th instar larvae was determined S. feltiae (Tokat-Bakışlı05) and S. carpocapsae (Tokat-Emir) isolates (98.87%) and (97.74%), respectively. Additionally, the highest larval mortality rate in the third stage by S. feltiae (Tokat-Bakışlı05) and S. carpocapsae (Tokat-Emir) was (100%) and (97.74%) at the highest concentration. Mortality rates of larvae were calculated at 3 different times after inoculation. The highest mortality rate counted in all isolates was determined 96 and 72 h after inoculation of EPNs.ConclusionsAll indigenous EPN isolates were found to be effective at different rates against S. littoralis. The results showed that these nematode species could be used against S. littoralis biological control programs.

Toxicities of three chitin synthesis inhibitors (diflubenzuron, nikkomycin Z and polyoxin D) were evaluated using second instars of the common malaria mosquito, Anopheles quadrimaculatus Say (Diptera: Culicidae). Neither nikkomycin Z nor polyoxin D at 50 microg/liter caused significant larval mortality, although they reduced the body weight of the survivors by 20.5 and 33.8%, respectively, in 48 h. In contrast, exposures of the larvae to diflubenzuron at 12.5 microg/liter for 48 h resulted in 86.7% larval mortality and reduced the body weight of the survivors by 29.1%. Exposure of the pupae (<12 h old) to diflubenzuron at 100 microg/liter for 48 h caused 18.9% pupal mortality and consequently reduced the adult emergence by 24.7% from the surviving pupae. Furthermore, exposure of third instars to diflubenzuron at 4, 20, 100, and 500 microg/liter for 24 h resulted in the reduction of larval chitin contents by 4.25, 33.2, 35.2, and 57.7%, respectively. Such an effect seemed to be associated with only cuticular chitin synthesis because the same exposures did not significantly affect chitin contents in the guts. Our results indicated that diflubenzuron was highly toxic to second instars by not only causing high larval mortality but also by affecting their growth. Diflubenzuron was also fairly toxic to pupae by not only causing pupal mortality but also affecting the adult emergence. Our results suggest that diflubenzuron might affect only chitin synthesis in the cuticle but not in the peritrophic matrix, which is probably due to diflubenzuron's direct contact to mosquito larvae in water, slow distribution in insect body, rapid degradation in the insect gut, or a combination.

BackgroundIn holometabolous insects, environmental factors experienced in pre-imaginal life stages affect the life-history traits within that stage and can also influence subsequent life stages. Here, I assessed tolerance to water immersion by the larval instars of the stable fly, Stomoxys calcitrans L. (Diptera: Muscidae) and its impact on the life-history traits of their subsequent life stages.ResultsAfter submerging the three larval instars of S. calcitrans in distilled water, I found that the first instar larvae remained active for longer as compared to the second and third instar larvae. Also, the first instar larvae took a longer period to recover from the stress-induced immobility when removed from the water and returned to ambient temperature. When I followed the development of individuals of each larval instar that survived from water immersion, I found that their developmental time, weight, pupation percentage, adult emergence percentage and adult weight were negatively affected by this stressor. However, the weight of S. calcitrans adults developed from immersed first larval instar individuals was not affected by water immersion whereas their counterparts developed from immersed second and third larval instars had lower body weight. This suggests that in S. calcitrans, water immersion stress at the earlier stage is less detrimental than that experienced at late stages.ConclusionThis study provides a comparative overview of the fitness consequences associated with water immersion stress during S. calcitrans larval ontogeny. The results prove that the fitness shift induced by water immersion in S. calcitrans is stage-specific. My results illustrate the importance of considering each larval instar when assessing the impact of environmental factors on holometabolous insect performance as these may be decoupled by metamorphosis.

In this laboratory study, insecticidal and inhibitory potential of two chitin synthesis inhibitors (CSIs) viz. Buprofezin and Lufenuron were evaluated against okra shoot and fruit borer (OSFB). CSIs were applied on three days old larvae through different bioassay methods like topical or direct, okra-dip or indirect and combined (topical + leaf-dip). Data were collected on larval mortality, weight reduction and deformations of larvae and adults. Results showed that larval mortality and weight reduction were clearly dose, application methods and time dependent. In case of both CSIs, the highest mortality was found @ 1.0 ml/L that was followed by 0.75 ml and 0.50 ml/L respectively. Likewise, the highest body weight reduction was also found from 1.0 ml/L. In both cases, the concentration 0.5 ml/L was found less effective. In case of application methods, combined bioassay method was found superior than that of rest of the two methods. Moreover, a significant percentage of deformed larvae and adults were developed when 3 days old larvae were treated with either Buprofezin or Lufenuron @ 1.0 ml/L through combined bioassay method. The concentration 0.5 ml/L had very less efficacy on the development of deformed larvae or adults. Therefore, based on the laboratory findings Buprofezin and Lufenuron may be the potential component in IPM programmed, rather than their individual application, for controlling okra shoot and fruit borer as well as production of safe okra.

Crocidolomia pavonana, commonly known as the cabbage cluster caterpillar, is a significant pest of cabbage plants, causing substantial damage and economic losses. One potential method to control C. pavonana is using the entomopathogenic fungus Trichoderma asperellum. This study aimed to identify the most effective isolate of T. asperellum in managing C. pavonana populations. Four isolates of T. asperellum were evaluated: T. asperellum SD324, T. asperellum SD327, T. asperellum A116, and T. asperellum AB2B3, with a conidial density of 108 conidia/ml. The conidial suspensions were applied to second instar larvae of C. pavonana. Observations included larval mortality, percentage of pupation, percentage of adult emergence, the pre-oviposition, oviposition, and post-oviposition periods of female adults, and number of egg batches. Data were analyzed using analysis of variance (ANOVA) followed by a 5% LSD test. Results indicated that all T. asperellum isolates demonstrated potential in controlling C. pavonana larvae, with the highest mortality rate (93.33%) observed in T. asperellum A116. Additionally, the application of T. asperellum isolates inhibited pupation rate (6.67%), adult emergence (10%), and inhibition of C. pavonana life cycle development.

chitinase as endo-type of chitinolytic enzymes was studied in the 4th larval instar of the black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae) to detect some of its physiological aspects. Results showed that, the chitinase activity was low till 18 h before ecdysis to the 4th instar, but the activity started increasing at 10 h and reached a peak at 2 h before the ecdysis. This confirms that the appearance of chitinase activity is restricted to the molting period and the main function of chitinase in larvae is the digestion of old cuticle. On the other hand, the enzyme activity in pupal stage was 1.73-fold as compared to that present in larvae, might to share in the intensive physiological processes in this stage. Results of enzyme localization revealed that larval integument had the highest chitinase activity (72.3%), while hemolymph had the lowest one (1.9%). Hence, we infer that hemolymph plays a minor role in transporting the enzyme, and chitinase localized there in the integument. The results showed that hyperchitinase activity in larvae was not a sign of LC50 treatment by nomolt. This led to the assumption that nomolt acts by inhibition of chitin synthesis not by enhancing of chitin degradation. Future studies concerned with factors affecting chitinase must take into consideration the fact that larval chitinase activity is rhytmic, i.e. precaution that the sampling time is critical.