Determination of Median Lethal Concentration (LC50) for Endosulfan, Heptachlor and Dieldrin Pesticides to African Catfish, Clarias gariepinus and Their Impact on Its Behavioral Patterns and Histopathological Responses

Pesticides such as endosulfan, heptachlor and dieldrin persist in aquatic environments as a result of their resistance to biodegradation. However, there is no adequate information about the toxicity of endosulfan, heptachlor and dieldrin to the aquatic organism, African catfish (Clarias gariepinus)—a high valued widely distributed commercially interesting species. The current experiment was performed with the aim to determine the median lethal concentration (LC50) of endosulfan, heptachlor and dieldrin to African catfish (Clarias gariepinus); their behavioral abnormalities and histopathological alterations in several vital organs. A total of 324 juvenile fish were exposed for 96 h to six concentrations of endosulfan and dieldrin at 0, 0.001, 0.002, 0.004, 0.008 and 0.016 ppm, and to heptachlor at concentrations of 0, 0.02, 0.04, 0.08, 0.16 and 0.32 ppm for dose-response tests. The study demonstrated that the species is highly susceptible to those contaminants showing a number of behavioral abnormalities and histopathological changes in gill, liver and muscle. The 96-h LC50 value of endosulfan, dieldrin and heptachlor for the African catfish was found as 0.004 (0.001−0.01) mg/L, 0.006 mg/L and 0.056 (0.006−0.144) mg/L, respectively. Abnormal behaviors such as erratic jerky swimming, frequent surfacing movement with gulping of air, secretion of mucus on the body and gills were observed in response to the increasing exposure concentrations. Histopathological alterations of liver, gill and muscle tissues were demonstrated as vacuolization in hepatocytes, congestion of red blood cells (RBCs) in hepatic portal vein; deformed secondary lamellae and disintegrated myotomes with disintegrated epidermis, respectively. These findings are important to monitor and responsibly manage pesticide use in and around C. gariepinus aquacultural areas.

Evaluation of Metarhizium rileyi Farlow (Samson) impregnated with azadirachtin and indoxacarb against Helicoverpa armigera (Hubner)

Background Entomopathogenic fungi are the most versatile having a wide host range, capable of infecting insects at different developmental stages. In the present study, Metarhizium rileyi, at the concentrations of 102, 103, 104, 105, 106, 107 and 108 conidia/ml and sub-lethal concentrations of azadirachtin (1.02 and 1.53 ppm) and indoxacarb (0.72 ppm) were evaluated against the 1st, 2nd, 3rd, 4th and 5th larval instars of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) under laboratory conditions. Results M. rileyi applied at 106 conidia/ml caused a maximum mortality of 83.33 and 80.00% of 1st and 2nd larval instars of H. armigera, respectively. The maximum mortality of 3rd, 4th and 5th larval instars of H. armigera with 108 conidia/ml of M. rileyi was 83.33, 76.67 and 53.33%, respectively. When M. rileyi blended with azadirachtin at 1.02 ppm, the highest mortality rate of 86.21% at 106 conidia/ml against 2nd instar larvae was resulted. Similarly, M. rileyi applied at 108 conidia /ml mixed with azadirachtin (1.53 ppm) showed 89.66% mortality of 3rd instar larvae. The 2nd instar larvae treated with M. rileyi at 106 conidia/ml, mixed with indoxacarb (0.72 ppm), the corrected mortality rate was 82.14%. Concentration mortality response of 3rd instar larvae to M. rileyi blended with indoxacarb (0.72 ppm) was 85.71% at 108 conidia/ml. The median lethal concentration (LC50) values were 5.51 × 103, 1.86 × 104, 2.81 × 105 and 5.55 × 105 conidia/ml for 1st, 2nd, 3rd and 4th larval instars, respectively, after 7 days of treatment. M. rileyi when mixed with sub-lethal concentrations of azadirachtin (1.02 ppm) and indoxacarb (0.72 ppm) resulted LC50 values of 1.09 × 104 conidia/ml and 1.37 × 104 conidia/ml against 2nd instar larvae, respectively, after 24 hours. Similarly, M. rileyi mixed with sub-lethal concentrations of azadirachtin (1.53 ppm) and indoxacarb (0.72 ppm) resulted LC50 values of 3.12 × 108 and 3.06 × 105 conidia/ml against 3rd instar larvae, respectively, after 24 hours. The study revealed that the susceptibility of larvae decreased in case of large larval instars. Conclusions M. rileyi can be utilized as one of the component of Integrated Pest Management Program for the eco-friendly management of H. armigera. As the application of M. rileyi @ 107 conidia/ml alone or in combination with azadirachtin (1.02 and 1.53 ppm) or indoxacarb (0.72 ppm) resulted to the highest mortality.

Lethal and sub-lethal toxicity of Verticillium lecanii on the biology of Mealybug (Pseudococcidae; Homoptera) under laboratory conditions

The Mealybug, Planococcus citri (Risso) (Hemiptera: Pseudococcidae) is a polyphagous pest of Citrus, grapevine, coffee and ornamental plants. The entomopathogenic fungus Beauveria bassiana, Metarhizium anisopliae, Verticillium lecanii and Isaria fumosorosea are very effective and commonly used as a biopesticide against various insect pests of different crops. The present study was conducted to check the lethal and sub-lethal effects of V. lecanii on mortality and life period of adult mealybug. Firstly LC50 value for nymph and adult was calculated by applying four different concentrations 1×105 , 1×106 , 1×107 and 1×108 CFU/ml. For nymphs, the median lethal concentration was 5.57×106 CFU/ ml, and for adults, 2.66×107 CFU/ml. After this, two lethal, 3.99×107 and 2.66×107 CFU/ml, and two sub-lethal concentrations, 1.33×107 and 6.65×106 CFU/ml, was prepared and tested against adult mealybug. Dose-dependent mortality was observed, and the highest mortality was due to the highest concentration of V. lecanii. The percentage mortality was 72.23, 52.78, 27.78 and 19.45% due to 3.99×107 , 2.66×107 , 1.33×107 and 6.65×106 CFU/ml, respectively, after 10 days of application. The weight of adult females was highest in the control treatment while lowest in high concentration, and it was gradually decreased after every day in the first three concentrations. The fecundity of mealybug also had an inverse relation with concentrations of V. lecanii, and the numbers of eggs were more in the control treatment than the other four treatments. The fecundity in lethal concentrations was reduced after the 8 th day of application. The life duration was most extended due to control treatment, 27 days, while the lowest period was due to the highest concentration, 13 days. Keywords: Verticillium lecanii, mealybug, mortality, weight, fecundity, life duration

Acute Cd Toxicity, Metal Accumulation, and Ion Loss in Southern Catfish (Silurus meridionalis Chen)

The amounts of cadmium in multiple organs and the amounts of Na+ and Ca2+ in the carcass were measured in dead and surviving southern catfish exposed to different concentrations of Cd. The 96 h median lethal concentration was 6.85 mg/L. The Cd content and Cd accumulation rate were positively correlated with Cd exposure concentrations, and there were significant differences between dead and surviving individuals, indicating that both Cd content in tissues and Cd accumulation rates were correlated with mortality. Cd levels in the liver of dead fish were saturated. A lethal threshold for Cd concentration in the whole fish was obtained. Bioconcentration factors for Cd did not decrease with increasing exposure. Acute exposure to waterborne Cd caused a significant decrease in the ion content of the fish carcass. There was a significant difference between the Na+ content of the carcass of dead fish (34.54 μmol/g wet weight) and surviving fish (59.34 μmol/g wet weight), which was not the case with the Ca2+ content, indicating that the lethal toxicity of Cd was probably related to the decrease in Na+ content. Collectively, these results suggest that whole-fish Cd concentration and carcass Na+ content can be useful indicators of fish acutely exposed to Cd.

New Hybrid Strains via Intraspecific Protoplast Fusion of the Entomopathogenic Fungi Lecanicillium spp.

The entomopathogenic fungal genus Lecanicillium Gams and Zare (formerly classified as the species Verticillium lecanii) includes species that are highly pathogenic to many insect genera. In this study, we identified six Lecanicilliumspp. isolated strains (designated as V1-V6) belonging to L. lecanii (V1, V3 and V5) and L. attenuatum (V2, V4 and V6). In addition, these strains were used to obtain new strains via protoplast fusion, and nit mutants were used for protoplast selection. Genetic recombination of the hybrid strains was determined using the random amplified polymorphic DNA(RAPD) technique. We obtained nine stable fusant strains from 176 new hybrid strains, which were termed V12-10, V14-3, V16-4, V23-6, V25-8, V34-14, V36-5, V45-16 and V56-7. Morphological characteristics varied between the hybrid and parental strains. Genomic DNA analysis of the fusants also showed genetic recombination. The median lethal concentration (LC50) for the fusants were lower than that for parental strains, and the median survival time (LT50) for the fusants were reduced compared with that for parental strains. Thus, these results showed that we produced new, more virulent hybrid Lecanicillium spp. strainsas biological control agents via intraspecific protoplast fusion.

Dynamics in the content of the cytokines in the bronchoalveolar lavage fluid in rats after acute inhalation intoxication by clorine and pyrolysis products, containing hydrogen chloride

It is known that inhalation exposure to chlorine and hydrogen chloride leads to damage to the respiratory system up to the development of acute pulmonary edema in victims. No data on the mechanisms of development of pulmonary edema upon exposure to hydrogen chloride have been found in the available literature. The study was carried out on white outbred male rats, which were divided into 3 groups: Group I control; Group II animals were intoxicated with chlorine at a dose of 1.5 median lethal concentration (30 min); Group III animals were intoxicated with hydrogen chloride at a dose of 1.5 median lethal concentration (30 min). Immediately after exposure to the studied toxicants, as well as after 1, 3 and 6 h, the lung coefficient and the content of cytokines (interleukins-1, 6, 10 and interferon-) in the bronchoalveolar lavage fluid were determined in animals. It was revealed that an increase in the lung coefficient (p 0.05) in animals in groups II and III was accompanied by a significant increase (1.5 times) in the content of the studied cytokines in the bronchial-alveolar lavage fluid compared with animals in group I. III an increase (p 0.05) in the content of cytokines is recorded later only 3 hours after exposure, while it is significantly lower than in animals of group II at all studied periods. Thus, intoxication with hydrogen chloride leads to a slower development of pulmonary edema and an increase in the content of both pro (interleukins-1, 6) and anti-inflammatory cytokines (interleukin-10, interferon-) in the bronchial-alveolar lavage fluid compared to animals, exposed to chlorine intoxication.

Viability and reconstitution of delta-endotoxins from Bacillus thuringiensis var. israelensis extracts after forty years of storage against Aedes aegypti (Diptera: Culicidae)

Background Bacillus thuringiensis subsp. israelensis (Bti) produces insecticidal endotoxins known as Cry and Cyt. Its efficiency and specificity make it the most widely used substance as a biopesticide for controlling disease from vector insects, such as mosquitoes, responsible for important human diseases such as malaria, filariasis, dengue, and yellow fevers. To date, it is proven difficult to develop a commercial product that has more than 2 years of shelf life, and there is little information on the viability of these commercial proteins under prolonged storage conditions. Results This study aimed to evaluate biological activity of reconstituted Bti endotoxins after 40 years of storage against the mosquito Aedes aegypti larvae. Five concentrations of Bti extracts were used for bioassays against 3rd and 4th instars of A. aegypti larvae. All reconstituted endotoxins from stored extracts showed a potency increase. The strain HD-500 from extract 3260 was the most effective insecticide (LC50 = 0.0014 mg/l), followed by 3756 (LC50 = 0.0037 mg/l). These strains were particularly notable, increasing their larvicidal potency one hundredfold and one thousandfold, respectively. Protein profiles in polyacrylamide gels revealed a greater presence of Cyt toxins compared to the stored Bti extracts, which maintained their activity at high concentrations. Conclusion The reconstituted Bti strains presented a great biological activity against A. aegypti larvae, specially extract 3260 (median lethal concentration (LC50) value = 0.0014 mg/l). This considerable larvicidal activity after 40 years under storage was an encouraging signal for the development of future formulation strategies regarding their useful life. The stability of extracts of stored endotoxins produced by Bti decreased significantly, particularly Cyt1A protein, which is responsible for their synergistic activity.

Mortality and immune challenge of a native isolate of Beauveria bassina against the larvae of Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)

Background Entomopathogenic fungi (EPF) attack a wide range of insects. They are considered environmental friendly alternatives to synthetic insecticides for pest control. In the present study, virulence of a native isolate of the EPF, Beauveria bassiana Vuillemin (Hypocreales: Cordycipitaceae) was evaluated against the least mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Crambidae), through bioassay, pathogenic pathways, and immune responses. Results The values of 2.6 × 104 conidia/ml and 3.54 days were determined as the median lethal concentration (LC50) and median lethal concentration (LT50) of AM-118 against the 4th instar larvae of G. pyloalis, respectively. The activities of proteases and chitinases in the culture medium containing the larval cuticle were higher than the control medium. Moreover, the total and the differential hemocyte counts of the larvae were significantly changed after injection with AM-118 spores. The highest numbers of total hemocytes and granulocytes were obtained 3 and 6 h post-injection, while the highest numbers of plasmatocytes and nodules were observed 6 h post-injection. The highest activity of phenoloxidase was determined 12 h post-injection by AM-118 spores. Conclusions The findings imply on virulence of the AM-118 isolate against the larvae of G. pyoalis although immune responses were triggered by the spores.

Morphological and molecular identification of four Purpureocillium isolates and evaluating their efficacy against the sweet potato whitefly, Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae)

Background Entomopathogenic fungi are widely distributed and well described within the fungal kingdom. This study reports the isolation, characterization, and virulence of 4 Purpureocillium lilacinum isolates against the sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Results Four strains of Purpureocillium lilacinum (XI-1, XI-4, XI-5, and J27) were isolated from soil samples from different localities of China. The morphological studies observed that four strains showed essentially the same morphological characteristics. After 7 days of cultivation, the colonies were purple, round, and bulged. Conidia were single-celled, oval to spindle-shaped, chain-like, and the spore size was about 2.0–2.3 × 3.1–4.0 μm. The genome-based identification results showed that ITS sequences of XI-1 (GenBank accession # MW386433), XI-4 (GenBank accession # MW386434), XI-5 (GenBank accession # MW386435), and J27 (GenBank accession # MW386436) were similar to another P. lilacinum. The newly identified strains of P. lilacinum proved pathogenicity to B. tabaci under laboratory conditions. In addition, the P. lilacinum isolate XI-5 was the most virulent one against different nymphal instars of whitefly having median lethal concentration (LC50) values of 4.99 × 106, 4.82 × 105, and 2.85 × 106 conidia/ml, respectively, 7 days post application. Conclusion The newly isolated strains of P. lilacinum can be developed as a potential biopesticide against the whitefly although extensive field bioassays as well as development of proper formulation are still required.

Characterization of alphabaculovirus: HearNPV-IIPR05 isolate infecting Helicoverpa armigera (Hubner) larvae

Background The alphabaculoviruses are lethal pathogens of lepidopteran caterpillars including a polyphagous and globally recognized pest, Helicoverpa armigera (Hubner) infesting economically important agriculture crops worldwide. The biological and molecular characterizations of indigenous nucleopolyhedrovirus of the genus Alphabaculovirus isolated from H. armigera in chickpea fields are described. Results The virulence of virus isolate was tested in 3rd instar H. armigera larvae, and LC50 (median lethal concentration) was estimated to be 2.69 × 104 OBs ml−1. The ST50 (median survival time) was 4 days post-inoculation, when the 3rd instar H. armigera larvae were inoculated by OB (occlusion body) concentration equivalent to LC90. An average incubation period of the virus isolate in 3rd instar ranged between 4 and 6 days post-inoculation. The OBs of a virus isolate appeared irregular in shape and variable in size with diameter ranging from 0.57 to 1.46 μm on the longest edge and average of 1.071 ± 0.068 μm (mean ± SE). On the basis of phylogenetic analysis of polh, pif-1, and lef-8 genes, the isolate was found to be a member of the genus Alphabaculovirus. The isolate showed a genetic affinity with species of group II Alphabaculoviruses and appeared to be a group II NPV. Conclusions On the basis of molecular phylogeny and associated host insect, this indigenous isolate was designated as HearNPV-IIPR05 isolate, which could be a potential candidate for the biological control of H. armigera infesting legumes and other commercial crops.