Growth inhibitory, immunosuppressive, cytotoxic, and genotoxic effects of γ-terpinene on Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae).

γ-Terpinene, a monoterpene widely present in essential oils of many medicinal and aromatic plants with numerous biological properties, was evaluated for its insecticidal activity against melon fruit fly, Zeugodacus cucurbitae (Coquillett). Different concentrations (5, 25, 125, 625, and 3125 ppm) of γ-terpinene along with control were fed to larvae of melon fly. The number of pupae formed and adults emerged declined significantly after treatment. Morphologically deformed adults and pupae were also observed. The developmental duration too prolonged in treated larvae. Food assimilated, mean relative growth rate, larval weight gain, and pupal weight also declined. In the larvae treated with LC30 and LC50 concentrations, there was a decline in the titers of phenoloxidase and total hemocyte count, and variations were observed in the differential hemocyte count, suggesting an immunosuppressive effect of γ-terpinene on melon fly. Both concentrations also led to an increase in the apoptotic and necrotic cells as well as decrease in the viable hemocytes in the circulating hemolymph of treated larvae. Comet parameters (tail length, % tail DNA, tail moment, and olive tail moment) of γ-terpinene fed larvae increased significantly. Given the observed effects of γ-terpinene on normal developmental and nutritional physiology, its immunosuppressive properties, and its potential for genome damage, it can be considered for incorporation into integrated pest management strategies for controlling Z. cucurbitae.

Insecticidal potential of endophytic Streptomyces sp. against Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) and biosafety evaluation.

Fruit flies of Tephritidae family pose a serious threat to cultivation of fruits and vegetables across the world. Among them, melon fruit fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) is a devastating pest of plants from Cucurbitaceae family. In a rising concern about the harmful effects associated with the use of chemical insecticides and development of resistance in pest insects, safer pest management strategies such as, use of biopesticides of microbial origin are being contemplated. Therefore, the present study aimed to evaluate the insecticidal potential of Streptomyces sp. SP5 protein extract against Z. cucurbitae. MTT assay, Ames mutagenicity, DNA nicking, and comet assay were conducted to determine the biosafety of protein extract. Second instar larvae of Z. cucurbitae were treated with various concentrations (1, 100, 200, 300, 400, and 500 µg/ml) of Streptomyces sp. SP5 protein extract. The protein extract showed significant larvicidal effects with LC50 value of 308.92 µg/ml. The percentage of adults emerged declined with increase in concentration. There was significant prolongation in developmental durations of the larvae. Various morphological aberrations in the form of deformed adults and pupae and decline in pupal weight were also observed. The nutritional physiology of the treated larvae was also adversely affected. The results from biosafety evaluation revealed antimutagenic and non-toxic nature of Streptomyces sp. proteins. This study indicates that Streptomyces sp. SP5 has the potential to be used as an ecologically safe biocontrol agent against Z. cucurbitae.

Immunomodulatory, cyto-genotoxic, and growth regulatory effects of nerolidol on melon fruit fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae).

Insects have evolved a robust immune system consisting of humoral and cellular branches and their orchestrated response enables insect to defend against exogenous stressors. Exploration of underlying immune mechanisms of insect pest under allelochemical stress can give us new insights on insect pest management. In this study, nerolidol, a plant sesquiterpene was evaluated for its insecticidal, growth regulatory, immunomodulatory, and cyto-genotoxic effects against melon fruit fly, Zeugodacus cucurbitae (Coquillett). First, second, and third instar larvae of Z. cucurbitae were fed on artificial diet containing different concentrations (5, 25, 125, 625, and 3125 ppm) of nerolidol. Results revealed a significant reduction in pupation and adult emergence as well as prolongation of developmental duration of treated larvae. Decline in growth indices showed remarkable growth inhibitory effects of nerolidol. Pupal weight and nutritional parameters viz. Larval weight gain, food assimilated, and mean relative growth rate declined after treatment. Immunological studies on second instar larvae depicted a drop in total hemocyte count and variations in proportions of plasmatocytes and granulocytes of LC30 and LC50 treated larvae. Phenoloxidase activity in nerolidol treated larvae initially increased but was suppressed after 72 h of treatment. The frequency of viable hemocytes decreased and that of apoptotic and necrotic hemocytes increased with both the lethal concentrations of nerolidol. Comet assay revealed a significant damage to DNA of hemocytes. The findings of the current study indicate that nerolidol exerts its insecticidal action through growth regulation, immunomodulation, and cyto-genotoxicity thus revealing its potential to be used as biopesticide against Z. cucurbitae.

Cosmopolitan honey bee, Apis mellifera, as quick and efficient marker of pesticide pollution in environment through RP-HPLC.

Pesticides are extremely hazardous to human health as well as various kinds of non-target organisms. The honey bee (Apis mellifera) is not only a dominant pollinator, but also a good indicator of pesticide residue and pollutants in the environment. At the time of sample collection in each village, the surrounding flora was Triticum aestivum and Brassica species. The area chosen for this study covered only 15% of the land in the state of Punjab, but pesticide consumption was approximately 75% of the state consumption. Pesticides in the collected samples (from six districts) were analyzed using RP-HPLC chromatography. The chemical methoxychlor (MC) was not found in any of the six villages' honey samples; however, spiromesifen (n = 5) and aldicarb (n = 5) were the most prevalent chemicals and were found in every honey sample. The pesticides carbendazim (n = 1) and parathion methyl (n = 1) were found only in Behman (longitude 29.9224° N and latitude 75.1137° E), and Malumazra (longitude 30.2468° N and latitude 75.8500° E). The carbofuran (n = 3) was discovered in Talwandi Sabo (longitude 29.984° N and latitude 75.8500° E), Himmatpura (longitude 30.5289° N and latitude 75.3616° E), and Malumazra, while atrazine (n = 4) was discovered in all except Malumazra. Three identical pesticides were observed in Chukrian (longitude 29.9759° N and latitude 75.4476° E) and Singo (longitude 29.9092° N and latitude 75.1589° E) indicating the same pesticides used in these villages. The given study gives a summary model to use A. mellifera as a quick monitoring bioindicator of the environment. This model helps to maintain a pesticide or pollutant database of selected areas for regular monitoring of the surrounding environment.

The genotoxic, cytotoxic and growth regulatory effects of plant secondary metabolite ß-caryophyllene on polyphagous pest Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae).

Use of secondary metabolites as an alternative to organic pesticides is an eco-friendly and safe strategy in pest management. ß-caryophyllene [(1R,4E,9S)-4,11,11-trimethyl-8-methylene bicyclo [7.2.0]undec-4-ene], a natural sesquiterpene is found as an essential oil in many plants like Syzygium aromaticum, Piper nigrum, Cannabis sativa. The present study aims at exploring the insecticidal, genotoxic and cytotoxic potential of ß-caryophyllene against common cutworm Spodoptera litura (Fab.), a major polyphagous pest. S. litura larvae were fed on different concentrations (5, 25, 125, 625 and 3125 ppm) of ß-caryophyllene. Results revealed delay in larval and pupal period with increase in concentration. Larval mortality increased and adult emergence declined significantly with increase in concentration. Higher concentrations of ß-caryophyllene caused pupal and adult deformities. A negative impact of ß-caryophyllene was also seen on the nutritional physiology of S. litura. Parameters such as relative growth rate, relative consumption rate, efficiency of conversion of ingested food, efficiency of conversion of digested food and approximate digestibility showed a significant reduction in a dose dependent manner. DNA damage assessed using comet assay revealed significant genotoxic effects at LC30 and LC50 concentrations. There was an increase in tail length, percent tail DNA, tail moment and olive tail moment. Phenol oxidase activity was suppressed at LC50 concentration with respect to control. Total hemocyte count also declined significantly at LC30 and LC50 concentrations as compared to control. ß-caryophyllene induced genotoxic and cytotoxic damage affecting the growth and survival of S. litura larvae. Our findings suggest that ß-caryophyllene has the potential to be used for the management of insect pests.

Appraisal of growth inhibitory, biochemical and genotoxic effects of Allyl Isothiocyanate on different developmental stages of Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae).

Allyl isothiocyanate (AITC), a glucosinolates' hydrolytic product, was studied for its anti-insect potential against an economically important, destructive tephritid pest, Zeugodacus cucurbitae (Coquillett). The first, second and third instar maggots of the pest were fed on artificial diets amended with varied concentrations of AITC viz. 5 ppm, 25 ppm, 50 ppm, 100 ppm, 150 ppm and 200 ppm with DMSO (0.5%) as control. Results revealed high larval mortality, alteration of larval period, prolongation of pupal and total developmental periods in all instars of the maggots treated with AITC as compared to controls. Percent pupation and percent adult emergence decreased in all larval instars. Growth indices viz. Larval Growth Index (LGI) and Total Growth Index (TGI) were negatively affected. Anti-nutritional/post ingestive toxicity of AITC was also revealed by the decrease in Food Assimilation (FA) and Mean Relative Growth rate (MRGR) values with respect to control. Profiles of PO (Phenol oxidase) and other detoxifying enzymes including SOD (Superoxide dismutases), CAT (Catalases), GST (Glutathione-S-transferases), EST (Esterases), AKP (Alkaline phosphatases) and ACP (Acid phosphatases) were also significantly influenced. The genotoxic effect of AITC was also evaluated by conducting comet assays at LC30 and LC50. Significant DNA damage in hemocytes was reflected by increase in Tail length (µm), Percent Tail DNA, Tail Moment (TM) and Olive Tail Moment (OTM) as compared to controls. The results indicated high potential of AITC as biopesticide for pest management.

Oviposition behaviour and biochemical response of an insect pest, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) to plant phenolic compound phloroglucinol.

Phenolic compounds are the secondary metabolites (SMs) present in plants carrying different bioactivities. In the present study, we explored the influence of a phenolic compound namely phloroglucinol on oviposition behaviour and different biochemical entities of an insect pest Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) using artificial diet. Phloroglucinol (IUPAC name: benzene-1,3,5-triol) affected the activity of antioxidant and detoxifying enzymes viz. superoxide dismutases (SOD), catalase (CAT), ascorbate peroxidases (APOX). dehydroascorbate reductase (DHAR), peroxidases (POX), phenol oxidase (PO), glutathione peroxidase (GPOX), glutathione S-transferase peroxidase (GSTpox), glutathione reductase (GR), glutathione S-transferase (GST) and esterases (EST) as well as the biological antioxidants viz. ascorbate content and glutathione. The lipid peroxide content (LP) and hydrogen peroxide content (H2O2) were significantly enhanced in the treated larvae indicating oxidative stress in the insect. Significant inhibition in oviposition was observed and effective repellency percentage increased with phloroglucinol treatment as compared to control. The oviposition deterrent activity and toxic effects of phloroglucinol on various biochemical parameters of Z. cucurbitae larvae revealed in the present study clearly confirms its suitability for use in pest management.

Toxic effects of purified phenolic compounds from Acacia nilotica against common cutworm.

Phenolics are the most abundant secondary metabolites of plants that are widely distributed in the plant kingdom. In the last few years, the development and identification of phenolic compounds from various plants have become a major area of the environment and health-related research. In the present study, different phenolic compounds were purified from the bark of the medicinally important plant Acacia nilotica which is rich in polyphenols and were evaluated for their insecticidal potential against a polyphagous pest, Spodoptera litura (Fab.). The compounds viz. Catechin, Chlorogenic acid, and Umbelliferone were purified from ethyl acetate-acetone (E-AE) fraction using Semi-preparative HPLC and were identified using melting point determination, Nuclear Magnetic Resonance (NMR), and Mass Spectroscopy (MS). These phenolic compounds recorded detrimental effects on the growth and development of second instar larvae (6 days old) of S. litura. The larval growth, survival, adult emergence, pupal weight, and different nutritional indices were adversely affected by the various concentrations of these purified compounds. The findings revealed the insecticidal potential of polyphenols obtained from the bark of A. nilotica, which can provide an alternative for resistance management, as these plant phytochemicals are highly effective against insecticide-resistant insect pests.

Broccoli (Brassica oleracea L. var. italica) cultivars, Palam Samridhi and Palam Vichitra affect the growth of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae).

Effect of the ethyl acetate seed extracts of two cultivars of broccoli, Brassica oleracea Italica, Palam Samridhi (PS) and Palam Vichitra (PV) on growth, development and nutritional physiology of an economically important insect pest, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) was evaluated by conducting bioassays and nutritional assays. The insect larvae were fed on diets amended with the seed extracts of two cultivars at different concentrations viz. 5, 25, 125, 625 and 3125 ppm and taking water as control. The response of the insect varied with plant varieties. The extracts disrupted the developmental cycle of the pest. Larval mortality and total adult emergence were negatively affected. Larval period and total development period were also negatively influenced. Nutritional indices of S. litura also showed considerable decrease in the RGR, RCR, ECI and ECD as compared to control. The AD values were also enhanced with both the cultivars. The findings of the study revealed a considerable anti-insect potential of the two extracts and needs to be further explored for identification and isolation of bioactive constituents from broccoli for efficient management of the pest population.

Exploration of insecticidal potential of Cry protein purified from Bacillus thuringiensis VIID1.

Insect pests are a threat to agriculture as they cause a loss of 15-22% to economically important crops every year. Bacillus thuringiensis produces parasporal crystal inclusions that have insecticidal 'Cry' proteins which are toxic to insect larvae of the order Lepidoptera, Coleoptera and Diptera, etc. In the present study, 40 different soil samples from Amritsar and its surrounding areas were selected for isolation of B. thuringiensis. The rod shaped, gram-positive bacterial isolates were further analyzed for characteristic crystal formation using phase contrast and scanning electron microscopy. 6 Bacillus samples containing cry genes were identified using the universal primers for cry genes, of which one isolate exhibited a protein band of ~95 kDa. This protein was purified using a Sephadex G-75 column. The insecticidal assays conducted with purified Cry protein on insect larvae of lepidopteran and dipteran orders viz. Spodoptera litura, Galleria malonella, Bactrocera cucurbitae and Culex pipens revealed considerable detrimental effects. A significant increase in larval mortality was observed for the larvae of all insects in a concentration dependent manner when treated with Cry protein purified from B. thuringenisis VIID1. The purified Cry protein did not have any significant effect on honey bee larvae.

Effect of gallic acid on the larvae of Spodoptera litura and its parasitoid Bracon hebetor.

The antibiosis effect of gallic acid on Spodoptera litura F. (Lepidoptera: Noctuidae) and its parasitoid evaluated by feeding six days old larvae on artificial diet incorporated with different concentrations (5 ppm, 25 ppm, 125 ppm, 625 ppm, 3125 ppm) of the phenolic compound revealed higher concentration (LC50) of gallic acid had a negative impact on the survival and physiology of S. litura and its parasitoid Bracon hebetor (Say) (Hymenoptera:Braconidae). The mortality of S. litura larvae was increased whereas adult emergence declined with increasing concentration of gallic acid. The developmental period was delayed significantly and all the nutritional indices were reduced significantly with increase in concentration. Higher concentration (LC50) of gallic acid adversely affected egg hatching, larval mortality, adult emergence and total development period of B. hebetor. At lower concentration (LC30) the effect on B. hebetor adults and larvae was non-significant with respect to control. Gene expression for the enzymes viz., Superoxide dismutase, Glutathione peroxidase, Peroxidase, Esterases and Glutathione S transferases increased while the total hemocyte count of S. litura larvae decreased with treatment. Our findings suggest that gallic acid even at lower concentration (LC30) can impair the growth of S. litura larvae without causing any significant harm to its parasitoid B. hebetor and has immense potential to be used as biopesticides.

In vitro screening of peptidase inhibitory activity in some plants of North India.

In the present study, trypsin and chymotrypsin inhibitory activity of some plants of different families was evaluated. A total of 55 plants were screened, out of which six showed the maximum trypsin inhibitory activity namely Acacia concinna, Caesalpinia bonducella, Lathyrus sativus, Mucuna pruriens, Psoralea corylifolia and Sapindus mukorossi. Results suggested that the plants showing trypsin inhibitory activity (TIA) also have chymotrypsin inhibitory activity (CIA). Both trypsin and chymotrypsin inhibitory activities were high in seeds compared to leaves followed by flowers. It was also observed that TIA was maximally present in Sapindaceae family whereas CIA was maximum in fabaceae family followed by others.

Development of mitochondrial replacement therapy: A review.

Mitochondrial replacement therapy (MRT) is a new form of reproductive invitro fertilization (IVF) which works on the principle of replacing a women's abnormal mitochondrial DNA (mt-DNA) with the donor's healthy one. MRT include different techniques like spindles transfer (ST), pronuclear transfer (PNT) or polar body transfer (PBT). Transmission of defective mitochondrial DNA to the next generation can also be prevented by using these approaches. The development of healthy baby free from genetic disorders and to terminate the lethal mitochondrial disorders are the chief motive of this technique. In aged individuals, through in vitro fertilization, MRT provides the substitution of defective cytoplasm with cured one to enhance the expectation of pregnancy rates. However, moral, social, and cultural objections have restricted its exploration. Therefore, this review summarizes the various methods involved in MRT, its global status, its exaggerated censure over the years which depicts a strong emphasis for social acceptance and clinical application in the world of medical science.

Deterioration of digestive physiology of Bactrocera cucurbitae larvae by trypsin inhibitor purified from seeds of Mucuna pruriens.

Peptidase inhibitors (PIs) are plant proteins that are found to be effective against various digestive peptidases of insects. The present study isolated and characterized a trypsin inhibitor from mature dry seeds of Mucuna pruriens and investigated its effect against Bactrocera cucurbitae larvae, a major pest of cucurbitaceae crops, for its inhibitory activity. The purified trypsin inhibitor from M. pruriens seeds gave a molecular weight of ~11 kDa on SDS-PAGE. M. pruriens trypsin inhibitor (MPTI) exhibited inhibitory effect on growth of melon fruit fly larvae (64-72 h old) as it resulted in prolongation of larval, pupal and total development period. There was a significant increase in larval mortality with increase in concentration of MPTI. Nutritional indices decreased significantly at all the concentrations of MPTI. Quantitative RT- PCR revealed that the mRNA expression level of trypsin and chymotrypsin genes was reduced while that of GST, esterases, AP, SOD and catalase were enhanced. It can therefore be inferred that MPTI can serve as a promising agent for biocontrol that can reduce the problems caused by fruit fly and other similar catastrophic pests. This study provides the fundamental information for future successful strategies for pest management.

Exploration of anti-insect potential of trypsin inhibitor purified from seeds of Sapindus mukorossi against Bactrocera cucurbitae.

Peptidase inhibitors (PIs) are defense proteins of plants which are active against gut peptidases of different insects. Sapindus mukorossi was identified as a source of bioactive PIs which could confer resistance against Bactrocera cucurbitae, a most devastating pest of several economically important crops. In the present study, a trypsin inhibitor was purified from mature dry seeds of S. mukorossi and characterized for its biochemical properties as well as its potential for bio control of B. cucurbitae. The purified fractions from RP- HPLC through SDS-PAGE gave an apparent molecular weight of ~29 kDa. S. mukorossi trypsin inhibitor (SMTI) was found to be a non-competitive inhibitor which was active over a broad range of temperature (10-100 °C) and pH (6-11). SMTI when incorporated in artificial diet inhibited the growth and development of B. cucurbitae larvae. Gene expression analysis of trypsin and chymotrypsin genes via qRT-PCR indicated that their mRNA expression was down-regulated while that of other genes namely, Catalase, Elastase, Superoxide Dismutase, Glutathione -S-transferase and Alkaline Phosphatase was up regulated. SMTI also showed deleterious effects against different bacterial strains. The results of this study indicated that S. mukorossi trypsin inhibitor has potential to be used as a bio control agent that can reduce the harm caused by melon fruit fly and other devastating pests.

Purification of a trypsin inhibitor from Psoralea corylifolia seeds and its influence on developmental physiology of Bactrocera cucurbitae.

A trypsin inhibitor was purified from the seeds of Psoralea corylifolia by ion-exchange and affinity chromatography. The purified fractions were subjected to RP- HPLC which resolved into a single peak. SDS-PAGE analysis gave an apparent molecular weight of 18 kDa. P. corylifolia trypsin inhibitor (PCTI) was found to be a competitive inhibitor and was active over a broad temperature (10-100 °C) and pH (6-11) range. It was shown to have a deleterious effect on growth and development of larvae of the melon fruit fly, Bactrocera cucurbitae, when incorporated in artificial diet using various concentrations. The larval, pupal, total development period and larval mortality significantly increased during the treatment. Inhibitory effects were also observed on percentage emergence which was significantly reduced. Nutritional indices namely food assimilated (FA) and mean relative growth rate (MRGR) also decreased significantly with increase in concentration of PCTI. qRT-PCR results indicated that the expression of trypsin and chymotrypsin genes were down-regulated while elastase, catalase, GST, SOD and AP were up-regulated. PCTI was also effective against certain bacterial strains. These results indicated that the peptidase inhibitor from P. corylifolia may be a potential bio-control agent which can decrease the damage caused by B. cucurbitae and other related destructive pests.

Insecticidal and growth inhibitory potential of Streptomyces hydrogenans DH16 on major pest of India, Spodoptera litura (Fab.) (Lepidoptera: Noctuidae).

BACKGROUND: Destructive impacts of insecticides on non targeted populations necessitate the development of an eco friendly pest control method. Streptomyces spp. are rich source of bioactive secondary metabolites which may provide valuable alternatives to chemical insect-control agents as they can be less toxic and readily biodegradable. Because of its potent biocontrol attributes, ethyl acetate extract of Streptomyces hydrogenans DH16, a soil isolate, was tested to assess its anti-insect potential against polyphagous noctuid, Spodoptera litura. RESULTS: The secondary metabolites in the ethyl acetate extract of S. hydrogenans DH16 exhibited larvicidal and growth inhibitory activities. The results indicated that highest concentration of 1600 µg/ml was significantly effective as 70% larval, 66.66% prepupal and 100% pupal mortality was noticed. The metabolites also prolonged the larval developmental period. The LC50 and LC90 values were 1337.384 and 2070.516 µg/ml, respectively for the insect. Negative effects of S. hydrogenans were also observed on development of the insect. Significant decline in adult emergence, adult longevity, fecundity and % hatching was recorded at higher concentrations along with morphological abnormalities as compared to control. Significant decrease in relative growth and consumption rate, efficiency of ingested and digested food and increase in approximate digestibility in larvae reared on diet supplemented with ethyl acetate extract accounts for the toxic as well as anti-nutritive nature of extract. CONCLUSION: Secondary metabolites in the fermentation broth from S. hydrogenans were toxic to the larvae at higher concentrations whereas lower concentrations significantly reduced the reproductive potential of S. litura. Therefore, these metabolites show considerable potential for incorporation in pest management programmes as new biopesticidal formulation.