Effect of male parental gamma irradiation on host suitability of its F1 progeny of a lepidopteran tropical pest, Spodoptera litura (Fabr.) towards development and virulence of entomopathogenic nematodes, Steinernema thermophilum.

The suitability of F1 progeny insect larvae of the irradiated male parent, Spodoptera litura (Fabr.) for infective juveniles (IJs) of entomopathogenic nematodes (EPN), Steinernema thermophilum was assessed to comprehend the feasibility of combining EPNs with nuclear pest control tactic. As compared to the control, the IJs induced faster host mortality with reduced proliferation in F1 host larvae. IJs derived from F1 host larvae exhibited almost similar proliferation capacity on normal hosts as in control. Further, the molecular basis of EPNs induced mortality in F1 host larvae was evaluated. Dual stress of EPN infection and irradiation induced downregulation of the relative mRNA expression of antimicrobial genes and upregulated expression of antioxidative genes. A pronounced effect of EPNs in association with irradiation stress was apparent on host mortality. Radiation induced sterile F1 insect larvae of S. litura acted as a reasonably suitable host for EPNs and also provided the environment for developing viable EPNs for their potential use as biocontrol agents.

Irradiation influence on the phenoloxidase pathway and an anti-oxidant defense mechanism in Spodoptera litura (Lepidoptera: Noctuidae) and its implication in radio-genetic 'F 1 sterility' and biorational pest suppression tactics.

The present study was conducted to appraise the ontogenic radio-sensitivity of a serious tropical pest, Spodoptera litura (Fabr.). The molecular responses pertaining to the phenoloxidase (PO) pathway and an anti-oxidant defense mechanism were evaluated in order to understand its implication in pest control at pre-harvest and post-harvest intervals. Irradiation exhibited an inverse relationship with age with respect to impact on developmental and transcriptional responses. Transcript abundance of PO cascade enzymes, prophenoloxidase (slppo-2), its activating enzyme (slppae-1) and free-radical scavenging enzymes, superoxide dismutase (slsod) and catalase (slcat) was evaluated upon gamma irradiation alone and the dual-stress of radiation plus microbial challenge. The slppo-2, slppae-1, slsod and slcat transcripts were significantly up-regulated in F 1 L6 larvae (6th-instar) resulting from 100 Gy sub-sterilized male adults and unirradiated female moths. The extent of upregulation was relatively higher in comparison with L6 survivors (6th-instar larvae) developed from irradiated neonates (L1) treated with 100 Gy. Upon Photorhabdus challenge, the transcripts were down-regulated in irradiated L1 suggesting increased larval susceptibility to bacterial infections. Radioresistance increased with the age of the insect, and molecular responses (transcript abundance) of insect defense mechanism were less influenced when older age (F 1 progeny) were irradiated. These findings will help to optimize the gamma dose to be employed in inherited sterility technique for (pre-harvest) pest suppression and (post-harvest) phytosanitation and quarantine, and suggest compatible integration of biorational tactics including nuclear technology.

Expression, purification and characterization of allelic variants of MSP-1(42) from Indian Plasmodium falciparum isolates.

The C-terminal 19 and 42 kDa fragments of Plasmodium falciparum merozoite surface protein 1 (MSP-1) have shown to be protective in animals against lethal parasite challenge. The MSP-1(19) being highly conserved may lack sufficient number of T-cell epitopes in order to elicit a broader response in genetically diverse populations. The inclusion of additional epitopes from the N-terminal MSP-1(42) has shown to enhance the protective efficacy of MSP-1(19) vaccine. In an attempt to examine the strain specific immunogenicity to MSP-1, we have cloned and expressed three diverse allelic variants of MSP-1(42) from Indian P. falciparum isolates in bacteria. Among three alleles, one was extremely rare and not been found before. These purified and refolded recombinant products were recognized by conformation specific monoclonal antibodies and hyper-immune sera. Immunization of mice and rabbits with the purified proteins generated high titer biologically active polyclonal antibodies supporting further development of this vaccine candidate antigen.

Impact of temperature on susceptible and resistant strains of Culex quinquefasciatus to synthetic pyrethroids.

Effect of temperature on the physiological activity and resistance status of Culex quinquefasciatus was studied. The LT(50) (lethal time) of deltamethrin, lambdacyhalothrin resistant and susceptible 4th-instar larvae decreased with increasing temperature. The LT(50) of deltamethrin resistant strains was significantly lower than the susceptible strains but not so in lambdacyhalothrin resistant strains. The developmental period of the thermal exposed and non-exposed larvae of resistant strains were nearly equal, however a significant difference was observed in case of susceptible strain. The thermal exposed susceptible larvae developed 3.3 days faster than the thermal un-exposed population. The percentage of survival of thermal exposed and un-exposed resistant strains were nearly equal. However, the thermal exposed population survived 12.9% less than the thermal un-exposed susceptible population. Longevity of thermal exposed deltamethrin and lambdacyhalothrin resistant females was significantly higher than the un-exposed population. The longevity of thermal exposed susceptible population was lower than the un-exposed population. The resistance of both the deltamethrin and lambdacyhalothrin resistant strains increased when exposed to high temperature for 3h. The difference in longevity between thermal un-exposed and exposed resistant male population was not significantly different. Among the un-exposed population, highest pupation was recorded in the susceptible strains, and lowest in the deltamethrin resistant strain. The fact that the thermal exposed resistant strains were more successful than the un-exposed population could be related to the presence of resistant gene.

Characterization of biochemical based insecticide resistance mechanism by thermal bioassay and the variation of esterase activity in Culex quinquefasciatus.

Biochemical mechanisms of insecticide resistance of thermal exposed and unexposed Culex quinquefasciatus strains are evaluated, which were not studied earlier. The activity of alpha- and beta-carboxylesterases and acetylcholinesterase of malathion susceptible and resistant strains were compared after thermal treatment. Three-day-old adult females were used for the malathion susceptibility test and biochemical assays, and males were used only for the susceptibility test. Thermal exposure brought about increase in resistance levels from 85% to 90% in males and 91% to 96.6% in females of resistant strain. The resistance status of the susceptibility strain was unchanged after thermal exposure. The activities of alpha- and beta-carboxylesterase of susceptible mosquitoes were within 800 and 700 U/mg protein, respectively. The alpha-carboxylesterase activity of the thermal exposed malathion-resistant population was significantly (t test, P < 0.05) higher than the unexposed resistant population, and the reverse was recorded in beta-carboxylesterase. The alpha-carboxylesterase activity of susceptible population was lower than the resistant population. The activity of alpha-carboxylesterase was higher than the beta-carboxylesterase in both the strains. Among the malathion resistant C. quinquefasciatus population, 2.3% population exhibited 30-40% inhibition which increased to 5.8% after the thermal exposure. Thermal exposure of mosquitoes increased the activity of both alpha-carboxylesterases and acetylcholinesterase but decreased the activity of beta-carboxylesterase.

Effect of temperature on development, eclosion, longevity and survivorship of malathion-resistant and malathion-susceptible strain of Culex quinquefasciatus.

Density of vectors and development of resistance against insecticides are two important aspects in the control of vector-borne diseases. Here, effect of temperature on different aspects of development of malathion-resistant and susceptible strains of Culex quinquefasciatus was evaluated in the laboratory. Fourth-instar larvae of C. quinquefasciatus were exposed to 37 degrees C, 39 degrees C, 40 degrees C, and 41 degrees C, and their LT(50) values calculated. The fourth-instar larvae were subjected to heat for 4 h at 39 degrees C and live larvae were reared until the completion of life cycle. The larvae of malathion-resistant strain were more tolerant to heat than the larvae of malathion-susceptible ones. The difference in mortality between 37 degrees C, 39 degrees C, 40 degrees C, and 41 degrees C of both the strains of C. quinquefasciatus were highly significant. Pupation percentage of heat-exposed larvae of both the strains was higher and delayed than the control ones. The percentage of adult emergence from the heat-exposed larvae was significantly lower than the control. The adult female of malathion-resistant strain showed increased longevity when exposed to heat at larval stage but reverse was true in case of malathion-susceptible. The highlights of the study would help in the management of resistance of mosquito vectors in the tropics where variable climatic condition is observed.

Naturally acquired immunity and reduced susceptibility to falciparum malaria in two subpopulations of endemic eastern India.

This study was aimed to assess the prevalence of naturally acquired humoral immune responses and their association with reduced susceptibility to malaria in children and adults with differential clinical conditions from an Indian zone where malaria is endemic. The study was undertaken in an eastern province of India (Keonjhar, Orissa) in a group of 341 children (both younger and older) and 98 adults living in two different areas, Town area and Forest area. They were studied for their parasitological and immunological profiles. Sera from different age-matched groups were screened by ELISA to measure IgG reactivities for characterizing humoral immune responses to the B-cell epitopes of Plasmodium falciparum MSP1, AMA1, RAP1 and EBA175 peptides and P. falciparum-infected erythrocyte lysate. In Town area, overall P. falciparum cases were 5.5%, whereas those in Forest area were 26.7%. We observed an age-wise increasing trend of immunity in these two populations. It was also noticed that the frequency of responders to stage-specific antigens was higher in individuals from the Town area where the frequency of malaria was lower. The naturally acquired humoral immune responses to different stage-specific antigens of P. falciparum reflect the reduced risk of malaria in the study groups. The higher frequency of seroresponders showed correlation with lower risk of developing malaria.

Effects of larval exposure to sublethal concentrations of the ecdysteroid agonists RH-5849 and tebufenozide (RH-5992) on male reproductive physiology in Spodoptera litura.

Sublethal concentrations of the bisacylhydrazine moulting hormone agonists, RH-5849, and tebufenozide (RH-5992) were fed to sixth (final) instar larvae of Spodoptera litura. Both RH-5849 and tebufenozide adversely affected the mating success of S. litura when the surviving treated males were crossed with normal females. The ecdysone agonists decreased the longevity of treated males and of untreated females when crossed with treated males. The number of eggs laid by untreated females mated to treated males was decreased, and the fertility (percentage of hatching success) of the resulting eggs was reduced. These effects on male reproductive success were at least in part explained by a reduction in the number of sperm transferred during mating. The adverse effects of tebufenozide on male reproductive function were qualitatively the same as those of RH-5849, but tebufenozide was active at lower concentrations. To understand the reason for these adverse effects on male reproduction, we investigated the effects of the insecticides on male reproductive physiology. Male reproductive tract development and testicular volume of resulting adult moths were adversely affected by sublethal larval exposure to the ecdysone agonists. Dose-dependent reductions occurred in the production of eupyrene and apyrene spermatozoa in the adult testes, and in the number of spermatozoa released from the testes into the male reproductive tract. The descent into the male tract of both eupyrene and apyrene sperm was found to start at the normal stage of development in both normal and treated insects, but the daily rhythm of sperm descent was subsequently disturbed in the insecticide-treated moths. This affected the numbers of sperm in the upper vas deferens (UVD), seminal vesicle (SV), and duplex (duplex). Injections of RH-5849 given to pharate adult or newly emerged adult S. litura also caused drastic reduction in the number of sperm in the upper regions of the male tract, when measured 24 h after injection. The possible importance of pest population reduction through the sublethal anti-reproductive effects of insecticides is discussed.

Radiation responses of Sf9, a highly radioresistant lepidopteran insect cell line.

PURPOSE: Lepidopteran insect cells are known to exhibit very high radioresistance. Although very effective DNA excision-repair has been proposed as a contributing factor, a detailed understanding of insect cell radiation responses has not yet been obtained. Therefore, the study was carried out to understand the in vitro radiation responses of Sf9 lepidopteran cells. MATERIALS AND METHODS: Exponentially growing asynchronous Sf9 cells (derived from ovaries of Spodoptera frugiperda) were exposed to gamma-radiation doses of 2-200 Gy. Cell survival, growth inhibition, cell cycle progression delay, alterations in cell morphology as well as induction of DNA damage, micronuclei and apoptosis were studied at various post-irradiation time intervals. RESULTS: Biphasic survival response curves were obtained with D0 rising from 20 Gy (at doses < or = 60 Gy) to 85 Gy (between 60 and 200 Gy), corroborating earlier reports on lepidopteran cells. An additional downward deviation at 2 Gy indicated a hypersensitive response. Dose-dependent growth inhibition with a transient G2 delay starting 12 h and extending up to 48-96 h was observed at doses of 10-200 Gy, while a brief G1/S transition delay was observed only at higher doses (> or = 100 Gy). Significant DNA damage was detected only at 20 Gy and higher doses, in contrast with human cells that showed similar damage at 2 Gy. Interestingly, micronuclei were not induced at any of the doses tested, although spontaneous micronucleation was evident in <1% of cells. Lack of micronucleus induction even at doses that induced significant DNA damage and a transient G2 block (20-50 Gy) strongly indicated a role of holocentric lepidopteran chromosomes. Apoptosis was detected only in a small proportion of cells (3%) exposed to 200 Gy, and cell/nucleus size and granularity increased by 72-96 h post-irradiation in a dose-dependent manner. Sf9 nucleoids extracted at 2 M NaCl showed higher compactness than the nucleoids prepared from human cells. CONCLUSIONS: It is clearly shown that lepidopteran cells are highly resistant to the induction of DNA damage and micronuclei, and display very low induction of apoptosis at doses up to 200 Gy. While the lack of micronucleus induction seems to be primarily due to the holocentric nature of their chromosomes, certain unique signalling pathways might be responsible for the low induction of apoptosis. Factors causing protection of Sf9 cellular DNA from radiation-induced damage are presently being investigated.

Movement of spermatozoa in the reproductive tract of adult male Spodoptera litura: daily rhythm of sperm descent and the effect of light regime on male reproduction.

Sperm production and movement from the fused testes into the male reproductive tract of the common cutworm Spodoptera litura were studied in insects maintained in a 12h:12h light dark (LD) regime. Two types of sperm bundles, eupyrene (nucleated) and apyrene (anucleate) were present in the adult testes. Eupyrene bundles constituted about 25% of the total. Descent of spermatozoa from the testes into the upper vas deferens (UVD) first occurred about 24-30h before adult eclosion. On entering the reproductive tract, eupyrene spermatozoa remained in bundles while apyrene bundles became dissociated before they reached the UVD. Downward movement of both eupyrene and apyrene spermatozoa within the male tract occurred in a daily rhythm. Sperm descent from the testes into the UVD occurred during the early scotophase, followed by their further descent into the seminal vesicle (SV) during the photophase. Spermatozoa remained in the SV for only a short duration, whence sperm quickly passed through the lower vas deferens into the duplex, which acted as the main sperm storage organ until mating was initiated. During mating 80% of sperm left the duplex, but mating did not influence the number of sperm bundles that subsequently descended into the duplex or the rate of their descent. There was no evidence of sperm reflux. Rearing in constant light (LL) and in constant dark (DD) reduced the number of eupyrene sperm present in the testes of adults that emerged in LL and DD compared to controls (LD), although there was no significant effect on the number of apyrene sperm in the testes. The rhythmic pattern of sperm descent was suppressed in both LL and DD regimes, and the number of sperm in the duplex was adversely affected, with a marked impact in LL reared insects. Male longevity, mating behaviour, oviposition and fertility were found to be more severely affected in LL than in DD.

Regulation of c-fos induction in lens epithelial cells by 12(S)HETE-dependent activation of PKC.

PURPOSE: 12(S)-Hydroxyeicosatetraenoic acid (12(S)HETE), a 12-lipoxygenase metabolite of arachidonic acid, is required for epidermal growth factor (EGF)-dependent DNA synthesis and c-fos induction in lens epithelial cells. The present study was undertaken to identify signal transduction events upstream of c-fos induction that may be regulated by 12(S)HETE. METHODS: The rabbit lens epithelial cell line, N/N1003A, was cultured in serum-free medium, with or without EGF. Activation of PKC and other selected enzymes was examined in the presence of the lipoxygenase inhibitor baicalein and/or exogenous 12(S)HETE. Relative abundance of PKC isoforms in subcellular fractions was determined by immunoblot analysis with isoform-specific antibodies. PKC activity in subcellular fractions was measured by peptide substrate phosphorylation, with and without pseudosubstrate peptide inhibitor. Phosphorylated enzymes were detected by immunoblot analysis. Relative levels of c-fos mRNA were determined by RT/PCR with internal standard. RESULTS: Baicalein blocked EGF-dependent translocation and activation of PKC, without affecting phosphorylation of Erk1/2. Of several PKC isoforms investigated (alpha, betaI, betaII, and gamma), only PKCalpha and betaII were significantly activated by EGF and inhibited by baicalein. 12(S)HETE, in combination with EGF, countered the effect of lipoxygenase inhibitors on PKC activation, and 12(S)HETE in the absence of EGF stimulated PKC translocation. Also of note, 12(S)HETE alone activated PKCgamma, an isoform that was not significantly activated by EGF. Inhibiting PKC activation with GF109203X blocked induction of c-fos by EGF but did not affect EGF-stimulated phosphorylation of Erk1/2, indicating that the effect of PKC on c-fos induction is independent of the Erk1/2 pathway. CONCLUSIONS: In lens epithelial cells, 12(S)HETE-dependent activation of PKCalpha and betaII acts in concert with other EGF-dependent signals to induce c-fos mRNA.

Protective function of alpha-tocopherol against the process of cataractogenesis in humans.

In a random trial 50 patients with unilateral/ bilateral idiopathic immature senile cataract (cortical n = 25, nuclear n = 25) requiring surgery at least in one eye were included in the present study. Reduced glutathione (GSH), vitamin E, malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were studied in these patients receiving either vitamin E (n = 12 in each subgroup) or placebo (n = 13 in each group) for 30 days. A comparable increase of vitamin E in both types of lens homogenates of cataractous patients was observed in the study group. The level of GSH in cortical cataractous lenses in the study group was significantly raised (p < 0.001) whereas it was not increased significantly in nuclear cataractous lenses (p > 0.05) as compared to the placebo group. The percentage decrease in MDA levels was greater in cortical cataracts (38.07%) than in the nuclear type (27.94%). The activity of GSH-Px in cortical lenses was higher than that in the nuclear cataractous lens. The change in the size of lens opacity in cortical cataractous patients receiving vitamin E therapy was significantly decreased as compared to the placebo group. There may be a direct modulating effect of vitamin E on some GSH-related enzymes and the cortex of the lens might be protected more than the nucleus.

Comparison of sodium nitroprusside added peritoneal dialysis and standard haemodialysis.

Thirty patients of acute or acute on chronic renal failure (ACRF) were randomly divided into two group of 15 cases each. Group A patients received 36 cycles of intermittent peritoneal dialysis (PD) with an exchange volume of one litre and duration of one hour per cycle. The 36 cycles of PD were divided into 12 clearance periods of 3 cycles each. Sodium Nitroprusside (SNP) was added in a dose of 4 mg/litre of dialysate in alternate clearance periods. Group B patients were given 4 hours of haemodialysis (HD) to compare the efficacy of two modes of dialysis. Symptomatic relief was observed in various uraemic signs and symptoms like vomiting, level of consciousness, fluid overload, hiccough and asterexis in most of the patients in both the groups. The percentage fall in blood urea and serum creatinine was 57.02 Vs 58.04 mg% and 46.9 Vs 47.8 mg% in group A and B respectively (P 70.5 each). Total dialysate urea removal following PD and HD was 118.8 +/- 57.3 gm and 98.5 +/- 37.0 gm respectively and also there was no significant difference in total creatinine removal. No untoward effects were observed with PD. However, following HD, 5 patients developed hypotension, supraventricular tachycardia was observed in one and disequilibrium syndrome in 8 of them. Therefore, it can be concluded that SNP added PD is comparable to 4 hours of haemodialysis both clinically as well as biochemically and in situations where facilities for HD do not exist or it is contraindicated, PD may be preferred mode of therapy.

Serum lipid peroxide and other enzyme levels of patients suffering from thermal injury.

The levels of the lipid peroxidation product malondialdehyde (MDA) and the activity of transaminases and alkaline phosphatases were estimated in the sera of 25 thermally injured patients at various time intervals after injury. The level of MDA was increased during the early postburn period, whereas the activities of transaminases and alkaline phosphatase became elevated later after injury. It is concluded that an increased concentration of lipid peroxidation product (MDA) in the early postburn period may affect the spleen, liver and kidney, resulting in the release of enzymes into blood stream. Such damage may be checked by the antioxidants superoxide dismutase or allopurinol.

Effect of atenolol and labetalol on serum lipids.

Adverse alterations in lipid profile suggesting higher atherogenicity were observed following 12 weeks treatment with atenolol in patients of hypertension. No significant alterations in lipid profile were observed with labetalol therapy.

Effect of vasodilator and surface active drugs on the efficacy of peritoneal dialysis.

The effect of sodium nitroprusside (SNP), a vasodilator drug, and chlorpromazine (CPZ), a surface active drug, on the efficacy of peritoneal dialysis was studied in 25 patients with acute or acute on chronic renal failure in a double blind fashion. Each drug was added to the dialysate during different sets of cycles. In each patient, six clearance periods of 3 cycles each were studied and peritoneal clearances of creatinine and urea and ultrafiltration rates were measured during each clearance period. SNP increased the peritoneal clearance of creatinine and urea by 28.8 percent each (p < 0.001) while CPZ increased the peritoneal creatinine and urea clearance by 17.7 and 26.0 percent respectively (p < 0.001 each). Both drugs significantly increased the ultrafiltration rates (p < 0.001). SNP was found to be superior to CPZ and had prolonged effect even after cessation of administration.

Adenosine deaminase levels in cerebrospinal fluid in tuberculosis and bacterial meningitis.

Adenosine deaminase activity was measured in cerebrospinal fluid of patients with confirmed tuberculous and bacterial meningitis. The values were compared with those of control subjects without meningitis. A statistically significant increase in the level of this enzyme was noted in the two types of meningitis, but no definite demarcation in the levels was observed between the two types. Therefore increases in adenosine deaminase activity may not be of such diagnostic significance as reported elsewhere.

Lipid profile in enteric fever.

Lipid profile is known to alter in patients with severe sepsis, but few studies regarding the status of lipid levels in enteric fever are available. Twenty patients with enteric fever, belonging to different age groups and both sexes, along with an equal number of matched patients with fever due to non-enteric causes, were studied with regard to alterations in lipid profile. We observed a severe and protracted hypertriglyceridaemia, decrease in HDL-cholesterol levels and increase in LDL-cholesterol levels in patients with enteric fever at the peak of fever. The values returned to normal on recovery and convalescence. This study serves to highlight the complexity of lipid variation during Salmonella typhi infection.