Diversity study of Beauveria bassiana species for finding the most virulent strain to manage Bemisia tabaci in cotton.

Beauveria bassiana (Bal.-Criv.) is an important entomopathogenic fungus being used for the management of various agricultural pests worldwide. However, all strains of B. bassiana may not be effective against whitefly, Bemisia tabaci, or other pests, and strains show diversity in their growth, sporulation, virulence features, and overall bioefficacy. Thus, to select the most effective strain, a comprehensive way needs to be devised. We studied the diversity among the 102 strains of B. bassiana isolated from 19 insect species based on their physiological features, virulence, and molecular phylogeny, to identify promising ones for the management of B. tabaci. Strains showed diversity in mycelial growth, conidial production, and their virulence against B. tabaci nymphs. The highest nymphal mortality (2nd and 3rd instar) was recorded with MTCC-4511 (95.1%), MTCC-6289 (93.8%), and MTCC-4565 (89.9%) at a concentration of 1 × 106 conidia ml-1 under polyhouse conditions. The highest bioefficacy index (BI) was in MTCC-4511 (78.3%), MTCC-4565 (68.2%), and MTCC-4543 (62.1%). MTCC-4511, MTCC-4565, and MTCC-4543 clustered with positive loading of eigenvalues for the first two principal components and the cluster analysis also corresponded well with PCA (principal component analysis) (nymphal mortality and BI). The molecular phylogeny could not draw any distinct relationship between physiological features, the virulence of B. bassiana strains with the host and location. The BI, PCA, and square Euclidean distance cluster were found the most useful tools for selecting potential entomopathogenic strains. The selected strains could be utilized for the management of the B. tabaci nymphal population in the field through the development of effective formulations. KEY POINTS: • 102 B. bassiana strains showed diversity in growth and virulence against B. tabaci. • Bioefficacy index, PCA, and SED group are efficient tools for selecting potential strains. • MTCC-4511, 4565, and 4543 chosen as the most virulent strains to kill whitefly nymphs.

Insufficient duration of insecticidal efficacy of Yahe® insecticide-treated nets in Papua New Guinea.

BACKGROUND: Insecticide-treated nets (ITNs) are the backbone of anti-malarial vector control in Papua New Guinea (PNG). Over recent years the quality and performance of ITNs delivered to PNG decreased, which has likely contributed to the stagnation in the malaria control effort in the country. The present study reports results from the first 24 months of a durability study with the ITN product Yahe LN® in PNG. METHODS: The durability study was conducted in four villages on the northern coast of PNG, in an area with high malaria parasite transmission, following WHO-recommended methodology adapted to the local scenario. A cohort of n = 500 individually identifiable Yahe® ITNs was distributed by the PNG National Malaria Control Programme from October to December 2021. Insecticidal efficacy of the ITNs was tested using cone bioassays with fully pyrethroid susceptible Anopheles farauti colony mosquitoes at baseline and at 6 months intervals, alongside evaluation of physical integrity and the proportion of ITNs lost to follow-up. A questionnaire was used to collect information on ITN end user behaviour, such as the frequency of use and washing. The observations from the durability study were augmented with simulated laboratory wash assays. RESULTS: Gradual uptake and replacement of previous campaign nets by the communities was observed, such that at 6 months 45% of all newly distributed nets were in use in their designated households. Insecticidal efficacy of the Yahe® nets, expressed as the percent 24 h mortality in cone bioassays decreased from 91 to 45% within the first 6 months of distribution, even though > 90% of study nets had never been washed. Insecticidal efficacy decreased further to < 20% after 24 months. ITNs accumulated physical damage (holes) at a rate similar to previous studies, and 35% were classified as 'too torn' by proportional hole index after 24 months. ITNs were lost to follow-up such that 61% of cohort nets were still present after 24 months. Laboratory wash assays indicated a rapid reduction in insecticidal performance with each consecutive wash such that average 24 h mortality was below 20% after 10 washes. CONCLUSION: Yahe® ITNs are not performing as per label claim in an area with fully pyrethroid susceptible vectors, and should be investigated more comprehensively and in other settings for compliance with currently recommended durability and efficacy thresholds. The mass distribution of low quality ITN products with variable performance is one of the major ongoing challenges for global malaria control in the last decade.

Temperature, mosquito feeding status and mosquito density influence the measured bio-efficacy of insecticide-treated nets in cone assays.

BACKGROUND: The WHO cone bioassay is routinely used to evaluate the bioefficacy of insecticide-treated nets (ITNs) for product pre-qualification and confirmation of continued ITN performance during operational monitoring. Despite its standardized nature, variability is often observed between tests. We investigated the influence of temperature in the testing environment, mosquito feeding status and mosquito density on cone bioassay results. METHODS: Cone bioassays were conducted on MAGNet (alphacypermethrin) and Veeralin (alphacypermethrin and piperonyl butoxide (PBO)) ITNs, using laboratory-reared pyrethroid-resistant Anopheles funestus sensu stricto (FUMOZ strain) mosquitoes. Three experiments were conducted using standard cone bioassays following WHO-recommended test parameters, with one variable changed in each bioassay: (i) environmental temperature during exposure: 22-23 °C, 26-27 °C, 29-30 °C and 32-33 °C; (ii) feeding regimen before exposure: sugar starved for 6 h, blood-fed or sugar-fed; and (iii) mosquito density per cone: 5, 10, 15 and 20 mosquitoes. For each test, 15 net samples per treatment arm were tested with four cones per sample (N = 60). Mortality after 24, 48 and 72 h post-exposure to ITNs was recorded. RESULTS: There was a notable influence of temperature, feeding status and mosquito density on An. funestus mortality for both types of ITNs. Mortality at 24 h post-exposure was significantly higher at 32-33 °C than at 26-27 °C for both the MAGNet [19.33% vs 7%; odds ratio (OR): 3.96, 95% confidence interval (CI): 1.99-7.87, P < 0.001] and Veeralin (91% vs 47.33%; OR: 22.20, 95% CI: 11.45-43.05, P < 0.001) ITNs. Mosquito feeding status influenced the observed mortality. Relative to sugar-fed mosquitoes, The MAGNet ITNs induced higher mortality among blood-fed mosquitoes (7% vs 3%; OR: 2.23, 95% CI: 0.94-5.27, P = 0.068) and significantly higher mortality among starved mosquitoes (8% vs 3%, OR: 2.88, 95% CI: 1.25-6.63, P = 0.013); in comparison, the Veeralin ITNs showed significantly lower mortality among blood-fed mosquitoes (43% vs 57%; OR: 0.56, 95% CI: 0.38-0.81, P = 0.002) and no difference for starved mosquitoes (58% vs 57%; OR: 1.05, 95% CI: 0.72-1.51, P = 0.816). Mortality significantly increased with increasing mosquito density for both the MAGNet (e.g. 5 vs 10 mosquitoes: 7% vs 12%; OR: 1.81, 95% CI: 1.03-3.20, P = 0.040) and Veeralin (e.g. 5 vs 10 mosquitoes: 58% vs 71%; OR 2.06, 95% CI: 1.24-3.42, P = 0.005) ITNs. CONCLUSIONS: The results of this study highlight that the testing parameters temperature, feeding status and mosquito density significantly influence the mortality measured in cone bioassays. Careful adherence to testing parameters outlined in WHO ITN testing guidelines will likely improve the repeatability of studies within and between product testing facilities.

Physical integrity and bioefficacy of used long-lasting insecticidal nets in Makenene, Centre Region of Cameroon.

Long-lasting insecticide nets (LLINs) are the recommended tools against mosquito-borne diseases. However, their physical integrity and bioefficacy in the field could be affected by several factors. This study evaluated the physical integrity and bioefficacy of nets used in Makenene since 2016. Cross-sectional field surveys were carried out after 6 y. A questionnaire was first administered to the heads of households, and then the physical integrity of the LLINs was determined by calculating the proportional hole index (pHI). WHO cone bioassays were conducted to determine the bioefficacy of LLINs currently being used against wild strains of Anopheles gambiae s.l., Culex pipiens s.l., and laboratory-reared pyrethroid-susceptible strain of Anopheles coluzzii (Ngousso). Of the 167 LLINs examined in households, 39.5% were fairly good, 26.4% were acceptable, and 34.1% were damaged. The most torn faces of the nets were the sides used for entering and exiting. None of the 30 LLINs used for WHO cone bioassays was still effective against An. gambiae s.l. and Cx. pipiens s.l. while up to 85.7% of these LLINs were at least effective against the susceptible strain after 24 h, with a significant difference observed when comparing the mortality rates between wild and laboratory-susceptible strain of Anopheles (P-value < 0.01). Anopheles gambiae s.l. were all (100%) identified as An. gambiae s.s. by PCR. The LLINs distributed in Makenene since the 2016 campaign are only effective on susceptible strain and should be replaced for a better control of residual malaria transmission and the nuisance by Culex mosquitoes in the locality.

Larvicidal activities of chinaberry, neem and Bacillus thuringiensis israelensis (Bti) to an insecticide resistant population of Anopheles arabiensis from Tolay, Southwest Ethiopia

Objective: To elucidate the larvicidal potency of neem, chinaberry and Bacillus thur-ingiensis israelensis (Bti) to larvae of Anopheles arabiensis under semi-field condition and adult susceptibility/resistance to the conventionally used insecticides in Tolay, Southwestern Ethiopia. Methods: Wild collected 3rd and 4th stage larvae were exposed to neem, and chinaberry seed powder dissolved in water and Bti in artificial containers at three treatment levels:0.2, 0.1 and 0.05 g/m2 and controls were free of treatments. Larval and pupal mortalities were monitored daily and residual activities were determined. The experiments were replicated three times. The World Health Organization tube test for all classes of in-secticides was conducted on adult Anopheles arabiensis reared from field collected larvae and pupae. Data were analyzed using STATA software version 11. Results: In the first application, neem powder caused 88.9%, 87.9%and 79.4%larval and pupal mortality at 0.2, 0.1 and 0.05 g/m2 after 4.3, 6.0 and 5.7 days, respectively. The cor-responding killing effect of chinaberry was 80.3%, 62.1%and 30.3%after 7.0, 7.7 and 8.3 days respectively. Bti at all treatments killed 100%after 24 h except 2.7 days for 0.05 g/m2. Adult mosquitoes were susceptible only for fenitrothion and pirimiphos-methyl with 100%mortality while resistant to deltamethrin, alpha-cypermethrin, etofenprox and dichloro-diphenyl-tricgloroethane with only 9.0%, 3.0%, 5.1%and 2.0%mortalities respectively. Conclusions: Neem, chinaberry and Bti showed potent larvicidal and pupicidal activ-ities. However, in the area, high level of mosquito resistance to pyrethroids and dichloro-diphenyl-tricgloroethane was seen which will pose serious challenge to vector control in the future. Therefore, using integrated approach including these botanical larvicides is warranted to manage insecticide resistance.

Larvicidal activities of chinaberry, neem and Bacillus thuringiensis israelensis (Bti) to an insecticide resistant population of Anopheles arabiensis from Tolay, Southwest Ethiopia

Objective To elucidate the larvicidal potency of neem, chinaberry and Bacillus thuringiensis israelensis (Bti) to larvae of Anopheles arabiensis under semi-field condition and adult susceptibility/resistance to the conventionally used insecticides in Tolay, Southwestern Ethiopia. Methods Wild collected 3rd and 4th stage larvae were exposed to neem, and chinaberry seed powder dissolved in water and Bti in artificial containers at three treatment levels: 0.2, 0.1 and 0.05 g/m

Monitoring the durability of long-lasting insecticidal nets in field conditions in Nepal

Understanding and improving the durability of long-lasting insecticidal nets (LLINs)in the field is critical for the success of malaria prevention using mosquito nets,as well as contributing to procurement decisions based on the number of yearsof protection, rather than the current practice of unit cost. Using the recentlypublished guidelines from the World Health Organization (WHO) some progresshas been made in the monitoring and assessment of performance of nets in thefield. This paper describes the protocol of an ongoing retrospective study of theattrition rate, physical integrity and bioefficacy of three polyester LLIN productsthat were distributed during 2010 to 2013 in Nepal. It is hoped that robust andauditable data on net survival (physical integrity and bioefficacy) of these threebrands in different environments will assist the Nepal National Malaria ControlProgramme in planning future LLIN-replacement strategies, including behaviourchange communication about LLIN care and maintenance. The advantages anddisadvantages of prospective and retrospective cross-sectional approachesare discussed, including appropriate strategies to validate the timing for massdistribution of nets. Similar studies should be done in other countries to (i) trackLLIN durability to support management of resupply, and (ii) inform procurementdecisions at the global level. New, more predictive, textile laboratory testing is alsourgently needed.

In vitro anti- bacterial activity of leaves extracts of Albizia lebbeck Benth against some selected pathogens

Objective: To screen the anti-bacterial activity of Albizia lebbeck (A. lebbeck) Benth leaves extract against the selected bacterial pathogens viz., Bacillus subtilis (MTCC441), Escherichia coli (MTCC443), Klebsiella pneumonia (MTCC 109), Proteus vulgaris (MTCC742), Pseudomonas aeruginosa (MTCC741), Salmonella typhii (MTCC733) and Staphylococus aureus (MTCC96).Methods:The leaves extracts of A. lebbeck was tested against bacteria by the agar disc diffusion method. Results: Results of the present study indicated that different extracts of A. lebbeck showed inhibitory effects against the pathogens. The present study results demonstrated that methanolic extracts of A. lebbeck conferred the widest spectrum activities that inhibited the growth of all studied pathogens with the maximum zone of inhibition. The methanolic extracts ofA. lebbeck illustrated the highest zone of inhibition against the pathogens Bacillus subtilis (16 mm), Escherichia coli (22 mm), Klebsiella pneumonia (11 mm), Proteus vulgaris (18 mm), Pseudomonas aeruginosa (22 mm), Salmonella typhii (23 mm) and Staphylococus aureus (17 mm). The ethyl acetate extracts demonstrated maximum zone of inhibition against Escherichia coli (26 mm), Pseudomonas aeruginosa (22 mm) and Klebsiella pneumonia (16 mm). Conclusions: It is expected that this study would direct to the establishment of some active compounds that could be used to formulate new and more potent anti-bacterial drugs of natural origin.

Preliminary Phytochemical, UV-VIS, HPLC and Anti-bacterial Studies on Gracilaria corticata J. Ag

Objective: The present study aimed to investigate the preliminary phytochemical analysis and UV-VIS, HPTC profiling and the antibacterial activity of Gracilaria corticata J. Ag extracts against the Gram positive and Gram negative bacteria. Methods: Preliminary phytochemical screening was carried out by Harborne method. The G. corticata extracts were tested against bacteria by the agar disc diffusion method. Results: The results of the presence study showed the presence of alkaloids, steroids, phenolic groups, saponins, tannin, flavonoids, terpenoids, glycosides and sugars. Proteins, xantoproteins, coumarins and catechin did not show any positive result for their presence in any of the six extracts of Gracilaria corticata tested. The result of the present study revealed the various behavior character of Gracilaria corticata crude drug. The UV-VIS spectrum profile of Gracilaria corticata methanolic, petroleum ether, benzene and aqueous extracts profiles were recorded. The HPLC profile of Gracilaria corticata petroleum ether benzene and aqueous extracts were tabulated. The maximum (9/12 bacterial pathogens) degree of antibacterial activity was observed in isopropanol soxhlet extracts followed by isopropanol cold extracts (7/12 bacterial pathogens). Conclusion: The results of the present study showed that G. corticata may be rich sources of phytoconstituents which can be isolated and further screened for different kinds of biological activities, depending on their reported therapeutic uses.

Bioefficacy ofMentha piperita essential oil against dengue fever mosquito Aedes aegypti L

Objective:To assess the larvicidal and repellent potential of the essential oil extracted from the leaves of peppermint plant,Mentha piperita (M. piperita) against the larval and adult stages of Aedes aegypti (Ae. Aegypti).Methods: The larvicidal potential of peppermint oil was evaluated against early fourth instar larvae ofAe. aegypti usingWHO protocol. The mortality counts were made after 24 and48 h, and LC50 and LC90values were calculated. The efficacy of peppermint oil as mosquito repellent was assessed using the human-bait technique. The measured area of one arm of a human volunteer was applied with the oil and the other arm was applied with ethanol. The mosquito bites on both the arms were recorded for3 min after every15 min. The experiment continued for 3 h and the percent protection was calculated.Results:The essential oil extracted fromM. piperita possessed excellent larvicidal efficiency against dengue vector. The bioassays showed an LC50 and LC90 value of111.9 and295.18 ppm, respectively after24 h of exposure. The toxicity of the oil increased11.8% when the larvae were exposed to the oil for48 h. The remarkable repellent properties ofM. piperita essential oil were established against adults Ae. aegypti. The application of oil resulted in100% protection till150 min. After next30min, only1-2 bites were recorded as compared with8-9 bites on the control arm.Conclusions:The peppermint essential oil is proved to be efficient larvicide and repellent against dengue vector. Further studies are needed to identify the possible role of oil as adulticide, oviposition deterrent and ovicidal agent. The isolation of active ingredient from the oil could help in formulating strategies for mosquito control.

Bioefficacy of Mentha piperita essential oil against dengue fever mosquito Aedes aegypti L

<p><b>OBJECTIVE</b>To assess the larvicidal and repellent potential of the essential oil extracted from the leaves of peppermint plant, Mentha piperita (M. piperita) against the larval and adult stages of Aedes aegypti (Ae. Aegypti).</p><p><b>METHODS</b>The larvicidal potential of peppermint oil was evaluated against early fourth instar larvae of Ae. aegypti using WHO protocol. The mortality counts were made after 24 and 48 h, and LC50 and LC90 values were calculated. The efficacy of peppermint oil as mosquito repellent was assessed using the human-bait technique. The measured area of one arm of a human volunteer was applied with the oil and the other arm was applied with ethanol. The mosquito bites on both the arms were recorded for 3 min after every 15 min. The experiment continued for 3 h and the percent protection was calculated.</p><p><b>RESULTS</b>The essential oil extracted from M. piperita possessed excellent larvicidal efficiency against dengue vector. The bioassays showed an LC50 and LC90 value of 111.9 and 295.18 ppm, respectively after 24 h of exposure. The toxicity of the oil increased 11.8% when the larvae were exposed to the oil for 48 h. The remarkable repellent properties of M. piperita essential oil were established against adults Ae. aegypti. The application of oil resulted in 100% protection till 150 min. After next 30 min, only 1-2 bites were recorded as compared with 8-9 bites on the control arm.</p><p><b>CONCLUSIONS</b>The peppermint essential oil is proved to be efficient larvicide and repellent against dengue vector. Further studies are needed to identify the possible role of oil as adulticide, oviposition deterrent and ovicidal agent. The isolation of active ingredient from the oil could help in formulating strategies for mosquito control.</p>

Comparative study on production, purification of penicillin by Penicillium chrysogenum isolated from soil and citrus samples

<p><b>OBJECTIVE</b>To explore various unexplored locations where Penicillium spp. would be available and study the production of penicillin from the isolated Penicillium spp. in different media with altered carbohydrate source.</p><p><b>METHODS</b>The collected soil samples were screened for the isolation of Penicillium chrysogenum (P. chrysogenum) by soil dilution plate. The isolated Penicillium species were further grown in different production media with changes in the carbohydrate source. The extracted penicillin from various isolates was analyzed by HPLC for the efficacy of the product. Further the products were screened with various bacterial species including methicillin resistant Staphylococcus aureus (MRSA). And the work was extended to find the possible action on MRSA, along with characterization using other pathogens.</p><p><b>RESULTS</b>From the various soil and citrus samples used for analysis, only the soil sample from Government General Hospital of Bangalore, India, and Sanjay Gandhi Hospital, Bangalore, India, showed some potential growth of the desired fungi P. chrysogenum. Different production media showed varied range of growth of Penicillium. Optimum production of penicillin was obtained in maltose which proved maximum zone of inhibition during assay. Characterization of penicillin on pathogens, like wild Escherichia coli strain, Klebsiella spp., and MRSA, gave quite interesting results such as no activity on the later strain as it is resistant. HPLC data provided the analytical and confirmation details of the penicillin produced. Accordingly, the penicillin produced from the soil sample of Government General Hospital had the high milli absorbance unit of 441.5 mAu compared with that of the penicillin produced from Sanjay Gandhi Hospital sample, 85.52 mAu. Therefore, there was a considerable change in quantity of the penicillin produced from both the samples.</p><p><b>CONCLUSIONS</b>The Penicillium spp. could be possibly rich in hospital contaminants and its environments. This research focuses on various unexplored sources of medical ailments, and also shows that the growth of penicillin is high in maltose rich media that could possibly enhance the growth.</p>