Introduction

Cereals grains are used to fulfill the dietary needs of mankind throughout the world1. In Pakistan, wheat and its products are consumed as the staple food2. Cereals grains are damaged by various insect pests both at pre-harvest and post-harvest stages3.

The red flour beetle, Tribolium castaneum (Herbst) 1797 (Coleoptera: Tenebrionidae), is a well-known insect pest that causes severe losses in broad range of stored grains worldwide4,5. The adult and larvae of the T. castaneum feed upon the wide range of edible materials like cereals products6. Severely infested flour becomes unfit for human consumption due to production of aflatoxins by T. castaneum7. Usage of chemicals including fumigants8 and contact insecticides9is a familiar practice for the control of insect pests of stored grains. Insecticides provide immediate result but continuous application leads to failure of product due to resistance development, pesticides residues and environmental issues10,11. In Pakistan, different pesticides like phosphine, malathion, methyl-bromide, chlorpyrifos-methyl, deltamethrin are commoly used for stored pests (supplentary file) pesticides used for stored product protection. There are no proper recomendations for the application of chemicals. Therefore, injudicious pesticides application is the most common practice12, which leads to pesticides resistance. Many scientists have studied resistance of different insecticides in stored product insect pests including T. castaneum13,14,15. Determining the susceptibility status is an important step. Such surveys help the scientists to develop proactive resistance management strategies, and judicious use of the insecticide which also protect the environment and also minimizes the resistance development. Variations in susceptibility is a regular phenomenon that occurs continuously depending upon the insecticide use patterns, type of insect species exposed, time and area16. Previously, susceptibility status of the T. castaneumin different insecticidesis determined by different authors17,18,19,20. Therefore reports manifesting the baseline susceptibility of a particular species to different commercially available synthetic insecticides are of dire need for its effective control.

Until now, there is no published report that provides the baseline data of the susceptibility of the T. castaneum populations collected from different districts of Punjab, Pakistan to different synthetic insecticides. Therefore, this study was aimed to monitor the geographical variation in the susceptibility levels in T. castaneum to 22 commercially available synthetic insecticides.

Materials and methods

Collection of the insects

Population of T. castaneum containing 100–150 adults were collected from five different district of Punjab, Pakistan. Insects were collected from different flour mills situated in Multan (30. 501100 N, 71. 3901500 E), Bahwalpur (29.3957° N, 71.6833° E), Lodhran (29. 1502500 N, 71. 3206000 E), Muzaffargarh (30. 40000 N, 71. 120000 E), and Vehari (30.0452° N, 72.3489° E).

Rearing of T. Castaneum in laboratory

Populations from respective localities were abbreviated as Multan (MLT), Bahwalpur (BWP), Lodhran (LDR), Muzaffargarh (MZG) and Vehari (VHR). All populations were reared and maintained separately in plastic jars of 1000 ml capacity covered with the muslin cloth. Populations were reared on 800gm wheat flour in combination with the yeast in 4:1 on weight basis (culture media). Insects were reared for one generation under laboratory conditions (27 ± 2 °C, 65%±5RH)21. The culture media containing adult T. castaneum was kept in darkness for mass production aimed at to obtain next generation22. After 7 days, adults (collected from field) were removed from culture media using 25mesh (600 μm) sieve23. Afterwards, culture media containing eggs and larvae was retained for rearing until they transform into adults (F1). Freshly emerged adults were separated to get homogenous population (F1) for bioassays. All the experiments were performed under above mentioned laboratory conditions.

Adulticide bioassay

Bioassays on all populations were carried out by using two to three days old adults (F1).An impregnated-paper assay method following Babarinde, Oyegoke and Adekunle4 was used to determine toxicity of different insecticides enlisted in (Table S1).

Five concentrations causing > 0 and < 100% mortality were prepared by serial dilution method. The What man’s paper No.2 of Petri-dish size was diluted with the aqueous solution of insecticide and was left exposed to open air for 3–4 min at 25–30 °C to evaporate extra moisture. Ten adults of T. castaneum were exposed in each Petri dish on filter paper treated with insecticides solution. Petri-dishes were covered with lid so that adults may not run out of dishes24. In Control group filter paper is dipped in distilled water only. One petri-dish was considered as one replicate for each treatment. Each treatment was replicated thrice. Mortality was determined after 48 and 72 h of treatment for conventional and new chemistry insecticides classified in (Table S1)16. Insects unable to move were considered dead.

Larvicidal bioassay

Larval toxicity of four insect growth regulators mentioned in (Table S1) was evaluated against the 5th larval instar of T. castaneum following Kostyukovsky and Trostanetsky25 with some modifications. Five concentrations (causing > 0 and < 100% mortality (determined first concentration by hit and trial method) mixed with water were prepared for each insecticide. To prepare the toxic arena, stock solution was made and prepared concentrations were mixed completely in wheat flour (20gram) and placed at room temperature until completely dried. Plastic cups were used for holding insecticide treated wheat flour to expose larvae. Plastic cups were considered as one replicate and each treatment was replicated three times. In this study, thirty 5th instar larvae were used per treatment. In control groups, untreated flour was used. The data was recorded at adult emergence. All those larvae which donot develop into adults were considered as dead.

Statistical analysis

The concentration response data was analyzed by probit analysis to determine the median lethal concentration (LC50) values, their 95% confidence intervals (CI), slope ± standard error (SE), and chi square (χ2). LC50values were considered significantly different when their 95% CI did not overlap26. The most susceptible population of the T. castaneum (out of the tested populations/collected from different locations) to a particular insecticide was considered as the reference strain27.

Results

Baseline susceptibility of field populations of T. castaneum against pyrethroids

Base-line susceptibility of field populations of T. castaneum to 22 synthetic insecticides was provided in (Table S2 and Table S3). Emamectin benzoate was the most toxic insecticide among all tested populations based on non-overlapping of 95% CI of LC50 values. There was non-significant difference in toxicity of deltamethrin and triazophos to all tested populations based on overlapping 95% Cl of LC50. Toxicity of bifenthrin was similar for LDR, VHR and BWP and significantly lower in MLT and MZG compared with LDR based on non-overlapping 95% Cl of LC50. LC50 value of lambda-cylothrin for BWP was similar to that of MLT and VHR based on overlapping 95% Cl. While LDR and MZG showed a significantly higher LC50 value than BWP (Fig. 1).

Fig. 1
figure 1

Baseline susceptibility of Tribolium castaneum populations at different locations of south Punjab Pakistan against pyrethroids.

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Baseline susceptibility of field populations of T. castaneum against organophosphates

Profenofos was similarly toxic to BWP, LDR and MZG and significantly lower toxicity to MLT and VHR as compared with BWP based on non-overlapping 95% Cl. Chlorpyrifos expressed similar level of toxicity to BWP, LDR, MZG and VHR based on LC50 (overlapping 95% CI). A significantly higher LC50 value was observed in MLT as compared with BWP based on non-overlapping 95% Cl. In case of diamethoate, LC50 value was similar in all tested populations based on overlapping 95% Cl of LC50. Methomyl showed similar toxicity to BWP, VHR and MZG based on overlapping 95% Cl of LC50. A low level of toxicity towards MLT was observed as compared with BWP based on non-overlapping 95% Cl (Fig. 2).

Fig. 2
figure 2

Baseline susceptibility of Tribolium castaneum populations at different locations of south Punjab Pakistan against organophosphates.

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Baseline susceptibility of field populations of T. castaneum against new chemistry insecticides

In case of spinosad, similar level of toxicity was observed for LDR, MZG, VHR and BWP based on overlapping 95% CI of LC50. However, a significantly lower toxicity to MTN was observed as compared with LDR based on non-overlapping 95% CI. Fipronil showed similar toxicity to VHR, BWP and MZG based on overlapping 95% CI of LC50. Moreover, fipronil expressed low toxicity to LDR and MTN as compared with VHR based on non-overlapping 95% CI. Clothianidin was similarly toxic to BWP, VHR, LDR and MZG based on overlapping 95% CI of LC50. However, significantly low toxicity to MTN as compared with BWP based on non-overlapping 95% CI. In case of acetamaprid, spiromesifen, nitenpyram, emamectin benzoate, spinetoram and spirotetramat, there was non- significant difference in LC50 values of the all tested strains of T. castaneum based on overlapping 95% CI. Toxicity of indoxacarb was similar for MTN, LDR and MZG based on overlapping 95% CI of LC50 and significantly lower in BWP and VHR as compared with MTN based on non-overlapping of 95% CI (Fig. 3).

Fig. 3
figure 3

Baseline susceptibility of Tribolium castaneum populations at different locations of south Punjab Pakistan against new chemistry insecticides.

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Toxicity of different insect growth regulators to larvae of T. Castaneum

There is non-significant difference in LC50 of lufenuron for all the tested populations based onoverlapping 95% Cl of LC50. Pyriproxyfen has similar toxicity for LDR, BWP and VHR based on overlapping 95% CI of LC50 (Table S2). While the MTN and MZG expressed significantly higher LC50 than LDR.VHR was the most susceptible population to methoxyfenozide compared with all tested populations based on non-overlapping 95% CI of LC50. Cyromazine has shown similar toxicity to VHR, MZG and BWP based on overlapping 95% CI of LC50. While, low toxicity to MTN and LDR was observed as compared with VHR due to non-overlapping of 95% CI (Fig. 4).

Fig. 4
figure 4

Baseline susceptibility of Tribolium castaneum populations at different locations of south Punjab Pakistan against organophosphates.

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Discussion

Our results indicate that emamactin benzoate was the most effective insecticide among all used insecticides against T. castaneum. Emamectin benzoate is a derivative of the natural product abamectin in the avermectin family of 16 membered macrocylic lactones leading to an increase in membrane chloride ion permeability due to a stimulator of the GABA receptors28,29. Previously, Ghelani, et al.30 reported that even after 12 months of storage, Pennisetum glaucum L seed treated with emamectin benzoate recorded the lowest damage and the highest germination .Thw findings of Study suggest that emamectin benzoate is an effective compound compared with other tested insecticides. It is a reduced risk insecticide and could be safely used in pest management programs of T. castaneum.

Organophosphates kill insects by inactivating the enzyme acetylcholinestrase31, an enzyme responsible for neurotransmitter degradation at the cholinergic nerve synapse. Among all the tested organophosphates, triazophos and dimethoate have similar toxicity to all the tested populations. Other tested organophosphates have differences in toxicity to the tested populations. Previously, Andric, Kljajic, Peric and Golic20 reported similar findings suggesting variations in toxicity of tested organophosphates. Toxicity of insecticide depends on many factors such as chemical composition of insecticide, frequency of using insecticide, weather conditions and on which development stages of insects being treated32.

Among the tested pyrethroids, deltamethrin has similar toxicity to all tested populations of T. castaneum. In many previous studies, deltamethrin showed high toxicity against T.castaneum20,33. In another study, field strains of T. castaneum showed reduced susceptibility to the mixture of cholopyrifos-methyl with deltamethrin and also to β-cyfluthrin34. Other tested pyrethroids showed differential toxicities to different tested populations. Previously, Lloyd35 reported dissimilar toxicity of tested pyrethroids to three different insect strains, T.castaneum, susceptible and pyrethrin-resistant Sitophilus granarius L 1875 (Coleoptera: Cruculionidae), using topical application techniques.

Neonicotinoids possess a mode of action as the nicotinic acetylcholine receptor blocker in the insect nervous system36. Neonicotinoids are systemic insecticides including acetamiprid and clothianidin and nitenpyram37.In the current study, all the neonicotinides have similar toxicity to all tested populations except clothianidin (low toxicity to MTN population compared with others). Previously, Daglish and Nayak38 reported that neonicotinides has potential to control stored product pests.

Spinosyns, nicotinic acetylcholine receptor allosteric modulators are active against insect pests especially lepidopterans and dipterans and have also an antagonistic effect39. In our results, spinosad was more toxic as compared with spinetoram. Previously, the lowest population of T. castaneum was observed in Pennisetumg laucum L.seed treated with spinosad30. Possible reason for the spinosad being the more toxic to T. castaneum as compared with the spintoram could be its comparatively less use in the chemical control management of the stored grains insect pests. Moreover, there is no knowledge about usage of chemical spray program and guidelines for the management of T. castaneum in Pakistan.

The ketoenols are derivatives of tetronic acid (spirocyclictetronic acids) and act via inhibition of lipid biosynthesis40,41. The effects of spiromesifen on the developmental stages of the whitefly Bemisi atabaci (Gennadius) were studied under laboratory conditions to generate baseline action thresholds for field evaluations of the compounds42. Overall, spiromesifen was less toxic as compared with the spirotetramat. Previously, contrary to our results, spiromesifen showed higher toxicity as compared to the spirotetramat against the adults of the Tetranychus urticae Koch 1836 (Acari: Tetranychidae)43. The toxicity of pesticides depends on microclimate condition, usage rates, chemistry of pesticides, and development stages of the insect pest under consideration44. Indoxacarb is a pyrazoline-type which blocks voltage-dependent sodium channels, preventing the influx of sodium into neurons against a wide range of lepidopteran, coleopteran, and sucking insect pests45.The suggested variable in toxicity of insecticide belonging to the same chemical groups has some practical implications. It is usually suggested to choose such chemical which is highly toxic to target pest specie. However, use of one chemical for target pest, for long term in pest control programs results in ultimate failure of product due to development of resistance against that product46. Therefore, spinosad and the most toxic insecticides from other groups should be used in combination with other insecticides for long term control.

Four IGRS, leufenuron, pyriproxyfen, methoxyfenozide and cyromazine as larvicides were tested to assess their toxicity. Leufenuron and pyriproxyfen were the most toxic among all the tested IGR’s. Use of IGR’s are the most efficacious method of killing immature larvae before developing into adult47. Among other Insecticides used against red flour beetle, the insect growth regulators are the strong candidates for IPM because of their tremendous selectivity, powerful pest controlling ability, and relatively less development of resistance in major insect pests48. Therefore, it is wiser choice to test IGRs for the management of stored products to reduce reliance on traditional chemicals available49.In present study, among tested IGRs, lufenuron and pyriproxyfen were the most toxic larvicides and therefore should be used in rotation with each other or with methoxyfenozide, a moderately toxic larvicide. Pyriproxyfen, a juvenile hormone mimic, an inhibitor of pupal adult metamorphosis, is a potent insecticide recommended for the control of medical pests like mosquitoes and house flies50. Previously, the pyriproxyfen showed the higher toxicity as compared with diflubenzuron and methoprene against A. albopictus 1894 (Diptera: Culicidae)51. Lufenuron is an inhibitor of the chitin synthesis and is also a potent chemical for integrated pest management (IPM) due to its unique mode of action and less toxic to environment50. However, the success of the insecticide rotation program depends upon the cross-resistance mechanism that is needed to be tested in the future to prolong the potential of these insecticides.

Conclusions

It is concluded that different strains of T. castaneum had differences in susceptibility to different tested insecticides. Emamectin benzoate was the most effective to control red flour beetle. Moreover, the baseline data presented here for different field strains can be used to monitor changes in susceptibility of the T. castaneum in future.