Given the threat presented by parasites and pathogens, insects employ various defences to protect themselves against infection, including chemical secretions. The red flour beetle Tribolium castaneum releases a secretion containing the benzoquinones methyl-1,4-benzoquinone (MBQ) and ethyl-1,4-benzoquinone (EBQ) into the environment. These compounds have known antimicrobial effects; however, their role in defence against macroparasites is not known. Entomopathogenic nematodes, such as Steinernema carpocapsae, present a serious threat to insects, with successful infection leading to death. Thus, quinone-containing secretions may also aid in host defence. We tested how exposure to the individual components of this quinone secretion, as well as a mix at naturally-occurring proportions, affected the survival and thrashing behaviour of S. carpocapsae, as well as their virulence to a model host (Galleria mellonella). Exposure to high concentrations of MBQ and EBQ, as well as the quinone mix, significantly increased nematode death but did not consistently reduce thrashing, which would otherwise be expected given their toxicity. Rather, quinones may act as a host cue to S. carpocapsae by triggering increased activity. We found that exposure to quinones for 24 or 72 hours did not reduce nematode virulence, and surviving nematodes remained infective after non-lethal exposure. Our results indicate that quinone secretions likely serve as a defence against multiple infection threats by reducing S. carpocapsae survival, but further research is required to contextualize their roles by testing against other nematodes, as well as other helminths using insects as hosts.

Zinc finger protein (Zelda) of Tribolium castaneum (TcZelda) has been showed to play pivotal roles in embryonic development and metamorphosis. However, the regulatory mechanism of TcZelda associated with these physiology processes is unclear. Herein, the developmental expression profile showed that Zelda of T. castaneum was highly expressed in early eggs. Tissue expression profiling revealed that TcZelda was mainly expressed in the larval head and adult ovary of late adults and late larvae. TcZelda knockdown led to a 95% mortality rate in adults. These results suggested that TcZelda is related to the activation of the zygote genome in early embryonic development. Furthermore, 592 differentially expressed genes were identified from the dsZelda treated group. Compared with the control group, altered disjunction (ALD) and AGAP005368-PA (GAP) in the dsZelda group were significantly down-regulated, while TGF-beta, propeptide (TGF) was significantly up-regulated, suggesting that TcZelda may be involved in insect embryonic development. In addition, the expression of Ubx ultrabithorax (UBX), Cx cephalothorax (CX), En engrailed (EN), and two Endocuticle structural glycoprotein sgabd (ABD) genes were significantly down-regulated, suggesting that they may cooperate with TcZelda to regulate the development of insect wings. Additionally, Elongation (ELO), fatty acid synthase (FAS), and fatty acyl-CoA desaturase (FAD) expression was inhibited in dsZelda insects, which could disturb the lipase signaling pathways, thus, disrupting the insect reproductive system and pheromone synthesis. These results may help reveal the function of TcZelda in insects and the role of certain genes in the gene regulatory network and provide new ideas for the prevention and control of T. castaneum.

Cytochrome P450 proteins (CYPs) in insects can encode various detoxification enzymes and catabolize heterologous substances, conferring tolerance to insecticides. This study describes the identification of a P450 gene (CYP6BQ8) from Tribolium castaneum (Herbst) and investigation of its spatiotemporal expression profile and potential role in the detoxification of terpinen-4-ol, a component of plant essential oils. The developmental expression profile showed that TcCYP6BQ8 expression was relatively higher in early- and late-larval stages of T. castaneum compared with other developmental stages. Tissue expression profiles showed that TcCYP6BQ8 was mainly expressed in the head and integument of both larvae and adults. The expression profiling of TcCYP6BQ8 in developmental stages and tissues is closely related to the detoxification of heterologous substances. TcCYP6BQ8 expression was significantly induced after exposure to terpinen-4-ol, and RNA interference against TcCYP6BQ8 increased terpinen-4-ol-induced larval mortality from 47.78 to 66.67%. This indicates that TcCYP6BQ8 may be involved in T. castaneum's metabolism of terpinen-4-ol. Correlation investigation between the CYP6BQ8 gene and terpinen-4-ol resistance in T. castaneum revealed that the TcCYP6BQ8 gene was one of the factors behind T. castaneum's resistance to terpinen-4-ol. This discovery may provide a new theoretical foundation for future regulation of T. castaneum.

Aldehyde oxidases (AOXs) are a group of metabolic enzymes that play critical roles in the degradation of xenobiotics and chemicals. However, the physiological function of this enzyme in insects remains poorly understood. In this study, three TcAOX genes (TcAOX1, TcAOX2, TcAOX3) were identified and characterized from Tribolium castaneum genome. Spatiotemporal expression profiling showed that TcAOX1 expression was most highly expressed at the early pupal stage and was predominantly expressed in the antennae of adults, indicating that TcAOX1 was involved in the degradation of chemical signals; TcAOX2 expression was most highly expressed at the late pupal stage and was mainly expressed in the fat body, epidermis of larvae and adults, respectively; and TcAOX3 expression was in all stages and was primarily expressed in the head of adults. Moreover, the transcripts of TcAOX2 and TcAOX3 were significantly induced after exposure to plant oil, and RNA interference (RNAi) targeting of each of them enhanced the susceptibility of beetles to this plant toxicant, suggesting that these two genes are associated with plant toxicant detoxification. Intriguingly, knockdown of the TcAOX1 led to reductions in female egg-laying but unchanged the hatchability and the development of genital organs, suggesting that this gene may mediate fecundity by effecting the inactivation of chemical signals in T. castaneum. Overall, these results shed new light on the function of AOX genes in insects, and could facilitate the development of research on pest control management.

This study was carried out to investigate the efficacy of the non-thermal atmospheric pressure plasma produced with dielectric barrier discharge (APPD) using air as a processing gas and microwave energy to control Tribolium castaneum and Trogoderma granarium adults and larvae in wheat grains. Insects’ mortality was found to be power and time-dependent. The results indicated that non-thermal APPD and the microwave have enough insecticidal effect on the target pests. From the bioassay, LT50's and LT90's levels were estimated, T. granarium larvae appeared more tolerant to non-thermal APPD and the microwave energy than adults 7 days post-exposure. The germination percentage of wheat grains increased as the time of exposure to the non-thermal APPD increased. On the contrary, the germination percentage of wheat grains decreased as the time of exposure to the microwave increased. In addition, changes in antioxidant enzyme activities, catalase (CAT), glutathione S-transferase (GST) and peroxidase, in adults and larvae were examined after 24 h post-treatment to non-thermal APPD at 15.9 W power level, which caused 50% mortality. The activity of CAT, GST and lipid peroxide in the treated larvae showed a significant increase post-exposure to the non-thermal APPD at 15.9 W power level. On the other hand, no significant change in GSH-Px activity was observed. Reductions in the level of glutathione (GSH) and protein content occurred in treated larvae in comparison with the control.

Tribolium castaneum is one of the most economically important insects that damages stored products. The effects of several infested or uninfested raw feed materials (wheat bran, coarse wheat meal, corn feed flour), feed products (compound feed for pigs and for laying hens) and flour mixed with brewer's yeast on the food-searching behaviour of T. castaneum adults were studied in a total of 48 combinations. Preference and olfactometer tests revealed that all the tested uninfested and intraspecific infested substrates were significantly more attractive to T. castaneum than the control (represented by part of an arena or olfactometer arm without substrate). We determined that all infested substrates were 2–9 times more attractive than uninfested in the preference test, while in the olfactometer test, they were 3–8 times more attractive. In comparing the attractiveness of the infested and uninfested substrates, in both tests wheat bran was found to be the most attractive substrate to T. castaneum adults and coarse wheat meal the least. The results of the present study contribute to our knowledge of how raw feed materials and products influence the behaviour of T. castaneum and their susceptibility to infestation, and indicate the possible utilization of wheat bran in monitoring processes of T. castaneum in pest management programmes.

Latrophilin (LPH) is known as an adhesion G-protein-coupled receptor which involved in multiple physiological processes in organisms. Previous studies showed that lph not only involved the susceptibility to anticholinesterase insecticides but also affected fecundity in Tribolium castaneum. However, its regulatory mechanisms in these biological processes are still not clear. Here, we identified two potential downstream carboxylesterase (cce) genes of Tclph, esterase4 and esterase6, and further characterized their interactions with Tclph. After treatment of T. castaneum larvae with carbofuran or dichlorvos insecticides, the transcript levels of Tcest4 and Tcest6 were significantly induced from 12 to 72 h. RNAi against Tcest4 or Tcest6 led to the higher mortality compared with the controls after the insecticides treatment, suggesting that these two genes play a vital role in detoxification of insecticides in T. castaneum. Furthermore, with insecticides exposure to Tclph knockdown beetles, the expression of Tcest4 was upregulated but Tcest6 was downregulated, indicating that beetles existed a compensatory response against the insecticides. Additionally, RNAi of Tcest6 resulted in 43% reductions in female egg laying and completely inhibited egg hatching, which showed the similar phenotype as that of Tclph knockdown. These results indicated that Tclph affected fecundity by positively regulating Tcest6 expression. Our findings will provide a new insight into the molecular mechanisms of Tclph involved in physiological functions in T. castaneum.

Cashew nut shell liquid (CNSL) is an economically important by-product obtained from the cashew nut Anacardium occidentale L. The liquid has been found to be versatile with a wide range of uses, including as a raw material in the polymer industry, in the development of drugs (antioxidants) and in pest control. One important chemical component of CNSL known for its bioactivity is cardanol. We isolated (8Z,11Z)-3-(8,11,14-pentadecatrienyl)phenol, a cardanol from CNSL, and determined its insecticidal and progeny growth and development inhibition activities against Tribolium castaneum Hebst. We used a bioassay-guided approach for the isolation. Exposure to cardanol produced 80% mortality of adult T. castaneum in the insecticidal bioassay. In the progeny growth and development inhibition bioassay, 46.7% of larvae were killed, and only 20.7% of adults emerged without deformities after the cardanol treatment. The amount of isolated cardanol from the 1 g equivalent/ml extracted CNSL was found to be 36.55 mg with LC50 value of 47.88 mg/ml and 13.28 mg/ml against adult T. castaneum and its larvae, respectively. We conclude that cardanol can potentially be useful in managing stored product insect pests, especially T. castaneum, but with the prerequisite that one should take much care when handling and when applying the compound.

Attempts have been made in recent years to reduce the use of synthetic pesticides and seek alternative and innovative methods for pest control. We conducted a study on the chemical composition of Eucalyptus occidentalis essential oil and investigated the fumigant, contact and repellent effects against Tribolium castaneum (Herbst) (Col: Tenebrionidae) and Rhyzopertha dominica (F.) (Col: Bostrichidae). The major components were τ-cadinol (17.20), 1,8-cineol (15.5%), α-cadinol (14%) and α-pinene (9.21%). The contact toxicity experiment showed that the oil was more toxic to R. dominica (LD50 value 0.82 μL/cm) than to T. castaneum (LD50 value 0.99 μL/cm) and showed the same fumigant toxicity for both insects according to the 95% confidence limit of LC50. The repellency against T. castaneum was more than for R. dominica and increased from 14 to 75% for R. dominica and 22 to 78% for T. castaneum after 2 h. These results suggest that E. occidentalis essential oil is a potential candidate for use as a natural repellent and insecticide for stored-product insect pests.

Food losses caused by insects during postharvest storage are of paramount economic importance worldwide, especially in Africa. Laboratory bioassays were conducted in stored grains to determine the toxicity of powders of Eugenia aromatica and Moringa oleifera alone or combined with enhanced diatomaceous earth (Probe-A® DE, 89.0% SiO2 and 5% silica aerogel) to adult Sitophilus granarius, Tribolium castaneum and Acanthoscelides obtectus. Adult mortality was observed up to 7 days, while progeny production was recorded at 6–10 weeks. LD50 and LT50 values for adult test insects exposed to plant powders and DE, showed that A. obtectus was the most susceptible towards the botanicals (LD50 0.179% and 0.088% wt/wt for E. aromatica and M. oleifera, respectively), followed by S. granarius. Tribolium castaneum was most tolerant (LD50 1.42% wt/wt and 1.40% wt/wt for E. aromatica and M. oleifera, respectively). The combined mixture of plant powders and DE controlled the beetles faster compared to the plant powders alone. LT50 ranged from 55.7 h to 62.5 h for T. castaneum exposed to 1.0% M. oleifera and 1.0% DE, and 0.5% E. aromatica and 1.0% DE, respectively. Botanicals caused significant reduction of F1 adults compared to the control. Combined action of botanical insecticides with DE as a grain protectant in an integrated pest management approach is discussed.

RNA interference (RNAi) is a very effective technique for studying gene function and may be an efficient method for controlling pests. Trehalose-6-phosphate synthase (TPS), which plays a key role in the synthesis of trehalose and insect development, was cloned in Tribolium castaneum (Herbst) (TcTPS) and the putative functions were studied using RNAi via the injection of double-stranded RNA (dsRNA) corresponding to conserved TPS and trehalose-6-phosphate phosphatase domains. Expression analyses show that TcTPS is expressed higher in the fat body, while quantitative real-time polymerase chain reaction results show that the expression of four trehalase isoforms was significantly suppressed by dsTPS injection. Additionally, the expression of six chitin synthesis-related genes, such as hexokinase 2 and glutamine-fructose-6-phosphate aminotransferase, was suppressed at 48 and 72 h post-dsTPS-1 and dsTPS-2 RNA injection, which were two dsTPS fragments that had been designed for two different locations in TcTPS open reading frame, and that trehalose content and trehalase 1 activity decreased significantly at 72 h post-dsRNA injection. Furthermore, T. castaneum injected with dsTPS-1 and dsTPS-2 RNA displayed significantly lower levels of chitin and could not complete the molting process from larvae to pupae, revealing abnormal molting phenotypes. These results demonstrate that silencing TPS gene leads to molting deformities and high mortality rates via regulation of gene expression in the chitin biosynthetic pathway, and may be a promising approach for pest control in the future.

Tribolium castaneum Herbst 1797 (Coleoptera: Tenebrionidae), an important pest of stored grains and byproducts, is naturally infected by Gregarina cuneata Stein 1848 (Apicomplexa: Gregarinidae). Changes in the life cycle of insects caused by the parasite development in the midgut were studied. Trophozoites, gamonts (solitary and associated), and gametocysts were present in the midgut of the insects. In young trophozoites, the apical region differentiated into an epimerite that firmly attached the parasite to the host epithelial cells. With maturation, trophozoites developed in gamonts that were associated with the initiation of sexual reproduction in the cell cycle, culminating in the formation of the spherical gametocyst. Morpho-functional analyses indicated that gregarines absorb nutrients from infected cells and can occlude the midgut as they develop. Consequently, nutritional depletion may interfere with the host's physiology, causing decreased growth, delayed development, and high mortality rates of the parasitized insects. These results suggest G. cuneata could be an important biological agent for controlling T. castaneum in integrated pest management programs.

The structure of two compounds extracted from the leaves of Vitex negundo L., their repellent activity and toxicity against three strains of Tribolium castaneum (Herbst) were elucidated. Two compounds were purified and a residual film bioassay was used to evaluate the toxicity to adult T. castaneum. Mortality was recorded 24, 48 and 72 h post-exposure to compounds. The repellency activity of methanol extract was tested by using nine concentrations. Each dose was applied on a half circle of the filter paper, keeping the other part untreated. Insects released on treated filter paper were assessed for repellency up to 5 h. Two bioactive compounds, 22,23-dihydro-α-spinasterol-β-d-glucoside and salicylic acid were isolated from the methanolic leaf extracts by column chromatography and thin-layer chromatography. Their structures were elucidated by various spectroscopic methods. Compound 22,23-dihydro-α-spinasterol-β-d-glucoside is the first report from this plant. Both compounds showed insecticidal activity and the leaf extracts caused 100% repellency in the three tested strains of T. castaneum. Both compounds showed toxicity to T. castaneum, but compound 22,23-dihydro-α-spinasterol-β-d-glucoside was more potent. Its potential future use as herbal insecticide against T. castaneum is discussed.

Insect pests cause major damage to stored grain and foodstuffs, reducing the products' weight, quality and market value. Eight spices commonly used to add flavour and colour to food were evaluated, singly and in combination, for their contact and fumigant toxicity, repellency and effects on progeny development against the cowpea weevil Callosobruchus maculatus F. and rust red flour beetle Tribolium castaneum Herbst. The powders of large cardamom, turmeric and ginger showed contact toxicity against the two test insects. Green cardamom powder was found to have fumigant toxicity against adults of both insects. Clove powder at 1.5 g/50 g dose showed 100% repellency against adults of T. castaneum. The powders of large cardamom, cinnamon and clove caused complete inhibition of progeny production in C. maculatus and T. castaneum at a dose of 5 g/100 g. A 1:1 mixture of green cardamom and turmeric showed both contact and fumigant toxicity against C. maculatus, while a mixture of clove and large cardamom showed repellency and inhibited progeny development in T. castaneum.

Perovskia abrotanoides Karel is a wild growing plant in Iran and has been used in traditional Iranian herbal medicine. The present study was conducted to investigate chemical composition and fumigant toxicity of the essential oil from P. abrotanoides against Sitophilus oryzae (L.) and Tribolium castaneum (Herbst). Dry flowering aerial parts of the plant were subjected to hydrodistillation using a modified Clevenger-type apparatus. The composition of the essential oil was analysed by gas chromatography (GC) and GC mass spectrophotometry. Twenty-four compounds representing 98.8% of total oil were identified. The predominant components in the oil were camphor (28.38%) and 1,8-cineole (23.18%). Fumigant toxicity was tested against 1- to 7-day-old adults of S. oryzae and T. castaneum with five replications at 25 ± 1 °C and 65 ± 5% relative humidity in dark conditions. The mortality was increased with concentrations of 32, 161, 322, 483 and 645 μl/l air and with exposure time from 2 to 15 h. The lowest concentration (32 μl/l air) of the oil induced 100% mortality of S. oryzae and T. castaneum after 15 and 8 h exposure, respectively. The oil at 322 μl/l air caused 100% mortality for S. oryzae and T. castaneum within 13 and 7 h exposure, respectively. At 645 μl/l air, the LT50 values (lethal time for 50% mortality) were 8 and 2.84 h for S. oryzae and T. castaneum, respectively. In the probit analysis, LC50 values (lethal concentration for 50% mortality) showed that T. castaneum (LC50 = 11.39 μl/l) was more susceptible than S. oryzae (LC50 = 18.75 μl/l). The essential oil of P. abrotanoides can play an important role in stored grain protection and reduce the need for the same, and also the risks associated with the use of synthetic insecticides.

The effects of methanol extract of Peganum harmala L. seeds were evaluated against Tribolium castaneum (Herbst). Larval growth was inhibited and food intake reduced when the extract was incorporated into the diet at concentrations of 2.5, 5 and 10%. All doses caused an increase in the duration of larval period and completely inhibited F1 progeny production. The main body metabolites were determined in last-instar larvae after feeding for 8 days on the diet treated with different doses. Protein and lipid contents were decreased with concentrations >2.5%. Glycogen content was reduced at a concentration of 10%. Tribolium castaneum larvae possessed two major and one minor α-amylase isoforms as determined by gel assays. Larvae fed the treated diet presented less activity of the major isoforms, while activity of the minor isoform was totally inhibited. The activity of this enzyme is not inhibited in vitro by seed extracts. Histological studies showed that P. harmala extracts caused a severe cytotoxicity on the epithelial cells of the midgut resulting in marked vacuolization of the cytoplasm and a large intercellular space. These findings suggest that seed extracts of P. harmala exert their toxic effect by severely damaging the midgut epithelial cells. This naturally occurring plant extract could be useful for control of T. castaneum.

Essential oils of three genotypes of Tagetes minuta, six of Tagetes patula and 15 of Tagetes erecta were screened for potential fumigant and contact toxicity against three stored product beetle species, Callosobruchus maculatus (Fabricius), Sitophilus oryzae (Linnaeus) and Tribolium castaneum (Herbst). Genotype 1 of T. minuta (TM-1), genotype 2 of T. patula (TP-2) and genotype 13 of T. erecta (TE-13) were the most toxic. These three genotypes were then evaluated for adult toxicity, oviposition deterrence, and ovicidal and population reduction activities. Essential oils of genotypes TM-1 and TP-2 induced 100% adult mortality for all three insect species at dosages of 50,000 ppm and 500 μg/insect in fumigant and contact toxicity bioassays, respectively. Adults of C. maculatus and S. oryzae were highly susceptible to the essential oils of the three genotypes in both fumigant and contact toxicity bioassays. The essential oil of genotype TM-1 deterred oviposition in T. castaneum by 81% and suppressed its egg hatchability by 91% when applied at a dosage of 70,000 ppm on filter paper.

l-menthol isolated from the essential oil of Mentha arvensis and seven of its acyl derivatives were tested for contact and vapour toxicity, and ovicidal and repellent activity against the storage pests Callosobruchus maculatus F., Rhyzopertha dominica F., Sitophilus oryzae L. and Tribolium castaneum Herbst. Menthyl propionate and l-menthol were highly toxic in contact assays and vapour toxicity assays respectively. l-menthol was also found to be strongly repellent (82–100% at 0.353 μg/cm2 dose) against all the insects, while menthyl acetate was highly ovicidal against T. castaneum.

Tannin extracts of two sorghum, Sorghum bicolor (L.) Moench. cultivars, BKS5 and BTX 623 were assessed for their bioactivity against the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and the angoumois grain moth, Sitotroga cerealella (Olivier) (Lepidoptera: Gelechiidae), in whole wheat flour pellets, and against the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) in whole wheat flour at three treatment levels. The number of adult progeny was reduced and number of days to emergence increased by tannins extracted from both sorghum cultivars at all treatment levels. However, the effects were not significant in all cases. In a short-term (25 days) feeding test using S.oryzae adults, the presence of sorghum tannins from cv. BKS 5 in the pellets significantly reduced feeding by the insects.

Postembryonic development and adult emergence of Tribolium castaneum (Herbst) were adversely affected in varying degrees when its larval stage was reared on wheat flour treated separately (at concentrations of 0.001–1.0%) with one of the following commercially available extracts: roots of both sweet flag, Acorus calamus Linn. and Java devil pep per, Rauwolfia serpentina Benth ex Kurz, fruit cortex of soap nut, Sapindus trifoliatus Linn. and gum of Indian Myrrh Commiphora mukul Jacq. A marked decline in the reproductive potential, in terms of female fecundity and egg hatchability was observed when the extracts were applied topically or administered orally through diet.