Flour Beetle Research Articles

Detection Limits of Insect Fragments in Spiked Whole Wheat Flour Using Multiplex Polymerase Chain Reaction (PCR)

The need for a sensitive molecular method to detect specific species of insect contaminants in food products remains a significant challenge in the food industry. This study evaluated the detection limit of a multiplex end-point PCR assay for detecting insects in food. The assay amplifies two fragments of the cytochrome oxidase subunit I gene (COI-Fa and COI-Fb) and one fragment of the protein-coding wingless (wg) gene found in insects. Five insect species, comprising three vectors of foodborne pathogens (the housefly, Musca domestica, the American cockroach, Periplaneta americana, and the pharaoh ant, Monomorium pharaonis) and two storage insect pests (the red flour beetle, Tribolium castaneum and the Indian meal moth, Plodia interpunctella), were spiked separately and in combination at levels of 1, 0.1, 0.01, and 0.001% in whole wheat flour. At spike levels greater than 0.01%, amplicon bands of expected sizes were seen in 100% of samples containing fragments from distinct insect species. At least 25% of spiked samples at the lowest spike level had amplicon bands, except for samples spiked with M. domestica. Results showed an 18.9% probability (with 11.3% and 30% lower and upper confidence limits, respectively) of detecting insect fragments at the lowest spike level (0.001%, corresponding to 3–22 fragments), which is far below the FDA’s regulatory level of less than 75 fragments per 50 g of wheat flour. The intensity of amplicon bands in the gel images was higher at higher spike levels. However, this method is not quantitative enough to extrapolate the intensity of the amplicon bands to the number of insect fragments present in a sample. This multiplex assay was also evaluated in a variety of market food samples derived from plants and animals, showing its potential use in various food types. Overall, the sensitivity and specificity of this molecular approach suggest that it could be used in the future as a screening tool for detecting insect contaminants in food.

Beyond adult models: Tribolium castaneum larval timekeeping reveals unexpected robustness and insights into circadian clock.

Circadian rhythms are self-sustained endogenous oscillations that are found in all living organisms. In insects, circadian rhythms control a wide variety of behavioral and physiological processes, including feeding, locomotion, mating, and metabolism. While the role of circadian rhythms in adult insects is well-understood, it is largely unexplored in larvae. This study investigates the potential for larval synchronized activity in the red flour beetle (Tribolium castaneum), a species exhibiting solitary and aggregation phases. We hypothesized that, similar to adults, larvae would exhibit a daily activity pattern governed by an endogenous circadian clock. We further predicted that the transition between the solitary and gregarious phases extends to unique temporal activity patterns. Our results revealed unique timekeeper gene expression in larvae, leading to a distinct daily rhythm characterized by nocturnal activity. Cues indicating on potential cannibalism did not change daily activity peak. However, the absence of these cues significantly reduced the proportion of rhythmic larvae and led to higher variation in peak activity, highlighting the crucial role of social interactions in shaping their rhythmicity. This study sheds light on the evolution and function of larval synchronization in group-living insects, offering novel insights into this complex behavior.

Effects of nutrition on recovery, mortality, and mobility of adult Tribolium castaneum after exposure to long-lasting insecticide-incorporated netting.

Long-lasting insecticide-incorporated netting (LLIN) has been used to deliver contact insecticides as an integrated pest management tool for stored product insect pests in food facilities. Although the presence of food is known to improve insect recovery after exposure, it is not clear whether food nutritional quality plays a role. Here, the red flour beetle Tribolium castaneum adults were exposed to two commercially available LLINs, Carifend (active ingredient α-cypermethrin) and D-Terrence (deltamethrin), then transferred to Petri dishes with foods with varying nutritional quality (e.g., 0-100% ratios of flour to non-nutritive cellulose). We investigated the effects of nutrition, LLIN type, and exposure time on post-exposure recovery, mortality, and mobility. After exposure for 2-168 h, the immediate mortality of T. castaneum adults ranged from 0.5% to 91.0% with Carifend and 0% to 75.3% with D-Terrence. Adult recovery and delayed mortality were significantly affected by nutritional quality, LLIN type, exposure time, and recovery time. For both LLINs, adult recovery increased over time, with a trend for higher recovery and lower mortality with increasing nutritional quality and decreasing exposure time. In addition, adult mobility decreased multiple-fold after Carifend or D-Terrence exposure for 30, 60 or 90 min compared to 10 min. This study shows nutrition significantly modulates the efficacy of LLIN against T. castaneum, and thus strengthens the rationale for implementing stringent sanitation protocols for food facility managers. © 2024 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Resources modulate developmental shifts but not infection tolerance upon coinfection in an insect system.

Energetic resources fuel immune responses and parasite growth within organisms, but it is unclear whether energy allocation is sufficient to explain changes in infection outcomes under the threat of multiple parasites. We manipulated diet in flour beetles ( Tribolium confusum ) infected with two natural parasites to investigate the role of resources in shifting metabolic and immune responses after single and co-infection. Our results suggest that gregarine parasites alter the within-host energetic environment, and by extension juvenile development time, in a diet- dependent manner. Gregarines do not affect host resistance to acute bacterial infection but do stimulate the expression of an alternative set of immune genes and promote damage to the gut, ultimately contributing to reduced survival regardless of diet. Thus, energy allocation is not sufficient to explain the immunological contribution to coinfection outcomes, emphasizing the importance of mechanistic insight for predicting the impact of coinfection across levels of biological organization.

Effect of temperature on sexual size dimorphism during the developmental period in the broad-horned flour beetle

Adult size in numerous insects is strongly dependent on temperature. In several cases, a temperature–size rule is observed in which developmental temperature and adult size tradeoff. Although several previous studies have demonstrated the temperature–size rule, only a few have explored the relationship between developmental temperature and weapon traits or sexual size dimorphism. This study was conducted to investigate the size of the broad-horned flour beetle Gnatocerus cornutus when it was developed under different temperatures. G. cornutus males possess weapon traits for male–male combat and exhibit sexual size dimorphism in other morphological traits. Results showed that male weapon size and body size complied with the temperature–size rule. Furthermore, the extent of sex dimorphism in genae width, a weapon-supportive trait, were larger at lower temperatures. Our findings suggest that the temperature–size rule also influences the size of sexual traits.

Deciphering a Beetle Clock: Individual and Sex-Dependent Variation in Daily Activity Patterns.

Circadian clocks are inherent to most organisms, including cryptozoic animals that seldom encounter direct light, and regulate their daily activity cycles. A conserved suite of clock genes underpins these rhythms. In this study, we explore the circadian behaviors of the red flour beetle Tribolium castaneum, a significant pest impacting stored grain globally. We report on how daily light and temperature cues synchronize distinct activity patterns in these beetles, characterized by reduced morning activity and increased evening activity, anticipating the respective environmental transitions. Although less robust, rhythmicity in locomotor activity is maintained in constant dark and constant light conditions. Notably, we observed more robust rhythmic behaviors in males than females with individual variation exceeding those previously reported for other insect species. RNA interference targeting the Clock gene weakened locomotor activity rhythms. Our findings demonstrate the existence of a circadian clock and of clock-controlled behaviors in T. castaneum. Furthermore, they highlight substantial individual differences in circadian activity, laying the groundwork for future research on the relevance of individual variation in circadian rhythms in an ecological and evolutionary context.

Impact of Different Food Sources on the Developmental Biology and Host Preferences of the Red Flour Beetle Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

An experiment conducted at the seed center laboratory at Tamil Nadu Agricultural University during June 2022 evaluated the host preferences and developmental biology of the red flour beetle (Tribolium castaneum) across three food sources: broken rice with rice flour, broken wheat with wheat flour, and tapioca flour. Beetle development was observed in ventilated containers under controlled conditions (28-30°C, 65% relative humidity) with 25 beetles per container. Significant differences in beetle survival and development. Wheat flour was the most favourable host, with 304 live adults, followed by broken wheat (145 live adults. Rice flour supported 20 adults and 11 larvae, indicating moderate suitability. Whole rice grains and tapioca flour were the least effective, with only dead adults and few larvae. Despite host quality variations, the developmental stages (egg incubation: 4-7 days, larval: 65-70 days, pupal: 6-9 days) were consistent across food sources. Wheat flour is the most supportive environment for T. castaneum development.

Relationship between walking movement and reproductive traits in the red flour beetle Tribolium castaneum

AbstractMany animal species have movement abilities. Behavior is important for evolutionary ecology because animal movement leads to dispersal, migration, search for food and mates, and escaping from enemies. However, individual differences in movement activity are found within a population. This phenomenon can be affected by various factors, one of which is suggesting that higher moving activity has fitness cost, whereas lower moving activity has benefits. Animal movement may also affect reproduction (e.g., resource allocation tradeoff between movement and reproduction as well as intra‐ and intersexual selection). Although many previous studies have investigated the relationship between movement and reproduction, less attention has been paid to walking movement. In this study, previous studies that investigated the relationship between movement and reproduction were reviewed using the flour beetle Tribolium castaneum, a model insect in behavioral ecology and genetics. Several previous studies suggest that beetle walking is strongly associated with male and female reproductive traits. In recent years, empirical studies on the correlation between walking and other traits have increased, particularly in T. castaneum. Although this species can fly and walk, the movement is often discussed without discriminating between flight and walk. Differences in modes of movement may affect the correlation between movement and other traits; thus, discussing each mode of movement separately is necessary.

Effects of sulfur dioxide fumigation on mortality of confused flour beetle (Coleoptera: Tenebrionidae) and rice weevil (Coleoptera: Curculionidae).

Sulfur dioxide (SO2) fumigation was studied in laboratory to determine its potential as an alternative treatment for postharvest control of stored product insects, confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae), and rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae). Three-hour fumigations with 0.1%-2.0% SO2 were conducted against eggs, immature stages, and adults of the 2 insects at 20 °C. Effective control of both insects was achieved. However, there were considerable variations between the 2 insects and among different life stages. Confused flour beetle was more susceptible to SO2 fumigation than rice weevil. Complete control of adults and all life stages of confused flour beetle was achieved in 3-h fumigations with 0.5% and 2.0% SO2, respectively. For rice weevil, 3-h fumigation with 1.5% SO2 resulted in 96.5% adult mortality and the fumigation with 2.0% SO2 resulted in 99.27% mortality of adults and 87.5% mortality of immature stages. Three-hour fumigations with 1% SO2 resulted in <5% egg survival to adults. The study demonstrated high efficacy of SO2 fumigation against the insects and suggested that SO2 fumigation has good potential for postharvest pest control on stored products.

Chemical Compositions and Fumigation Effects of Essential Oils Derived from Cardamom, Elettaria cardamomum (L.) Maton, and Galangal, Alpinia galanga (L.) Willd, against Red Flour Beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae).

This study explores the use of essential oils from cardamom (Elettaria cardamomum (L.) Maton) and galangal (Alpinia galanga (L.) Willd) as alternatives to synthetic insecticides for controlling the red flour beetle, Tribolium castaneum (Herbst). The chemical compositions of these oils were analyzed using GC-MS, and their fumigation effects were tested in a vapor-phase bioassay. The experiment followed a factorial design with four types of essential oils, namely, those manually extracted from cardamom leaves (MCL) and galangal leaves (MGL) and those commercially produced from cardamom seeds (CCS) and galangal rhizomes (CGR), at seven concentrations (0, 50, 100, 150, 200, 250, and 300 µL/L air). The manually extracted oils yielded 0.6% from cardamom leaves and 0.25% from galangal leaves. MCL contained 28 components, with eucalyptol (25.2%) being the most abundant, while CCS had 34 components, primarily α-terpinyl acetate (46.1%) and eucalyptol (31.2%). MGL included 25 components, mainly caryophyllene (28.7%) and aciphyllene (18.3%), whereas CGR comprised 27 components, with methyl cis-cinnamate (47.3%) and safrole (19.8%) as the major constituents. The fumigation bioassay results revealed that CGR was the most effective, demonstrating the highest mortality rates of T. castaneum across all the tested periods and concentrations, achieving up to 96% mortality at 168 h with a concentration of 300 µL/L air. Statistical analyses showed significant differences in mortality based on the type and concentration of essential oil, particularly after 96 h. These findings highlight the potential of CGR, with its advantages and differences in chemical composition, as an effective biopesticide against T. castaneum, with increasing efficacy over time and at higher concentrations.

Efficiency of Alkaloid and Phenolic Extracts of Seeds and Leaves Sesbania Plant on Adults Red Flour Beetle Tribolium castaneum Herbst (Coleoptera : Tenebrionidae)

Efficiency of Alkaloid and Phenolic Extracts of Seeds and Leaves Sesbania Plant on Adults Red Flour Beetle Tribolium castaneum Herbst (Coleoptera : Tenebrionidae)

Characterization of double-stranded RNA and its silencing efficiency for insects using hybrid deep-learning framework.

RNA interference (RNAi) technology is widely used in the biological prevention and control of terrestrial insects. One of the main factors with the application of RNAi in insects is the difference in RNAi efficiency, which may vary not only in different insects, but also in different genes of the same insect, and even in different double-stranded RNAs (dsRNAs) of the same gene. This work focuses on the last question and establishes a bioinformatics software that can help researchers screen for the most efficient dsRNA targeting target genes. Among insects, the red flour beetle (Tribolium castaneum) is known to be one of the most sensitive to RNAi. From iBeetle-Base, we extracted 12027 efficient dsRNA sequences with a lethality rate of ≥20% or with experimentation-induced phenotypic changes and processed these data to correspond to specific silence efficiency. Based on the first complied novel benchmark dataset, we specifically designed a deep neural network to identify and characterize efficient dsRNA for RNAi in insects. The dna2vec word embedding model was trained to extract distributed feature representations, and three powerful modules, namely convolutional neural network, bidirectional long short-term memory network, and self-attention mechanism, were integrated to form our predictor model to characterize the extracted dsRNAs and their silencing efficiencies for T. castaneum. Our model dsRNAPredictor showed reliable performance in multiple independent tests based on different species, including both T. castaneum and Aedes aegypti. This indicates that dsRNAPredictor can facilitate prescreening for designing high-efficiency dsRNA targeting target genes of insects in advance.

The effects of loose, firm, fine, and coarse substrates on the movement of the red flour beetle

The effects of loose, firm, fine, and coarse substrates on the movement of the red flour beetle

Artemisia fragrans Willd. Essential Oil: Chemical Profile and Insecticidal Potential against the Confused Flour Beetle, Tribolium confusum du Val.

The confused flour beetle, Tribolium confusum du Val, is one of the cosmopolitan and polyphagous storage insect pests. The frequent application of chemical insecticides has resulted in several side effects, including threats to human health and non-target organisms and the resistance of insect pests. In the current study, the fumigant toxicity and feeding deterrence potential of Artemisia fragrans Willd. essential oil on T. confusum adults were investigated. The essential oil was rich in terpenic compounds, in which α-thujone (27.8%) and 1,8-cineole (22.8%) were dominant. The essential oil displayed significant fumigant toxicity on T. confusum, where a concentration of 35.3 μL/L caused 100% mortality of the treated adults after 48 h. The LC30 and LC40 values (lethal concentrations to kill 30% and 40% of tested insects: 15.1 and 18.4 μL/L, respectively) significantly decreased the nutritional indices of the pest, including the consumption index, relative consumption rate, and relative growth rate. The feeding deterrence index of the essential oil were calculated as being 62.29 and 48.66% for the concentrations of 15.1 and 18.4 μL/L after 5 days, respectively. Accordingly, A. fragrans essential oil can be considered an efficient, available, and natural alternative to detrimental chemical pesticides in the management of T. confusum.

A new trypsin inhibitor from Centrosema plumieri effective against digestive proteases from Tribolium castaneum, an eco-friendly alternative

Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.

Effect of zinc fortified, arid and irrigated wheat varieties flour upon life history parameters of red flour beetle, Tribolium castaneum (Herbest) (Coleoptera: Tenebrionidae)

Red flour beetle, Tribolium castaneum (Herbest) (Coleoptera: Tenebrionidae) is a stored grain pest. Various fortified wheat varieties have been produced but data about the impact of different wheat varieties flour on life history parameters of T. castaneum are poorly understood. This study evaluated the effect of fortified (Zincol-16 T1), arid zone [(BARS-09 T2), and (Fatehjang-2016 T3)] and irrigated zone [(Faisalabad-2008 T4), (Gold-2016 T5), (Manthar-03 T6), (Fareed-06 T7), (Mairaj-08 T8)] wheat varieties flour upon some life history parameters of T. castaneum in the laboratory using age-stage two sex life table. The egg incubation period, adult male and female longevities, and total male and female longevities of T. castaneum were shortest in T5 and T6 as compared to all other treatments. The T5 presented shorter larval and pre-adult duration. Egg incubation, larval, and pupal durations and total male and female longevities were higher in T1. Total longevity (egg to adult) of T. castaneum was lowest in T1 as compared to T3 and T4 but similar to other treatments. Pre-adult duration was longer in T1 as compared to all other treatments. Pre-adult survival was significantly lower in T1 (53%) than all the treatments in which it remained at par. Lowest proportion of female adult (Nf/N) was found in T1 as compared to T3, T4, and T7. Age stage specific survival rate (sxj) was lower on T1, whereas life expectancy (exj) was higher than other varieties. These results suggest that T. castaneum develop poorly upon zinc fortified wheat variety, Zincol as compared to other varieties thus it can be a good integrated pest management tool against this pest.

Temporal control of RNAi reveals both robust and labile feedback loops in the segmentation clock of the red flour beetle

Animals from all major clades have evolved a segmented trunk, reflected in the human spine or the insect segments. These units emerge during embryogenesis from a posterior segment addition zone (SAZ), where repetitive gene activity is regulated by a mechanism described by the clock and wavefront/speed gradient model. In the red flour beetle Tribolium castaneum, RNA interference (RNAi) has been used to continuously knock down the function of primary pair-rule genes (pPRGs), caudal or Wnt pathway components, which has led to the complete breakdown of segmentation. However, it has remained untested, if this breakdown was reversible by bringing the missing gene function back to the system. To fill this gap, we established a transgenic system in T. castaneum, which allows blocking an ongoing RNAi effect with temporal control by expressing a viral inhibitor of RNAi via heat shock. We show that the T. castaneum segmentation machinery was able to reestablish after RNAi targeting the pPRGs Tc-eve, Tc-odd, and Tc-runt was blocked. However, we observed no rescue after blocking RNAi targeting Wnt pathway components. We conclude that the insect segmentation system contains both robust feedback loops that can reestablish and labile feedback loops that break down irreversibly. This combination may reconcile conflicting needs of the system: Labile systems controlling initiation and maintenance of the SAZ ensure that only one SAZ is formed. Robust feedback loops confer developmental robustness toward external disturbances.

Insecticidal evaluation of various plant extracts against the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae); A major stored grains insect pest

Insecticidal evaluation of various plant extracts against the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae); A major stored grains insect pest

Environmental impact assessment of dicationic ionic liquids with ammonium-phosphonium cations and amino acid anions

Progress in the development of biodegradable or biobased ionic liquids (ILs) has led to the design of green compounds for several applications. Herein, four biocompatible dicationic ionic liquids (DILs) with ammonium-phosphonium cations and amino acid anions were synthesized and investigated their environmental impact. The structures of the DILs were confirmed by spectral analyses (1H, 13C and 31P NMR). Furthermore, physicochemical properties such as density, viscosity and refractive index were determined. Water content, bromide content and solubility were thereafter determined as the parameters needed for further studies. Subsequently, their antifeedant activity towards economically important pests of grain in storage warehouses: the granary weevil, the confused flour beetle, and the khapra beetle was examined, showing the dependence on structure. Moreover, selected DILs were investigated for toxicity towards white mustard, Daphnia magna, and Artemia franciscana to specify the environmental impact. These studies were complemented by understand the biodegradation of DILs by bacterial communities derived from soil at the agricultural land. The result was DILs with limited environmental footprints that have great potential for further application studies.

Insecticidal activity on Tribolium castaneum and Phenacoccus solenopsis and characterization of active phytonanoparticles

Phytonanoparticles can be used as an alternative method for pest control in agriculture. The objective of this study was to evaluate the insecticidal activity of mono, bi and trimetallic phytonanoparticles (PNPs) synthesized from cachanilla (Pluchea sericea Nutt. Coville) and epazote (Chenopodium ambrosioides L.) against the red flour beetle (Tribolium castaneum) and the cotton mealybug (Phenacoccus solenopsis). Overall, T. castaneum showed greater sensitivity to PNPs than P. solenopsis. The trimetallic PNPs, Zn/Cu/Mn of P. sericea showed significantly higher effect against T. castaneum than the crude aqueous extract of P. sericea. No other PNPs showed more effect than their respective aqueous plant extract. Physicochemical characterization of the most active PNPs showed that the Zn/Cu/Mn NP of P. sericea had a strong zeta potential of +200 mV, hydrodynamic size of 16 nm, and contains zinc (6.73%), copper (0.13%), and manganese (4.69%). This study suggests that the trimetallic Zn/Cu/Mn PNPs of P. sericea have high insecticidal effect on T. castaneum, but low toxicity on P. solenopsis. Additional research on the mode of action of PNPs on insect targets is required.