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Waprin, a WAP (Whey acidic protein) domain‐containing extracellular secretory protein, is widely known for its antibacterial properties. In this study, a waprin homologue (Tc_wapF) expressing in a female‐specific manner was identified in Tribolium castaneum, through the analysis of sex‐specific transcriptomes. Developmental‐ and tissue‐specific profiling revealed the widespread expression of Tc_wapF in adult female tissues, particularly in the ovary, gut and fatbody. This female‐specific expression of Tc_wapF is not regulated by the classical sex‐determination cascade of T. castaneum, as we fail to get any attenuation in Tc_wapF transcript levels in Tcdsx and Tctra (key players of sex determination cascade of T. castaneum) knockdown females. RNA interference‐mediated knockdown of Tc_wapF in females led to the non‐hatching of eggs laid by these females, suggesting the crucial role of Tc_wapF in the embryonic development in T. castaneum. This is the first report on the identification of a sex‐specific waprin homologue in an insect and its involvement in embryonic development. Future investigations on the functional conservation of insect waprins and their mechanistic role in embryonic development can be exploited for improving pest management strategies. [ABSTRACT FROM AUTHOR]

Plant extracts offer an alternative approach to safeguarding stored food products. Our research is focused on assessing the insecticidal properties of aqueous and ethanolic extracts containing bioactive compounds obtained from two species of Azolla; A. pinnata and A. Microphylla against pests that affect stored food items. Two drying processes were conducted in order to compare the yield of bioactive compounds (freeze-drying and oven-drying). We employed two extraction methods (maceration and decoction), using ethanol and water as solvents. Phytochemical screening of both extracts was carried out by CG-MS analysis. The insecticidal properties of the obtained extracts were assessed using the spraying method on larvae and adults of Tribolium castaneum. The results indicated that the freeze-dried method was more efficient comparing to oven-drying, recording the highest value of 26.07%, with the ethanolic extract by decoction. GC–MS analysis for both species confirmed the existence of primarily fatty acid, terpenoid, steroid, coumarin, and flavonoid derivatives compounds. Overall, the various extracts exhibited high toxicity against both larvae and adults. The mortality rate was increased according to the increasing concentration of extracts. The lowest LC50 of A. pinnata and A. microphylla extracts were found to be 872.42 and 894.65 µg/mL, respectively. The aqueous extract caused higher toxicity, reaching 96%. The findings of this study suggest that the dry matter from both azolla species exhibited significant toxicity, evidenced by the mortality of both the larvae and adults. Extracts from A. pinnata were found to be more toxic to T. castaneum in comparison with A. microphylla extracts. [ABSTRACT FROM AUTHOR]

As the catalytic subunit of the Elongator complex, Elongator protein 3 (Elp3) plays a crucial role in multiple physiological processes, including growth, development and immune responses. Previous studies on Elp3 have focused on Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens (human) or Mus musculus (mouse), whereas there are few reports on Elp3 in agricultural pests. Here, the role of TcElp3 in reproduction in the red flour beetle, Tribolium castaneum, was investigated, and the underlying mechanisms were explored. The results showed that RNA interference (RNAi)‐mediated knockdown of TcElp3 in female pupae led to decreased fecundity in female adults. Consistently, the injection of dsTcElp3 into female pupae decreased the mRNA levels of the vitellogenin (Vg)‐encoding genes TcVg1 and TcVg2 in female adults. Notably, knockdown of TcElp3 upregulated the expression of forkhead box protein O (FOXO) at both the mRNA and the protein level in T. castaneum, and promoted the nuclear translocation of TcFOXO. Additionally, TcElp3 directly interacts with TcFOXO and the silencing of TcElp3 significantly decreased the acetylation level of TcFOXO. Overall, our studies reveal that Elp3 regulates beetle reproduction by interacting with FOXO and modulating its acetylation status. [ABSTRACT FROM AUTHOR]

Eucalyptol is one of the major insecticidal active ingredients in a variety of plant essential oils, and has good killing and avoidance effects on Tribolium castaneum. The presence of detoxifying enzymes glutathione S-transferase (GST) in T. castaneum makes it resistant to a variety of insecticides. However, whether GST is involved in regulating the sensitivity of eucalyptol by T. castaneum is not well understood. In our previous study, a glutathione S-transferase, TcGSTu1, was significantly up-regulated in RNA sequencing data when T. castaneum was exposed to eucalyptol. Therefore, in this study, the role of TcGSTu1 in the regulating the sensitivity of T. castaneum to eucalyptol was studied. The enzyme activities of GST and the transcription levels of TcGSTu1 were significantly increased following stimulation with eucalyptol. When using RNA interference technology knockdown TcGSTu1 heightens the sensitivity of T. castaneum to eucalyptol, demonstrating a link between TcGSTu1 and eucalyptol detoxification metabolism. Furthermore, TcGSTu1 is expressed in all developmental stages of T. castaneum, with higher expression levels observed particularly in the late egg stage. There was significant expression of TcGSTu1 in various tissues of different organisms, including larval head, fat body, and adult head. This observation indicated a possible connection between high TcGSTu1 expression and eucalyptol detoxification. The present findings suggest that TcGSTu1 may be involved in regulating the sensitivity and response of T. castaneum to treatment with eucalyptol, providing new research insight into pest control. [ABSTRACT FROM AUTHOR]

The red flour beetle, Tribolium castaneum, is an agricultural and storage pest with a global distribution. Studies have shown that eucalyptol has strong contact toxicity against larvae of this beetle, whereas odorant binding proteins (OBPs) are known to contribute to larval defenses against this phytochemical toxin. However, the mechanisms underlying the protective effect of insect OBPs against eucalyptol remain unclear. Here, TcOBPC02 from T. castaneum was cloned and characterized. Gene expression profile analysis showed that TcOBPC02 is highly expressed at early larval and early pupal stages. Additionally, tissue expression profiling revealed that, in the adult, TcOBPC02 was most highly expressed in the head, followed by the epidermis, whereas in larvae, TcOBPC02 was mainly expressed in hemolymph and the epidermis. These developmental stages and tissues that exhibit high TcOBPC02 expression are closely related to the detoxification of heterologous substances. Furthermore, the mRNA level of TcOBPC02 was significantly increased after exposure to eucalyptol, whereas TcOBPC02‐targeted RNA interference increased the susceptibility of T. castaneum to eucalyptol, indicating that TcOBPC02 participates in the tolerance of this beetle to eucalyptol. Additionally, recombinant TcOBPC02 was expressed in Escherichia coli and isolated, enabling a straightforward fluorescence competition binding assay. In combination, these results have demonstrated that TcOBPC02 is required for defenses against phytochemicals in T. castaneum. This study provides a theoretical basis for understanding the mechanisms underlying the degradation of exogenous toxicants in insects and adds to the repertoire of potential target genes for pest control. [ABSTRACT FROM AUTHOR]

Several ecological processes – from population dynamics to species co‐existence – are driven by density dependence (DD) in population growth rate. Thus, to predict and manage ecological outcomes, we need a deep understanding of which factors and demographic traits drive variation in DD. In the insect pest Tribolium castaneum, we found large variation in DD across habitats but not across source populations. We modeled DD in population growth as the product of DD in fecundity and survival, experimentally estimating each parameter. Across habitats, survival parameters varied more than fecundity, including in simulations with varying parameter values. Thus, DD in survival drives variation in density‐dependent population growth. Hence, under strong density‐dependent selection, we expect evolutionary change in density‐dependent survival, provided sufficient genetic variance. Our general framework combining detailed experiments and simulations with a simple model can be used for other species to better understand the causes and consequences of density dependent population growth. [ABSTRACT FROM AUTHOR]

Using a new type of insect probe trap, the activity and distribution of Tribolium castaneum in wheat grain bulks inside experimental silos were collected and analyzed to investigate its distribution patterns and main influencing factors within the grain bulks. An equation for estimating insect population density was established to provide a scientific basis for accurate monitoring and early warning. The results showed that the daily capture of Tribolium castaneum did not change significantly over time. When the insect population density was low, Tribolium castaneum was mainly distributed in the top and middle layers of the grain bulk. When the insect population density was high, Tribolium castaneum was uniformly distributed within the grain bulk. Tribolium castaneum exhibited thermophilic and hygrophilic behaviors, with an optimal activity temperature range of 22~30 ℃ and a relative humidity range of 40%~53%. By comprehensively analyzing the spatial and temporal distribution patterns of the insect population and the effects of temperature and humidity, the actual insect population density could be estimated based on the number of insects captured by the probe traps. The equation for estimating the insect population density was: y = 10.566 – 0.321 × x1 – 0.017 × x2 + 0.026 × x3, x1: temperature (℃), x2: relative humidity (%), x3: number of insects captured.

This study was conducted to evaluate the efficacy of four entomopathogenic fungi against the red flour beetle, Tribolium castaneum (Herbst) as an alternative to chemical pesticides. The adult stage inside stored wheat was subjected to different concentrations of Beauveria bassiana (Balsamo), Metarhizium anisopliae (Metsch), Verticillium lecanii (Zimmerman), and Paecilomyces ilacinus (Thoms) in either Petri-plates at 7, 15 and 22 days or bag storage at 20, 40 and 60 days exposure times. The first experiment indicated that V. lecanii achieved maximum mortality (58.89 and 57.5%) via interaction with the highest dose of 0.748 £ 107 and maximum exposure time of 22 days respectively. The adults required the lowest concentration of V. lecanii to achieve an average LC50 (6,233,697 mg) within various exposure times. Regarding the latter experiment (liquid assay), the least survivorship (41.25) was observed by P. ilacinus followed by V. lecanii (56.25) during 60 days of exposure time. On the other hand, the powder assay revealed that the highest mortality (57.08 and 48.75) of adults inside bags was recorded respectively via the interaction terms between V. lecanii with both the highest concentration (1000 mg) and exposure times 60 days and the LC50 value of the aforementioned bioagent was (240.02) for 60 days treatment followed by B. bassiana. The study suggests that the use of biopesticides can provide significant insight in the management of pests while minimize the weight loss of stored wheat and thus, can be used as an effective alternative or integrated with chemical pesticides in pest management strategies.

Abstract Background Numerous insect pests attack stored grains causing both qualitative and quantitative losses. The most damaging pest that infests dry stored produce is the red flour beetle, Tribolium castaneum, a secondary pest of stored goods. This pest, especially in its adult stage, exhibits resistance to chemical insecticides, thereby rendering the traditional pesticides ineffective in controlling it. Phyto-derivatives, which are strong insecticides and also ecologically benign, have gained interest as non-chemical solutions for controlling this pest. Hence, the objective of this study was to investigate the potential of Piper longum leaf extract insecticidal action as an environmentally benign insecticide for the first time against the adults of T. castaneum. In this study, P. longum leaf ethanol extract was tested against the adults of T. castaneum by petri dish bioassay method. Ad hoc studies to verify significant mortality for the initial confirmation of adulticidal activity were conducted for 24 h at different dosages of 62.5, 125, 250, 500 and 1000 mg/L of P. longum leaf ethanol extract. Thereafter, dosages set at 10, 20, 30 and 40 mg/L for the fractions of P. longum leaf ethanol extract were conducted. Prior to this, the leaf extract of this plant was subjected to column chromatography for fractionation. The fractions tested for adulticidal activity were subjected to gas chromatography–mass spectroscopy. Results Significant adulticidal action with 100% adult mortality was observed in ethanol extract of P. longum leaves. Among the fourteen fractions (F0–F13) obtained tested, only fractions, F5, F10 and F13, demonstrated adulticidal activity, and the remaining fractions displayed poor activity. One hundred per cent morality was noted in T. castaneum adults after 96 h at 40 mg/L in F5 and F10, and in F13 at 20 mg/L, and their respective LD50 values were 17.6, 26.6 and 10.0 mg/L. The fractions F5, F10 and F13 contained fatty acids, viz., hexadecanoic acid, dotriacontane and heptacosane in F5; tetradecanoic acid and nonadecanoic acid in F10; and octadecanoic acid, aspartame and tridecanoic acid in F13, revealed through gas chromatography–mass spectroscopy. Conclusions The results of the study showed that P. longum ethanol leaf extract revealed significant adulticidal activity and is a promising toxic agent to the adults of T. castaneum. The fatty acids in the ethanolic leaf extract fractions of P. longum could have caused toxicity to the adults of T. castaneum. According to the current literature survey, this is the first research report on the adulticidal activity of P. longum leaf extracts against the adults of T. castaneum.

The use of synthetic pesticides carries a significant risk of pests developing resistance, leading to decreased pesticide effectiveness. ATP‐binding cassette (ABC) transporters, especially the ABCC subfamily members, have been suggested to act as efflux pumps for various pesticides, thereby contributing to pesticide resistance. So far, the identification of potential pesticide substrates of insect ABC transporters is most often based on the quantification of transcript in arthropods. Here, we screened and identified the potential pesticide substrates of ABCC‐9C from Tribolium castaneum based on an in vitro ATPase activity assay. Together with affinity evaluation‐, cytotoxicity analysis‐, and RNA interference‐based bioactivity tests, we revealed that the insecticides, carbofuran, and buprofezin, are potential substrates of TcABCC‐9C. Additionally, we identified an amphipathic translocation channel in the transmembrane domain of TcABCC‐9C formed by 8 transmembrane helices. Molecular docking suggested that both carbofuran and buprofezin bind at the same site within the translocation channel via hydrophobic interactions. These findings indicate that TcABCC‐9C might play a critical role in multi‐pesticide resistance, providing a potential target for managing pesticide resistance and laying the groundwork for future pest control strategies. Given the conservations among ABCC subfamily members, the experimental model we developed in this study can be also applied to identify the potential substrates of other ABCC transporters, as well as to predict insecticide resistance mediated by ABCC transporters. [ABSTRACT FROM AUTHOR]

Copyright of Science & Technology of Cereals, Oils & Foods is the property of Science & Technology of Cereals, Oils & Foods Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

ABSTRACT Arthropods have integrated digestive and renal systems, which function to acquire and maintain homeostatically the substances they require for survival. The cryptonephridial complex (CNC) is an evolutionary novelty in which the renal organs and gut have been dramatically reorganised. Parts of the renal or Malpighian tubules (MpTs) form a close association with the surface of the rectum, and are surrounded by a novel tissue, the perinephric membrane, which acts to insulate the system from the haemolymph and thus allows tight regulation of ions and water into and out of the CNC. The CNC can reclaim water and solutes from the rectal contents and recycle these back into the haemolymph. Fluid flow in the MpTs runs counter to flow within the rectum. It is this countercurrent arrangement that underpins its powerful recycling capabilities, and represents one of the most efficient water conservation mechanisms in nature. CNCs appear to have evolved multiple times, and are present in some of the largest and most evolutionarily successful insect groups including the larvae of most Lepidoptera and in a major beetle lineage (Cucujiformia + Bostrichoidea), suggesting that the CNC is an important adaptation. Here we review the knowledge of this remarkable organ system gained over the past 200 years. We first focus on the CNCs of tenebrionid beetles, for which we have an in‐depth understanding from physiological, structural and ultrastructural studies (primarily in Tenebrio molitor), which are now being extended by studies in Tribolium castaneum enabled by advances in molecular and microscopy approaches established for this species. These recent studies are beginning to illuminate CNC development, physiology and endocrine control. We then take a broader view of arthropod CNCs, phylogenetically mapping their reported occurrence to assess their distribution and likely evolutionary origins. We explore CNCs from an ecological viewpoint, put forward evidence that CNCs may primarily be adaptations for facing the challenges of larval life, and argue that their loss in many aquatic species could point to a primary function in conserving water in terrestrial species. Finally, by considering the functions of renal and digestive epithelia in insects lacking CNCs, as well as the typical architecture of these organs in relation to one another, we propose that ancestral features of these organs predispose them for the evolution of CNCs. [ABSTRACT FROM AUTHOR]

Hemocytes are pivotal in the immune response of insects against invasive pathogens. However, our knowledge of hemocyte types and their specific function in Tribolium castaneum, an increasingly important Coleoptera model insect in various research fields, remains limited. Presently, a combination of morphological criteria and dye‐staining properties were used to characterize hemocyte types from T. castaneum larvae, and 4 distinct types were identified: granulocytes, oenocytoids, plasmatocytes and prohemocytes. Following different immune challenges, the total hemocyte counts declined rapidly in the initial phase (at 2 h), then increased over time (at 4 and 6 h) and eventually returned to the naive state by 24 h post‐injection. Notably, the morphology of granulocytes underwent dramatic changes, characterized by an expansion of the surface area and an increased production of pseudopods, and with the number of granulocytes rising significantly through mitotic division. Granulocytes and plasmatocytes, the main hemocyte types in T. castaneum larvae, can phagocytose bacteria or latex beads injected into the larval hemolymph in vivo. Furthermore, these hemocytes participate in the encapsulation and melanization processes in vitro, forming capsules to encapsulate and melanize nickel–nitrilotriacetic acid (Ni–NTA) beads. This study provides the first comprehensive characterization of circulating hemocytes in T. castaneum larvae, offering valuable insights into cell‐mediated immunity in response to bacterial infection and the injection of latex beads. These results deepen our understanding of the cellular response mechanisms in T. castaneum larvae and lay a solid foundation for subsequent investigations of the involvement of T. castaneum hemocytes in combating pathogens. [ABSTRACT FROM AUTHOR]

The insecticidal activity of anise, thyme, and coriander essential oils in bulk and prepared nano-emulsions formulations were evaluated against the adults of stored grain pests Sitophilus oryzae and Tribolium castaneum. The prepared essential oil nano-emulsions exhibited poly dispersity indices ranging from 0.209 to 0.37, and zeta potentials between −14.5 and −27.8 mV. The smallest oil nano-emulsion droplet diameter was coriander (88.83 nm) and the largest was anise (103.57 nm). Although no significant differences in the toxicity of the three oils against T. castaneum were detected, anise and thyme oils were significantly more toxic than coriander oil to S. oryzae. Preparing the anise and thyme oils as nano-emulsion formulations significantly increased the toxicity by 1.50- and 1.47-fold, respectively, against T. castaneum. The nano-emulsions were also significantly more toxic (1.41–1.48 times) than the bulk essential oils to S. oryzae. The anise nano-emulsion showed the highest toxicity against S. oryzae and T. castaneum, with LC50 values of 3858.88 and 4985.1 mg/L respectively. GC–MS analysis of anise essential oil revealed that ultra-sonication caused a 2.73-fold decrease in the anethole level in the nano-emulsion and completely removed nine minor compounds. The anise oil nano-emulsion was the most effective insecticide, reducing S. oryzae emergence by 94.64% and protecting nearly 84.5% of wheat grains from T. castaneum infestation. Finally, the tested NEs or EO had not any negatively significant effect on wheat seed germination rate. [ABSTRACT FROM AUTHOR]

The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is among the global storage pests that lead to the deterioration of grains and stored products. A series of laboratory experiments were conducted to evaluate the efficiency of different concentrations of Mentha piperita L. (Lamiaceae) oil and leaf methanol extracts against the rust-red flour beetle in the different instars. The results of the present study showed that the mint, M. piperita, oil treatments at all concentrations used (8%, 10%, or 12%) were significantly effective, resulting in remarkable individual mortality of T. castaneum third instar larvae and adult mortality compared to the treatment of the peppermint methanol leaf extract at the highest concentration. Peppermint oil showed insecticidal effect against the third instar larvae of the red flour beetle, especially where sprayed at the highest concentration of 12%. The post-treatment results after seventh days showed 94% adult mortality and 82% mortality in the third instar of larvae. This study confirmed the possibility of using mint essential oil at relatively low concentrations as environmentally friendly product in controlling the rust-red flour beetle T. castaneum. [ABSTRACT FROM AUTHOR]

The repellent activity of two odourless vegetable oils (sweet almond—SAO and soybean—SO) applied on filter paper and four types of food packaging materials (polyethylene film, plastic gunnysacks, cardboard, and wood) was tested against three species of stored product pests (Sitophilus granarius, Tribolium castaneum, and Oryzaephilus surinamensis). The repellence of three doses was evaluated after 24 h, and after 7, 14, 21, and 28 days. Both oils exhibited a repellent effect when applied to filter paper, but this effect decreased with time and concentration. The most susceptible species was T. castaneum. The maximum repellence was 84% and 76% for SO and SAO, respectively. On packaging materials, the repellent effect diminished after a certain period of time, ranging from 7 to 28 days. Subsequently, the oils even became attractive. Generally, the repellent effects of both SO and SAO were higher on cardboard and wood slab than on polyethylene film and plastic gunnysacks. As such, in the case of SO, the repellence against T. castaneum was 88% 24 h after application on cardboard, whilst 68% on plastic bag. This work demonstrated the repellent potential of vegetable oils in the short term for the protection of packaged food against stored product pests. [ABSTRACT FROM AUTHOR]

The laboratory studies on the biology of the rust red flour beetle, Tribolium castaneum (Herbst) (Tenebrionidae: Coleoptera) conducted on stored wheat revealed that adult females deposited the scattered eggs within the food material preferably on the upper layer with an average fecundity of 54.3±5.63 eggs / month while the incubation period was 3.45±0.68 days with 80.2±7.59 per cent hatching. The average larval period of the first, second, third, fourth, fifth, and sixth larval instar was 4.85±0.74, 3.60±0.75, 4.40±0.99, 5.40±1.39, 7.70 ±1.52 and 5.95±1.09 days, respectively. After completion of the last larval instar, it formed the pre-pupal stage before transforming into a complete pupa. The pre-pupal period lasted for 2.90±1.07 days and the duration of the pupal period was 6.45±1.05 days. Newly emerged adults were yellowish brown in colour, but later itturned into reddish brown. The total life cycle of males with and without food was 106.15±28.19 days and 64.7±12.48 days, respectively whereas in the case of adult females, it was 119.2±22.04 days with food and without food was 68.35 ±15.49 days. [ABSTRACT FROM AUTHOR]

Background: Numerous insect pests attack stored grains causing both qualitative and quantitative losses. The most damaging pest that infests dry stored produce is the red flour beetle, Tribolium castaneum, a secondary pest of stored goods. This pest, especially in its adult stage, exhibits resistance to chemical insecticides, thereby rendering the traditional pesticides ineffective in controlling it. Phyto-derivatives, which are strong insecticides and also ecologically benign, have gained interest as non-chemical solutions for controlling this pest. Hence, the objective of this study was to investigate the potential of Piper longum leaf extract insecticidal action as an environmentally benign insecticide for the first time against the adults of T. castaneum. In this study, P. longum leaf ethanol extract was tested against the adults of T. castaneum by petri dish bioassay method. Ad hoc studies to verify significant mortality for the initial confirmation of adulticidal activity were conducted for 24 h at different dosages of 62.5, 125, 250, 500 and 1000 mg/L of P. longum leaf ethanol extract. Thereafter, dosages set at 10, 20, 30 and 40 mg/L for the fractions of P. longum leaf ethanol extract were conducted. Prior to this, the leaf extract of this plant was subjected to column chromatography for fractionation. The fractions tested for adulticidal activity were subjected to gas chromatography–mass spectroscopy. Results: Significant adulticidal action with 100% adult mortality was observed in ethanol extract of P. longum leaves. Among the fourteen fractions (F0–F13) obtained tested, only fractions, F5, F10 and F13, demonstrated adulticidal activity, and the remaining fractions displayed poor activity. One hundred per cent morality was noted in T. castaneum adults after 96 h at 40 mg/L in F5 and F10, and in F13 at 20 mg/L, and their respective LD50 values were 17.6, 26.6 and 10.0 mg/L. The fractions F5, F10 and F13 contained fatty acids, viz., hexadecanoic acid, dotriacontane and heptacosane in F5; tetradecanoic acid and nonadecanoic acid in F10; and octadecanoic acid, aspartame and tridecanoic acid in F13, revealed through gas chromatography–mass spectroscopy. Conclusions: The results of the study showed that P. longum ethanol leaf extract revealed significant adulticidal activity and is a promising toxic agent to the adults of T. castaneum. The fatty acids in the ethanolic leaf extract fractions of P. longum could have caused toxicity to the adults of T. castaneum. According to the current literature survey, this is the first research report on the adulticidal activity of P. longum leaf extracts against the adults of T. castaneum. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR‐associated nuclease 9 (Cas9)‐mediated gene editing technology has revolutionized the study of fundamental biological questions in various insects. Diverse approaches have been developed to deliver the single‐guide RNA (sgRNA) and Cas9 to the nucleus of insect embryos or oocytes to achieve gene editing, including the predominant embryonic injection methods and alternative protocols through parental ovary delivery. However, a systematic comparative study of these approaches is limited, especially within a given insect. Here, we focused on revealing the detailed differences in CRISPR/Cas9‐mediated gene editing between the embryo and ovary delivery methods in the beetle Tribolium castaneum, using the cardinal and tyrosine hydroxylase (TH) as reporter genes. We demonstrated that both genes could be efficiently edited by delivering Cas9/sgRNA ribonucleoproteins to the embryos by microinjection, leading to the mutant phenotypes and indels in the target gene sites. Next, the Cas9/sgRNA complex, coupled with a nanocarrier called Branched Amphiphilic Peptide Capsules (BAPC), were delivered to the ovaries of parental females to examine the efficacy of BAPC‐mediated gene editing. Although we observed that a small number of beetles’ progeny targeting the cardinal exhibited the expected white‐eye phenotype, unexpectedly, no target DNA indels were found following subsequent sequencing analysis. In addition, we adopted a novel approach termed “direct parental” CRISPR (DIPA‐CRISPR). However, we still failed to find gene‐editing events in the cardinal or TH gene‐targeted insects. Our results indicate that the conventional embryonic injection of CRISPR is an effective method to initiate genome editing in T. castaneum. However, it is inefficient by the parental ovary delivery approach. [ABSTRACT FROM AUTHOR]

Tribolium castaneum is a challenging pest of stored products, causing significant economic losses. The present study explored the efficacy of Coridothymus capitatus essential oil and its primary constituent, carvacrol, as eco-friendly alternatives for managing this pest. To evaluate their insecticidal potential, repellency, fumigant toxicity, and antifeedant properties, progeny inhibition assays were performed. Carvacrol exhibited superior repellency compared to the essential oil, achieving a 92% repellency rate at 2 mg/cm2. Both compounds demonstrated significant fumigant toxicity against T. castaneum, with LC50 values of 168.47 and 106.5 μL/L for the essential oil and carvacrol, respectively, after 24 h. Carvacrol also outperformed the essential oil in antifeedant activity, inducing an 80.7% feeding deterrence at 1.17 mg/g. Moreover, both treatments effectively suppressed the development of the pest's progeny. These results collectively underscore the potent insecticidal properties of C. capitatus essential oil and carvacrol, particularly carvacrol, as promising candidates for the sustainable management of T. castaneum in stored product protection. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Henan University of Technology Natural Science Edition is the property of Henan University of Technology Journal Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This work focuses on the profiling of semiochemicals (SCS) from Callosobruchus maculatus, Sitophilus oryzae and Tribolium castaneum by headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography–mass spectrometry (GC–MS) technique. Totally, 6, 9 and 8 volatile compounds (VCS) were detected from C. maculatus, S. oryzae and T. castaneum, respectively. As a result of pherobase analysis and preference bioassay, stearic acid (C. maculatus); nonanal, lauric acid and stearic acid (S. oryzae); stearic acid (T. castaneum) were identified as new SCS that could be useful for integrated pest management (IPM) practices. [ABSTRACT FROM AUTHOR]

Abstract The thrust of the study was to determine the chemical composition of the essential oils extracted from Thymus pallescens de Noé and Cymbogon citratus Stapf. as well as to evaluate their efficacy in controlling Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst) in either single or combined populations. Carvacrol (56.04%) and geraniol (20.86%) were identified as the major constituents of T. pallescens and C. citratus respectively. The tested essential oils showed pronounced insecticidal activity against the pest species in relation with the applied doses. T. pallescens EO had the highest efficacy and S. zeamais was found to be more susceptible to both individual and combined treatments. With reference to the contact and fumigation assessments, T. pallescens EO effectuated corrected mortality rates ranging from 42.5–100% to 25–100% in S. zeamais with corresponding lethal concentration (LC50) values of 17.7 µl/ml and 15µL/L air respectively. Whereas, the T. pallescens EO exhibited corrected mortality rates of 42.5–100% and 20–100% with corresponding LC50 values of 18.1 µl/ml and 15.5 µL/L air against T. castaneum in contact and fumigation assessments, respectively. The corrected mortality rates increased for both insect species when using combination treatments, with significant increases in the LC50 values, ranging from 8.59 to 49.9% for both pest species. Analysis of energy biomarkers in the treated insects indicate significantly increased protein and carbohydrate contents and decreased lipids levels. The study therefore demonstrated the bio-insecticidal toxicity of the EOs from T. pallescens and C. citratus against two important maize post-harvest pests, concurrently revealing significant positive and negative insecticidal activity gradients in relation to single or combined populations.

BACKGROUND: Actin‐related protein 2/3 complex regulates actin polymerization and the formation of branched actin networks. However, the function and evolutionary relationship of this complex subunit 2 (Arpc2) has been poorly understood in insects. RESULTS: To address these issues, we performed comprehensive analysis of Arpc2 in Tribolium castaneum. Phylogenetic analysis revealed that Arpc2 was originated from one ancestral gene in animals but evolved independently between vertebrates and insects after species differentiation. T. castaneum Arpc2 has a 906‐bp coding sequence and consists of 4 exons. Arpc2 transcripts were abundantly detected in embryos and pupae but less so in larvae and adults, while it had high expression in the gut, fat body and head but low expression in the epidermis of late‐stage larvae. Knockdown of it at the late larval stage inhibited the pupation and resulted in arrested larvae. Silencing it in 1‐day pupae impaired eclosion, which caused adult wings to fail to close. Injection of Arpc2 dsRNAs into 5‐day pupae made adults have smaller testis and ovary and could not lay eggs. The expression of vitellogenin 1 (Vg1), Vg2 and Vg receptor (VgR) was downregulated after knocking down Arpc2 5 days post‐adult emergence. Arpc2 silencing reduced 20‐hydroxyecdysone titer by affecting the enzymes of its biosynthesis and catabolism but increased juvenile biosynthesis via upregulating JHAMT3 expression. CONCLUSION: Our results indicate that Arpc2 is associated with the metamorphosis and reproduction by integrating ecdysone and juvenile hormone metabolism in T. castaneum. This study provides theoretical basis for developing Arpc2 as a potential RNA interference target for pest control. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

This study, conducted at Department of Plant Protection, Faculty of Agriculture, Ankara University, aimed to evaluate the efficacy of Spinetoram (Radiant 120 SC) against the red flour beetle, Tribolium castaneum, across three temperatures (20, 25, and 30 °C) and at 65% relative humidity. Adults of T. castaneum were exposed to soft wheat treated with Spinetoram at concentrations of 0.01, 0.25, 0.50, 0.75, 1.00, 2.00, 5.00, and 10.00 ppm in PVC vials for of 1, 2, 3, 7, and 15 days. Bioassays at each dose rates were repeated 3 times with three replicates each. Adult mortality data were analyzed by repeated measures analysis of variance technique and the differences between the means were determined by Tukey's test. Mortality rates were found to be proportional to the dose, temperature, and exposure time. At 0.01, 0.25, 0.5, and 0.75 ppm doses, mortality rates were similar to those of the controls. At 1 ppm dose, mortality rates were 37.04 %, 52.59 %, and 72.59 % after 15 days of exposure at 20, 25 and 30 °C, respectively. The mortalities were 80.74 %, 68.52 % and 81.85 % at 2 ppm dose, 92.59 %, 98.15 %, 90.37 % at 5 ppm dose, and 98.89 %, 99.26 % and 99.63 % at 10 ppm dose, respectively. The development of F1 adults was significantly suppressed at 2 ppm, with the suppression rates of 80.0 %, 63.7 %, and 45.3 % at 20, 25, and 30 °C, respectively. In conclusion, Spinetoram can be considered as an alternative to chemical protectants in the control of T. castaneum. [ABSTRACT FROM AUTHOR]

Simple Summary: Due to issues with pesticide residues and resistance caused by traditional pesticides, the use of modified atmosphere storage technology has become increasingly popular. However, research has shown that prolonged exposure to high concentrations of CO2 for pest control in this technology can lead to insect resistance to hypoxia. Therefore, it is necessary to find out the resistance mechanism. This study identifies TcCYP6K1 and TcCYP9F2 as key factors in the response to high CO2 in Tribolium castaneum, suggesting that these genes may affect the resistance to high CO2 by influencing in the synthesis or breakdown of the carbohydrate metabolism pathways. These findings provide a theoretical basis for the combined use of novel nucleic acid pesticides and modified atmosphere treatment. Cytochrome P450 monooxygenases (CYP), crucial detoxification enzymes in insects, are involved in the metabolism of endogenous substances as well as the activation and degradation of exogenous compounds. In this study, T. castaneum was utilized to investigate the roles of TcCYP6K1 and TcCYP9F2 genes influencing in the trehalose metabolism pathway under high-CO2 stress. By predicting the functional sequences of TcCYP6K1 and TcCYP9F2 genes and analyzing their spatiotemporal expression patterns, it was discovered that both genes belong to the CYP3 group and exhibit high expression levels during the larval stage, decreasing during the pupal stage, while showing high expression in the fatty body, intestine, and malpighian tubules. Furthermore, following the knockdown of TcCYP6K1 and TcCYP9F2 genes in combination with treating larvae with 75% CO2, it was observed that larval mortality increased, and glycogen content significantly decreased, while trehalose content increased significantly. Additionally, membrane-bound trehalase enzyme activity declined, TPS gene expression was significantly upregulated, GS gene expression was significantly downregulated, and ATP content showed a marked decrease. In conclusion, CYP genes are critical responsive genes of T. castaneum to high CO2 levels, potentially impacting the insect's resistance to carbon dioxide through their involvement in the synthesis or breakdown of the carbohydrate metabolism pathway. These findings could serve as a theoretical basis for the utilization of novel pesticides in low-oxygen grain storage techniques and offer new insights for environmentally friendly pest control strategies in grain storage. [ABSTRACT FROM AUTHOR]

The research was conducted with meticulous attention to detail. The study aimed to discern the food precedence and investigate the various life stages of Tribolium castaneum. The research was carried out in two flour media, wheat (Triticum indicum) and rice (Oryza sativa), at an average temperature of 30°C and 50% R.H. The study found that larval and pupal development time was comparatively faster in wheat flour (23 to 27 days) as compared to rice (33 to 37). Oviposition was observed higher in rice flour than in wheat flour. Instars were discerned as six and seven in wheat and rice flour, respectively. The pace of reproduction, development, and growth was higher in the warmer seasons from April till the start of September, especially in June and July, but with the cessation of all these activities in fall and cold seasons, even adult beetles were found dead in extreme cold. [ABSTRACT FROM AUTHOR]

Many species often show male–male combat for mating opportunities and resources within the species. Sexual selection through this radical combat leads to the evolution of males with exaggerated traits used as weapons, such as horns or mandibles, that often result in victory during combat. However, heterospecific interaction due to errors in species identification has often been observed, which results in decreased mating opportunities within the same species and fewer fertilized eggs. Males with exaggerated weapons may show dominance in resource acquisition over males without weapons and may decrease the reproductive success of the latter due to competition between the two. However, few studies have examined heterospecific interaction focusing on males with or without weapons. In this study, we investigated the effects of the male weapon on reproductive traits in heterospecific interaction in two species: the broad‐horned flour beetle (Gnatocerus cornutus), in which males have exaggerated weapon traits; and the red flour beetle (Tribolium castaneum), in which males have no weapon traits. Both species are closely related and use the same food resources. G. cornutus males interfered with the resource acquisition and reproductive opportunities of T. castaneum by attacking T. castaneum. The reproductive success of T. castaneum decreased when they cohabited with G. cornutus males. These findings show that male weapon traits, which are important for sexual selection within the same species, can also greatly influence reproduction in other species. [ABSTRACT FROM AUTHOR]

The thrust of the study was to determine the chemical composition of the essential oils extracted from Thymus pallescens de Noé and Cymbogon citratus Stapf. as well as to evaluate their efficacy in controlling Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst) in either single or combined populations. Carvacrol (56.04%) and geraniol (20.86%) were identified as the major constituents of T. pallescens and C. citratus respectively. The tested essential oils showed pronounced insecticidal activity against the pest species in relation with the applied doses. T. pallescens EO had the highest efficacy and S. zeamais was found to be more susceptible to both individual and combined treatments. With reference to the contact and fumigation assessments, T. pallescens EO effectuated corrected mortality rates ranging from 42.5–100% to 25–100% in S. zeamais with corresponding lethal concentration (LC50) values of 17.7 µl/ml and 15µL/L air respectively. Whereas, the T. pallescens EO exhibited corrected mortality rates of 42.5–100% and 20–100% with corresponding LC50 values of 18.1 µl/ml and 15.5 µL/L air against T. castaneum in contact and fumigation assessments, respectively. The corrected mortality rates increased for both insect species when using combination treatments, with significant increases in the LC50 values, ranging from 8.59 to 49.9% for both pest species. Analysis of energy biomarkers in the treated insects indicate significantly increased protein and carbohydrate contents and decreased lipids levels. The study therefore demonstrated the bio-insecticidal toxicity of the EOs from T. pallescens and C. citratus against two important maize post-harvest pests, concurrently revealing significant positive and negative insecticidal activity gradients in relation to single or combined populations. [ABSTRACT FROM AUTHOR]

The present study investigated the insecticidal efficacy of Diaterra®, a native diatomaceous earth, against two significant stored grain pests, the grain weevil, Sitophilus granarius (Linnaeus, 1758), (Coleoptera: Dryophthoridae) and the red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae). To control of insect pests in stores or warehouses, grain industry relies on synthetic insecticides, but they have significant environmental and health risks. Diatomaceous earth offers a natural and environmentally friendly alternative. In addition, it is safe for wildlife, leaves no residues, and effectively controls pests. In the study, different doses (0.25, 0.5, 1, 2, and 4 g/m2) of diatomaceous earth were applied on concrete and wooden surface. A mortality of 100.0% was achieved for S. granarius on the wooden surface at a dose of 4 g/m2 during the 96-hour and subsequent application periods, whereas for T. castaneum, this rate was recorded as 88.9% under the same dose and a 192-hour application period. On the concrete surface, a mortality rate of 100.0% was achieved for S. granarius at a dose of 4 g/m2 after 120 h of exposure. In contrast, for T. castaneum, this rate did not exceed 59.5% even with the same dose and a 192-hour application period. Under controlled laboratory conditions, a dose of 4 g/m2 of Diaterra® showed efficacy in managing S. granarius populations on wooden and concrete surfaces. The desired level of efficacy was not achieved for T. castaneum on either surface type. Based on the data obtained from this study, it can be concluded that prolonged exposure to diatomaceous earth leads to enhanced insecticidal efficacy against both S. granarius and T. castaneum on different surface types. This study emphasizes the importance of considering application duration and surface type as critical factors in maximizing the effectiveness of diatomaceous earth applications. [ABSTRACT FROM AUTHOR]

The red flour beetle, Tribolium castaneum, is the most common coleopteran pest that infests different kinds of stored grains and flours. Under natural conditions, T. castaneum adults secrete benzoquinones as cuticular semiochemicals for aggregation and defensive purposes. In this study, we have examined the physico-chemical effects on the secretion of cuticular semiochemicals in T. castaneum. Adult beetles were exposed to different food flours, starvation, cold, heat and chemical treatments. After 24 h of exposures, cuticular surface chemicals were extracted and analyzed using gas chromatography–mass spectrometry (GC–MS) and gas chromatography–flame ionization detector (GC–FID) techniques. Totally, 14 compounds were identified as cuticular surface chemicals from the body surface of beetles. When exposed to different food flours (chickpea, green gram, rice and wheat), there were no remarkable variations in the secretion of cuticular surface chemicals. Whereas under starvation, cold, heat and chemical stress conditions, remarkable variations (significantly at P ≤ 0.05) were observed in 2-methyl-p-benzoquinone, 2-ethyl-p-benzoquinone and 1-pentadecene secretions. Remarkably, maximum 99.30, 98.79 and 77.41% reductions were observed in 2-methyl-p-benzoquinone, 2-ethyl-p-benzoquinone and 1-pentadecene secretions due to the effect of diallyl disulfide chemical stress, respectively, with the significance of P ≤ 0.05. In conclusion, it was predicted that starvation, cold, heat and chemical stress conditions alter 2-methyl-p-benzoquinone, 2-ethyl-p-benzoquinone and 1-pentadecene secretions in T. castaneum adults rather than food flours. [ABSTRACT FROM AUTHOR]

In today's rapidly changing world, it is critical to examine how animal populations will respond to severe environmental change. Following events such as pollution or deforestation that cause populations to decline, extinction will occur unless populations can adapt in response to natural selection, a process called evolutionary rescue. Theory predicts that immigration can delay extinction and provide novel genetic material that can prevent inbreeding depression and facilitate adaptation. However, when potential source populations have not experienced the new environment before (i.e. are naive), immigration can counteract selection and constrain adaptation. This study evaluated the effects of immigration of naive individuals on evolutionary rescue using the red flour beetle, Tribolium castaneum , as a model system. Small populations were exposed to a challenging environment, and 3 immigration rates (0, 1, or 5 migrants per generation) were implemented with migrants from a benign environment. Following an initial decline in population size across all treatments, populations receiving no immigration gained a higher growth rate one generation earlier than those with immigration, illustrating the constraining effects of immigration on adaptation. After 7 generations, a reciprocal transplant experiment found evidence for adaptation regardless of immigration rate. Thus, while the immigration of naive individuals briefly delayed adaptation, it did not increase extinction risk or prevent adaptation following environmental change. [ABSTRACT FROM AUTHOR]

The essential oil (EO) extraction is one of the ways to utilize of grapefruit (Citrus paradisi L.) peel waste. However, the low stability and penetration problems of EO limit their use. These limitations can be overcome by nanoemulsions. Therefore, this study aimed to form and characterize of the grapefruit peel essential oil nanoemulsion (EO N) and investigate its insecticidal properties against Sitophilus granarius (Linnaeus, 1875) and Tribolium castaneum (Herbst, 1797) for an alternative bio-economic way. The grapefruit peel EO N (4:3:3:90 w/w, EO:Tween 80:Ethanol:water) was formed by using the grapefruit peel EO with major chemical components such as d-limonene (85.70 %), myrcene (3.57 %), α-pinene (1.17 %). The grapefruit peel EO N was characterized by TEM (50–120 nm), Zeta sizer (the mean droplet particle size: 109.3 nm, ζ-potential value:-20.6 mV, polydispersity index:0.265) and FT-IR analysis. 500 ppm of the grapefruit peel EO N caused 50 % and 20 % mortality in S. granarius and T. castaneum after 7 days, respectively. 1000 ppm of the grapefruit peel EO N caused 85 % and 50 % mortality in S. granarius and T. castaneum after 7 days, respectively. Based on the findings, the grapefruit peel EO N may be considered having the potential to integrate management strategies for these pests and open a new bio-economic way. [Display omitted] • One of the ways to evaluate grapefruit peel waste is essential oil (EO) extraction. • Limitations on the use of EO can be overcome with nanoemulsion (N) form. • Grapefruit peel EO N (50–120 nm and −20.6 mV of ζ potential) was formed. • Grapefruit peel EO N showed a higher toxicity against S. granarius than T. castaneum. • EO N can be one of bio-economic ways of grapefruit peel waste in pest management. [ABSTRACT FROM AUTHOR]

Tribolium castaneum is a significant pest in grain storage, causing considerable economic impact globally. Plant-derived insecticides are being employed as alternatives to chemical insecticides and have shown strong insecticidal activity against T. castaneum. However, the mechanism underlying the insecticidal effects of plant-derived insecticides on T. castaneum remains unclear. In this study, we investigated the repellent, fumigation, and contact activities of R. anthopogonoides essential oil (EO) and its four main chemical components against T. castaneum. The results demonstrated that both the EO and its main chemical component, benzylacetone, exhibited potent insecticidal activity against T. castaneum. Benzylacetone may be the primary active component of R. anthopogonoides EO against T. castaneum. Subsequently, transcriptome sequencing of T. castaneum treated with Benzylacetone, along with negative controls, revealed 1616 differentially expressed genes (DEGs), with 758 up-regulated and 858 down-regulated genes. GO analysis indicated that the DEGs were mainly enriched in "cellular process," "metabolic process," "cell," "cell part," "catalytic activity," "binding," and other categories. KEGG pathway analysis revealed that the 417 DEGs were distributed across 217 different pathways, with several pathways related to xenobiotic or drug metabolism significantly enriched. This suggests that Benzylacetone likely disrupts metabolic and detoxication processes. Additionally, qRT-PCR validation of the TcOBP-4E and TcCYP450-6BK11 genes exhibited consistent results with the transcriptome data. Homology modeling and molecular docking results indicated the presence of a binding cavity formed by numerous hydrophobic amino acid residues in TcOBPs, with possible hydrogen bonds and hydrophobic interaction forces between the protein and ligand. These findings suggest that OBP and CYP450 play crucial roles in the resistance to foreign substances and provide a theoretical basis for understanding the insecticidal mechanisms of plant-derived insecticides at the molecular level. [ABSTRACT FROM AUTHOR]

Numerous species of beetle attack agricultural storage items, causing significant economic losses in terms of quantity and quality. The most harmful pest infesting dry stored produce is the red flour beetle, Tribolium castaneum, which is a well-known secondary pest of stored products. This insect pest show resistance to chemical insecticides, making it difficult to control this pest species with conventional pesticides. Search for non-chemical alternatives for managing this beetle pest has drawn attention for phyto derivatives, many of which are potent as insecticide and yet environmentally friendly. In this study Piper longum leaf extracts in ethyl acetate and methanol were used against eggs of different age, and fourth and fifth instar larvae of Tribolium castaneum. In petri dish bioassay method, ethyl acetate and methanol extracts showed significant ovicidal action with 100% mortality on '0', '2', and '4' days old eggs at concentrations ranging from 300 to 500 µg/dL after 72 and 96 h. The ethyl acetate extract caused 100% mortality of fourth instar larvae at 10, 9 and 8 µg/dL; and on the fifth instar at 10, 8 and 7 µg/dL after 48, 72 and 96 h, respectively. The methanol extract exhibited a similar pattern to that shown in the ethyl acetate extract on the fourth instar larvae after 48, 72 and 96 h of exposure, while for the fifth instar larvae, 100% mortality was recorded at concentrations of 9, 8 and 7 µg/dL, respectively. The results of the study showed that Piper longum leaf extracts is a promising toxic agent to Tribolium castaneum. According to the existence literature survey, this is the first research report on the ovicidal and larvicidal activity of Piper longum leaf extracts against Tribolium castaneum. [ABSTRACT FROM AUTHOR]

A study was conducted to classify the red flour beetle, Tribolium castaneum, using the molecular diagnostic method employing the polymerase chain reaction technique. Five samples were collected from different locations in Baghdad, Iraq. Electrophoresis results using agarose gel revealed the presence of a DNA band of 420 base pairs when using the primers LCO1490 and HCO2198. Furthermore, nucleotide sequence analysis results indicated that all samples belonged to the T. castaneum family. These sequences were deposited in GenBank under the accession number OR912519 and exhibited close similarity to Indian (MN888508) and Thai (MK649853) insects, with 100% and 99% similarity, respectively. The COI region is considered a crucial genetic region that can be effectively applied to measure heterogeneity among closely related species within the same genus. This identification is significant from the point of view of quantifying the infestations by this pest of the grains and subsequent utilization of eco-friendly biopesticide strategies for their control in candidate locations. [ABSTRACT FROM AUTHOR]

Abstract Comparative analyses between traditional model organisms, such as the fruit fly Drosophila melanogaster, and more recent model organisms, such as the red flour beetle Tribolium castaneum, have provided a wealth of insight into conserved and diverged aspects of gene regulation. While the study of trans-regulatory components is relatively straightforward, the study of cis-regulatory elements (CREs, or enhancers) remains challenging outside of Drosophila. A central component of this challenge has been finding a core promoter suitable for enhancer-reporter assays in diverse insect species. Previously, we demonstrated that a Drosophila Synthetic Core Promoter (DSCP) functions in a cross-species manner in Drosophila and Tribolium. Given the over 300 million years of divergence between the Diptera and Coleoptera, we reasoned that DSCP-based reporter constructs will be useful when studying cis-regulation in a variety of insect models across the holometabola and possibly beyond. To this end, we sought to create a suite of new DSCP-based reporter vectors, leveraging dual compatibility with piggyBac and PhiC31-integration, the 3xP3 universal eye marker, GATEWAY cloning, different colors of reporters and markers, as well as Gal4-UAS binary expression. While all constructs functioned properly with a Tc-nub enhancer in Drosophila, complications arose with tissue-specific Gal4-UAS binary expression in Tribolium. Nevertheless, the functionality of these constructs across multiple holometabolous orders suggests a high potential compatibility with a variety of other insects. In addition, we present the piggyLANDR (piggyBac-LoxP AttP Neutralizable Destination Reporter) platform for the establishment of proper PhiC31 landing sites free from position effects. As a proof-of-principle, we demonstrated the workflow for piggyLANDR in Drosophila. The potential utility of these tools ranges from molecular biology research to pest and disease-vector management, and will help advance the study of gene regulation beyond traditional insect models.