Coccinellidae Research Articles

An Advanced Energy Efficient Resource Allocation for Software‐Defined WSN Using Hybrid Optimization Algorithm

ABSTRACTSoftware‐Defined Wireless Sensor Networking (SDWSN) is termed a rising network structure that has become more important to the Internet of Things (IoT). In this network structure, the control planes effectively manage the sensor plane. Due to these kinds of separation, the management of networks became easier and also their efficacies are enhanced in the self‐motivated atmosphere. The common issue presented in the sensor atmosphere is minimal life in the network devices inspired by the maximal level of energy consumption rate. The current study provides a system architecture that intends to increase efficiency in an SDWSN by incorporating optimization techniques. A novel hybrid optimization strategy is created by merging the Election‐Based Optimization Algorithm (EBOA) with the Ladybird Beetle Optimization Algorithm (LBOA), and the result is known as Hybrid Election‐based Ladybird Beetle Optimization (HELBO). This work examines an energy‐efficient resource allocation mechanism in SDWSNs that have substantial computing power and memory. These algorithms optimize the bandwidth along with power allocation in the SDWSN to attain a considerable Signal to Interference plus Noise Ratio (SINR) under the individual constraint of quality of service. Finally, empirical findings show that the recommended HELBO method outperforms other present algorithms.

Seasonal dynamics of arthropods in Lucerne (Medicago sativa L.) and nutritional link to rodent foraging

Lucerne (Medicago sativa L.) is a highly nutritive crop with more vitamins and minerals used as a fodder legume since it is available throughout the year. In the Coimbatore district farmers prefer lucerne as a fodder crop because of its quality, year-round production, and seed yield fetches a high price. The monitoring study on lucerne crop pests, natural enemies and pollinators was conducted at Tamil Nadu Agricultural University, Coimbatore for three seasons from October 2021 to June 2024. The major sucking pests observed in Lucerne were aphids (Aphis craccivora Koch) and stink bugs (Nezara virudula, (L.)) and the natural enemy observed was coccinellid predators (Cheilomenes sexmaculata (Fabricius)). Natural enemies such as the Ladybird beetle (Coccinella repanda Thunberg) and Paper wasp (Polistes exclamans Vierick) were also documented as crucial in regulating these pest populations. The study also observed a diverse range of pollinators including the Alfalfa Leafcutter bee (Megachile rotundata (Fabricius)), various butterflies, and several species of bees that are vital for effective pollination and enhanced crop quality. The population of aphids and stink bugs ranged from 0.00 to 1.47 and from 0.00 to 1.35, respectively, from April to May. In contrast, from November to February, the aphid population varied from 0.07 to 0.45, while the stink bug population ranged from 0.02 to 0.38. The maximum population of coccinellids was recorded at 3.16 in October 2023. The correlation of pest and predator population with weather parameters showed maximum and minimum temperatures favoured the population development while it was negatively correlated with relative humidity and rainfall. Profiling of root nutrients showed lucerne roots contain a significant amount of essential nutrients that meet the dietary requirements of mice.

Crop Association and Predator-Prey Relationship of Predaceous Coccinellid Beetles (Coleoptera: Coccinellidae) from Aurangabad District, Maharashtra, India

A field study was conducted to explore the crop association and predator-prey relationship of predaceous coccinellid beetles of Aurangabad district, starting from May 2022 to April 2024. All the specimens of coccinellid beetles were collected from different agricultural fields. In the family Coccinellidae, 13 species belonging to 11 genera and 3 subfamilies were found during the study period. The collected specimens were found to be associated with various agricultural crops and insect pests.

InsectNet: Real-time identification of insects using an end-to-end machine learning pipeline

Abstract Insect pests significantly impact global agricultural productivity and crop quality. Effective integrated pest management strategies require the identification of insects, including beneficial and harmful insects. Automated identification of insects under real-world conditions presents several challenges, including the need to handle intraspecies dissimilarity and interspecies similarity, life-cycle stages, camouflage, diverse imaging conditions, and variability in insect orientation. An end-to-end approach for training deep-learning models, InsectNet, is proposed to address these challenges. Our approach has the following key features: (a) uses a large dataset of insect images collected through citizen science along with label-free self-supervised learning to train a global model; (b) fine-tuning this global model using smaller, expert verified regional datasets to create a local insect identification model; (c) which provides high prediction accuracy even for species with small sample sizes, (d) is designed to enhance model trustworthiness, and (e) democratizes access through streamlined machine learning operations (MLOps). This global-to-local model strategy offers a more scalable and economically viable solution for implementing advanced insect identification systems across diverse agricultural ecosystems. We report accurate identification (>96% accuracy) of numerous agriculturally and ecologically relevant insect species, including pollinators (e.g., butterflies, bees), parasitoids (e.g., some wasps and flies), predators (e.g., lady beetles and mantises), and harmful insects (e.g., armyworms, cutworms, grasshoppers, stink bugs). InsectNet provides fine-grained insect species identification, works effectively in challenging backgrounds, and avoids making predictions when uncertain, increasing its utility and trustworthiness. The model and associated workflows are available through a web-based portal accessible through a computer or mobile device. We envision InsectNet to complement existing approaches, and be part of a growing suite of AI technologies for addressing agricultural challenges.

Beyond Hunger: A Connection Between Diet and Gonadal Development of a Ladybird Beetle.

Food availability shapes morphology, physiology, growth, reproduction, and overall fitness of insects. Countless research in coccinellids reported the effect of diet in terms of quality and quantity on reproductive output. But fewer studies have reported the direct effects of food on the gonadal development in both sexes. In nature, ladybird beetle Cheilomenes sexmaculata exhibit food preference, favouring Aphis craccivora (nutritious prey) over Aphis nerii (poor prey). We hypothesized that beetles consuming nutritious prey in abundant quantity will have larger and heavier gonads. The current study assessed the effect of food preferred and its fluctuations on the gonadal development of C. sexmaculata. First instars were collected randomly from experimental stock and reared on two different diet regimes for a lifetime. After eclosion, beetles from each dietary regime were dissected daily to assess gonadal development until maturity. Here, we documented total developmental duration, body weight, weight of gonads, GSI, total area of gonads, length, and number of testicular lobules/ovarioles across different treatments. Both quality and quantity of diet significantly affected development period, body weight, and gonadal parameters in both sexes except for GSI in males and number of ovarioles in females that reared on varied diet quality, which were found to be similar. Aphis craccivora in abundant supply positively affected the growth and development of the gonads in C. sexmaculata. Beetles reared on the abundant supply of nutritious prey with larger and heavier gonads than those reared on limited supply or poor prey. This study improves our understanding of the development of gonads in ladybird beetles, which may be helpful in improving the mass rearing of ladybird beetles.

Evaluation of olfactory and visual cues for conservation biological control of crape myrtle bark scale in urban landscapes.

Acanthacoccus lagerstroemiae (crape myrtle bark scale, CMBS) is an exotic scale insect that feeds on the sap of crape myrtle trees. Heavy infestations of CMBS reduce flowering and honeydew promotes sooty mold growth on the leaves and branches, reducing the aesthetic value of crape myrtle trees in urban landscapes. Lady beetles (Coleoptera: Coccinellidae) are generalist predators that feed on CMBS. Several laboratory and field studies have demonstrated the attraction of lady beetles to olfactory and visual cues. We evaluated lady beetles' responses to olfactory lures and yellow visual attractants on infested potted and landscape trees to increase natural enemy recruitment and reduce dependence on chemical control methods. Gas chromatography mass spectrometry analysis showed that limonene is the dominant volatile compound released by crape myrtles infested with CMBS. Limonene and methyl salicylate lures, alone or in combination, failed to reduce CMBS or recruit additional lady beetles to infested trees in all experiments. Yellow rectangular panels placed 1 m above the base of an infested tree recruited up to twofold more lady beetles than control trees; however, this was not statistically significant. A significant reduction in CMBS was observed on infested trees with yellow rectangular panels placed in the canopy. Yellow rectangular panels are more likely to recruit lady beetles than limonene or methyl salicylate lures in an urban landscape. Management of CMBS is currently achieved using systemic insecticides. This study provides a basis for conservation biological control of CMBS through the recruitment of lady beetles, a significant advancement toward integrated management of this exotic pest. © 2024 Society of Chemical Industry.

Biodiversity of predators and parasitoids of greenideine aphids (Insecta: Hemiptera: Aphididae: Greenideinae) and their host plant associations in India

The present article lists the predators and parasitoids of the Greenideinae aphids (Insecta: Hemiptera: Aphididae: Greenideinae) infesting different food plants and their distribution in various states and union territories of India. Predators belong to one order of class Arachnida, the order Araneae (spiders) (2 families) and five orders of class Insecta: Coleoptera (family Coccinellidae), Dermaptera (family Forficulidae), Diptera (family Syrphidae), Hemiptera (family Anthocoridae) and Neuroptera (families Chrysopidae, Hemerobiidae). A total of 43 species of predators from various taxa were recorded to feed 15 species of the greenideine aphids on 12 food plant species in 14 states of India. The highest number of predators belonged to the families Coccinellidae (28 species) and Syrphidae (5 species). Most of the tritrophic associations (triplets, predators-preys-host plants) of these predators were reported in Manipur (41 triplets), Uttarakhand (22 triplets), West Bengal (10 triplets) and in other states with fewer than 10 triplets. All the parasitoids of greedineine aphids belong to a single subfamily Aphelininae (Aphelinidae: Hymenoptera) and Aphidiinae (Braconidae: Hymenoptera) in India. A total of 23 species of parasitoids were observed parasitising these aphids infesting on 19 host plants in 12 states of India. Most of the tritrophic associations (triplets, parasitoids-hosts-host plants) of these parasitoids were reported from Meghalaya (15 triplets), followed by West Bengal (9 triplets), Manipur (8 triplets), and in other states with fewer than 8 triplets. Indeed, minimal attempts have been made to record the natural enemies of these aphids in India, and an intensive and extensive survey plan is warranted to record them in unexplored areas.

Molecular characterization of aphid and their mutualistic and antagonistic interactions with co-occurring herbivore in country bean

The bean aphid is a serious pest due to its ability to drain plant sap and spread viruses and controlling it remains challenging in Bangladesh. Ants are known to establish antagonistic and mutualistic interactions with honeydew-producing sucking pests, and their potential varies with the presence of different cooccurring herbivores. This study aims to infer the role that such interactions play in the population dynamics of ants, ladybird beetles, and aphids in country beans in a multilayered correspondence toward developing an effective biocontrol strategy. The molecular characterization of aphids was performed via the use of the cytochrome oxidase subunit-1 gene of mtDNA along with a taxonomic study of ants. The seven treatments (T1 to T7) included periodic culture and mass release of the ladybird beetle (LBB), which induced ants and aphids. Two subspecies of bean aphids, namely, Aphis fabae fabae (Scopoli, 1763) and Aphis fabae cirsiiacanthoidis (Scopoli 1763), were identified in reference to the GenBank accessions OQ892174-OQ892178 and OQ892199-OQ89220. This is the first sequencing of aphid colonies in Bangladesh, which identified two subspecies of black bean aphids. Among the identified ants, Tapinoma simrothi (Emery, 1925) and Monomorium libanicum (Emery, 1925) were found to be effective under both natural and induced LBB conditions. These findings hold promise for the development of sustainable pest management strategies for Bangladesh’s country bean ecosystems.

Do more food choices lead to bad decisions? A case study in predaceous ladybird beetle, Propylea dissecta.

Understanding why animals choose one food over another is one of the key questions underlying the fields of behaviour ecology. This study aims to test if ladybird beetle, Propylea dissecta Mulsant (Coleoptera: Coccinellidae) can forage selectively for nutrients in order to redress specific nutritional imbalances to maximise their fitness. We hypothesised that the presence of more food choices leads to bad decisions in terms of their food selection which ultimately negatively affects the mating and reproductive parameters of P. dissecta. To test this, we first manipulated the predator's nutritional status by rearing them in five separate dietary groups from first instar larvae to newly emerged adult stage. Thereafter, we tested their food choice between five different foods, i.e. Aphis craccivora Koch, Aphis nerii Boyer de Fonsclombe, conspecific eggs, heterospecific eggs and mixed pollen grains, equidistantly placed in a Petri dish. Based on the food choice of the newly emerged adults, they were reared on the chosen diet for 10 days. Thereafter, adults were paired with their opposite sex (collected from stock culture reared on A. craccivora) and mating and reproductive parameters were recorded. Our results suggested that the variety of food did not affect the food choice of ladybird beetle, P. dissecta. They tend to choose their natural diet, i.e. aphid in each dietary regime. We found that previous dietary regime, i.e. larval dietary regime, significantly influences the mating and reproductive parameters of both the male and female except for the time to commence mating by the male. Food choices of adult beetles were found to significantly influence the time to commence mating, average fecundity and per cent egg viability in males and only mating duration in females. Our findings suggest that P. dissecta consistently made optimal decisions when facing various food choices. They consistently preferred their natural and preferred food choice over others, indicating a strong food selection behaviour.

Pathogenicity of Beauveria bassiana against Chnootriba similis Thunberg (Coleoptera: Coccinellidae) under laboratory and screen house conditions

The herbivorous ladybird beetle, tef epilachna Chnootriba similis Thunberg (Coleoptera Coccinellidae), is a serious pest of cereal crops in many African countries. In Ethiopia, it was reported as a pest of tef (Eragrostis tef (Zuccagni) Trotter (Poaceae) and caused severe damage to all cereal crops in the country. This study was designed to investigate the pathogenicity of different Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) isolates (1x108 conidia mL-1 concentration) against egg, larvae and adults of C. similis under laboratory and screen house conditions. All isolates of B. bassiana showed pathogenicity in all life stages of C. similis. Among the tested isolates, PPRC-56 caused the highest mortality in eggs (76%), first instar larvae (36%), fourth instar larvae (57%) and adults (78%) of C. similis, 11, 6, 8 and 16-days post treatments, respectively. The first and fourth instar larvae were more susceptible than the eggs and adults of C. similis. Our study showed that all B. bassiana isolates were highly effective at killing all stages of C. similis in both laboratory and screen house conditions. The study suggests using the entomopathogenic fungus B. bassiana as a biocontrol agent to control C. similis is very impressive.

Predatory efficiency of six-spotted ladybird beetle, Cheilomenes sexmaculata (Fabricius) against the mixed prey densities: a functional response analysis

Predatory efficiency of six-spotted ladybird beetle, Cheilomenes sexmaculata (Fabricius) against the mixed prey densities: a functional response analysis

Distributions of agrobiont lady beetles (Coleoptera: Coccinellidae) among crop and non-crop habitats within an agricultural landscape in east-central South Dakota, USA

Distributions of agrobiont lady beetles (Coleoptera: Coccinellidae) among crop and non-crop habitats within an agricultural landscape in east-central South Dakota, USA

Population Dynamics of Arthropods in Green Gram [Vigna radiata (L.)] and their Correlation with Meteorological Data at Jabalpur District of Madhya Pradesh, India

Field experiment was carried out to study the population dynamics of arthropods on green gram [Vigna radiata (L.)] at Jabalpur district of Madhya Pradesh, India during the kharif season 2022-23. The result revealed that the highest population of whitefly, jassid and ladybird beetle were observed in the 37th standard meteorological week (SMW) with a population of 6.87 adult / plant, 6.56 adult / 6 leaves and 0.76 adult / plant respectively. The highest population of yellow mosaic virus and damselfly were observed during the 43rd and 40th SMW respectively, with a population of 22.87% and 1.83 adult / sweep respectively. Spider and dragonfly were during the 41st SMW with a population of 0.36 adult / plant and 1.5 adult / sweep respectively. Correlation studies revealed that the whitefly showed a significant positive correlation with wind speed jassid showed a significant positive correlation with evening relative humidity, wind speed and rainy days while the dragonfly showed a significant positive correlation with evaporation. Damselfly showed a significant positive correlation with maximum temperature and ladybird beetle showed a significant positive correlation with whitefly, suggesting potential biocontrol relationships. These findings provide valuable insights into how climate variables influence arthropod populations, which can inform more targeted pest management strategies for green gram cultivation.

Structure, relationships and diversity in the community of aphids and aphidophagous species in alfalfa.

There is a need for comprehensive research on the species structure and the population dynamics of the most common aphidophagous species. A critical factor of the effectiveness of aphid biocontrol is the ratio of beneficial polyphagous (generalist) to oligo- or monophagous (specialist) species within the various trophic groups. Aphids' population density and environmental conditions influence the development and potential feeding of useful insects. The present study aimed to determine the community structure, relationships and diversity between aphids and their aphidophagous species in alfalfa fields using the following methods: sweeping with an entomological net, the quadratic method, coloured sticky board method, route survey method and visual observations. Research on the structure of the aphid-aphidophagous community revealed that aphidophagous species belong to three groups: (1) polyphagous predatory bugs from the families Anthocoridae and Nabidae, (2) oligophagous and polyphagous predators from the families Coccinellidae, Syrphidae and Chrysopidae; and (3) monophagous and oligophagous parasitoids, primarily from the families Aphidiidae and Ichneumonidae. From mid-May to June, there was a sufficiently large potential for aphidophagous species (Coccinellidae, Syrphidae, Chrysopidae, Anthocoridae and Nabidae) to control aphids, while in September, predatory ladybirds from the Coccinellidae family were the main biological control agents. Coccinellidae (Coleoptera) exhibited the highest values of diversity, dominance and richness indices among insect groups in the aphid-aphidophagous community. The existence of diverse aphidophagous species in alfalfa fields suggests that these predators can complement each other, leading to effective biological control of aphids. The synergy among different predator species holds promise for enhancing the overall efficacy of integrated pest management strategies.

Gut bacteria facilitate pollinivory of the ladybird beetle Micraspis discolor.

The ladybird beetle Micraspis discolor plays an important role as a predator of various arthropods within Asia's rice ecosystems. While pollen could serve as an alternative diet for this beetle, facilitating mass-rearing, its pollinivory might inadvertently result in attacks on crop pollen. This study aims to explore the role of gut bacteria on pollinivory of M. discolor. We found that antibiotic treatment significantly reduced the performance of M. discolor when fed Brassica campestris pollen. However, the treatment did not significantly affect their performance when fed an alternative diet of Ephestia kuehniella eggs. Further, we found that antibiotics can eliminate a strain of Serratia marcescens, SmMd, which is a specific gut bacterium in M. discolor. Moreover, the performance of M. discolor showed some degree of recovery when SmMd was reintroduced into its diet. Therefore, we propose that gut bacteria, particularly SmMd, play a significant role in pollen use by this ladybird beetle. This insight enhances our understanding of the important role of gut bacteria in insect adaptation to diverse diets and can potentially optimize the utilization of M. discolor in biological control strategies.

Evaluation of Biopesticides for Fall Armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) Control and Predator Safety in Maize Field Condition

Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a major pest of maize worldwide. The objective of present study was to evaluate the effectiveness of various biopesticides for managing S. frugiperda infesting maize in vegetative (whorl) and reproductive (cob) stages, their effect on the predator, and their effects on yield in Indian conditions. A field trial was conducted in 2019-20 and 2020-21 using a randomized complete block design. The effect of biopesticides against S. frugiperda infesting maize indicated that all the treatments were found effective against fall armyworm as compared to untreated control in management practices applied at the vegetative (whorl) as well as reproductive (cob) stage of maize. Among the biopesticides, spinosad 45 SC was found most effective against fall armyworm followed by Nomuraea rileyi 2×108cfu/g. Among biopesticides treatments, pongamia oil 1% was safe for lady beetles and spiders followed by Bacillus thuringienesis 0.5% WP, Metarhizium anisopliae 2×108cfu/ml, Beauveria bassiana 2×108cfu/ml, Azadirachtin 10000 ppm and Nomuraea rileyi 2×108cfu/g. Economics of bio-pesticides management practices revealed that Spinosad 45 SC @ 0.3 ml/l was the most economical one recording the highest yield (130.63 q/ha) and ICBR 1:36.07. It was followed by Nomuraea rileyi @ 4 g/l, Beauveria bassiana @ 5 ml/l, and Metarhizium anisopliae @ 3 ml/l recording ICBR of 1:12.72, 1:11.73, and 1:11.57 respectively. This study has shown the potential use of biopesticides for the management of FAW. This would offer the farmers a sustainable and affordable option for the management of FAW.

Seasonal Incidence of Sucking Pests and Their Natural Enemies on Moth Bean [Vigna aconitifolia(Jacq.) Marechal

A field experiment was conducted at the College of Agriculture, NAU, Bharuch (Gujarat) to study the seasonal incidence of sucking pests and their natural enemies and their correlation with weather parameters on moth bean during kharif -2023. The studies on seasonal incidence indicated that jassid infestation began in the 1st week of August (31st SMW) and peaked during the 1st week of September (35th SMW). Whitefly infestation also began in the 1st week of August (31st SMW) achieved its peak during 2nd week of September (36th SMW). The population of thripscommenced from 1st week of August (31st SMW) reached its peak during2nd week of September (36th SMW). The population of ladybird beetle appeared from 2nd week of August (32th SMW) and reached its peak during 2nd week of September (36th SMW). The population of chrysoperla appeared from 2nd week of August(32nd SMW) and reached its peak population during 2nd week of September (36th SMW). Among the different environmental factors, morning relative humidity had a significant positive impact on the jassid and whitefly population. Bright sunshine hours showed significant positive influence on thrips and whitefly population. However no significant correlation was observed between ladybird beetle and Chrysoperla population with weather parameters.

Pleiotropic effects of Ebony on pigmentation and development in the Asian multi-coloured ladybird beetle, Harmonia axyridis (Coleoptera: Coccinellidae).

Melanin plays a pivotal role in insect body pigmentation, significantly contributing to their adaptation to diverse biotic and abiotic environmental challenges. Several genes involved in insect melanin synthesis showed pleiotropic effects on insect development and reproduction. Among these, the N-β-alanyl dopamine synthetase gene (Ebony) is integral to the pigmentation process. However, the full spectrum of its pleiotropic impacts is not yet thoroughly understood. In this study, we identified and characterised the HaEbony gene in the Asian multi-coloured ladybird beetle (Harmonia axyridis) and found that HaEbony gene is a conserved gene within the Coleoptera order. We aimed to further explore the multiple roles of HaEbony in the physiology and behaviour in H. axyridis. The CRISPR/Cas9 system was applied to generate multiple HaEbony knockout allele (HaEbony+/-), showing nucleotide deletion in the G0 and G1 generations. Remarkably, the resultant HaEbony+/- mutants consistently displayed darker pigmentation than their wild-type counterparts across larval, pupal and adult stages. Furthermore, these HaEbony+/- individuals (G0) demonstrated an enhanced predatory efficiency, evidenced by a higher number of aphids consumed compared to the wild type. A significant finding was the reduced egg hatchability in both G0 and G1 generations of the HaEbony+/- group, highlighting a potential reproductive fitness cost associated with HaEbony deficiency. In conclusion, our study not only sheds light on the multifaceted roles of HaEbony in H. axyridis but also highlights the potential of employing CRISPR/Cas9-targeted modifications of the Ebony gene. Such genetic interventions could enhance the environmental adaptability and predatory efficacy of ladybirds, presenting a novel strategy in biological control application.

Dietary Ecology of Predatory Insects Examining the Role of Prey and Non Prey Foods of Ladybird Beetle

Ladybird beetles consume a wide variety of non-prey items in their natural habitats, including aphids, fruits, plants, fungi, honeydew, and nectar. These non-prey foods play several critical roles, such as boosting fertility, decreasing mortality during dormancy, supporting migration, and enhancing survival during times when prey is scarce. Larval ladybird beetles have more specialized nutritional needs compared to adults, with simple carbohydrates being crucial nutrients that can improve adult beetle function and reproductive success. Non-prey foods often provide similar or greater nutritional and caloric content compared to prey. Each non-prey item has distinct defense mechanisms and nutritional benefits, influencing its suitability for ladybird beetles. Research comparing diets consisting solely of prey with mixed diets that include non-prey foods revealed that while pollen improved larval and adult performance, a diet high in sugar alone was less effective for reproduction compared to an aphid-based diet. Wheat, which lacks essential nutrients such as proteins, could not adequately support egg production and larval development. Although pollen did enhance overall performance, it could not completely offset the nutritional shortcomings of a diet high in sugar. Adding sugar to a diet did not markedly enhance the performance of ladybird beetles compared to a diet composed entirely of prey. This review underscores the need to understand the varied diet of ladybird beetles within their ecological context, as it has significant implications for the success of conservation-oriented biological control effort.

Selection and validation of reference genes for quantitative expression analysis of regeneration-related genes in Cheilomenes sexmaculata by real-time qRT-PCR.

Regeneration is a fascinating phenomenon that has intrigued scientists for a long time. Cheilomenes sexmaculata (Fabricius), a zig-zag ladybird beetle, possesses a high capacity for limb regeneration. The molecular mechanics of the zig-zag ladybird beetle are under-explored. Current research trends are focused on uncovering functional genes associated with limb regeneration. Most of these investigations involve quantitative real-time PCR (qRT-PCR) for their rapid and accurate analysis of gene expression levels. Hence, a stable and suitable reference gene is required to normalize the gene expression data. In this study, five housekeeping genes were selected from the transcriptomics data (in-house unpublished data) of C. sexmaculata (Fabricius). The expression stabilities of the selected genes were evaluated under different time intervals post-amputation using geNorm, normFinder, and refFinder software. Actin was revealed to be the most stable housekeeping gene, along with elongation factor 2 and glyceraldehyde-3-phosphate dehydrogenase. A target gene named engrailed (an important segment-forming gene) was used to validate the selected reference genes. The expression levels were found to be consistent with the transcriptomics results. According to our study, actin, along with elongation factor 2 and glyceraldehyde-3-phosphate dehydrogenase, serve as the most stable reference genes and are suitable for regeneration-related research. This study is a groundbreaking effort to identify the most stable reference gene for limb regeneration in C. sexmaculata (Fabricius), and the findings can be applied to other related insect species.