Pupal Exuviae of Culex Pipiens L. (Diptera: Culicidae) Can be Utilised as a Non-Invasive Method of Biotype Differentiation.

BACKGROUND: Culex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay. RESULTS: Genomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55-80%. CONCLUSIONS: This study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.

A trade-off between desiccation resistance and developmental humidity for pupation height in the North Indian seasonal population of Drosophilid-Zaprionus indianus.

Drosophila larvae and pupae are vulnerable to seasonal abiotic stressors such as humidity and temperature. In wild low-humidity habitats, desiccation stress can occur as Drosophila larvae forsake wet food in search of a drier pupation site. Henceforth, the hypothesis that developmental humidity impacts pupation height, affecting larval and pupae water balance and fitness-related traits, was examined. Accordingly, warm-adapted Drosophilid- Zaprionus indianus from two seasons were reared under season-specific simulated conditions, with significantly varying relative humidity (summer RH: 40%; rainy RH: 80%), but nearly identical temperatures. A trade-off between pupation height and developmental humidity was observed. Drier summer conditions lead to pupae wandering farther from drier glass surfaces, resulting in higher pupation height (17.3 cm) while rainy pupae prefer wet food, resulting in lower pupation height (7.12 cm). Additionally, density-dependent pupation height was developmental humidity-specific, with most rainy-season pupae pupated on wetter food, while dry summer pupae pupated on glass surfaces or cotton. Nevertheless, flies from far pupation exhibited greater desiccation resistance, fecundity, and copulation duration than those from near pupation. The cuticular lipid mass of larvae and pupae was higher during far-than-near pupation, indicating decreased water loss rates compared to near-pupation. Finally, pupae eclosion (%) was unaffected by greater humidity (85%) in either season. Still, it considerably decreased at lower humidity (RH: 0% and 38%) for rainy pupae, further supporting the selection of low-humidity desiccation resistance in pupae. In conclusion, low humidity is crucial for survival of pre-adult stages of Zaprionus indianus under desiccation stress and for preference of pupation site.

Development of a Fully Protective Pandemic Avian Influenza Subunit Vaccine in Insect Pupae.

In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines.

The combined effects of ultrasound and plasma-activated water on silkworm pupae:Physicochemical properties, microbiological diversity and ultrastructure.

A novel technique was proposed for processing silkworm pupae by combining plasma- activated water (PAW) with ultrasound (US). The microbial diversity and quality characteristics of the silkworm pupae were also evaluated. The results of the microbial diversity analysis indicated that PAW combined with US treatment significantly reduced the relative abundance of Streptococcaceae, Leuconostocaceae, and Acetobacteraceae from 32%, 18% and 16% to 27%, 11% and 11%, respectively. Microstructural analysis demonstrated that the collapse of the internal structure of chitin in silkworm pupae facilitated the release of nutrients and flavour compounds including fatty acids, water-soluble proteins (WSP), amino acids, phenolics, and volatile compounds. Furthermore, the increase in antioxidant capacity and the decrease in catalase activity and malondialdehyde content confirmed the mechanism of quality change. These findings provide new insights into the possible mechanism of PAW combined with US to improve the quality of edible insects.

An In-Depth Insight into the Profile, Mechanisms, Functions, and Transfer of Essential Amino Acids from Mulberry Leaves to Silkworm Bombyx mori L. Pupae and Fish.

The silkworm Bombyx mori, the second most varied group of insects, is a fascinating insect that belongs to the Lepidoptera species. We aimed to deepen our knowledge about the composition and significance of amino acids (AA) from the sericulture chain to fish. AAs are the most prevalent molecules throughout the growth process of silkworms. We described AAs classification, occurrence, metabolism, and functions. Online datasets revealed that the essential AAs (EAA) level in fish meal and silkworm pupae (SWP) is comparable. SWP have a high content of methionine and lysine, which are the principal limiting AAs in fish diets, indicating that SWP have nutritional potential to be added to fish diets. Additionally, an overview of the data analyzed displays that SWP have a higher protein efficiency ratio than fish meal, the classical protein-rich source (>1.19 times), and compared to soybean meal, the second-most preferred source of protein in aquaculture (>2.08 times), indicating that SWP can be considered effective for animal feeding. In this study, we provide an overview of the current knowledge concerning AAs, paying special emphasis to EAAs and explaining, to some extent, certain mechanisms and functions of these compounds, from mulberry leaves to larvae-pupae and fish diets.

Novel report on soil infection with Metarhizium rileyi against soil-dwelling life stages of insect pests.

Entomopathogenic fungi are the most effective control remedy against a wide range of medical and agricultural important pests. The present study aimed to isolate, identify, and assess the virulence of Metarhizium rileyi against Spodoptera litura and Spodoptera frugiperda pupae under soil conditions. The biotechnological methods were used to identify the isolate as M. rileyi. The fungal conidial pathogenicity (2.0 × 107, 2.0 × 108, 2.0 × 109, 2.0 × 1010, and 2.0 × 1011 conidia/mL-1) was tested against prepupae of S. litura and S. frugiperda at 3, 6, 9, and 12 days after treatments. Additionally, the artificial soil-conidial assay was performed on a nontarget species earthworm Eudrilus eugeniae, using M. rileyi conidia. The present results showed that the M. rileyi caused significant mortality rates in S. litura pupae (61-90%), and S. litura pupae were more susceptible than S. frugiperda pupae (46%-73%) at 12 day posttreatment. The LC50 and LC90 of M. rileyi against S. litura, were 3.4 × 1014-9.9 × 1017 conidia/mL-1 and 6.6 × 105-4.6 × 1014 conidia/mL-1 in S. frugiperda, respectively. The conidia of M. rileyi did not exhibit any sublethal effect on the adult stage of E. eugeniae, and Artemia salina following a 12-day treatment period. Moreover, in the histopathological evaluation no discernible harm was observed in the gut tissues of E. eugeniae, including the lumen and epithelial cells, as well as the muscles, setae, nucleus, mitochondria, and coelom. The present findings provide clear evidence that M. rileyi fungal conidia can be used as the foundation for the development of effective bio-insecticides to combat the pupae of S. litura and S. frugiperda agricultural pests.

Intrapuparial development of Sarcophaga (Liopygia) ruficornis (Diptera: Sarcophagidae), a species of medical-veterinary and forensic importance, under laboratory conditions.

Sarcophaga (=Parasarcophaga) (Liopygia) ruficornis (Fabricius, 1794) is a species of medical-veterinary and forensic importance, as its immatures cause myiasis in humans and animals and colonize carcasses and cadavers. Therefore, investigations into the biology and morphology of this species, with a particular focus on pupae that constitute ≥50% of the developmental period for the immatures, are pertinent. Although there are biological and morphological studies of pupae, the intrapuparial development at different temperatures has not yet been analysed. Therefore, the present study aimed to describe how temperature (22, 27 and 32 ± 1°C) affects the development and morphology of S. (L.) ruficornis pupae at 60 ± 10% relative humidity and a 12-h photoperiod. Ten pupae were collected, euthanized and fixed every 4 h from pupariation until 24 h and every 8 h until the emergence of the first adult. Emergence occurred at 440, 272 and 232 h at 22, 27 and 32°C, with 590, 380 and 330 pupae, respectively. The highest mortality occurred at 32°C. Eight periods were defined, and sex was determined in pharate adult stage; in addition, 40 key morphological characteristics to estimate pupal age were presented. These findings can serve as a basis for studies on the biology and morphology of the pupa of S. (L.) ruficornis, particularly in bionomics, control and forensics, helping researchers and experts.

Sarcophaga (=Parasarcophaga) (Liopygia) ruficornis (Fabricius, 1794) é uma espécie de importância médico­veterinária e forense, pois seus imaturos causam miíase em humanos e animais e colonizam carcaças e cadáveres. Portanto, são pertinentes as investigações sobre a biologia e morfologia dessa espécie, com foco especial nas pupas que constituem ≥50% do período de desenvolvimento dos imaturos. Embora existam estudos biológicos e morfológicos das pupas, o desenvolvimento intrapuparial em diferentes temperaturas ainda não foi analisado. Logo, o presente estudo teve como objetivo descrever como a temperatura (22, 27 e 32 ± 1°C) afeta o desenvolvimento e a morfologia das pupas de S. (L.) ruficornis a 60 ± 10% de umidade relativa e fotoperíodo de 12 horas. Dez pupas foram coletadas, eutanasiadas e fixadas a cada quatro horas desde a pupariação até 24 horas e a cada oito horas até a emergência do primeiro adulto. A emergência ocorreu em 440, 272 e 232 horas a 22, 27 e 32°C, com 590, 380 e 330 pupas, respectivamente. A maior mortalidade ocorreu a 32°C. Foram definidos oito períodos e o sexo foi determinado na fase de adulto farato; além disso, foram apresentadas 40 características morfológicas importantes para estimar a idade das pupas. Essas descobertas podem servir de base para estudos sobre a biologia e a morfologia da pupa de S. (L.) ruficornis, especialmente em bionomia, controle e ciência forense, ajudando pesquisadores e especialistas.

Purification and identification of novel antioxidant peptides derived from Bombyx mori pupae hydrolysates.

The biological importance of antioxidant peptides was the focus of new natural sources of food preservatives. Bombyx mori pupae are considered a valuable by-product of the silk-reeling industry due to their high-quality protein content. This study aimed to purify and identify the antioxidant peptides obtained from enzymatically hydrolyzed B. mori pupae, which could be used as new sources of natural food preservatives. Among the prepared hydrolysates, pepsin hydrolysate with the highest antioxidant activities was purified sequentially using ultrafiltration and reversed-phase high-performance liquid chromatography (RP-HPLC). The DPPH radical scavenging and ferrous ion chelating activity were used to evaluate antioxidant activity. Fractions with high activity were further analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Three peptides were identified as Glu-Asn-Ile-Ile-Leu-Phe-Arg (ENIILFR), Leu-Asn-Lys-Asp-Leu-Met-Arg (LNKDLMR), and Met-Leu-Ile-Ile-Ile-Met-Arg (MLIIIMR), respectively. All three novel identified peptides exhibited significantly stronger antioxidant capacity than synthetic antioxidants used in the food industry, including butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT). ENIILFR showed the best antioxidant activity. These findings indicate that the three peptides have potential applications as natural antioxidants in the food industry.

New records of immature aquatic Diptera from the Foulden Maar Fossil-Lagerstätte, New Zealand, and their biogeographic implications.

Background: The biogeographical and ecological history of true flies (Diptera) in New Zealand is little known due to a scarcity of fossil specimens. Here, we report a fauna of immature aquatic dipterans from freshwater diatomites of the early Miocene Foulden Maar Fossil-Lagerstätte in southern New Zealand. Methods: We document 30 specimens of immature dipterans, mostly pupae, and compare their external morphology to extant aquatic Diptera. Based on the reconstructed paleoenvironment of Foulden Maar, we discuss taxonomic, ecological and taphonomic implications of this early Miocene fauna. Results: Among Chironomidae, one pupal morphotype is attributed to Tanypodinae, one pupal morphotype and one larval morphotype are placed into Chironomus (Chironominae) and a further morphotype into Chironominae incertae sedis. Chaoboridae are represented by a pupal morphotype congeneric or very close to the extant Chaoborus, today globally distributed except for New Zealand. Additional immature specimens are likely larvae and puparia of brachyceran flies but cannot be identified to a narrower range. These finds document an aquatic dipteran fauna in New Zealand in the earliest Miocene and highlight Neogene extinction as a factor in shaping the extant Diptera fauna in New Zealand. Immature aquatic dipterans were a common and likely ecologically important component of the early Miocene Foulden Maar lake. Preservation of larvae and pupae may have been promoted by diatomaceous microbial mats and the light colour of the diatomite likely facilitated spotting of these minute fossils in the field.

Deep orange gene editing triggers temperature-sensitive lethal phenotypes in Ceratitis capitata.

BACKGROUND: The Mediterranean fruit fly, Ceratitis capitata, is a significant agricultural pest managed through area-wide integrated pest management (AW-IPM) including a sterile insect technique (SIT) component. Male-only releases increase the efficiency and cost-effectiveness of SIT programs, which can be achieved through the development of genetic sexing strains (GSS). The most successful GSS developed to date is the C. capitata VIENNA 8 GSS, constructed using classical genetic approaches and an irradiation-induced translocation with two selectable markers: the white pupae (wp) and temperature-sensitive lethal (tsl) genes. However, currently used methods for selecting suitable markers and inducing translocations are stochastic and non-specific, resulting in a laborious and time-consuming process. Recent efforts have focused on identifying the gene(s) and the causal mutation(s) for suitable phenotypes, such as wp and tsl, which could be used as selectable markers for developing a generic approach for constructing GSS. The wp gene was recently identified, and efforts have been initiated to identify the tsl gene. This study investigates Ceratitis capitata deep orange (Ccdor) as a tsl candidate gene and its potential to induce tsl phenotypes. RESULTS: An integrated approach based on cytogenetics, genomics, bioinformatics, and gene editing was used to characterize the Ccdor. Its location was confirmed on the right arm of chromosome 5 in the putative tsl genomic region. Knock-out of Ccdor using CRISPR/Cas9-NHEJ and targeting the fourth exon resulted in lethality at mid- and late-pupal stage, while the successful application of CRISPR HDR introducing a point mutation on the sixth exon resulted in the establishment of the desired strain and two additional strains (dor 12del and dor 51dup), all of them expressing tsl phenotypes and presenting no (or minimal) fitness cost when reared at 25 °C. One of the strains exhibited complete lethality when embryos were exposed at 36 °C. CONCLUSIONS: Gene editing of the deep orange gene in Ceratitis capitata resulted in the establishment of temperature-sensitive lethal mutant strains. The induced mutations did not significantly affect the rearing efficiency of the strains. As deep orange is a highly conserved gene, these data suggest that it can be considered a target for the development of tsl mutations which could potentially be used to develop novel genetic sexing strains in insect pests and disease vectors.

Radiation dose fractionation and its potential hormetic effects on male Glossina palpalis gambiensis (Diptera: Glossinidae): a comparative study of reproductive and flight quality parameters.

One of the most critical factors for implementing the sterile insect technique for the management of tsetse is the production of large quantities of highly competitive sterile males in the field. Several factors may influence the biological quality of sterile males, but optimizing the irradiation protocols to limit unwanted somatic cell damage could improve male performance. This study evaluated the effect of fractionation of gamma radiation doses on the fertility and flight quality of male Glossina palpalis gambiensis. Induced sterility was assessed by mating irradiated males with virgin fertile females. Flight quality was assessed using a standard protocol. The male flies were irradiated as pupae on day 23-27 post larviposition with 110 Gy, either in a single dose or in fractionations of 10 + 100 Gy and 50 + 60 Gy separated by 1-, 2- and 3-day intervals or 55 + 55 Gy separated by 4-, 8-, and 24-hour intervals. All treatments induced more than 90% sterility in females mated with irradiated males, as compared with untreated males. No significant differences were found in emergence rate or flight propensity between fractionated and single radiation doses, nor between the types of fractionations. Overall, the 50(D0) + 60(D1) Gy dose showed slightly higher induced sterility, flight propensity, and survival of males under feeding regime. Dose fractionation resulted in only small improvements with respect to flight propensity and survival, and this should be traded off with the required increase in labor that dose fractionation entails, especially in larger control programs.

Title: Fractionnement de la dose de rayonnement et ses effets hormétiques potentiels sur les Glossina palpalis gambiensis mâles (Diptera : Glossinidae) : une étude comparative des paramètres de reproduction et de qualité de vol. Abstract: L'un des facteurs les plus critiques pour la mise en œuvre de la technique de l'insecte stérile pour la gestion des glossines est la production de grandes quantités de mâles stériles hautement compétitifs sur le terrain. Plusieurs facteurs peuvent influencer la qualité biologique des mâles stériles, mais l'optimisation des protocoles d'irradiation pour limiter les dommages indésirables aux cellules somatiques pourrait améliorer les performances des mâles. Cette étude a évalué l'effet du fractionnement de la dose d'irradiation gamma sur la fertilité et la qualité de vol des mâles de Glossina palpalis gambiensis. La stérilité induite a été évaluée en accouplant des mâles irradiés avec des femelles vierges et fertiles. La qualité du vol a été évaluée à l'aide d'un protocole standard. Les mouches mâles ont été irradiées sous forme de pupes agées de 23 à 27 jours après la larviposition avec 110 Gy, soit en dose unique, soit en fractions de 10 + 100 Gy et 50 + 60 Gy séparées par 1, 2 et 3 jours ou 55 + 55 Gy séparés par des intervalles de 4, 8 et 24 heures. Tous les traitements ont induit plus de 90 % de stérilité chez les femelles accouplées avec des mâles irradiés par rapport aux mâles non traités. Aucune différence significative n'a été trouvée dans le taux d'émergence ou la propension au vol entre les doses d'irradiation fractionnées et uniques ni entre les types de fractionnements. Dans l'ensemble, la dose de 50 (J0) + 60 (J1) Gy a montré une stérilité induite, une propension à voler et une survie légèrement plus élevées chez les mâles sous régime alimentaire. Le fractionnement de dose n'a entraîné que de légères améliorations en ce qui concerne la propension à voler et la survie, et cela devrait être compensé par l'augmentation nécessaire du travail qu'implique le fractionnement de dose, en particulier dans les programmes de contrôle de grande envergure.

Silkworm pupae protein co-degradation by magnetic nanoparticles immobilized proteinase K and Mucor circinelloides aspartic protease for further utilization of sericulture by-products.

Silkworm pupae, by-product of sericulture industry, is massively discarded. The degradation rate of silkworm pupae protein is critical to further employment, which reduces the impact of waste on the environment. Herein, magnetic Janus mesoporous silica nanoparticles immobilized proteinase K mutant T206M and Mucor circinelloides aspartic protease were employed in the co-degradation. The thermostability of T206M improved by enhancing structural rigidity (t1/2 by 30 min and T50 by 5 °C), prompting the degradation efficiency. At 65 °C and pH 7, degradation rate reached the highest of 61.7%, which improved by 26% compared with single free protease degradation. Besides, the immobilized protease is easy to separate and reuse, which maintains 50% activity after 10 recycles. Therefore, immobilized protease co-degradation was first applied to the development and utilization of silkworm pupae resulting in the release of promising antioxidant properties and reduces the environmental impact by utilizing a natural and renewable resource.

The Pupa Stage Is the Most Sensitive to Hypoxia in Drosophila melanogaster.

Hypoxia not only plays a critical role in multiple disease conditions; it also influences the growth and development of cells, tissues and organs. To identify novel hypoxia-related mechanisms involved in cell and tissue growth, studying a precise hypoxia-sensitive time window can be an effective approach. Drosophila melanogaster has been a useful model organism for studying a variety of conditions, and we focused in this study on the life cycle stages of Drosophila to investigate their hypoxia sensitivity. When normoxia-grown flies were treated with 4% O2 at the pupa stage for 3, 2 and 1 day/s, the eclosion rates were 6.1%, 66.7% and 96.4%, respectively, and, when 4% O2 was kept for the whole pupa stage, this regimen was lethal. Surprisingly, when our hypoxia-adapted flies who normally live in 4% O2 were treated with 4% O2 at the pupa stage, no fly eclosed. Within the pupa stage, the pupae at 2 and 3 days after pupae formation (APF), when treated for 2 days, demonstrated 12.5 ± 8.5% and 23.6 ± 1.6% eclosion, respectively, but this was completely lethal when treated for 3 days. We conclude that pupae, at 2 days APF and for a duration of a minimum of 2 days, were the most sensitive to hypoxia. Our data from our hypoxia-adapted flies clearly indicate that epigenetic factors play a critical role in pupa-stage hypoxia sensitivity.

Characterization of intrapuparial development of Musca domestica (Diptera: Muscidae), under different temperatures in laboratory conditions.

Musca domestica (Linnaeus, 1758) (Diptera: Muscidae), popularly known as "housefly", is a highly synanthropic species, with economic, medical-sanitary, veterinary, and forensic importance. It is able to cause damage to health, transmit pathogenic agents, cause infection in domestic animals, and, in its immature stage, cause secondary myiasis. The scavenging habit of its immature stages makes these flies pioneers in colonizing both human and animal carcasses, from the initial stages of corpse decomposition to the final stages. Intrapuparial development studies of all stages of the biological cycle of these insects help estimate pupal age, being useful to forensic entomology to aid in determining the minimum postmortem interval (minPMI). This study describes, morphologically, the external structures of the pupae, under temperatures of 23, 27, and 30 ±â€…1 °C aiming to identify the characteristics that define their developmental stages and estimation of the pupae age of M. domestica. The whole experiment was carried out under laboratory conditions, with relative humidity 60 ±â€…10% and 12 hours of photoperiod. The process of pupariation and pupation including pre-pupae phases were observed; larvae pupae apolysis; early cryptocephalic pupae; late cryptocephalic pupae; phanerocephalic pupae; pharate adult, discriminated by eye color (transparent eyes, pink eyes, and red eyes); and the emergency of adults, which occurred in the intervals of 162-180; 138-144, and 96-102 hr, respectively, being described throughout the metamorphosis of the external morphological characteristics of the pupal stage of M. domestica.

Integrated Analysis of Machine Learning and Deep Learning in Silkworm Pupae (Bombyx mori) Species and Sex Identification.

Hybrid pairing of the corresponding silkworm species is a pivotal link in sericulture, ensuring egg quality and directly influencing silk quantity and quality. Considering the potential of image recognition and the impact of varying pupal postures, this study used machine learning and deep learning for global modeling to identify pupae species and sex separately or simultaneously. The performance of traditional feature-based approaches, deep learning feature-based approaches, and their fusion approaches were compared. First, 3600 images of the back, abdomen, and side postures of 5 species of male and female pupae were captured. Next, six traditional descriptors, including the histogram of oriented gradients (HOG), and six deep learning descriptors, including ConvNeXt-S, were utilized to extract significant species and sex features. Finally, classification models were constructed using the multilayer perceptron (MLP), support vector machine, and random forest. The results indicate that the {HOG + ConvNeXt-S + MLP} model excelled, achieving 99.09% accuracy for separate species and sex recognition and 98.40% for simultaneous recognition, with precision-recall and receiver operating characteristic curves ranging from 0.984 to 1.0 and 0.996 to 1.0, respectively. In conclusion, it can capture subtle distinctions between pupal species and sexes and shows promise for extensive application in sericulture.

Salivary Cystatin-L2-like of Varroa destructor Causes Lower Metabolism Activity and Abnormal Development in Apis mellifera Pupae.

Varroa destructor injects a salivary secretion into honeybees during their feeding process. The salivary secretion plays a vital role in mite-bee interactions and is the main cause of honeybee illness. To determine the biological function of cystatin-L2-like, one of the components of V. destructor salivary secretion, its gene expression in mites during the reproductive phase and dispersal phase was quantified using RT-qPCR, respectively. Moreover, the E. coli-expressed and -purified cystatin was injected into the white-eyed honeybee pupae, and its effects on the survival, the weight of the newly emerged bee, and the transcriptome were determined. The results showed that cystatin was significantly upregulated in mites during the reproductive phase. Cystatin significantly shortened the lifespan of pupae and decreased the weight of the newly emerged bees. Transcriptome sequencing showed that cystatin upregulated 1496 genes and downregulated 1483 genes in pupae. These genes were mainly enriched in ATP synthesis, the mitochondrial respiratory chain, and cuticle structure and function. Cystatin comprehensively downregulated the metabolism of carbohydrates, fatty acids, and amino acids, and energy production in the pupae. The downregulation of metabolic activity could save more nutrients and energy for V. destructor, helping it to maximize its reproduction potential, implying that the mite could manipulate the metabolism of host bees through the injected salivary secretion. The results provide new insights into mite-bee interactions, providing a basis for related studies and applications.

Immature stages of Phloeosinus tacubayae (Curculionidae: Scolytinae): morphology and chaetotaxy of larva and pupa, sexual dimorphism of adults, and developmental time.

The current knowledge of morphology and chaetotaxy of the different developmental stages within the subfamily Scolytinae presents an information deficit that needs to be addressed. Thus, the objective of the present study was to describe, the chaetotaxy and morphology of larvae and pupae, and determine the number of larval instars, the sexual dimorphism in adults, and the development time in Phloeosinus tacubayae. The number of larval instars was determined using traditional morphometry of cephalic capsule and multivariate analysis; description of morphology and chaetotaxy of larvae and pupae, and sexual dimorphism in adults was based on light microscopy and scanning electron microscopy photographs; finally, we quantified development time by mean reviews of 10 gallery systems selected randomly in infested logs, in the laboratory. Morphometric analysis of the cephalic capsule allowed the recognition of 3 different instars. Our results showed that the larvae of P. tacubayae have unique attributes in the body that differentiate them from other genera of the subfamily for example the epicranial suture is not marked, and differentiated from Phloeosinus canadensis, such as a smaller number of setae in the maxillae and without a tergal plate. The pupa had a smaller number of setae on the whole body. The most useful morphological characters to identify a sexual dimorphism in adults were found in the shape and relative position of the seventh and eighth tergites; development time lasted 40 days in total, being the pupal stage the one that took the longest to complete.

Simultaneous Extraction of Oil and Protein from Silkworm (Bombyx mori L.) Pupae (Lueng Parroj var.) and Their In Vitro Skin Moisturization.

Oil and protein from silkworm (Bombyx mori var. Leung Pairoj) pupae, by-product from sericulture, were extracted and evaluated for their potential uses as skin biomoisturizer. The silkworm pupae (SWP) oil and protein were simultaneously extracted by using three-phase partitioning (TPP) method and determined for their physicochemical properties including fatty acid and amino acid content, respectively. The highest yields of oil and protein at 8.24 ± 0.21% and 8.41 ± 0.26% w/w, respectively were obtained from 18 h extraction. Fatty acid analysis of SWP oil was rich in linolenic acid (37.81 ± 0.34%), oleic acid (28.97 ± 0.13%), palmitic acid (21.27 ± 0.05%), stearic acid (6.60 ± 0.09%) and linoleic acid (4.73 ± 0.21%). The clear yellow SWP oil possessed saponification value of 191.51 mg/g, iodine value of 119.37 g I2/g and peroxide value of 2.00 mg equivalent O2/kg. The SWP protein composed of 17 amino acids which aspartic acid, glutamic acid, glycine and serine were the major residues. SDS-PAGE analysis revealed that the SWP protein consisted of distinct protein at around 51, 70, 175 and over 175 kDa. Cytotoxicity of the SWP oil and protein was evaluated by using MTT assay and they showed low cytotoxicity toward keratinocyte cell (HaCat cell line). The SWP oil provided moisturizing effect on pig skin comparable to olive oil, while 1% and 2% of SWP protein showed higher moisturizing efficacy than 3% hydrolyzed collagen. The study indicated that the SWP oil and protein could be potential biomoisturizers for cosmetic products.

Architecture, construction, retention, and repair of faecal shields in three tribes of tortoise beetles (Coleoptera, Chrysomelidae, Cassidinae: Cassidini, Mesomphaliini, Spilophorini).

Animal constructions are the outcomes of complex evolutionary, behavioural, and ecological forces. A brief review of diverse animal builders, the materials used, and the functions they provide their builders is provided to develop approaches to studying faecal-based constructions and faecal-carrying in leaf beetles (Coleoptera: Chrysomelidae). Field studies, rearing, dissections, photography, and films document shields constructed by larvae in two species in two tribes of the subfamily Cassidinae, Calyptocephalaattenuata (Spaeth, 1919) (Spilophorini), and Cassidasphaerula Boheman, 1853 (Cassidini). Natural history notes on an undetermined Cassidini species and Stolascucullata (Boheman, 1862) (Tribe Mesomphaliini) outline the life cycle of tortoise beetles and explain terms. Commonly, the cassidine shield comprises exuviae onto which faeces are daubed, producing a pyramidal-shaped shield that can cover most of the body (up to the pronotum). In Cal.attenuata the larval shield comprises only exuviae, while in Cass.sphaerula, instar 1 initiates the shield by extending its telescopic anus to apply its own faeces onto its paired caudal processes; at each moult the exuvia is pushed to the caudal process base but remains attached, then more faeces are applied over it. The larva's telescopic anus is the only tool used to build and repair the shield, not mouthparts or legs, and it also applies chemicals to the shield. Pupae in Cal.attenuata retain part of the exuviae-only shield of instar VI, while pupae in Cass.sphaerula retain either the entire 5th instar larval shield (faeces + all exuviae) or only the 5th larval exuvia. The caudal processes are crucial to shield construction, shield retention on the body, and as materials of the central scaffold of the structure. They also move the shield, though the muscular mechanism is not known. Altogether the faecal + exuviae shields may represent a unique morpho-behavioural synapomorphy for the crown-clade Cassidinae (10 tribes, ~ 2669 species) and may have been a key innovation in subsequent radiation. Defensive shields and domiciles may help explain the uneven radiation of chrysomelid subfamilial and tribal clades.

Effect of silkworm pupae (Bombyx mori) protein on colon cancer in nude mice: inhibition of tumor growth, oxidative stress and inflammatory response.

Silkworm pupa (bombyx mori) protein (SPP) is a potential therapeutic bioactive substance that has anti-tumor activity against breast, liver, and gastric cancers. The aim of this study was to investigate the antitumor effect of SPP on colon cancer nude mice. Using a subcutaneous tumor formation method, we validated the therapeutic effect of SPP on colon cancer nude mice in vivo. Results showed that SPP was cytotoxic to tumor cells. SPP could protect the liver of the nude mice by lowering hepatic oxidative stress and regulating serum inflammation levels by decreasing TNF-α and IL-2 levels while in-creasing INF-γ levels. In addition, diminished Ki-67 protein, enhanced cleaved caspase-3 protein, di-minished Vimentin, enhanced E-cadherin. These findings suggested that SPP's antitumor activity may be achieved by reducing inflammation, inhibiting tumor proliferation and metastasis, and inducing apoptosis in cancer cells. In the future, SPP could be used as an anticancer drug, potentially providing a new source of drugs for the treatment of colon cancer.