Enterococcus casseliflavus regulates amino acid metabolism in edible insect Clanis bilineata tsingtauica: a functional metagenomics study.

Introduction: The soybean hawkmoth, Clanis bilineata tsingtauica, is an edible insect that possesses high nutritional, medicinal and economic value. It has developed into a characteristic agricultural industry in China. Methods: The dominant gut bacterium in diapause larvae of soybean hawkmoths was identified by metagenomics, and the effect of diapause time on gut microbiome composition, diversity and function was investigated. Results: Enterococcus and Enterobacter were measured to be the dominant genera, with Enterococcus casseliflavus and Enterococcus pernyi being the dominant species. Compared to the controls, the relative abundance of E. casseliflavus and E. pernyi on day 14 was lower by 54.51 and 42.45%, respectively. However, the species richness (including the index of Chao and ACE) of gut microbiota increased on day 28 compared to controls. The gene function was mainly focused on carbohydrate and amino acid metabolism. Metabolic pathways annotated for amino acids on day 14 increased by 9.83% compared to controls. It is speculated that diapause soybean hawkmoths may up-regulate amino acid metabolism by reducing E. casseliflavus abundance to maintain their nutritional balance. Additionally, tetracycline, chloromycetin and ampicillin were screened as the top three antibiotics against E. casseliflavus. Discussion: This study not only extends our knowledge of gut microbiome in soybean hawkmoths at the species level, but also provides an initial investigation of gene functionality in interaction with insect hosts.

Morphological Characterization of the Antenna and Scent Patch of Three Danaus Species (Papilionoidea: Nymphalidae, Danainae).

The scent system of Danaus is important for the study of butterfly sexual communication and relevant investigations in biomimetics due to its involvement with mimicry. Using light, scanning, and transmission electron microscopy, the morphological characteristics of Danaus' antennae and scent patches of the scent system for three species, D. chrysippus, D. genutia, and D. plexippus, were investigated herein. Their apical clubs of the flagellums contain sensilla trichodea, sensilla chaetica, and sensilla coeloconica. The scent patch scales typically have a tree-like structure in its lumen at the nano-scale. Comparisons were made between the androconial scales and the other scales in scent patches. Rank sum tests showed significant differences in scent patch scales' characteristics between the species, as well as in the ultrastructure of antennal segments between species and sexes. Spearman's correlation tests showed significant correlations between the morphological characteristics of androconial scales in scent patches. Moreover, the antennal characteristics were significantly correlated. The morphological characteristics of the females' antennae were significantly correlated with those of the males' antennae and androconial scales. However, the significance and coefficient of these correlations were inconsistent across species and sexes. This study provides fundamental morphological information that helps in understanding the pheromone recognition system of Danaus.

Nutritional and Feeding Adaptability of Clanis bilineata tsingtauica Larvae to Different Cultivars of Soybean, (Glycine max).

The larvae of Clanis bilineata tsingtauica, a special species of Chinese edible insect, are of great nutritional, medicinal and economic value to humans. This study aimed to clarify the effect of different soybean varieties (Guandou-3 (G3), Ruidou-1 (R1), September cold (SC)) on the nutritional quality and feeding selection behavior of C. bilineata tsingtauica larvae. The results showed that soybean isoleucine (Ile) and phenylalanine (Phe) were positively correlated with larval host selection (HS) and protein content. The order of soybean plants selected by C. bilineata tsingtauica larvae was R1 > SC > G3, and they selected R1 significantly higher than SC and G3 by 50.55% and 109.01%, respectively. The protein content of the larvae fed on R1 was also the highest among the three cultivars. In addition, a total of 17 volatiles belonging to 5 classes were detected from soybeans: aldehydes, esters, alcohols, ketones, and heterocyclic compounds. Pearson's analysis showed that soybean methyl salicylate was positively correlated with larval HS and their protein content, and soybean 3-octenol was negatively correlated with larval HS and their palmitic acid content. In conclusion, C. bilineata tsingtauica larvae are more adapted to R1 than to the other two soybean species. This study provides a theoretical basis for the production of more protein-rich C. bilineata tsingtauica in the food industry.

TM7 (Saccharibacteria) regulates the synthesis of linolelaidic acid and tricosanoic acid, and alters the key metabolites in diapause Clanis bilineata tsingtauica.

Good exploitation and utilization of edible insects can effectively alleviate global food security crisis in years. The study on diapause larvae of Clanis bilineata tsingtauica (DLC) was conducted to explore how gut microbiota regulate the nutrients synthesis and metabolism of edible insects. The results showed that C. bilineata tsingtauica maintained a total and stable nutrition levels at early phase of diapause. The activity of instetinal enzymes in DLC fluctuated markedly with diapause time. Additionally, Proteobacteria and Firmicutes were the predominant taxa, and TM7 (Saccharibacteria) was the marker species of gut microbiota in DLC. Combined the gene function prediction analysis with Pearson correlation analysis, TM7 in DLC was mainly involved in the biosynthesis of diapause-induced differential fatty acids, i.e., linolelaidic acid (LA) and tricosanoic acid (TA), which was probably regulated by changing the activity of protease and trehalase, respectively. Moreover, according to the non-target metabolomics, TM7 might regulate the significant differential metabolites, i.e., D-glutamine, N-acetyl-d-glucosamine and trehalose, via the metabolism of amino acid and carbohydrate pathways. These results suggest that TM7 increased LA and decreased TA via the intestinal enzymes, and altered intestinal metabolites via the metabolism pathways, maybe a key mechanism for regulating the nutrients synthesis and metabolisms in DLC.

Universal cooling patterns of the butterfly wing scales hierarchy deduced from the heterogeneous thermal and structural properties of Tirumala limniace (Lepidoptera: Nymphalidae, Danainae).

The radiative cooling of butterfly wing scales hierarchy has great value in understanding how poikilotherms adapt to the environment and developing bionic materials. However, it remains unclear what the cooling system is like and how the variation of hierarchy affects the cooling efficiency. Therefore, the correlation between the variations of the structure and emissivity of scale hierarchy is thoroughly investigated in Tirumala limniace (Cramer, 1775), whose thermal properties are highly heterogeneous among different wings and regions but similar between males and females. Patterns were deduced from the biological and model simulation experiments. The scale hierarchy varies at the micro- to nanolevel on both surface and section, corresponding to the variating emissivity. Scales on wing veins and margins have large nanostructured units with small lumens and are distinctly thickened, which bring extraordinarily high emissivity. The variations of light and dark scales, respectively, lead to the high emissivity of the middle region of wings and the front wings. Generally, the elevation of the inner surface area and the thickness of the chitin is the key to enhancing the cooling efficiency. For the first time, the effects of the variation of hierarchy toward emissivity of the mid-infrared spectrum are systematically clarified. It is demonstrated that wing scales integrally differentiate in coping with the heterogeneous cooling needs, which may benefit in balancing multifunctions and the development toward the adaptation to the abiotic environment. The study provides insights into the comprehensive thermoregulation system of butterflies and the further development of radiative cooling materials.

Nutritional Properties of Larval Epidermis and Meat of the Edible Insect Clanis bilineata tsingtauica (Lepidoptera: Sphingidae).

Insects represent a sustainable, protein-rich food source widely consumed in Asia, Africa, and South America. Eating Clanis bilineata tsingtauica Mell is common in the eastern part of China. A comparative characterization of nutrients in the meat and epidermis of C. bilineata tsingtauica was performed in this study. The results showed this insect to be high in nutrients, particularly in the epidermis where protein total was 71.82%. Sixteen different amino acids were quantified in C. bilineata tsingtauica, and the ratio of essential to nonessential amino acids in the epidermis and meat was 68.14% and 59.27%, respectively. The amino acid composition of C. bilineata tsingtauica is balanced, representing a high-quality protein source. Eight minerals were quantified in C. bilineata tsingtauica, including four macro and four trace elements. Fe in the epidermis and Zn in the meat were abundant at 163.82 and 299.31 µg/g DW, respectively. The presence of phytic acid impacted the absorption of mineral elements in food. We also detected phytic acid in C. bilineata tsingtauica. The molar ratio of phytic acid to zinc (PA/Zn) in C. bilineata tsingtauica was very low (3.28) compared to Glycine max and Cryptotympana atrata, which indicated that mineral utilization was high. In conclusion, this study confirms that C. bilineata tsingtauica is a highly nutritious food source for human consumption, and the results provide a basis for further consumption and industrialization of this edible insect.

Edible Aquatic Insects: Diversities, Nutrition, and Safety.

Edible insects have great potential to be human food; among them, aquatic insects have unique characteristics and deserve special attention. Before consuming these insects, the nutrition and food safety should always be considered. In this review, we summarized the species diversity, nutrition composition, and food safety of edible aquatic insects, and also compared their distinguished characteristics with those of terrestrial insects. Generally, in contrast with the role of plant feeders that most terrestrial edible insect species play, most aquatic edible insects are carnivorous animals. Besides the differences in physiology and metabolism, there are differences in fat, fatty acid, limiting/flavor amino acid, and mineral element contents between terrestrial and aquatic insects. Furthermore, heavy metal, pesticide residue, and uric acid composition, concerning food safety, are also discussed. Combined with the nutritional characteristics of aquatic insects, it is not recommended to eat the wild resources on a large scale. For the aquatic insects with large consumption, it is better to realize the standardized cultivation before they can be safely eaten.

Capacity for heat absorption by the wings of the butterfly Tirumala limniace (Cramer).

Butterflies can directly absorb heat from the sun via their wings to facilitate autonomous flight. However, how is the heat absorbed by the butterfly from sunlight stored and transmitted in the wing? The answer to this scientific question remains unclear. The butterfly Tirumala limniace (Cramer) is a typical heat absorption insect, and its wing surface color is only composed of light and dark colors. Thus, in this study, we measured a number of wing traits relevant for heat absorption including the thoracic temperature at different light intensities and wing opening angles, the thoracic temperature of butterflies with only one right fore wing or one right hind wing; In addition, the spectral reflectance of the wing surfaces, the thoracic temperature of butterflies with the scales removed or present in light or dark areas, and the real-time changes in heat absorption by the wing surfaces with temperature were also measured. We found that high intensity light (600-60,000 lx) allowed the butterflies to absorb more heat and 60-90° was the optimal angle for heat absorption. The heat absorption capacity was stronger in the fore wings than the hind wings. Dark areas on the wing surfaces were heat absorption areas. The dark areas in the lower region of the fore wing surface and the inside region of the hind wing surface were heat storage areas. Heat was transferred from the heat storage areas to the wing base through the veins near the heat storage areas of the fore and hind wings.