ABSTRACT

Circadian rhythms are indispensable intrinsic programs that regulate the daily rhythmicity of physiological processes, such as feeding and sleep. The cricket has been employed as a model organism for understanding the neural mechanisms underlying circadian rhythms in insects. However, previous studies measuring rhythm-controlled behaviours only analysed locomotive activity using seesaw-type and infrared sensor-based actometers. Meanwhile, advances in deep learning techniques have made it possible to analyse animal behaviour and posture using software that is devoid of human bias and does not require physical tagging of individual animals. Here, we present a system that can simultaneously quantify multiple behaviours in individual crickets – such as locomotor activity, feeding, and sleep-like states – in the long-term, using DeepLabCut, a supervised machine learning-based software for body keypoints labelling. Our system successfully labelled the six body parts of a single cricket with a high level of confidence and produced reliable data showing the diurnal rhythms of multiple behaviours. Our system also enabled the estimation of sleep-like states by focusing on posture, instead of immobility time, which is a conventional parameter. We anticipate that this system will provide an opportunity for simultaneous and automatic prediction of cricket behaviour and posture, facilitating the study of circadian rhythms.

Abstract

Background

Cerebral organoids are three-dimensional in vitro cultured brains that mimic the function and structure of the human brain. One of the major challenges for cerebral organoids is the lack of functional vasculature. Without perfusable vessels, oxygen and nutrient supplies may be insufficient for long-term culture, hindering the investigation of the neurovascular interactions. Recently, several strategies for the vascularization of human cerebral organoids have been reported. However, the generalizable trends and variability among different strategies are unclear due to the lack of a comprehensive characterization and comparison of these vascularization strategies. In this study, we aimed to explore the effect of different vascularization strategies on the nervous system and vasculature in human cerebral organoids.

Results

We integrated single-cell RNA sequencing data of multiple vascularized and vascular organoids and fetal brains from publicly available datasets and assessed the protocol-dependent and culture-day-dependent effects on the cell composition and transcriptomic profiles in neuronal and vascular cells. We revealed the similarities and uniqueness of multiple vascularization strategies and demonstrated the transcriptomic effects of vascular induction on neuronal and mesodermal-like cell populations. Moreover, our data suggested that the interaction between neurons and mesodermal-like cell populations is important for the cerebrovascular-specific profile of endothelial-like cells.

Conclusions

This study highlights the current challenges to vascularization strategies in human cerebral organoids and offers a benchmark for the future fabrication of vascularized organoids.

Abstract

The insect gut is colonized by microbes that confer a myriad of beneficial services to the host, including nutritional support, immune enhancement, and even influence behavior. Insect gut microbes show dynamic changes due to the gut compartments, sex, and seasonal and geographic influences. Crickets are omnivorous hemimetabolous insects that have sex-specific roles, such as males producing chirping sounds for communication and exhibiting fighting behavior. However, limited information is available on their gut bacterial communities, hampering studies on functional compartmentalization of the gut and sex-specific roles of the gut microbiota in omnivorous insects. Here, we report a metagenomic analysis of the gut bacteriome of the field cricket Teleogryllus occipitalis using 16S rRNA V3-V4 amplicon sequencing to identify sex- and compartment-dependent influences on its diversity and function. The structure of the gut microbiota is strongly influenced by their gut compartments rather than sex. The species richness and diversity analyses revealed large difference in the bacterial communities between the gut compartments while minor differences were observed between the sexes. Analysis of relative abundance and predicted functions revealed that nitrogen- and oxygen-dependent metabolism and amino acid turnover were subjected to functional compartmentalization in the gut. Comparisons between the sexes revealed differences in the gut microbiota, reflecting efficiency in energy use, including glycolytic and carbohydrate metabolism, suggesting a possible involvement in egg production in females. This study provides insights into the gut compartment dependent and sex-specific roles of host-gut symbiont interactions in crickets and the industrial production of crickets.

Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics.

Abstract

Significant advances in biophysical methods such as next-generation sequencing technologies have now opened the way to conduct evolutionary and applied research based on the genomic information of greatly diverse insects. Crickets belonging to Orthoptera (Insecta: Polyneoptera), one of the most flourishing groups of insects, have contributed to the development of multiple scientific fields including developmental biology and neuroscience and have been attractive targets in evolutionary ecology for their diverse ecological niches. In addition, crickets have recently gained recognition as food and feed. However, the genomic information underlying their biological basis and application research toward breeding is currently underrepresented. In this review, we summarize the progress of genomics of crickets. First, we outline the phylogenetic position of crickets in insects and then introduce recent studies on cricket genomics and transcriptomics in a variety of fields. Furthermore, we present findings from our analysis of polyneopteran genomes, with a particular focus on their large genome sizes, chromosome number, and repetitive sequences. Finally, how the cricket genome can be beneficial to the food industry is discussed. This review is expected to enhance greater recognition of how important the cricket genomes are to the multiple biological fields and how basic research based on cricket genome information can contribute to tackling global food security.

A cross-sectional survey was conducted to evaluate the market channels, relationship between market intermediates and their functions along with the marketing cost and profit margin of edible wild field cricketBrachytrupes portentosus in Bangladesh. Male respondents were dominantly involved in the insect collection procedure and female were widely engaged in the market. The field cricket trading totally depends on its availability from nature. Generally, this insect becomes available in the winter season (November to February). It was found that insect collectors, wholesaler cum retailers, and local trader cum retailers are the key market intermediates of the four different marketing channels of wild field cricket in Bandarban district of Bangladesh. Among the four observed marketing channels, the channel from insect collectors to the consumer held the big share in the local market. Approximately 43% of the total collection of the wild field crickets occurred through this channel. Both local trader cum retailers and wholesaler cum retailers processed and graded theB. portentosus on the basis of size and sex with the demand of consumers. The net marketing margin was the highest in insect collectors followed by wholesaler cum retailers and local trader cum retailers. The returns on operating capital of insect collectors, wholesaler cum retailers, and local trader cum retailers were 283.7, 48.7 and 42.9%, respectively. The major constraint of edible insect marketing was poor transportation and processing.

Species of infraorder Gryllidea, or crickets, are useful invertebrate models for studying developmental biology and neuroscience. They have also attracted attention as alternative protein sources for human food and animal feed. Mitochondrial genomic information on related invertebrates, such as katydids, and locusts, has recently become available in attempt to clarify the controversial classification schemes, although robust phylogenetic relationships with emphasis on crickets remain elusive. Here, we report newly sequenced complete mitochondrial genomes of crickets to study their phylogeny, genomic rearrangements, and adaptive evolution. First, we conducted de novo assembly of mitochondrial genomes from eight cricket species and annotated protein-coding genes (PCGs) and transfer and ribosomal RNAs using automatic annotations and manual curation. Next, by combining newly described PCGs with public data of the complete Gryllidea genomes and gene annotations, we performed phylogenetic analysis and found gene order rearrangements in several branches. We further analyzed genetic signatures of selection in ant-loving crickets (Myrmecophilidae), which are small wingless crickets that inhabit ant nests. Three distinct approaches revealed two positively selected sites in the cox1 gene in these crickets. Protein 3D structural analyses suggested that these selected sites could influence the interaction of respiratory complex proteins, conferring benefits to ant-loving crickets with a unique ecological niche and morphology. These findings enhance our understanding of the genetic basis of cricket evolution without relying on estimates based on a limited number of molecular markers.

Endocannabinoid system activity contributes to the homeostatic defense against aging and thus may counteract the progression of brain aging. The cannabinoid type 1 (CB1) receptor activity declines with aging in the brain, which impairs neuronal network integrity and cognitive functions. However, the underlying mechanisms that link CB1 activity and memory decline remain unknown. Mitochondrial activity profoundly influences neuronal function, and age-dependent mitochondrial activity change is one of the known hallmarks of brain aging. As CB1 receptor is expressed on mitochondria and may regulate neuronal energy metabolism in hippocampus, we hypothesized that CB1 receptors might influence mitochondria in hippocampal neurons. Here, we found that CB1 receptor significantly affected mitochondrial autophagy (mitophagy) and morphology in an age-dependent manner. Serine 65-phosphorylated ubiquitin, a key marker for mitophagy, was reduced in adult CB1-deficient mice (CB1-KO) compared to those in wild type controls, particularly in CA1 pyramidal cell layer. Transmission electron microscopy (TEM) analysis showed reduced mitophagy-like events in hippocampus of adult CB1-KO. TEM analysis also showed that mitochondrial morphology in adult CB1-KO mice was altered shown by an increase in thin and elongated mitochondria in hippocampal neurons. 3D reconstruction of mitochondrial morphology after scanning electron microscopy additionally revealed an enhanced density of interconnected mitochondria. Altogether, these findings suggest that reduced CB1 signaling in CB1-KO mice leads to reduced mitophagy and abnormal mitochondrial morphology in hippocampal neurons during aging. These mitochondrial changes might be due to the impairments in mitochondrial quality control system, which links age-related decline in CB1 activity and impaired memory.

Ge-132 is a synthetic organic germanium that is used as a dietary supplement. The antioxidant activity of Ge-132 on cultured mammalian cells was investigated in this study. First, Ge-132 cytotoxicity on mammalian cultured cells was determined by measuring lactate dehydrogenase (LDH) levels. Ge-132 had no cytotoxic effect on three different cell lines. Second, the cell proliferative effect of Ge-132 was determined by measuring ATP content of whole cells and counting them. Ge-132 treatment of Chinese hamster ovary (CHO-K1) and SH-SY5Y cells promoted cell proliferation in a dose-dependent manner. Finally, antioxidant activity of Ge-132 against hydrogen peroxide-induced oxidative stress was determined by measuring the levels of intracellular reactive oxygen species (ROS) and carbonylated proteins. Pre-incubation of CHO-K1 and SH-SY5Y cells with Ge-132 suppressed intracellular ROS production and carbonylated protein levels induced by hydrogen peroxide. Our results suggest that Ge-132 has antioxidant activity against hydrogen peroxide-induced oxidative stress.

Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we determined if CRBN has a protective function against oxidative stress, similar to Lon protease. We report that CRBN partially distributes in mitochondria, suggesting it has a mitochondrial function. To specify the mitochondrial role of CRBN, we mitochondrially expressed CRBN in human neuroblastoma SH-SY5Y cells. The resulting stable SH-SY5Y cell line showed no apparent effect on the mitochondrial functions of fusion, fission, and membrane potential. However, mitochondrially expressed CRBN exhibited protease activity, and was induced by oxidative stress. In addition, stably expressed cells exhibited suppressed neuronal cell death induced by hydrogen peroxide. These results suggest that CRBN functions specifically as a Lon-type protease in mitochondria.