Whole-Genome Resequencing-Based Qualitative Trait Locus Mapping Correlated yellow with the Mutant Color in Honeybees, Apis cerana cerana.

The honeybee, Apis cerana cerana (Ac), is an important pollinator and has adapted to the local ecological environment with relevant coloration. The cuticle coloration of the brown (br) mutant is brown instead of black in wild-type individuals. Therefore, this study aimed to identify and characterize the gene responsible for the br mutation. Genome resequencing with allele segregation measurement using Euclidean distance followed by Lowess regression analysis revealed that the color locus linked to the mutation was located on chromosome 11. A 2-base deletion on exon 4 was identified in the g7628 (yellow) gene after genome assembly and sequence cloning. In addition, the cuticle color of the abdomen of worker bees changed from black to brown when a defect was induced in the yellow gene using short interfering RNA (siRNA); however, the survival rate did not decrease significantly. These results indicate that the yellow gene participated in the body pigmentation, and its defect was responsible for the br mutation. This study promotes the understanding of the molecular basis of body coloration in honeybees, enriching the molecular mechanisms underlying insect pigmentation.

The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees.

Honey bees are typical model organisms for the study of caste differentiation, and the juvenile hormone (JH) is a crucial link in the regulatory network of caste differentiation in honey bees. To investigate the mechanism of JH-mediated caste differentiation, we analyzed the effect of the JH response gene AmKr-h1 on this process. We observed that AmKr-h1 expression levels were significantly higher in queen larvae than in worker larvae at the 48 h, 84 h, and 120 h larval stages, and were regulated by JH. Inhibiting AmKr-h1 expression in honey bee larvae using RNAi could lead to the development of larvae toward workers. We also analyzed the transcriptome changes in honey bee larvae after AmKr-h1 RNAi and identified 191 differentially expressed genes (DEGs) and 682 differentially expressed alternative splicing events (DEASEs); of these, many were related to honey bee caste differentiation. Our results indicate that AmKr-h1 regulates caste differentiation in honey bees by acting as a JH-responsive gene.

Honey bee maternal effects improve worker performance and reproductive ability in offspring.

Maternal effects are an evolutionary strategy used to improve offspring quality. In an example of maternal effects in honey bees (Apis mellifera), mother queens produce larger eggs in queen cells than in worker cells in order to breed better daughter queens. In our current study, morphological indexes, reproductive tissues, and the egg-laying ability of newly reared queens reared with eggs laid in queen cells (QE), eggs laid in worker cells (WE), and 2-day-old larvae in worker cells (2L) were evaluated. In addition, morphological indexes of offspring queens and working performance of offspring workers were examined. The thorax weight, number of ovarioles, egg length, and number of laid eggs and capped broods of QE were significantly higher than those of WE and 2L, indicating that the reproductive capacity of QE group was better than that of other groups. Furthermore, offspring queens from QE had larger thorax weights and sizes than those from the other two groups. Offspring worker bees from QE also had larger body sizes and greater pollen-collecting and royal jelly-producing abilities than those of other two groups. These results demonstrate that honey bees display profound maternal effects on queen quality that can be transmitted across generations. These findings provide a basis for improving queen quality, with implications in apicultural and agricultural production.

In Situ Polymerized MXene/Polypyrrole/Hydroxyethyl Cellulose-Based Flexible Strain Sensor Enabled by Machine Learning for Handwriting Recognition.

Flexible strain sensors have significant progress in the fields of human-computer interaction, medical monitoring, and handwriting recognition, but they also face many challenges such as the capture of weak signals, comprehensive acquisition of the information, and accurate recognition. Flexible strain sensors can sense externally applied deformations, accurately measure human motion and physiological signals, and record signal characteristics of handwritten text. Herein, we prepare a sandwich-structured flexible strain sensor based on an MXene/polypyrrole/hydroxyethyl cellulose (MXene/PPy/HEC) conductive material and a PDMS flexible substrate. The sensor features a wide linear strain detection range (0-94%), high sensitivity (gauge factor 357.5), reliable repeatability (>1300 cycles), ultrafast response-recovery time (300 ms), and other excellent sensing properties. The MXene/PPy/HEC sensor can detect human physiological activities, exhibiting excellent performance in measuring external strain changes and real-time motion detection. In addition, the signals of English words, Arabic numerals, and Chinese characters handwritten by volunteers measured by the MXene/PPy/HEC sensor have unique characteristics. Through machine learning technology, different handwritten characters are successfully identified, and the recognition accuracy is higher than 96%. The results show that the MXene/PPy/HEC sensor has a significant impact in the fields of human motion detection, medical and health monitoring, and handwriting recognition.

Phenotype-Based Genetic Analysis Reveals Missing Heritability of KIF11-Related Retinopathy: Clinical and Genetic Findings.

The purpose of this study was to detect the missing heritability of patients with KIF11-related retinopathy and to describe their clinical and genetic characteristics. We enrolled 10 individuals from 7 unrelated families harboring a pathogenic monoallelic variant in KIF11. All subjects underwent ophthalmic assessment and extraocular phenotype evaluations, as well as comprehensive molecular genetic analyses using next-generation sequencing. Minigene assays were performed to observe the effects of one novel deep intron variant (DIV) and one novel synonymous variant on pre-mRNA splicing. We detected 6 novel different disease-causing variants of KIF11 in the seven pedigrees. Co-segregation analysis and ultra-deep sequencing results indicated that 5 variants arose de novo in 5 families (71%). Functional validation revealed that the synonymous variant leads to an exon skip, while the DIV causes a pseudoexon (PE) inclusion. The patients presented with high variations in their phenotype, and two families exhibited incomplete penetrance. Ocular manifestations and characteristic facial features were observed in all patients, as well as microcephaly in seven patients, intellectual disability in five patients, and lymphedema in one patient. The key retinal features for KIF11-related retinopathy were retinal folds, tractional retinal detachment, and chorioretinal dysplasia. All seven probands had more severe visual detects than other affected family members. Our findings widen the genetic spectrum of KIF11 variants. DIV explained rare unresolved cases with KIF11-related retinopathy. The patients displayed a variable phenotype expressivity and incomplete penetrance, indicating the importance of genetic analysis for patients with KIF11-related retinopathy.

Flexible Pressure Sensors Based on Molybdenum Disulfide/Hydroxyethyl Cellulose/Polyurethane Sponge for Motion Detection and Speech Recognition Using Machine Learning.

Flexible pressure sensors with excellent performance have broad application potential in wearable devices, motion monitoring, and human-computer interaction. In this paper, a flexible pressure sensor with a porous structure is proposed by coating molybdenum disulfide (MoS2) and hydroxyethyl cellulose (HEC) on a polyurethane (PU) sponge skeleton. The obtained sensor has excellent sensitivity (0.746 kPa-1), a wide detection range (250 kPa), fast response (120 ms), and outstanding repeatability over 2000 cycles. It is proven that the sensor can realize human motion detection and distinguish the touch of varying strength. In addition, a pressure sensing array was fabricated to reflect the pressure distribution and recognize the writing of Arabic numerals. Finally, the sensor performs speech detection through throat muscle movements, and high-accuracy (97.14%) speech recognition for seven words was achieved by a machine learning algorithm based on the support vector machine (SVM). This work provides an opportunity to fabricate simple flexible pressure sensors with potential applications in next-generation electronic skin, health detection, and intelligent robotics.

The adverse impact on lifespan, immunity, and forage behavior of worker bees (Apis mellifera Linnaeus 1758) after exposure to flumethrin.

Several pyrethroids (such as flumethrin and fluvalinate) with low toxicity to honey bees and comparable high toxicity to mites are used worldwide as acaricides. However, flumethrin has been used for a long time in colonies to control Varroa destructor and the honey bees might be exposed to flumethrin cumulatively, which could affect the health of honey bee colonies. This study evaluated the potential adverse effects of direct flumethrin exposure on worker bees under laboratory and colony conditions. Under laboratory conditions, downregulation of genes related to immune was observed when worker bees were exposed to flumethrin above 1/16 LD50; at levels above 1/8 LD50, olfactory learning was impaired, and genes related to learning memory were downregulated; and at >1/4 LD50, their lifespan was shortened. Monitoring with radio frequency identification (RFID) revealed that worker bees in a colony exposed to flumethrin above 1/8 LD50 had a shortened lifespan and reduced foraging ability. When worker bees are exposed to >1/4 LD50 of flumethrin, it can lead to excessive rest day behavior. These results indicate that applying flumethrin in colonies may pose a severe health risk to honey bees and reveal the urgent need to develop non-toxic and highly effective acaricides.

Guar Gum/Ethyl Cellulose-Polyvinyl Pyrrolidone Composite-Based Quartz Crystal Microbalance Humidity Sensor for Human Respiration Monitoring.

In this work, the guar gum (GG) and the electrospinned ethyl cellulose-polyvinyl pyrrolidone (EC-PVP) nanofibers were used as humidity-sensitive materials for fabricating a quartz crystal microbalance (QCM) sensor. Fourier transform infrared spectroscopy, scanning electron microscopy, water contact angle test, and X-ray photoelectron spectra were used to characterize the synthesized GG/EC-PVP composite material, confirming its successful preparation and good hydrophilicity. The humidity sensitivity experiments were performed at room temperature. The GG/EC-PVP-coated QCM sensor has high sensitivity (55.72 Hz/%RH) and low hysteresis (2.8% RH) in a wide relative humidity range (0-97% RH), short response/recovery time (26/2 s), excellent selectivity, good repeatability, and stability. The combined action of hydrophilic groups and porous structure enhances the humidity sensitivity. The GG/EC-PVP sensor can be used to capture and measure typical breathing patterns in different human basic emotions due to its good performance. Furthermore, a lie-detector system was also designed for judging the lying through detecting the emotional breathing pattern of the subjects.

Engineered chimeric insecticidal crystalline protein improves resistance to lepidopteran insects in rice (Oryza sativa L.) and maize (Zea mays L.).

The insecticidal crystalline proteins (Crys) are a family of insect endotoxin functioning in crop protection. As insects keep evolving into tolerance to the existing Crys, it is necessary to discover new Cry proteins to overcome potential threatens. Crys possess three functional domains at their N-termini, and the most active region throughout evolution was found at the domain-III. We swapped domain-IIIs from various Cry proteins and generated seven chimeric proteins. All recombinants were expressed in Escherichia coli and their toxicity was assessed by dietary exposure assays. Three of the seven Crys exhibited a high toxicity to Asian corn borer over the controls. One of them, Cry1Ab-Gc, a chimeric Cry1Ab being replaced with the domain-III of Cry1Gc, showed the highest toxicity to rice stem borer when it was over-expressed in Oryza sativa. Furthermore, it was also transformed into maize, backcrossed into commercial maize inbred lines and then produced hybrid to evaluate their commercial value. Transgenic maize performed significant resistance to the Asian corn borer without affecting the yield. We further showed that this new protein did not have adverse effects on the environment. Our results indicated that domain III swapped of Crys could be used as an efficient method for developing new engineered insecticidal protein.

High-Quality Queens Produce High-Quality Offspring Queens.

Honey bees, rather than rear queens with eggs and larvae from worker cells, prefer to rear new queens with eggs form queen cells, if available. This may be a result of long-term evolutionary process for honey bee colonies. However, the exact mechanism of this phenomenon is unclear. In this study, queens were reared with eggs from queen cells (F1-QE), eggs from worker cells (F1-WE), and two-day-old larvae from worker cells (F1-2L). Physiological indexes and the expression of the development-related genes ((Hexamerin (Hex110, Hex70b), Transferrin (Trf), and Vitellogenin (Vg)) of reared F1 generation queens were measured and compared. Furthermore, F2 generation queens were reared with one-day-old larvae from F1 queens, and the weight and ovariole count of reared F2 generation daughter queens were examined. Meanwhile, the expression of the development- and reproduction-related genes (Hex110, Hex70b, Trf, Vg, and Juvenile Hormone (Jh)) and immune detoxication-related genes (Hymenoptaecin, Abeacin, and CytP450) of reared F2 queens were further explored. We found that the F1-QE queens had the highest physiological indexes and higher Hex110 and Trf expression levels, while no significant difference was found in the expression of Hex70b and Vg among the three groups of F1 queens. In addition, the reared queens of F2-QE had the highest quality, with the highest development, reproduction, immune-detoxication genes' expression levels. Our results revealed that the quality of reared offspring queens from high-quality mother queens was also high. These findings inform methods for rearing high-quality queens and highlight that a high-quality queen is essential for offspring colony growth and survival.

Sublethal fluvalinate negatively affect the development and flight capacity of honeybee (Apis mellifera L.) workers.

Fluvalinate has been heavily used to control the pest Varroa destructor and residues in honeybee colony causing long-term exposure threat for bees. But, little is known about the lifetime trips and homing ability of worker bees under fluvalinate stresses during the development period. In this study, honeybees from 2-day-old larvae to 7-day-old adults were continuously fed with different concentrations of fluvalinate (0, 0.5, 5 and 50 mg/kg) and the effects of fluvalinate on the development of larvae were examined. And then, all the treated bees were reintroduced into the original source colony and were monitored, and the homing ability of 20 days old bees at 1000 and 2000 m away from the beehive were tested using the radio frequency identification (RFID). We found that fluvalinate significantly activates the superoxide dismutase (SOD) activities of larvae and 5 mg/kg fluvalinate reduced the homing rate of workers at 2000 m away from colony. 50 mg/kg fluvalinate reduced proportion of capped worker cells, activated Cytochrome P450 (CYP450) activity of larvae, affected the foraging times, influenced the homing rate and homing time of one trip at 2000 m away from colony. Our results showed that the larvae can activate the activities of SOD and detoxification enzymes in detoxification of fluvalinate and reduce the influence on honeybees. But, when the concentration is higher than 5 mg/kg fluvalinate, it is difficult for bees to detoxify fluvalinate completely, which affect the homing rate. The results reflect the potential risk for honeybees in the development stage continuously exposed to fluvalinate.

Honey bee Apis mellifera larvae gut microbial and immune, detoxication responses towards flumethrin stress.

Mites are considered the worst enemy of honey bees, resulting in economic losses in agricultural production. In apiculture, flumethrin is frequently used to control mites. It causes residues of flumethrin in colonies which may threaten honey bees, especially for larvae. Still, the impact of flumethrin-induced dysbiosis on honey bees larval health has not been fully elucidated, and any impact of microbiota for decomposing flumethrin in honey bees is also poorly understood. In this study, 2-day-old larvae were fed with different flumethrin-sucrose solutions (0, 0.5, 5, 50 mg/kg) and the dose increased daily (1.5, 2, 2.5 and 3 µL) until capped, thereafter the expression level of two immune genes (hymenoptaecin, defensin1) and two detoxication-related genes (GST, catalase) were measured. Meanwhile, the effect of flumethrin on honey bee larvae (Apis mellifera) gut microbes was also explored via 16S rRNA Illumina deep sequencing. We found that flumethrin at 5 mg/kg triggered the over expression of immune-related genes in larvae, while the larval detoxification-related genes were up-regulated when the concentrations reached 50 mg/kg. Moreover, the abundance and diversity of microbes in flumethrin-treated groups (over 0.5 mg/kg) were significantly lower than control group, but it increased with flumethrin concentrations among the flumethrin-treated groups. Our results revealed that microbes served as a barrier in the honey bee gut and were able to protect honey bee larvae to a certain extent, and reduce the stress of flumethrin on honey bee larvae. In addition, as the concentration of flumethrin increases, honey bee larvae activate their immune system then detoxification system to defend against the potential threat of flumethrin. This is the first report on the impact of flumethrin on gut microbiota in honey bees larvae. The findings revealed new fundamental insights regarding immune and detoxification of host-associated microbiota.

Sublethal acetamiprid doses negatively affect the lifespans and foraging behaviors of honey bee (Apis mellifera L.) workers.

The neonicotinoid insecticide acetamiprid is applied widely for pest control in agriculture production. However, little is known about the effects of acetamiprid on the foraging behavior of nontarget pollinators. This study aims to investigate effects of sublethal acetamiprid doses on lifespans and foraging behaviors of honey bees (Apis mellifera L.) under natural swarm conditions. Newly emerged worker bees of each treatment received a drop of 1.5 µL acetamiprid solution (containing 0, 0.5, 1, and 2 µg/bee acetamiprid, diluted by water) on the thorax respectively. Bees from 2-day-old to deadline were monitored on foraging behaviors involving the age of bee for first foraging flights, rotating day-off status and the number of foraging flights using the radio frequency identification (RFID) system. We found that acetamiprid at 2 µg/bee significantly reduced the lifespan, induced precocious foraging activity, influenced the rotating day-off status and decreased foraging flights of worker bees. The abnormal behaviors of worker bees may be associated with a decline in lifespan. This work may provide a new perspective into the neonicotinoids that accelerate the colony failure.

A Chromosome-Scale Assembly of the Asian Honeybee Apis cerana Genome.

Apis cerana is one of the main honeybee species in artificial farming, which is widely distributed in Asian countries. The genome of A. cerana has been sequenced by several different research groups using second generation sequencing technologies. However, it is still necessary to obtain more complete and accurate genome sequences. Here we present a chromosome-scale assembly of the A. cerana genome using single-molecule real-time (SMRT) Pacific Biosciences sequencing and high-throughput chromatin conformation capture (Hi-C) genome scaffolding. The updated assembly is 215.67 Mb in size with a contig N50 of 4.49 Mb, representing an 212-fold improvement over the previous Illumina-based version. Hi-C scaffolding resulted in 16 pseudochromosomes occupying 97.85% of the assembled genome sequences. A total of 10,741 protein-coding genes were predicted and 9,627 genes were annotated. Besides, 314 new genes were identified compared to the previous version. The improved high-quality A. cerana reference genome will provide precise sequence information for biological research of A. cerana.

The role of M2-2 PDZ-binding motifs in pulmonary innate immune responses to human metapneumovirus.

Human metapneumovirus (HMPV) is a leading cause of lower respiratory tract infection (LRTI) in pediatric and geriatric populations. We recently found that two PDZ-binding motifs of the M2-2 protein, 29-DEMI-32 and 39-KEALSDGI-46, play a significant role in mediating HMPV immune evasion in airway epithelial cells (AECs). However, their role in the overall pulmonary responses to HMPV infection has not been investigated. In this study, we found that two recombinant HMPVs (rHMPV) lacking the individual M2-2 PDZ-binding motif are attenuated in mouse lungs. Mice infected with mutants produce more cytokines/chemokines in bronchoalveolar lavage (BAL) fluid compared to mice infected with wild-type rHMPV. In addition, both mutants are able to enhance the pulmonary recruitment of dendritic cells (DCs) and T cells and induce effective protections against the HMPV challenge. The DC maturation is also significantly improved by the motif mutation. Taken together, our data provide proof-of-principle for two live-attenuated M2-2 mutants to be promising HMPV vaccine candidates that are effective in inducing higher pulmonary innate immunity and generating protection against HMPV infection.

Assessment of macular function in patients with idiopathic Epiretinal membrane by multifocal Electroretinography: correlation with visual acuity and optical coherence tomography.

BACKGROUND: This study evaluates the macular function changes in patients with idiopathic macular epiretinal membrane (ERM) by multifocal electroretinography (mfERG) and their correlations with visual acuity and central macular thickness (CMT) by optical coherence tomography (OCT). METHODS: Twenty eyes of 20 patients with ERM underwent OCT and mfERG examinations. The response amplitude densities and implicit times of mfERG were compared to the control fellow eyes. Correlation analyses among visual acuity, central macular thickness and mfERG values in the central two concentric rings were performed. RESULTS: The mfERG P1 response amplitude densities in ring 1-2 and P1 implicit time in ring1 were significantly changed in epiretinal membrane eyes compared with controls (P < 0.05). Multivariate stepwise linear regression analyses showed LogMAR visual acuity was significantly correlated with CMT (P = 0.004), and also with the P1 amplitude density in ring 1 (P = 0.002). CMT showed significant correlation with P1 implicit time in ring 2 (P = 0.013). CONCLUSIONS: The mfERG abnormalities show macular function changes and correlate with visual acuity and central macular thickness in eyes with ERM. In first-order mfERG responses, P1 wave changes may be a sensitive functional measurement for ERM patients.

Fundus Autofluorescence Imaging in the Assessment of Acute Zonal Occult Outer Retinopathy.

PURPOSE: To evaluate the fundus autofluorescence (FAF) findings in eyes with acute zonal occult outer retinopathy (AZOOR) and the relationships between the FAF, spectral-domain optical coherence tomography (SD-OCT), and Humphrey visual field (HVF) findings. METHODS: We performed a retrospective review of the clinical features and FAF, SD-OCT, and HVF findings of 23 eyes of 22 consecutive patients with AZOOR. The mean deviation (MD) values of HVF were compared between areas of normal and abnormal FAF. RESULTS: FAF abnormalities were detected in 21 (91.3%) eyes, with diffuse hyperautofluorescence in 19 (82.6%) eyes and mottling autofluorescence in 2 (8.7%) eyes. In all eyes with FAF abnormality, a clear boundary between the affected and unaffected retina was observed. SD-OCT revealed loss of ellipsoid zone in retinal regions corresponding to the dysfunctional areas demonstrated by HVF. The FAF abnormality also corresponded with the boundaries demonstrated by SD-OCT and/or HVF. The differences in HVF MD values between areas with normal and abnormal FAF in AZOOR eyes were significantly higher than in normal unaffected eyes (p < 0.05). CONCLUSIONS: FAF abnormalities were detected in most AZOOR eyes, and the former corresponded to the abnormal retinal regions revealed by SD-OCT and HVF. The findings suggested that FAF is a useful investigation for the assessment of AZOOR.

Differential protein expression analysis following olfactory learning in Apis cerana.

Studies of olfactory learning in honeybees have helped to elucidate the neurobiological basis of learning and memory. In this study, protein expression changes following olfactory learning in Apis cerana were investigated using isobaric tags for relative and absolute quantification (iTRAQ) technology. A total of 2406 proteins were identified from the trained and untrained groups. Among these proteins, 147 were differentially expressed, with 87 up-regulated and 60 down-regulated in the trained group compared with the untrained group. These results suggest that the differentially expressed proteins may be involved in the regulation of olfactory learning and memory in A. cerana. The iTRAQ data can provide information on the global protein expression patterns associated with olfactory learning, which will facilitate our understanding of the molecular mechanisms of learning and memory of honeybees.

Transcriptome differences in the hypopharyngeal gland between Western Honeybees (Apis mellifera) and Eastern Honeybees (Apis cerana).

BACKGROUND: Apis mellifera and Apis cerana are two sibling species of Apidae. Apis cerana is adept at collecting sporadic nectar in mountain and forest region and exhibits stiffer hardiness and acarid resistance as a result of natural selection, whereas Apis mellifera has the advantage of producing royal jelly. To identify differentially expressed genes (DEGs) that affect the development of hypopharyngeal gland (HG) and/or the secretion of royal jelly between these two honeybee species, we performed a digital gene expression (DGE) analysis of the HGs of these two species at three developmental stages (newly emerged worker, nurse and forager). RESULTS: Twelve DGE-tag libraries were constructed and sequenced using the total RNA extracted from the HGs of newly emerged workers, nurses, and foragers of Apis mellifera and Apis cerana. Finally, a total of 1482 genes in Apis mellifera and 1313 in Apis cerana were found to exhibit an expression difference among the three developmental stages. A total of 1417 DEGs were identified between these two species. Of these, 623, 1072, and 462 genes showed an expression difference at the newly emerged worker, nurse, and forager stages, respectively. The nurse stage exhibited the highest number of DEGs between these two species and most of these were found to be up-regulated in Apis mellifera. These results suggest that the higher yield of royal jelly in Apis mellifera may be due to the higher expression level of these DEGs. CONCLUSIONS: In this study, we investigated the DEGs between the HGs of two sibling honeybee species (Apis mellifera and Apis cerana). Our results indicated that the gene expression difference was associated with the difference in the royal jelly yield between these two species. These results provide an important clue for clarifying the mechanisms underlying hypopharyngeal gland development and the production of royal jelly.