E93 is indispensable for reproduction in ametabolous and hemimetabolous insects.

Ecdysone-induced protein 93 (E93), known as the "adult-specifier" transcription factor in insects, triggers metamorphosis in both hemimetabolous and holometabolous insects. While E93 is conserved in ametabolous insects, its spatiotemporal expression and physiological function remain poorly understood. In this study, we first discovered that in the ametabolous firebrat Thermobia domestica, the previtellogenic ovary exhibits cyclically high E93 expression, and E93 mRNAs are broadly distributed in previtellogenic ovarioles. E93 homozygous mutant females of T. domestica exhibit severe fecundity deficiency due to impaired previtellogenic development of the ovarian follicles, likely because E93 induces the expression of genes involved in ECM (extracellular matrix)-receptor interactions during previtellogenesis. Moreover, we revealed that in the hemimetabolous cockroach Blattella germanica, E93 similarly promotes previtellogenic ovarian development. In addition, E93 is also essential for vitellogenesis to guarantee ovarian maturation and promotes the vitellogenesis-previtellogenesis switch in the fat body of adult female cockroaches. Our findings deepen the understanding of the roles of E93 in controlling reproduction in insects and of E93 expression and functional evolution, which are proposed to have made crucial contributions to the origin of insect metamorphosis.

High-throughput extraction and automatic purification of alternariol from edible and medicinal herbs based on aptamer-functionalized magnetic nanoparticles.

Mycotoxin contamination is widespread in plants and herbs, posing serious threats to the consumer and human health. Of them, alternariol (AOH) has attracted great attention as an "emerging" mycotoxin in medicinal herbs. However, a specific and high-throughput extraction method for AOH is currently lacking. Thus, developing an efficient pre-treatment technique for AOH detection is extremely vital. Here, a novel automated magnetic solid-phase extraction method was proposed for the highly efficient extraction of AOH. Combining the aptamer-functionalized magnetic nanoparticles (AMNPs) and the automatic purification instrument, AOH could be extracted in medicinal herbs in high throughput (20 samples) and a short time (30 min). The main parameters affecting extraction were optimized, and the method was finally carried out by incubation AMNPs with 3 mL of sample solution for 10 min, and then desorption in 75% methanol for liquid-phase detection. Under optimal conditions, good reproducibility, stability, and selectivity were realized with an adsorption capacity of 550.84 ng/mg. AOH extraction in three edible herbs showed good resistance to matrix interference with recovery rates from 86% to 111%. In combination with AMNPs and the automatic purification instrument, high-throughput and labor-free extraction of AOH in different complex matrices was achieved, which could be extended in other complex matrices.

Dissipation and Dietary Risk Assessment of Prochloraz in Strawberries under Greenhouse Conditions.

Prochloraz and its metabolites in strawberries have not been determined until now. Meanwhile, few reports in the literature have concerned the dissipation behavior and risk assessment of prochloraz and its metabolites in strawberries under greenhouse conditions in Beijing. A method for the determination of prochloraz and its metabolites in strawberries was developed using QuEChERS in combination with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Prochloraz and its metabolites recovered from strawberries were present in concentrations of 73.06% to 116.01%, their RSDs ranged from 1.12% to 9.17%, and their limits of detection ranged from 0.1 to 1 µg kg-1. Then, a study was conducted on the dissipation of prochloraz in strawberries under greenhouse conditions. The dissipation of prochloraz in strawberries followed the first-order kinetic equation, and its half-life was 8.06 days. The health risk associated with prochloraz in strawberries was evaluated using the target hazard quotient (THQ) method and EFSA PRIMo model. The results showed that the THQ values, %ARfD values, and %ADI values were less than 1. These results indicate that no health concerns of prochloraz are associated with the consumption of the studied strawberries. The government can use the results of this study to support the establishment of a maximum residue level for prochloraz in strawberries.

A novel electrochemical aptasensor for ultrasensitive detection of herbicide prometryn based on its highly specific aptamer and Ag@Au nanoflowers.

Herbicide prometryn has become a common pollutant in aquatic environments and caused adverse impacts on ecosystems. This study developed an ultrasensitive electrochemical aptasensor for prometryn based on its highly affinitive and specific aptamer and Ag@Au nanoflowers (Ag@AuNFs) for signal amplification. Firstly, this study improved the Capture-SELEX strategy to screen aptamers and obtained aptamer P60-1, which had a high affinity (Kd: 23 nM) and could distinguish prometryn from its structural analogues. Moreover, the typical stem-loop structure in aptamer P60-1 was found to be the binding pocket for prometryn. Subsequently, an electrochemical aptasensor for prometryn was established using multiwalled carbon nanotubes and reduced graphene oxide as electrode substrate, Ag@Au NFs as signal amplification element, and aptamer P60-1 as recognition element. The aptasensor had a detection range of 0.16-500 ng/mL and a detection limit of 60 pg/mL, which was much lower than those of existing detection methods. The aptasensor had high stability and good repeatability, and could specifically detecting prometryn. Furthermore, the utility of the aptasensor was validated by measuring prometryn in environmental and biological components. Therefore, this study provides a robust and ultrasensitive aptasensor for accurate detection for prometryn pollution.

300 Million years of coral treaders (Insecta: Heteroptera: Hermatobatidae) back to the ocean in the phylogenetic context of Arthropoda.

Among hundreds of insect families, Hermatobatidae (commonly known as coral treaders) is one of the most unique. They are small, wingless predaceous bugs in the suborder Heteroptera. Adults are almost black in colour, measuring about 5 mm in body length and 3 mm in width. Thirteen species are known from tropical coral reefs or rocky shores, but their origin and evolutionary adaptation to their unusual marine habitat were unexplored. We report here the genome and metagenome of Hermatobates lingyangjiaoensis, hitherto known only from its type locality in the South China Sea. We further reconstructed the evolutionary history and origin of these marine bugs in the broader context of Arthropoda. The dated phylogeny indicates that Hexapoda diverged from their marine sister groups approximately 498 Ma and that Hermatobatidae originated 192 Ma, indicating that they returned to an oceanic life some 300 Myr after their ancestors became terrestrial. Their origin is consistent with the recovery of tropical reef ecosystems after the end-Triassic mass extinction, which might have provided new and open niches for them to occupy and thrive. Our analyses also revealed that both the genome changes and the symbiotic bacteria might have contributed to adaptations necessary for life in the sea.

Recent progress in aptamer and CRISPR-Cas12a based systems for non-nucleic target detection.

Efficient and sensitive detection of targets is one of the motivations for constant development and innovation of various biosensors. CRISPR-Cas12a, a new generation of gene editing tools, has shown excellent application potential in biosensor design and construction. By combining with the specific recognition element-aptamer, a single-stranded oligonucleotide obtained by systematic evolution of ligands by exponential enrichment (SELEX) in vitro screening, CRISPR-Cas12a also shows superior performance non-nucleic acid targets detection, such as small molecules, proteins, virus and pathogenic bacteria. However, aptamer and CRISPR-Cas12a (CRISPR-Cas12a/Apt) still face some problems in non-nucleic acid target detection, such as single signal response mode and narrow linear range. The development of diverse CRISPR-Cas12a/Apt biosensors is necessary to meet the needs of various detection environments. In this review, the working principle of CRISPR-Cas12a/Apt was introduced and recent progress in CRISPR-Cas12a/Apt in the application of non-nucleic acid target detection was summarized. Moreover, the requirements of critical parameters such as crRNA sequence, activator sequence, and reaction system in the design of CRISPR-Cas12a/Apt biosensors were discussed, which could provide the reference for the design of efficient and sensitive novel non-nucleic acid target biosensors. In addition, the challenges and prospects of CRISPR-Cas12a/Apt-based biosensor were further presented.

High-quality genomes reveal significant genetic divergence and cryptic speciation in the model organism Folsomia candida (collembola).

The collembolan Folsomia candida Willem, 1902, is widely distributed throughout the world and has been frequently used as a test organism in soil ecology and ecotoxicology studies. However, it is questioned as an ideal "standard" because of differences in reproductive modes and cryptic genetic diversity between strains from various geographical origins. In this study, we obtained two high-quality chromosome-level genomes of F. candida, for a parthenogenetic strain (named FCDK, 219.08 Mb, 25,139 protein-coding genes) and a sexual strain (named FCSH, 153.09 Mb, 21,609 protein-coding genes), reannotated the genome of the parthenogenetic strain reported by Faddeeva-Vakhrusheva et al. in 2017 (named FCBL, 221.7 Mb, 25,980 protein-coding genes) and conducted comparative genomic analyses of the three strains. High genome similarities between FCDK and FCBL based on synteny, genome architecture, mitochondrial and nuclear gene sequences suggest that they are conspecific. The seven chromosomes of FCDK are each 25%-54% larger than the corresponding chromosomes of FCSH, showing obvious repetitive element expansions and large-scale inversions and translocations but no whole-genome duplication. The strain-specific genes, expanded gene families and genes in nonsyntenic chromosomal regions identified in FCDK are highly related to the broader environmental adaptation of parthenogenetic strains. In addition, FCDK has fewer strain-specific microRNAs than FCSH, and their mitochondrial and nuclear genes have diverged greatly. In conclusion, FCDK/FCBL and FCSH have accumulated independent genetic changes and evolved into distinct species after 10 million years ago. Our work provides important genomic resources for studying the mechanisms of rapidly cryptic speciation and soil arthropod adaptation to soil ecosystems.

Monitoring leaching of Cd2+ from cadmium-based quantum dots by an Cd aptamer fluorescence sensor.

Quantum Dots (QDs) have been demonstrated with outstanding optical properties and thus been widely used in many biological and biomedical studies. However, previous studies have shown that QDs can cause cell toxicity, mainly attributable to the leached Cd2+. Therefore, identifying the leaching kinetics is very important to understand QD biosafety and cytotoxicity. Toward this goal, instrumental analyses such as inductively coupled plasma mass spectrometry (ICP-MS) have been used, which are time-consuming, costly and do not provide real-time or spatial information. To overcome these limitations, we report herein a fast and cost-effective fluorescence sensor based a Cd2+-specific aptamer for real-time monitoring the rapid leaching kinetics of QDs in vitro and in living cells. The sensor shows high specificity towards Cd2+ and is able to measure the Cd2+ leached either from water-dispersed CdTe QDs or two-layered CdSe/CdS QDs. The sensor is then used to study the stability of these two types of QDs under conditions to mimic cellular pH and temperature and the results from the sensor are similar to those obtained from ICP-MS. Finally, the sensor is able to monitor the leaching of Cd2+ from QDs in HeLa cells. The fluorescence aptamer sensor described in this study may find many applications as a tool for understanding biosafety of numerous other Cd-based QDs, including leaching kinetics and toxicity mechanisms in living systems.

IFT52 plays an essential role in sensory cilia formation and neuronal sensory function in Drosophila.

Cilia are microtubule-based, hair-like organelles involved in sensory function or motility, playing critical roles in many physiological processes such as reproduction, organ development, and sensory perception. In insects, cilia are restricted to certain sensory neurons and sperms, being important for chemical and mechanical sensing, and fertility. Although great progress has been made regarding the mechanism of cilia assembly, the formation of insect cilia remains poorly understand, even in the insect model organism Drosophila. Intraflagellar transport (IFT) is a cilia-specific complex that traffics protein cargos bidirectionally along the ciliary axoneme and is essential for most cilia. Here we investigated the role of IFT52, a core component of IFT-B, in cilia/flagellar formation in Drosophila. We show that Drosophila IFT52 is distributed along the sensory neuronal cilia, and is essential for sensory cilia formation. Deletion of Ift52 results in severe defects in cilia-related sensory behaviors. It should be noted that IFT52 is not detected in spermatocyte cilia or sperm flagella of Drosophila. Accordingly, ift52 mutants can produce sperms with normal motility, supporting a dispensable role of IFT in Drosophila sperm flagella formation. Altogether, IFT52 is a conserved protein essential for sensory cilia formation and sensory neuronal function in insects.

Convergent Adaptation of Ootheca Formation as a Reproductive Strategy in Polyneoptera.

Insects have evolved numerous adaptations and colonized diverse terrestrial environments. Several polyneopterans, including dictyopterans (cockroaches and mantids) and locusts, have developed oothecae, but little is known about the molecular mechanism, physiological function, and evolutionary significance of ootheca formation. Here, we demonstrate that the cockroach asymmetric colleterial glands produce vitellogenins, proline-rich protein, and glycine-rich protein as major ootheca structural proteins (OSPs) that undergo sclerotization and melanization for ootheca formation through the cooperative protocatechuic acid pathway and dopachrome and dopaminechrome subpathway. Functionally, OSP sclerotization and melanization prevent eggs from losing water at warm and dry conditions, and thus effectively maintain embryo viability. Dictyopterans and locusts convergently evolved vitellogenins, apolipoprotein D, and laminins as OSPs, whereas within Dictyoptera, cockroaches and mantids independently developed glycine-rich protein and fibroins as OSPs. Highlighting the ecological-evolutionary importance, convergent ootheca formation represents a successful reproductive strategy in Polyneoptera that promoted the radiation and establishment of cockroaches, mantids, and locusts.

Shortened and multivalent aptamers for ultrasensitive and rapid detection of alternariol in wheat using optical waveguide sensors.

Alternariol (AOH) is one of the common mycotoxins existing in a variety of foods at low level. Aptamers hold great promise for the development of sensitive and rapid aptasensors, but suffer from the excessive length and the difficulty in identification of critical binding domains (CBDs). In this study, the 5 nt CBD of the original 59-nt AOH aptamer (AOH-59, KD = 423 nM) was identified to be a 'C' bulge in between two A-T base pairs. AOH-59 was successfully shortened to a 23 nt aptamer (AOH 6C, KD = 701 nM). A 30 nt bivalent aptamer B-2-3 (KD = 445 nM) and a 39 nt trivalent aptamer T-2-3 (KD = 274 nM) were obtained by simply incorporating one or two CBDs into AOH 6C. The AOH 6C-, B-2-3-, and T-2-3-based optical waveguide aptasensors possessed the unprecedented detection of limits (LODs, S/N = 3) of 42 ± 3, 6 ± 1 and 2 ± 1 fM, respectively. Using the AOH 6C-based sensor as an example, we further demonstrated the detection of AOH spiked in wheat powder with a LOD of 37 pg/g, 20-230-fold lower than those achieved by ELISAs. The sensor was capable for 35 times 2-min regeneration and the assay time including the extraction of AOH from wheat was only about 1 h. We not only devised the first aptasensors for AOH detection, but also provided a simple strategy to design multivalent aptamers for small molecule targets.

Evaluation of Reference Genes for Transcriptional Profiling in Two Cockroach Models.

The German cockroach, Blattella germanica, and the American cockroach, Periplaneta americana are the most common and synanthropic household pests of interest to public health. While they have increasingly served as model systems in hemimetabolous insects for studying many biological issues, there is still a lack of stable reference gene evaluation for reliable quantitative real-time PCR (qPCR) outputs and functional genomics. Here, we evaluated the expression variation of common insect reference genes, including the historically used actin, across various tissues and developmental stages, and also under experimental treatment conditions in these two species by using three individual algorithms (geNorm, BestKeeper, and NormFinder) and a comprehensive program (RefFinder). RPL32 in B. germanica and EF1α in P. americana showed the overall lowest variation among all examined samples. Based on the stability rankings by RefFinder, the optimal but varied reference genes under specific conditions were selected for qPCR normalization. In addition, the combination of RPL32 and EF1α was recommended for all the tested tissues and stages in B. germanica, whereas the combination of multiple reference genes was unfavorable in P. americana. This study provides a condition-specific resource of reference gene selection for accurate gene expression profiling and facilitating functional genomics in these two important cockroaches.

The mitochondrial genome of a minute springtail species Megalothorax incertus (Collembola: Neelipleona: Neelidae).

In this study, the complete mitochondrial genome for the Wolbachia infected parthenogenetic collembola Megalothorax incertus Börner, 1903 was determined. It represents the first report of a complete mitochondrial genome from Neelipleona, one of the four orders of Collembola. The circularized 14,994 bp mitochondrial genome sequence consists of canonical 37 mito-genes, including 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes. The base composition of the majority strand (same direction as most of the mitochondrial genes) is 32.0% for A, 24.1% for C, 11.9% for G, and 32.1% for T. The phylogenetic trees inferred from 13 PCGs using the Maximum-likelihood (ML) and Bayesian inference (BI) methods suggested that Neelidae is basal to the remaining springtails. This mitochondrial genome provides new insights to decipher the phylogeny of Collembola.

An aptamer affinity column for purification and enrichment of lactoferrin in milk.

As an active glycoprotein with high nutritional value, lactoferrin is widely used in food and medical treatment. Therefore, it is very important to establish an accurate and efficient detection method for lactoferrin. At present, the detection of lactoferrin in milk faces many challenges, such as low separation degree and poor parallelism. To address this issue, we developed an aptamer affinity column (AAC) for purification and enrichment of lactoferrin in milk. The column was prepared by covalent conjugation of an amino-modified aptamer with NHS-activated Sepharose. The washing buffer type (0.01 mol/L phosphate buffer) and volume (10 mL) and the sodium chlorideconcentration (1 mol/L) in the elution buffer were optimized for the AAC method. The performance of the AAC was then evaluated in terms of the column capacity, specificity, stability, and reusability. The column capacity was 500 ± 13.7 µg and the column could be reused up to ten times with a large loss in performance. The AAC method combined with high-performance liquid chromatography gave excellent linearity over a wide range, good sensitivity with a limit of detection of 3 µg/mL, and acceptable recoveries for different concentrations of lactoferrin spiked in real raw milk samples from cattle. Finally, the AAC was successfully applied to analyze lactoferrin in milk. This method could be applied to routine analysis of samples for lactoferrin in testing laboratories and dairy factories.

DNAzyme Amplified Aptasensing Platform for Ochratoxin A Detection Using a Personal Glucose Meter.

Aptamer-based sensors have emerged as a major platform for detecting small-molecular targets, because aptamers can be selected to bind these small molecules with higher affinity and selectivity than other receptors such as antibodies. However, portable, accurate, sensitive, and affordable detection of these targets remains a challenge. In this work, we developed an aptasensing platform incorporating magnetic beads and a DNAzyme for signal amplification, resulting in high sensitivity. The biosensing platform was constructed by conjugating a biotin-labeled aptamer probe of small-molecular targets such as toxins and a biotin-labeled substrate strand on magnetic beads, and the DNAzyme strand hybridized with the aptamer probe to block the substrate cleavage activity. The specific binding of the small-molecular target by the aptamer probe can replace the DNAzyme strand and then induce the hybridization between the DNAzyme strand and substrate strand, and the iterative signal amplification reaction of hydrolysis and cleavage of the substrate chain occurs in the presence of a metal ion cofactor. Using invertase to label the substrate strand, the detection of small molecules of the toxin is successfully transformed into the measurement of glucose, and the sensitive analysis of small molecules such as toxins can be realized by using the household portable glucose meter as a readout. This platform is shown to detect ochratoxin, a common toxin in food, with a linear detection range of 5 orders of magnitude, a low detection limit of 0.88 pg/mL, and good selectivity. The platform is easy to operate and can be used as a potential choice for quantitative analysis of small molecules, at home or under point-of-care settings. Moreover, by changing and designing the aptamer probe and the arm of DNAzyme strand, it can be used for the analysis of other analytes.

Grainy head signaling regulates epithelium development and ecdysis in Blattella germanica.

The transcription factor grainy head (Grh) functions in the protection of the epithelium against the external environment by generating strongly adhesive layers, and this function is conserved in vertebrates and invertebrates. In Drosophila, the top model for holometabolous insects, Grh is necessary during embryonic development, epidermal differentiation, central nervous system specification and epithelial repair. However, the function of this gene in hemimetabolous insect epithelia remains unknown. To examine the function of Grh signaling in regulating epithelium development in Hemimetabola, we focused on the Blattella germanica epidermal layer using a gene knockdown strategy. The spatiotemporal expression pattern of BgGrh was detected, and knockdown of BgGrh and BgCad96ca, which provide positive feedback to BgGrh, caused severe defects in new epithelium development and impeded the molting process required to discard the old integument. Knockdown of the expression of BgGrh and BgCad96ca caused increased expression of chitin synthase gene (BgCHS1) and chitinase gene (BgCht5), the upregulations of which should be mediated by the higher level of hormone receptor 3 (BgHr3) gene. In conclusion, epithelium development is regulated by Grh signaling, which might represent a potential target for the control of urban pest cockroaches.

Selection of Reference Genes for Normalization of Gene Expression in Thermobia domestica (Insecta: Zygentoma: Lepismatidae).

Zygentoma occupies a key evolutionary position for understanding the evolution of insect metamorphosis but has received little attention in terms of genetic analysis. To develop functional genomic studies in this insect, we evaluated five candidate internal reference genes for quantitative RT-PCR (qPCR) studies from Thermobia domestica, a representative species of Zygentoma, including Actin 5C (Actin5C), Elongation factor-1 alpha (EF1A), Ribosome protein S26 (RPS26), Ribosome protein L32 (RPL32), and Superoxide dismutase 2 (SOD2), at different developmental stages, in various body parts, and under dsRNA microinjection and starvation stresses, using four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) and a comparative algorithm (RefFinder). Specific suitable reference genes were recommended across specific experimental conditions, and the combination of RPS26 and RPL32 was appropriate for all tested samples. Employing our selected reference gene combination, we investigated the gene expression pattern of Myoglianin (Myo), a crucial gene-regulating insect metamorphosis, in ametabolous T. domestica, and demonstrated the efficiency of RNA interference (RNAi) in firebrat nymphs. This study provides a basis for reliable quantitative studies of genes and greatly benefits evolutionary and functional genomics studies in Zygentoma.

Advances in the Application of Aptamer Biosensors to the Detection of Aminoglycoside Antibiotics.

Antibiotic abuse is becoming increasingly serious and the potential for harm to human health and the environment has aroused widespread social concern. Aminoglycoside antibiotics (AGs) are broad-spectrum antibiotics that have been widely used in clinical and animal medicine. Consequently, their residues are commonly found in animal-derived food items and the environment. A simple, rapid, and sensitive detection method for on-site screening and detection of AGs is urgently required. In recent years, with the development of molecular detection technology, nucleic acid aptamers have been successfully used as recognition molecules for the identification and detection of AGs in food and the environment. These aptamers have high affinities, selectivities, and specificities, are inexpensive, and can be produced with small batch-to-batch differences. This paper reviews the applications of aptamers for AG detection in colorimetric, fluorescent, chemiluminescent, surface plasmon resonance, and electrochemical sensors for the analysis in food and environmental samples. This study provides useful references for future research.

New and little known Isotomidae (Collembola) from the shore of Lake Baikal and saline lakes of continental Asia.

Collembola of the family Isotomidae from the shores of Lake Baikal and from six saline lake catenas of the Buryat Republic (Russia) and Inner Mongolia Province (China) were studied. Pseudanurophorus barathrum Potapov & Gulgenova, sp. nov. and Parisotoma baicalica Potapov & Gulgenova, sp. nov. from Baikal and Ephemerotoma buryatica Potapov, Huang & Gulgenova, sp. nov. and Folsomia mongolica Huang & Potapov, sp. nov. from saline lakes are described here. A morphological description of epitokous males of Scutisotoma acorrelata Potapov, Babenko & Fjellberg, 2006 is given. A list of 23 species of the family Isotomidae found in the shores of studied lakes is provided based on literature sources and newly collected material.