Screening of reference genes for gene expression study in different tissues from the transcriptome data of the vector leafhopper Psammotettix striatus.

Selecting appropriate reference genes is crucial for ensuring the accuracy and reliability of gene expression study using reverse transcription-quantitative PCR (RT-qPCR). To screen the optimal reference genes for analyzing gene expression in different tissues of the vector leafhopper Psammotettix striatus which causes extensive damage to a wide range of crops by vectoring multiple plant pathogenic microorganisms, the transcriptome data from Malpighian tubules (MTs) of P. striatus were mined. Twenty alternative candidate reference genes were initially selected for screening, among which seven genes with diverse Gene Ontology (GO) annotations were choosed as candidate reference genes, i.e., ribosomal protein L7A (RPL7A), ribosomal protein S28 (RPS28), ribosomal protein L22 (RPL22), ribosomal protein LP2 (RPLP2), H3 histone family 3A (H3F3A), elongation factor 1γ (EF-1γ), and elongation factor 1α (EF-1α). Gene expression levels in different tissues of P. striatus adults were examined using RT-qPCR, and their expression stability was analyzed using multiple reference gene screening software. This study revealed EF-1α as the most abundantly expressed gene, while RPL22 exhibited the lowest expression levels. EF-1α showed the most stable expression, whereas RPS28 showed the least stability. Various software tools confirmed EF-1α as the most stable single reference gene, and EF-1α and RPLP2 an optimal combination. This study provides a foundation for future investigation of the transmission of pathogenic microorganisms mediated by the vector leafhoppers, the function of the MTs, the biosynthesis of brochosomes, the coevolutionary processes and nutritional interactions of symbionts and host insects, and the gene expression study of other sap-sucking insects.

Functional and evolutionary implications of protein and metal content of leafhopper brochosomes.

Brochosomes derived from the specialized glandular segments of the Malpighian tubules (MTs) form superhydrophobic coatings for insects of Membracoidea, and have multiple hypothetical functions. However, the constituents, biosynthesis and evolutionary origin of brochosomes remain poorly understood. We investigated general chemical and physical characteristics of the integumental brochosomes (IBs) of the leafhopper Psammotettix striatus, determined the constituents of IBs, identified the unigenes involved in brochosomal protein synthesis, and investigated the potential associations among brochosomal protein synthesis, amino acid composition of food source, and the possible roles of endosymbionts in brochosome production. The results show that IBs are mainly composed of glycine- and tyrosine-rich proteins and some metal elements, which contain both essential and non-essential amino acids (EAAs and NEAAs) for insects, including EAAs deficient in the sole food source. All 12 unigenes involved in synthesizing the 12 brochosomal proteins (BPs) with high confidence are exclusively highly expressed in the glandular segment of MTs, confirming that brochosomes are synthesized by this segment. The synthesis of BPs is one of the key synapomorphies of Membracoidea but may be lost secondarily in a few lineages. The synthesis of BPs might be related to the symbiosis of leafhoppers/treehoppers with endosymbionts that provide these insects with EAAs, including those are deficient in the sole diet (i.e., plant sap) and could only be made available by the symbionts. We hypothesize that the functional modification of MTs have combined with the application of BPs enabling Membracoidea to colonize and adapt to novel ecological niches, and evolve to the dramatic diversification of this hemipteran group (in particular the family Cicadellidae). This study highlights the importance of evolutionary plasticity and multiple functions of MTs in driving the adaptations and evolution of sap-sucking insects of Hemiptera.

A Non-Gradual Development Process of Cicada Eyes at the End of the Fifth-Instar Nymphal Stage to Obtain Visual Ability.

Insects' visual system is directly related to ecology and critical for their survival. Some cicadas present obvious differences in color and ultrastructure of compound eyes between nymphal and adult stages, but little is known about when cicadas obtain their visual ability to deal with the novel above-ground habitat. We use transcriptome analyses and reveal that cicada Meimuna mongolica has a trichromatic color vision system and that the eyes undergo a non-gradual development process at the end of the 5th-instar nymphal stage. The white-eye 5th-instar nymphs (i.e., younger 5th-instar nymphs) have no visual ability because critical components of the visual system are deficient. The transformation of eyes toward possessing visual function takes place after a tipping point in the transition phase from the white-eye period to the subsequent red-eye period, which is related to a decrease of Juvenile Hormone. The period shortly after adult emergence is also critical for eye development. Key differentially-expressed genes related to phototransduction and chromophore synthesis play positive roles for cicadas to adapt to above-ground habitat. The accumulation of ommochromes corresponds to the color change of eyes from white to red and dark brown during the end of the 5th-instar nymphal period. Cuticle tanning leads to eye color changing from dark-brown to light-brown during the early adult stage. We hypothesize that the accumulation of ommochromes occurring at the end of 5th-instar nymphal stage and the early adult stage is not only for cicadas to obtain visual ability, but also is a secure strategy to cope with potential photodamage after emergence.

Evaluating the influence of financial investment in compulsory education on the health of Chinese adolescents: a novel approach.

BACKGROUND: With China's aging and declining fertility rate, the importance of population quality is increasing. As the main force of the labor market in the future, the Chinese government tries to promote the development of adolescents by increasing the financial investment in compulsory education, so as to improve the future population quality of China and enhance the national competitiveness. Therefore, the aim of this study was to investigate the relationship between financial investment in compulsory education and the health of Chinese adolescents. METHODS: This study specifically uses data obtained from China Education Panel Survey (CEPS). The data were nationally representative, which covered families, schools, and communities. For the CEPS data obtained, the 2013-2014 school year was the baseline, and two cohorts of 7th and 9th graders were the starting point of the survey. In the 2014-2015 school year, 8th-grade students who participated in the baseline survey (7th-grade students in the 2013-2014 school year) were followed up. Since the second period only began to investigate the data on financial investment in compulsory education, this article uses the data from the 2014-2015 academic year for research. OLS and Ordered Probit models were used to investigate the impact of financial investment in compulsory education on adolescent health. RESULTS: With the doubling of financial investment in compulsory education, self-rated health increased by 0.021, frequency of illness decreased by 0.03, the number of sick leave days decreased by 0.207, and depression decreased by 0.191. The heterogeneity analysis shows that compared to only-child, high-income and nonagricultural groups, the financial investment in compulsory education has a greater impact on the health of adolescents with the characteristics of agriculture hukou, non-only-child and low-income families. Further analysis of the impact mechanism shows that financial investment in compulsory education exerts a significant influence on the health of adolescents by easing family budget constraints, improving school sports facilities, and increasing the expected return of health investment and social capital. CONCLUSIONS: Financial investment in compulsory education can enhance the health of adolescents, and vulnerable groups benefit more, which is conducive to promoting health equity.

Gene expression profiles in Malpighian tubules of the vector leafhopper Psammotettix striatus (L.) revealed regional functional diversity and heterogeneity.

BACKGROUND: Many leafhoppers are known as pests and disease vectors of economically important plants. Previous studies of the physiological functions of vector leafhoppers have mainly focused on the salivary glands and the alimentary tract that are deemed to be associated with digestion, host defense and phytoplasma and/or virus transmission. By contrast, the significance of Malpighian tubules (MTs) is less studied. To clarify the physiological function of MTs of the vector leafhopper Psammotettix striatus that transmits phytoplasma triggering the wheat blue dwarf disease, we performed a transcriptome study on P. striatus MTs and compared gene expression profiles among different anatomical regions in the tubules (i.e., MT1+2, the anterior segment together with the sub-anterior segment; MT3, the inflated segment; and MT4, the distal segment). RESULTS: Transcriptome of P. striatus MTs generate a total of 42,815 high-quality unigenes, among which highly expressed unigenes are mainly involved in organic solute transport, detoxification and immunity in addition to osmoregulation. Region-specific comparative analyses reveal that all these MT regions have functions in osmoregulation, organic solute transport and detoxification, but each region targets different substrates. Differential expression and regional enrichment of immunity-related effector activities and molecules involved in phagocytosis and the biosynthesis of antimicrobial peptides among different regions indicate that MT1+2 and MT4 have the ability to eliminate the invading pathogens. However, in MT3 which secrets brochosomes to the integument and eggs as physical barriers, disulfide-isomerase, acidic ribosomal protein P and many other unigenes were highly expressed, which can be attractive candidate genes for future studies of the biosynthesis and the origin of brochosomes. CONCLUSIONS: Psammotettix striatus MTs perform multiple physiological functions as versatile organs than just excretory organs with osmoregulatory function. Heterogeneity of physiological functions among different MT regions is related to organic solute transport, detoxification, immunity and brochosome biosynthesis in addition to osmoregulation, and each region targets different substrates. These functions may be helpful for P. striatus to resist pathogens from habitats and to utilize a wider range of host plants, which may assist the transmission and spread of phytoplasmas. The results provide potential molecular targets for the exploit of chemical and/or gene-silencing insecticides.