Selection and validation of optimal reference genes for RT-qPCR analyses in Aphidoletes aphidimyza Rondani (Diptera: Cecidomyiidae).

Aphidoletes aphidimyza is a predator that is an important biological agent used to control agricultural and forestry aphids. Although many studies have investigated its biological and ecological characteristics, few molecular studies have been reported. The current study was performed to identify suitable reference genes to facilitate future gene expression and function analyses via quantitative reverse transcription PCR. Eight reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RPS13, RPL8, RPS3, α-Tub, ß-actin, RPL32, and elongation factor 1 alpha (EF1-α) were selected. Their expression levels were determined under four different experimental conditions (developmental stages, adult tissues, sugar treatment, and starvation treatment) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper, and RefFinder). The results showed that GAPDH, RPL32, and EF1-α were ranked as the best reference gene combinations for measuring gene expression levels among different developing stages and in various starvation treatments. RPL8 and RPS3 were recommended to normalize the gene expression levels among different adult tissues. RPL32, ß-actin, and EF1-α were recommended sugar-feeding conditions. To validate the utility of the selected reference pair, RPL8, and RPS3, we estimated the tissue-biased expression level of a chemosensory protein gene (AaphCSP1). As expected, AaphCSP1 is highly expressed in the antennae and lowly expressed in the abdomen. These findings will lay the foundation for future research on the molecular physiology and biochemistry of A. aphidimyza.

Effects of cold storage after cold acclimation on the fitness of Habrobracon hebetor (Hymenoptera: Braconidae).

Habrobracon hebetor (Say) (Hymenoptera: Braconidae) is a cosmopolitan, idiobiont, and gregarious ectoparasitoid, which can parasitize the larvae of several pyralid and noctuid moths. However, adult parasitoids require cold storage to ensure that adequate individuals are available when a pest outbreak occurs. To understand the effects of cold storage after acclimation on offspring fitness of H. hebetor, the development, fecundity, population parameters, and paralysis rate of the F1 generation were evaluated using an age-stage, two-sex life table. Four pairing treatments were used in this study, with refrigerated males and females (ReF×ReM), unrefrigerated females and refrigerated males (UnF×ReM), refrigerated females and unrefrigerated males (ReF×UnM), and unrefrigerated females and males (UnF×UnM, control). Cold storage after acclimation had no significant effect on the fecundity or oviposition period of F0-generation H. hebetor. Moreover, the survival rate (Sa = 61.43%), proportion of females (Nf/N = 0.41), intrinsic rate of increase (r = 0.3450), finite rate of increase (λ = 1.4121), net reproduction rate (R0 = 149.47), and net paralysis rate (C0 = 74.52) of ReF×UnM and UnF×UnM (Sa = 50.00%, Nf/N = 0.34, r = 0.3297, λ = 1.3881, R0 = 155.69, C0 = 62.90, respectively) treatments were significantly higher than those of the ReF×ReM treatment (Sa = 45%, Nf/N = 0.16, r = 0.2277, λ = 1.2558, R0 = 68.81, C0 = 31.61, respectively) (except for the Sa of UnF×UnM treatment), and there was no significant difference between the 2 treatments. Overall, it is advisable to avoid simultaneous cold storage of female and male parasitoids or to add unrefrigerated males appropriately when using cold-stored parasitoids to control pests.

Transgenerational effects of multiple mating in Spodoptera litura Fabricius (Lepidoptera: Noctuidae).

Polyandrous mating can result in sexual conflict and/or promote the evolution of mating patterns. Does multiple mating by females support the genetic benefits hypothesis and can it be validated as an evolutionary strategy? If we are to decipher the consequences of sexual interactions and understand the interplay of sexual conflict and multiple generational benefits, the transgenerational effects need to be followed over multiple generations. We investigated the effects of three mating patterns, single mating, repeated mating, and multiple mating, on parental Spodoptera litura copulation behavior, and then identified the impact on the development, survival, and fecundity of the F1 and F2 generations. Fecundity was not significantly affected in the F1 generation but was substantially enhanced in the F2 generation. There was a reversal of offspring fitness across the F2 generations from the F1 generations in progeny produced by multiple mating. In addition, the intrinsic rate of increase, finite rate of increase and net reproductive rate in the F1 generation the multiple mating treatment was significantly lower than in the single mating treatment, but there was no apparent effect on the F2 generation. Repeated mating had no significant effects on progeny fitness. We postulate that multiple mating imposes cross-transgenerational effects and may ultimately influence multigenerational fitness in S. litura.

Three new species of Atkinsoniella (Arthropoda, Insecta, Hemiptera, Cicadellidae, Cicadellinae) from China, with an updated checklist to the known species worldwide.

The sharpshooter genus Atkinsoniella Distant, 1908 includes 99 valid species worldwide. Here, three new species from China are described and illustrated: Atkinsoniellastenopyga, A.wangi, and A.yingjiangensisspp. nov. An updated checklist of the known Atkinsoniella species worldwide based on the data of previous literature and studied materials is also provided. All the type specimens of three new species are deposited at the Institute of Entomology, Guizhou University, Guiyang, China.

[Effects of chitosan oligosaccharide on bone metabolism and IKK/NF-κB pathway in rats with osteoporosis and periodontitis].

PURPOSE: To investigate the effects of chitosan oligosaccharide on bone metabolism and IKK/NF-κB pathway in mice with osteoporosis and periodontitis. METHODS: Thirty rats were randomly divided into 3 groups, with 10 rats in each group. They were divided into control group, ovariectomized periodontitis group and chitosan oligosaccharide treatment group. Except for the control group, the other two groups were ovariectomized and smeared with Porphyromonas gingivalis fluid to establish the model of osteoporosis with periodontitis. Four weeks after ligation, the rats in chitosan oligosaccharide treatment group were gavaged with 200 mg/kg chitosan oligosaccharide, and the other two groups were gavaged with equal volume of normal saline once a day for 90 days. The periodontal tissues of each group were observed before administration, and the bone mineral density of rats was detected by dual energy X-ray animal bone mineral density and body composition analysis system. After 90 days of administration, the bone mineral density was detected again. After administration, blood was collected from tail vein, and the contents of serum alkaline phosphatase (ALP), bone Gla protein (BGP) and tartrate resistant acid phosphatase 5b (TRACP5b) were measured by enzyme-linked immunodeficient assay. The gingival index and periodontal attachment loss of rats in each group were obtained by visual examination and exploratory examination. The maxilla was removed, and the distance from the enamel cementum boundary to the alveolar crest was measured to obtain alveolar bone absorption value. H-E staining was used to observe the pathology of maxilla in each group. RT-PCR and Western blot were used to detect the nuclear factors in periodontal tissue of rats in each group. SPSS 22.0 software package was used for statistical analysis. RESULTS: Before administration, the gums of the control group were pink without bleeding, and the gums of the other two groups were red and swollen with slight bleeding. After administration, compared with the control group, the bone mineral density, serum ALP, BGP of ovariectomized periodontitis group decreased significantly(P＜0.05); while TRACP5b, gingival index, loss of periodontal attachment and alveolar bone resorption, NF-κB and IKK mRNA and protein expression in periodontal tissue increased significantly(P＜0.05). Compared with the ovariectomized periodontitis group, the bone mineral density, serum ALP, BGP were significantly increased(P＜0.05); while TRACP5b, gingival index, periodontal attachment loss and alveolar bone resorption, NF-κB and IKK mRNA and protein expression in periodontal tissue were significantly decreased (P＜0.05). In the ovariectomized periodontitis group, the periodontal tissue combined with epithelium was separated from the tooth surface, the dental pocket was obvious and deep, and the height of alveolar bone decreased. Although dental pocket could be observed in the periodontal tissue of rats treated with chitosan oligosaccharide, it was not obvious, and new bone appeared around the alveolar bone. CONCLUSIONS: Chitosan oligosaccharide can induce biochemical indexes of bone metabolism to become normal, alleviate the symptoms of periodontitis, this may be related to the inhibition of IKK/NF-κB pathway by chitosan oligosaccharide.

Complete mitochondrial genome of Gortyna guizhouensis Wu, Yang, and Han, 2022 (Lepidoptera, Noctuidae) from China.

Gortyna guizhouensis Wu, Yang, and Han, 2022 (Lepidoptera, Noctuidae) is a newly discovered moth species that feeds on tobacco piths during the larval stage. In this study, we sequenced and analyzed the complete mitochondrial genome (mitogenome) of G. guizhouensis larvae via next-generation sequencing. The mitogenome was 15,441 bp long with an overall A + T content of 79.1%, 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA subunit genes and one control region. The phylogenetic tree, based on the nucleic acid sequences of 13 shared PCGs of 20 Noctuidae species, revealed that G. guizhouensis is in a well-supported clade with Striacosta albicosta.

Naoxintong Capsule for Secondary Prevention of Ischemic Stroke: A Multicenter, Randomized, and Placebo-Controlled Trial.

OBJECTIVE: To examine whether the combination of Naoxintong Capsule with standard care could further reduce the recurrence of ischemic stroke without increasing the risk of severe bleeding. METHODS: A total of 23 Chinese medical centers participated in this trial. Adult patients with a history of ischemic stroke were randomly assigned in a 1:1 ratio using a block design to receive either Naoxintong Capsule (1.2 g orally, twice a day) or placebo in addition to standard care. The primary endpoint was recurrence of ischemic stroke within 2 years. Secondary outcomes included myocardial infarction, death due to recurrent ischemic stroke, and all-cause mortality. The safety of drugs was monitored. Results were analyzed using the intention-to-treat principle. RESULTS: A total of 2,200 patients were enrolled from March 2015 to March 2016, of whom 143 and 158 in the Naoxintong and placebo groups were lost to follow-up, respectively. Compared with the placebo group, the recurrence rate of ischemic stroke within 2 years was significantly lower in the Naoxintong group [6.5% vs. 9.5%, hazard ratio (HR): 0.665, 95% confidence interval (CI): 0.492-0.899, P=0.008]. The two groups showed no significant differences in the secondary outcomes and safety, including rates of severe hemorrhage, cerebral hemorrhage and subarachnoid hemorrhage (P>0.05). CONCLUSION: The combination of Naoxintong Capsule with standard care reduced the 2-year stroke recurrence rate in patients with ischemic stroke without increasing the risk of severe hemorrhage in high-risk patients. (Trial registration No. NCT02334969).

Description and complete mitochondrial genome of Atkinsoniella zizhongi sp. nov. (Hemiptera: Cicadellidae: Cicadellinae) from China and its phylogenetic implications.

A new species, Atkinsoniella zizhongi sp. nov. of the subfamily Cicadellinae, was described and illustrated from China. The new species is similar to A. nigrominiatula (Jacobi, 1944), A. limba Kuoh, 1991, A. dormana Li, 1992, A. peaka Yang, Meng et Li, 2017, and A. divaricata Yang, Meng et Li, 2017. But the characteristics of aedeagus and pygofer process can be used to distinguish them easily. The complete mitochondrial genome of the paratype was sequenced and assembled. The mitogenome of A. zizhongi sp. nov. was 16,483 bp in length, with an A+T content of 75.9%, containing 37 typical genes and a control region (CR). The gene order was consistent with the inferred insect ancestral mitochondrial genome. All of the PCGs were determined to have the typical stop codon TAA or TAG, while COX2 and ND5 ended with incomplete termination codons T and TA, respectively. In addition, phylogenetic trees were reconstructed based on PCGs and rRNAs using both the maximum likelihood (ML) and Bayesian inference (BI) methods. The results showed that the intergeneric and interspecific relationships within the subfamily Cicadellinae were completely consistent in all of the phylogenetic trees, except that the different interspecific relationships within the genus Bothrogonia were detected in the ML analysis based on the amino acid sequences. This study enriches the species diversity of Cicadellinae and further promotes research on its phylogeny.

Comparative Analysis of Mitochondrial Genomes among Twelve Sibling Species of the Genus Atkinsoniella Distant, 1908 (Hemiptera: Cicadellidae: Cicadellinae) and Phylogenetic Analysis.

The herbivorous leafhopper genus Atkinsoniella Distant, 1908 (Hemiptera: Cicadellidae: Cicadellinae), a large genus of subfamily Cicadellinae, consists of 98 valid species worldwide and 88 species recorded in China. Some species of the genus are very similar in morphological characteristics, so they are difficult to identify accurately. In this study, 12 mitochondrial genomes of Atkinsoniella species with similar morphological characteristics were first obtained through high-throughput sequencing, which featured a typical circular molecule of 15,034-15,988 bp in length. The arrangement and orientation of 37 genes were identical to those of typical Cicadellidae mitogenomes. The phylogenetic relationship within the subfamily Cicadellinae was reconstructed using maximum-likelihood (ML) and Bayesian inference (BI) methods based on three concatenated datasets. The topological structures of the six obtained phylogenetic trees were highly consistent. The results suggested that Atkinsoniella was recovered as a monophyletic group and emerged as a sister group with the monophyletic clade of Bothrogonia, Paracrocampsa (part), and Draeculacephala (part). The branches of the 12 newly sequenced species were clearly separated, with most nodes receiving strong support in all analyses. In addition, the key to the 12 Atkinsoniella species was provided to identify species according to morphological characteristics. This study further promotes research on the classification, genetics, evolution, and phylogeny of the genus Atkinsoniella and subfamily Cicadellinae.

Antimicrobial mexicanolide limonoids from the seeds of Khaya senegalensis.

Three new mexicanolide limonoids were obtained from the 90% ethanol extract of the seeds of Khaya senegalensis. Their structures were elucidated as senegalenines A-C (1-3) by analysing their 1D/2D NMR and MS spectroscopic analysis. In addition, the isolated limonoids were tested in vitro for antimicrobial potentials against 5 pathogenic microorganisms. Consequently, compounds 1-3 exhibited antimicrobial activity against the tested Gram negative bacteria at the minimum inhibitory concentration values less than 40 µg/ml.

Host Deprivation Effects on the Functional Response and Parasitism Rate of Habrobracon hebetor (Hymenoptera: Braconidae) on Ephestia elutella (Lepidoptera: Pyralidae) in the Laboratory.

Habrobracon hebetor (Say) is an important biological control agent for lepidopteran pests of stored products. In this study, the age-specific functional response, paralysis rate, and parasitism rate of H. hebetor under different host deprivation treatments (PC: without host deprivation, used as the control, P1d: host deprivation, but the host was removed after 1 d contact, and PW: host deprivation from beginning) were evaluated at different larval densities (5, 10, 20, 40, and 80) of the Ephestia elutella (Hübner) at 28 ± 1°C, 75 ± 5% RH and 16:8 h L:D. Ages of parasitoid females used were 2, 5, 10, and 20 d old. The logistic regression results indicated that the functional response of H. hebetor females under different host deprivation treatments was type II. The longest handling time was observed in 20-d old females, while the shortest handling time and highest maximum attack rate (T/Th) were estimated at the age of 2 d in all treatments. The paralysis and parasitism rates of H. hebetor were the highest at 2, 5, and 10-d old in all treatments. The results of this study suggest that H. hebetor females up to 10-d old can be used as an efficient biological control agent against E. elutella. The data of this study can also be used to predict the efficacy of different aged H. hebetor females in controlling E. elutella populations.

The Biocontrol Agent Pyemotes zhonghuajia Has the Highest Lethal Weight Ratio Compared with Its Prey and the Most Dramatic Body Weight Change during Pregnancy.

Pyemotes spp. are small, toxic, ectoparasitic mites that suppress Coleoptera, Hemiptera, and Lepidoptera plant pests. To explore their potential use as a biocontrol agent, we studied the reproductive development, paralytic process, time to lethality and mortality, and searching ability of Pyemotes zhonghuajia on different developmental stages of the oriental leafworm moth, Spodoptera litura. Pyemotes zhonghuajia gained 14,826 times its body weight during pregnancy. One single P. zhonghuajia female could rapidly kill one S. litura egg and first to third instar larvae, but not fourth to sixth instar larvae, prepupae, or pupae within 720 min. Pyemotes zhonghuajia could develop on eggs, first to sixth larvae, and pupae, but only produced offspring on the eggs and pupae. A single P. zhonghuajia female (an average weight of 23.81 ng) could paralyze and kill one S. litura third instar larvae (an average weight of 16.29 mg)-680,000 times its own weight. Mites significantly affected the hatch rate of S. litura eggs, which reduced with increasing mite densities on S. litura eggs. Releasing 50 or 100 P. zhonghuajia in a 2 cm searching range resulted in significantly higher mortality rates of S. litura first instar larvae within 48 h compared to second and third instar larvae in searching ranges of 4.5 and 7.5 cm within 24 h. To the best of our knowledge, this is the first study to reveal that P. zhonghuajia undergoes the greatest changes in weight during pregnancy of any adult female animal and has the highest lethal weight ratio of any biocontrol agent.

Outside-in catalytic graphitization method for synthesis of dispersible and uniform graphitic porous carbon nanospheres.

Common strategies to synthesize graphitic porous carbon nanospheres suffer from energy consumption, exorbitant cost and harsh condition, and lead to closed pore and polydisperse particles. The successful manipulation of adjustable graphitic skeleton, developed porosity, good monodispersity and dispersity of carbon nanospheres is essential to meet their structural varieties and practical applications. Herein, an outside-in catalytic graphitization method is reported to synthesize carbon nanospheres with abovementioned properties, which involves interfacial assembly between layered double hydroxides nanosheets and polymer nanospheres, in-situ generation of nickel nanoparticles, and outside-in catalytic graphitization. The unusual phenomenon is that the in-situ generated nickel nanoparticles are preferentially oriented to the carbon side rather than to the free open space. The interface reactions between nickel nanoparticles and amorphous carbons drive continuous etching of carbon species to form graphitic structure in the interior of spheres. The graphitic structure can be tuned by changing effective charge ratio and pyrolysis conditions and obtained carbon nanospheres possessed good dispersibility in water and ethanol. Moreover, such carbon nanospheres exhibited good performance when used as anodes in lithium-ion batteries. These findings may pave new ways for synthesizing multifarious carbon nanomaterials with adjustable graphitic skeleton, developed porosity, good monodispersity and dispersibility for various applications.

Characterization of Two Complete Mitochondrial Genomes of Atkinsoniella (Hemiptera: Cicadellidae: Cicadellinae) and the Phylogenetic Implications.

The complete mitochondrial genomes of Atkinsoniella grahami and Atkinsoniella xanthonota were sequenced. The results showed that the mitogenomes of these two species are 15,621 and 15,895 bp in length, with A+T contents of 78.6% and 78.4%, respectively. Both mitogenomes contain 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a control region (CR). For all PCGs, a standard start ATN codon (ATT, ATG, or ATA) was found at the initiation site, except for ATP8, for which translation is initiated with a TTG codon. All PCGs terminate with a complete TAA or TAG stop codon, except for COX2, which terminates with an incomplete stop codon T. All tRNAs have the typical cloverleaf secondary structure, except for trnS, which has a reduced dihydrouridine arm. Furthermore, these phylogenetic analyses were reconstructed based on 13 PCGs and two rRNA genes of 73 mitochondrial genome sequences, with both the maximum likelihood (ML) and Bayesian inference (BI) methods. The obtained mitogenome sequences in this study will promote research into the classification, population genetics, and evolution of Cicadellinae insects in the future.

Does Larval Rearing Diet Lead to Premating Isolation in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)?

Host plant preference during the larval stage may help shape not only phenotypic plasticity but also behavioral isolation. We assessed the effects of diet on population parameters and mate choice in Spodoptera litura. We raised larvae fed on tobacco, Chinese cabbage, or an artificial diet, and we observed the shortest developmental time and highest fecundity in individuals fed the artificial diet. However, survival rates were higher for larvae on either of the natural diets. Population parameters including intrinsic rate of increase and finite rate of increase were significantly higher with the artificial diet, but this diet led to a lower mean generation time. Copulation duration, copulation time, and number of eggs reared significantly differed between diets. In terms of mate choice, females on the artificial diet rarely mated with males fed on a natural host. Our results support the hypothesis that different diets may promote behavioral isolation, affecting mating outcomes. Thus, findings for populations fed an artificial diet may not reflect findings for populations in the field.

Three new species of Mileewa (Hemiptera: Cicadellidae: Mileewinae) from Tibet, China.

Three new species of Mileewini, Mileewa digitata sp. nov., M. furcata sp. nov., M. nigricincta sp. nov., from Tibet, China are described and illustrated.

The complete mitochondrial genomes of Bothrogonia tongmaiana and Bothrogonia yunana (Hemiptera: Cicadellidae) from China.

The complete mitochondrial genomes of Bothrogonia tongmaiana and Bothrogonia yunana are sequenced and annotated, which are 15,539 and 15,585 bp in length, respectively. Bothrogonia tongmaiana has an A + T content of 79.4% (A 55.7%; C 13.5%; G 7.1%, and T 23.7%), while B. yunana has an A + T content of 80.6% (A 37.5%; C 5.6%; G 13.8%, and T 43.1%). Every mitogenome encodes 13 proteins, 2 ribosomal RNAs, 22 tRNAs, and a control region. The phylogenetic tree was reconstructed based on them and 27 reference species, which could further confirm the status of these two species.

Host deprivation effects on population performance and paralysis rates of Habrobracon hebetor (hymenoptera: Braconidae).

BACKGROUND: Habrobracon hebetor (Hymenoptera: Braconidae) is a gregarious ectoparasitoid that attacks the larvae of several species of pyralid and noctuid moths. The reproduction and population dynamics of parasitoids in general are affected by host deprivation. However, how host deprivation affects H. hebetor is unknown. The effect of host deprivation on the parental generation, life table parameters, and the paralysis rate of the F1 generation of H. hebetor were evaluated using the age-stage, two-sex life table under laboratory conditions. RESULTS: The results indicated that the greatest longevity and the least lifetime fecundity of the F0 generation occurred after 19 days of host deprivation (PW-20 treatment). The life table parameters (intrinsic rate of increase, r; finite rate of increase, λ; and net reproductive rate, R0 ) and the paralysis rate parameters (net paralysis rate, C0 ; transformation rate, Qp ; stable paralysis rate, ψ; and finite paralysis rate, ω) of F1 individuals after PW-20 treatment were significantly higher than those of individuals subjected to the control treatment (no host deprivation). However, no difference was detected between the two host deprivation treatments: host deprivation after 1 day of host contact and immediate host deprivation (PW treatment). CONCLUSION: Our results demonstrated that the effectiveness of H. hebetor did not decrease even during host deprivation for 19 days. Meanwhile, it was observed that mass rearing of the parasitoid could be improved by providing 10 individuals of 5th instar larvae of Ephestia elutella (Lepidoptera: Pyralidae) with a 20% honey-water solution. © 2020 Society of Chemical Industry.

Mitochondrial genome of Bolanusoides shaanxiensis (Cicadellidae: Typhlocybinae: Typhlocybini), with its phylogenetic analysis.

The mitochondrial genome of one leafhopper species Bolanusoides shaanxiensis was sequenced and annotated. The mitogenome is 15,724 bp in length, containing 37 typical genes and a control region. The A + T content of the whole mitogenome is 78.9%. Most of PCGs started with ATN and stopped with TAA, except for ATP8 started with TTG, COX2, COX3 and ND5 used incomplete T as stop codon. The phylogeny tree is monophyletic among 31 related species. The relationships of B. shaanxiensis and Typhlocyba sp. were closer than others. This study further enriched mitogenome database of the tribe Typhlocybini.

The mitochondrial genome of Cofana yasumatsui (Hemiptera: Cicadellidae: Cicadellinae).

The complete mitochondrial genome of Cofana yasumatsui (Hemiptera: Cicadellidae: Cicadellinae) is sequenced. Based on annotation, the mitochondrial genome is 15,019 bp, has an A + T content of 77.2% (A = 42.0%, T = 35.2%, G = 10.0%, C = 12.8%), which is the classical structure for insect mitogenome. All PCGs started with ATN, except ATP8 and ND5 starting with TTG. All PCGs used TAN as stop codon, except ND5 stopping with single T. The phylogenetic relationship of C. yasumatsui clustered together with Cicadella viridis, Homalodisca coagulata, Homalodisca vitripennis, and Bothrogonia ferruginea from Cicadellinae. This is identical with the result of the traditional taxonomy.