Cuticular proteins in codling moth (Cydia pomonella) respond to insecticide and temperature stress.

The insect cuticle consists of chitin and cuticular proteins (CPs), which stabilize the body shape and provide an effective physical barrier against the external environment. They are also potential target sites for developing environmentally friendly insect management through the utilization of physiology-based methods. The codling moth, Cydia pomonella, is a pest afflicting fruit orchards worldwide. This study used a comparative genomic approach, whole-genome resequencing, and transcriptome data to understand the role that CPs played in the environmental adaptation of the codling moth. A total of 182 putative CPs were identified in the codling moth genome, which were classified into 12 CP families. 119 CPR genes, including 54 RR-1, 60 RR-2, and 5 RR-3 genes were identified and accounted for 65.4% of the total CPs. Eight and seven gene clusters are formed in RR1 and RR2 subfamily and the ancestor-descendant relationship was explained. Five CPAP genes were highly expressed during the egg stage and exposed to high temperature, which indicated their potential role in aiding codling moth eggs in acclimating to varying external heat conditions. Moreover, six CPs belonging to the CPR and CPLCP families were identified in association with insecticide resistance by population resequencing. Their expression levels increased after exposure to insecticides, suggesting they might be involved in codling moth resistance to the insecticides azinphos-methyl or deltamethrin. Our results provide insight into the evolution of codling moth CPs and their association with high temperature adaptation and insecticide resistance, and provide an additional information required for further analysis of CPs in environmental adaptation.

Determining the complete mitochondrial genome of Tethea albicostata (Lepidoptera: Drepanidae) and phylogenetic analysis.

Tethea albicostata is a widely distributed insect species in northern and central China. To date, few studies have been conducted on this species, with the exception of morphological taxonomy studies. Here, we report the complete mitochondrial genome of T. albicostata collected in China. The circular-mapping mitogenome is 15,308 bp in length, with an overall A + T content of 80.52%, encoding 2 ribosomal RNA genes, 22 transfer RNA genes, and 13 protein-coding genes. The gene arrangement and components of T. albicostata are identical to those of most other Lepidopteran insects. Phylogenetic analysis based on mitogenomes showed that T. albicostata is grouped with Drepana pallida, which belongs to the same family as Drepanidae. The family Drepanidae formed a separate branch from other families in the phylogenetic tree. This study determined the second mitochondrial genome of the Drepanidae species.

Global genomic signature reveals the evolution of fall armyworm in the Eastern hemisphere.

The major plant pest fall armyworm (FAW), Spodoptera frugiperda, is native to the Americas and has colonized Africa and Asia within the Eastern hemisphere since 2016, causing severe damage to multiple agricultural crop species. However, the genetic origin of these invasive populations requires more in-depth exploration. We analysed genetic variation across the genomes of 280 FAW individuals from both the Eastern hemisphere and the Americas. The global range-wide genetic structure of FAW shows that the FAW in America has experienced deep differentiation, largely consistent with the Z-chromosomal Tpi haplotypes commonly used to differentiate 'corn-strain' and 'rice-strain' populations. The invasive populations from Africa and Asia are different from the American ones and have a relatively homogeneous population structure, consistent with the common origin and recent spreading from Africa to Asia. Our analyses suggest that north- and central American 'corn-strain' FAW are the most likely sources of the invasion into the Eastern hemisphere. Furthermore, evidence based on genomic, transcriptomic and mitochondrial haplotype network analyses indicates an earlier, independent introduction of FAW into Africa, with subsequent migration into the recent invasive population.

Investigation of Immune Responses in Giant African Snail, Achatina immaculata, against a Two-Round Lipopolysaccharide Challenge.

As one of the 100 most-threatening invasive alien species, the giant African snail (Achatina immaculata) has successfully invaded and established itself in most areas of southern China. Protection against recurrent pathogen infections is vital to biological invasion. Enhanced immune protection has been previously found in other invertebrates, but not in the unique immune system of the giant African snail. In the present study, the survival rate of the giant African snail was recorded following a second infection with lethal doses of Escherichia coli after a previous first injection using lipopolysaccharide (LPS), and the mechanism of immune enhancement was investigated by examining the cellular and transcriptomic response of the giant African snail after two successive stimuli using LPS. Snails injected first with LPS, sterilized physiologic (0.9%) saline (SPS), phosphate-buffered saline (PBS) or untreated (Blank) were rechallenged at 7d with E. coli (Ec), and were named as LPS + Ec, SPS + Ec, PBS + Ec, Ec, and Blank. The log-rank test shows the survival rate of the LPS + Ec group as significantly higher than that of other control groups after the second injection (p < 0.05). By performing cell counting and BrdU labeling on newly generated circulating hemocytes, we found that the total hemocyte count (THC) and the ratio of BrdU-positive cells to total cells increased significantly after primary stimulation with LPS and that they further increased after the second challenge. Then, caspase-3 of apoptosis protease and two antioxidant enzyme activities (CAT and SOD) increased significantly after infection, and were significantly higher in the second response than they had been in the first round. Moreover, transcriptome analysis results showed that 84 differentially expressed genes (DEGs) were expressed at higher levels in both the resting and activating states after the second immune response compared to the levels observed after the first challenge. Among them, some DEGs, including Toll-like receptor 4 (TLR4) and its downstream signaling molecules, were verified using qRT-PCR and were consistent with the transcriptome assay results. Based on gene expression levels, we proposed that these genes related to the TLR signaling cascade participate in enhanced immune protection. All results provide evidence that enhanced immune protection exists in the giant African snail.

Characterization of the mitochondrial genome of the lawn cutworm Spodoptera depravata (Lepidoptera: Noctuidae).

The lawn cutworm, Spodoptera depravata, is one of the most important pests that causes economic damage to grass crops. This study reports the complete mitochondrial genome of an S. depravata sample collected in China. The genome is a circular molecule 15,460 bp in length with an overall A + T content of 81.6%. It contains 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes. The gene content and organization of the mitogenome of S. depravata are identical to those of other Spodoptera species. Maximum-likelihood phylogenetic analysis based on mitogenomes showed a close evolutionary relationship between S. depravata and S. exempta. This study provides new molecular data for the identification and further phylogenetic analyses of Spodoptera species.

Inhibition of invasive plant Mikania micrantha rapid growth by host-specific rust (Puccinia spegazzinii).

Mikania micrantha Kunth is a fast-growing global invasive weed species that causes severe damage to natural ecosystems and very large economic losses of forest and crop production. Although Puccinia spegazzinii can effectively inhibit the growth of M. micrantha and is used as a biological control strain in many countries, the mechanism of inhibiting the growth of M. micrantha is not clear. Here, we used a combination of phenotypic, enzyme activity, transcriptomic, and metabolomic approaches to study the response of M. micrantha after infection by P. spegazzinii. In the early stages of rust infection, jasmonic acid (JA), jasmonoyl-isoleucine (JA-Ile), and salicylic acid (SA) levels in infected leaves were significantly lower than those in uninfected leaves. In teliospore initial and developed stages of P. spegazzinii, JA and JA-Ile levels substantially increased by more than 6 times, which resulted in a significant decrease in the accumulation of defense hormone SA in infected leaves of M. micrantha. The contents of plant growth-promoting hormones were significantly reduced in the infected plants as a result of substantial downregulation of the expression of key genes related to hormone biosynthesis. Furthermore, rust infection led to high levels of reactive oxygen species in chloroplasts and the destruction of chlorophyll structure, which also led to decreased photosynthetic gene expression, net photosynthetic rate, activity of Rubisco, and levels of important organic acids in the Calvin cycle. We hypothesized that after P. spegazzinii infection, JA or JA-Ile accumulation not only inhibited SA levels to promote rust infection and development, but also impeded the rapid growth of M. micrantha by affecting plant growth hormones, carbon, and nitrogen metabolic pathways.

A chromosome-level genome assembly of the beet armyworm Spodoptera exigua.

BACKGROUND: The beet armyworm Spodoptera exigua is a polyphagous caterpillar that causes serious damage to many species of crops and vegetables. To gain insight into how this polyphagous insect differs from less harmful oligophagous species, we generated a chromosome-level assembly and compared it to closely related species with the same or different feeding habits. RESULTS: Based on Illumina and Pacific Biosciences data and Hi-C technology, 425.6 Mb of genome sequences were anchored and oriented into 31 linkage groups, with an N50 length of 14.8 Mb. A total of 24,649 gene models were predicted, of which 97.4% were identified in the genome assembly. Chemosensory genes are vital for locating food: of the four main families, odorant-binding proteins, chemosensory proteins and olfactory receptors showed little difference, whereas gustatory receptors are greatly expanded in S. exigua. Examination of other polyphagous insects confirmed this difference from oligophagous congeners and further identified the bitter receptor subfamily as being particularly affected. CONCLUSION: Our high-quality genome sequence for beet armyworm identified a key expansion of the bitter gustatory receptor subfamily in this and other pests that differs crucially from more benign relatives and offers insight into the biology and possible future means of control for these economically important insects.

Genomic insights into longan evolution from a chromosome-level genome assembly and population genomics of longan accessions.

Longan (Dimocarpus longan) is a subtropical fruit best known for its nutritious fruit and regarded as a precious tonic and traditional medicine since ancient times. High-quality chromosome-scale genome assembly is valuable for functional genomic study and genetic improvement of longan. Here, we report a chromosome-level reference genome sequence for longan cultivar JDB with an assembled genome of 455.5 Mb in size anchored to fifteen chromosomes, representing a significant improvement of contiguity (contig N50 = 12.1 Mb, scaffold N50 = 29.5 Mb) over a previous draft assembly. A total of 40 420 protein-coding genes were predicted in D. longan genome. Synteny analysis suggests longan shares the widespread gamma event with core eudicots, but has no other whole genome duplications. Comparative genomics showed that D. longan genome experienced significant expansions of gene families related to phenylpropanoid biosynthesis and UDP-glucosyltransferase. Deep genome sequencing analysis of longan cultivars identified longan biogeography as a major contributing factor for genetic diversity, and revealed a clear population admixture and introgression among cultivars of different geographic origins, postulating a likely migration trajectory of longan overall confirmed by existing historical records. Finally, genome-wide association studies (GWAS) of longan cultivars identified quantitative trait loci (QTL) for six different fruit quality traits and revealed a shared QTL containing three genes for total soluble solid and seed weight. The chromosome-level reference genome assembly, annotation and population genetic resource for D. longan will facilitate the molecular studies and breeding of desirable longan cultivars in the future.

Excessive Nitrogen Fertilization Favors the Colonization, Survival, and Development of Sogatella furcifera via Bottom-Up Effects.

Fertilization can trigger bottom-up effects on crop plant-insect pest interactions. The intensive use of nitrogen fertilizer has been a common practice in rice production, while the yield has long been challenged by the white-backed planthopper, Sogatella furcifera (Horváth). High nitrogen fertilization can facilitate S. furcifera infestation, however, how nitrogen fertilizer leads to high S. furcifera infestation and the nutritional interactions between rice and S. furcifera are poorly understood. Here, we evaluated the effects of various levels of nitrogen fertilizer application (0-350 kg/ha) on rice, and subsequently on S. furcifera performance. We found that higher nitrogen fertilizer application: (1) increases the preference of infestation behaviors (feeding and oviposition), (2) extends infestation time (adult lifespan), and (3) shortens generation reproduction time (nymph, pre-oviposition, and egg period), which explain the high S. furcifera infestation ratio on rice paddies under high nitrogen conditions. Moreover, high nitrogen fertilizer application increased all tested rice physical indexes (plant height, leaf area, and leaf width) and physiological indexes (chlorophyll content, water content, dry matter mass, and soluble protein content), except for leaf thickness, which was reduced. Correlation analysis indicated that the specific rice physical and/or physiological indexes were conducive to the increased infestation behavior preference, extended infestation time, and shortened generation reproduction time of S. furcifera. The results suggested that nitrogen fertilizer triggers bottom-up effects on rice and increases S. furcifera populations. The present study provides an insight into how excess nitrogen fertilization shapes rice-planthopper interactions and the consequent positive effect on S. furcifera infestation.

Gene expression patterns of Cydia pomonella granulovirus in codling moth larvae revealed by RNAseq analysis.

The Cydia pomonella granulovirus (CpGV) has been used as a biological control agent of codling moth (Cydia pomonella), a severe global pest on pome fruit. Despite the economic importance, our knowledge of its molecular biology is still limited and a detailed picture of its gene expression is still missing. Here, we sequenced the transcriptome of codling moth larvae infected with the Mexican isolate CpGV-M and analyzed the expression of viral genes at 12, 48, and 96 h post infection (hpi). The results showed that two genes (p6.9 and pp31/39K) related to DNA binding of virus production, were highly expressed at 48 and 96 hpi. From 48 to 96 hpi, the expression of genes associated with virus replication and dissemination decreased, whereas the expression of genes related to infectious virion production and per os infectivity increased. This study provides a comprehensive view of CpGV gene expression patterns in host larvae.

Characterization of the mitochondrial genome of Spodoptera exempta (Lepidoptera: Noctuidae) from South Africa.

The African armyworm, Spodoptera exempta, is an episodic migratory crop pest with an expanding distribution worldwide. This is the first report of the circular mitochondrial genome of S. exempta, with a length of 15,457 bp and an A + T content of 81.7%. It encoded a common set of 37 genes, including 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes, and contained a putative control region of 379 bp (94.7% in A + T proportion). The maximum-likelihood phylogenetic tree based on the complete mitogenome demonstrated that five species belonging to the Spodoptera genus formed one clade, in which S. exempta was the most isolated branch, followed by Spodoptera exigua. This data will contribute for the identification and phylogenetic analyses of S. exempta, providing useful information for its comprehensive control.

Complete mitochondrial genome of Spodoptera littoralis (Lepidoptera: Noctuidae) from Egypt.

The polyphagous cotton leafworm (Spodoptera littoralis) is one of the most destructive herbivorous insects worldwide. The present study reports the complete mitochondrial genome of S. littoralis collected from Egypt. The circular-mapping mitogenome was 15,408 bp in length with an overall A + T content of 81.1%, encoding a common set of 37 genes, including 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes. Most PCGs were found to use conventional ATN as the start codon and TAN as the stop codon. The phylogenetic tree based on the nucleic acid sequences of 13 shared PCGs of 29 Noctuidae species revealed that S. littoralis and Spodoptera litura are sister species. The data in this study will be helpful to understand geographical genetic variations, phylogenetic relationships, and species identification of S. littoralis.

Giant African snail genomes provide insights into molluscan whole-genome duplication and aquatic-terrestrial transition.

Whole-genome duplication (WGD), contributing to evolutionary diversity and environmental adaptability, has been observed across a wide variety of eukaryotic groups, but not in molluscs. Molluscs are the second largest animal phylum in terms of species numbers, and among the organisms that have successfully adapted to the nonmarine realm through aquatic-terrestrial (A-T) transition. We assembled a chromosome-level reference genome for Achatina immaculata, a globally invasive species, and compared the genomes of two giant African snails (A. immaculata and Achatina fulica) to other available mollusc genomes. Macrosynteny, colinearity blocks, Ks peak and Hox gene clusters collectively suggested a WGD event in the two snails. The estimated WGD timing (~70 million years ago) was close to the speciation age of the Sigmurethra-Orthurethra (within Stylommatophora) lineage and the Cretaceous-Tertiary (K-T) mass extinction, indicating that the WGD may have been a common event shared by all Sigmurethra-Orthurethra species and conferred ecological adaptability allowing survival after the K-T extinction event. Furthermore, the adaptive mechanism of WGD in terrestrial ecosystems was confirmed by the presence of gene families related to the respiration, aestivation and immune defence. Several mucus-related gene families expanded early in the Stylommatophora lineage, and the haemocyanin and phosphoenolpyruvate carboxykinase families doubled during WGD, and zinc metalloproteinase genes were highly tandemly duplicated after WGD. This evidence suggests that although WGD may not have been the direct driver of the A-T transition, it played an important part in the terrestrial adaptation of giant African snails.

Genetic structure and insecticide resistance characteristics of fall armyworm populations invading China.

The rapid wide-scale spread of fall armyworm (Spodoptera frugiperda) has caused serious crop losses globally. However, differences in the genetic background of subpopulations and the mechanisms of rapid adaptation behind the invasion are still not well understood. Here we report the assembly of a 390.38-Mb chromosome-level genome of fall armyworm derived from south-central Africa using Pacific Bioscience (PacBio) and Hi-C sequencing technologies, with scaffold N50 of 12.9 Mb and containing 22,260 annotated protein-coding genes. Genome-wide resequencing of 103 samples and strain identification were conducted to reveal the genetic background of fall armyworm populations in China. Analysis of genes related to pesticide- and Bacillus thuringiensis (Bt) resistance showed that the risk of fall armyworm developing resistance to conventional pesticides is very high. Laboratory bioassay results showed that insects invading China carry resistance to organophosphate and pyrethroid pesticides, but are sensitive to genetically modified maize expressing the Bt toxin Cry1Ab in field experiments. Additionally, two mitochondrial fragments were found to be inserted into the nuclear genome, with the insertion event occurring after the differentiation of the two strains. This study represents a valuable advance toward improving management strategies for fall armyworm.

Effects of Water Immersion and Soil Moisture Content on Larval and Pupal Survival of Bactrocera minax (Diptera: Tephritidae).

Bactrocera minax, one of the most devastating citrus pests in Asia, has two developmental stages (mature larva and pupa) that complete their life cycle in the soil. Currently, southern China has a climate with abundant autumn rains, and soil moisture can be a major factor affecting the survival of larvae and pupae of B. minax. In the present study, we evaluated the effects of water immersion and high soil moisture content on the development of mature larvae and pupae of B. minax. When immersed in water for 1 d, 100% of mature larvae of B. minax were knocked out. When larvae were immersed for less than 6 d, however, more than 92% of knocked-out larvae recovered within 24 h. The days of water immersion with 50% and 90% recovery ratios (indicated as RD50 and RD90) were 10.3 d and 6.4 d, respectively. When larvae were immersed less than 6 d, the mortality ratios of larvae were not significantly different from those that were not immersed at all. The days of immersion causing 50% and 90% mortality of larvae (MD50 and MD90, respectively) were 7.6 d and 11.1 d, respectively. The pupation ratios of larvae were also observed to be not significantly different compared to non-immersion, and the days of immersion causing 50% and 90% pupation (PD50 and PD90, respectively) were 6.6 d and 0.8 d, respectively. Larval respiration rates were reduced after water immersion as a strategy for larval survival. High water content was not detrimental to pupae of B. minax. Adult emergence did not significantly decrease in soil with high water content, even though pupae were under those conditions for 161-175 d. The respiration rates of pupae were lower in soil with different moisture levels and were not significantly different, which ensured the survival of pupae in high water content. Reduced respiration rate is a strategy for survival of larvae and pupae, and remarkable tolerance to high moisture conditions could explain the high rate of spread and geographical distribution of B. minax. The results of this study provide a reference for the occurrence and control of B. minax.

Influence of Supplemental Protein on the Life Expectancy and Reproduction of the Chinese Citrus Fruit Fly, Bactrocera minax (Enderlein) (Tetradacus minax) (Diptera: Tephritidae).

Bactrocera minax (Enderlein) (Diptera: Tephritidae) is a major citrus pest in China, whose artificial rearing technology of the adult is not well documented to date. In this study, we tried to determine if supplementing proteins to the adult diet could result in the enhancement of some fitness parameters of B. minax. Four feeds with varying protein source were provided as F0 (water), F1 (sucrose), F2 (sucrose + yeast), and F3 (sucrose + peptone). F0 and F1 being the control, F2 and F3 were protein food types. The results showed that adults fed by F2 and F3 lived longer with 40.1 d and 32.8 d, respectively, had reduced death rates (death peaks were delayed for 5.6 d and 4.1 d, respectively), increased mating frequencies (8.1 and 5.3 per females, 4.7 and 7.3 per males, respectively), and longer mating durations (with 42 d and 34 d). In addition, females recorded an increased adult ovary development, more egg load (with 94.8 and 77.3 brood eggs per ovary) and to greater oviposition rates of 63.2 eggs/female and 19.3 eggs/female. Based on our results, protein supplements enhanced B. minax survival, mating, and fecundity. This study does not only provide basic knowledge to implement artificial rearing of B. minax, but also deepens our understanding on its physiology that could be used to enhance the management of the pest.

[Summary of professor YANG Jun's experience for intractable facial paralysis].

Professor YANG Jun's experience of diagnosis and treatment for intractable facial paralysis is introduced. Professor YANG focuses on the thinking model that combines TCM, western medicine and acupuncture, and adopts the differentiation system that combines disease differentiation, syndrome differentiation and meridian differentiation; he adopts the treatment integrates etiological treatment, overall regulation, symptomatic treatment as well as acupuncture, moxibustion, medication and flash cupping. The acupoints of yangming meridians are mostly selected, and acupoints of governor vessel such as Dazhui (GV 14) and Jinsuo (GV 8) are highly valued. The multiple-needles shallow-penetration-insertion twirling lifting and thrusting technique are mostly adopted to achieve slow and mild acupuncture sensation; in addition, the facial muscles are pulled up with mechanics action. The intensive stimulation with electroacupuncture is recommended at Qianzheng (Extra), Yifeng (TE 17) and Yangbai (GB 14), which is given two or three treatments per week.