Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a Technology for Rapid and Highly Sensitive Detection of Heterodera avenae and Heterodera filipjevi.

The cereal cyst nematodes Heterodera avenae and Heterodera filipjevi are recognized as cyst nematodes that infect cereal crops and cause severe economic losses worldwide. Rapid, visual detection of cyst nematodes is essential for more effective control of this pest. In this study, recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (formerly known as cpf1) was developed for the rapid detection of H. avenae and H. filipjevi from infested field samples. The RPA reaction was performed at a wide range of temperatures from 35 to 42°C within 15 min. There was no cross-reactivity between H. avenae, H. filipjevi, and the common closely related plant-parasitic nematodes, indicating the high specificity of this assay. The detection limit of RPA-Cas12a was as low as 10-4 single second-stage juvenile (J2), 10-5 single cyst, and 0.001 ng of genomic DNA, which is 10 times greater than that of RPA-lateral flow dipstick (LFD) detection. The RPA-Cas12a assay was able to detect 10-1 single J2 of H. avenae and H. filipjevi in 10 g of soil. In addition, the RPA-LFD assay and RPA-Cas12a assays could both quickly detect H. avenae and H. filipjevi from naturally infested soil, and the entire detection process could be completed within 1 h. These results indicated that the RPA-Cas12a assay developed herein is a simple, rapid, specific, sensitive, and visual method that can be easily adapted for the quick detection of H. avenae and H. filipjevi in infested fields.

Meloidogyne graminicola Population Structure in China Suggests a South-to-North Expansion.

The distribution range of root-knot nematode Meloidogyne graminicola is rapidly expanding, posing a severe threat to rice production. In this study, the sequences of cytochrome oxidase subunit I (COI) genes of rice M. graminicola populations from all reported provinces in China were amplified and sequenced by PCR. The distribution pattern and phylogenetic tree showed that all 54 M. graminicola populations in China have distinct geographical distribution characteristics; specifically, cluster 1 (southern China), cluster 2 (central south and southwest China), and cluster 3 (central and eastern China). The high haplotype diversity (Hd = 0.646) and low nucleotide diversity (π = 0.00682), combined with the negative value of Tajima's D (-1.252) and Fu's Fs (-3.06764), suggested that all nematode populations were expanding. The existence of high genetic differentiation (Fst = 0.5933) and low gene flow (Nm = 0.3333) indicated that there was a block of gene exchange between most populations. Mutation accumulation with population expansion might be directly responsible for the high genetic differentiation; therefore, the tested nematode population showed high within-group genetic variation (96.30%). The haplotype Hap8 was located at the bottom of the network topology, with the widest distribution and the highest frequency (59.26%), indicating that it was the ancestral haplotype. The populations in cluster 3 were newly invasive according to the lowest frequency of occurrence of Hap8, the highest number of endemic haplotypes, and the highest total haplotype frequency (60%). In contrast, cluster 1 having the highest genetic diversity (Hd = 0.772, π = 0.01127) indicated that it was the most primitive. Interestingly, the highest gene flow (Nm > 1), lowest genetic differentiation (Fst ≤ 0.33), and closest genetic distance (0.000) only occurred between the Guangdong/Hainan population and others, which suggested that there might be channels for gene exchange between them and that long-distance dispersal occurred. This suggestion is further confirmed by the weak correlation between genetic distance and geographical distance. Based on these data, a hypothesis can be drawn that M. graminicola populations in China were spreading from south to north, specifically from Guangdong and Hainan Provinces to other regions. Natural selection (including anthropogenic) and genetic drift were the main drivers of their evolution. Coincidentally, this hypothesis was consistent with the gradual warming trend and the chronological order of reporting these populations. The main factors influencing current M. graminicola population expansion and distribution patterns might be geography, climate, long-distance seedling transport, interregional operations of agricultural machinery, and rotation mode. It reminds human beings of the necessity to be vigilant about preventing nematode disease according to local conditions all year round.

Origin and Phylogeography of Chinese Cereal Cyst Nematode Heterodera avenae Revealed by Mitochondrial COI Sequences.

Heterodera avenae, a globally distributed plant-parasitic nematode, is one of the most significant pests on cereal crops. In China, it is widely distributed in cereal-growing areas of 16 provinces and causes serious yield losses. In the present study, a total of 98 populations of H. avenae were collected from major wheat-growing regions in China and six other countries. The mitochondrial COI genes were amplified and analyzed. Forty-one mitochondrial COI haplotypes were identified, suggesting a high genetic diversity and endemism level of H. avenae in China. Phylogenetic analysis showed that H. avenae populations in China were divided into four clades. Significant evolutionary and genetic differences were found between Chinese (except Hubei) and foreign populations. Hap1, the most widely distributed haplotype, was considered to be a separate evolutionary origin in China. The gene flow of H. avenae from the northwestern region to the north China region and Huang-Huai-Hai region was significant, so as the direction between north China and Huang-Huai-Hai region. We speculate that water flowing from the Yellow River and mechanical harvesters promoted gene exchange among these groups. A distance-based redundancy analysis showed that genetic distances observed among H. avenae populations were explained foremost not only by geographic distance but also by temperature and precipitation. This study provides theoretical support for the origin and spread of H. avenae populations in China and elsewhere in the world.

Molecular identification and lipid mobilization role of adipokinetic hormone receptor in Spodoptera litura (F.).

Energy homeostasis is essential for organisms to maintain fluctuation in energy accumulation, mobilization. Lipids as the main energy reserve in insects, their metabolism is under the control of many physiological program. This study aimed to determine whether the adipokinetic hormone receptor (AKHR) was involved in the lipid mobilization in the Spodoptera litura. A full-length cDNA encoding AKHR was isolated from S. litura. The SlAKHR protein has a conserved seven-transmembrane domain which is the character of a putative G protein receptor. Expression profile investigation revealed that SlAKHR mRNA was highly expressed in immatural stage and abundant in fat body in newly emerged female adults. Knockdown of SlAKHR expression was achieved through RNAi by injecting double-stranded RNA (dsRNA) into the 6th instar larvae. The content of triacylgycerol (TAG) in the fat body increased significantly after the SlAKHR gene was knockdown. And decrease of TAG releasing to hemolymph with increase of free fatty acid (FFA) in hemolymph were observed when the SlAKHR gene was knowned-down. In addition, lipid droplets increased in fat body was also found. These results suggested that SlAKHR is critical for insects to regulate lipids metabolism.

Evaluating the Effects of Cu2+ on the Development and Reproduction of Spodoptera litura (Lepidoptera: Noctuidae) Based on the Age-Stage, Two-Sex Life Table.

Copper (Cu2+) is a micronutrient that promotes the development and reproduction of organisms. However, with the rapid expansion of modern industry and agriculture, Cu2+ concentrations are increasing, which might have negative impacts on biological and ecological safety. Spodoptera litura is not only an intermittent outbreak pest but also can be used as a model organism to assess environmental and ecological risks. Therefore, the effects of the life history and population parameters of S. litura fed on artificial diets with different Cu2+ concentrations were analyzed using the age-stage, two-sex life table. Our results showed that not only the preadult survival rate but also the intrinsic rate of increase (r) and the finite rate of increase (λ) were significantly increased under exposure to low Cu2+ concentrations (2, 4, and 8 mg/kg). In addition, the population growth of S. litura was significantly faster, indicating that S. litura can adapt well to low concentrations and is likely to undergo outbreaks of damage. Whereas, in addition to a significant reduction in preadult survival rate, population growth rate, pupal weight, pupal length, adult body weight, and oviposition were also significantly reduced under exposure to high Cu2+ concentration (32 mg/kg). And when the concentration reached 64 mg/kg, the survival rate of adults was extremely low, suggesting a decrease in the adaptation of S. litura. These results can help to understand the population dynamics of S. litura and predict potential ecological risks.

Transcriptome analysis of resistant and susceptible mulberry responses to Meloidogyne enterolobii infection.

BACKGROUND: Mulberry (Morus alba L.) is an important sericulture crop; however, root-knot nematode infection seriously limits its production. Understanding the mechanism of interaction between mulberry and nematode is important for control of infection. RESULTS: Using sequencing and de novo transcriptome assembly, we identified 55,894 unigenes from root samples of resistant and susceptible mulberry cultivars at different stages after infection with the nematode Meloidogyne enterolobii; 33,987 of these were annotated in the Nr, SWISS-PROT, KEGG, and KOG databases. Gene ontology and pathway enrichment analyses of differentially expressed genes (DEGs) revealed key genes involved in hormone metabolic processes, plant hormone signal transduction, flavonoid biosynthesis, phenylpropanoid biosynthesis, and peroxisomal and photosynthetic pathways. Analysis of key trends in co-expression networks indicated that expression of unigenes 0,015,083, 0,073,272, 0,004,006, and 0,000,628 was positively correlated with resistance to M. enterolobii. Unigene 0015083 encodes tabersonine 16-O-methyltransferase (16OMT), which is involved in alkaloid biosynthesis. Unigene 0073272 encodes a transcription factor contributing to nitric oxide accumulation during plant immune responses. Unigenes 0,004,006 and 0,000,628 encode ERF and MYB transcription factors, respectively, involved in plant hormone signaling. We verified the accuracy of transcriptome sequencing results by RT-qPCR of 21 DEGs. CONCLUSIONS: The results of this study increase our understanding of the resistance mechanisms and candidate genes involved in mulberry-M. enterolobii interaction. Thus, our data will contribute to the development of effective control measures against this pathogen.

Comparative transcriptome analysis of differentially expressed genes in Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae) at different acute stress temperatures.

The gnat, Bradysia odoriphaga Yang et Zhang, is an important underground pest in Asia. B. odoriphaga differ in heat and cold tolerance and exhibit quite different developmental strategies. To understand the underlying mechanisms, we sequenced and compared the transcriptome of B. odoriphaga under 40 °C (a stressful high temperature), 25 °C, and 4 °C (a stressful low temperature) for 1 h. We found that metabolism- and ribosome-related genes were modulated. In high temperature (40 °C), heat shock protein (HSP) genes, detoxication genes, metabolism genes, protein turnover genes, and stress signal transduction genes were differentially expressed. In low temperature (4 °C), genes related with heat shock protein (HSP) and detoxication were differentially expressed. Our study increases our understanding of the complex molecular mechanisms involved in the responses of B. odoriphaga to acute temperature stress and provides a potential strategy for pest management.

The Influence of Gut Microbiota on the Fecundity of Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae).

The gut microbiota of insects usually plays an important role in the development and reproduction of their hosts. The fecundity of Henosepilachna vigintioctopunctata (Fabricius) varies greatly when they develop on different host plants. Whether and how the gut microbiota regulates the fecundity of H. vigintioctopunctata was unknown. To address this question, we used 16S rRNA sequencing to analyze the gut microbiomes of H. vigintioctopunctata adults fed on two host plant species (Solanum nigrum and Solanum melongena) and one artificial diet. The development of the ovaries and testes was also examined. Our results revealed that the diversity and abundance of gut microorganisms varied significantly in insects reared on different diets. The gut microbiota of H. vigintioctopunctata raised on the two host plants was similar, with Proteobacteria being the dominant phylum in both groups, whereas Firmicutes was the dominant phylum in the group reared on the artificial diet. The predominant microbiota in the S. nigrum group were Acinetobacter soli and Acinetobacter ursingii (Acinetobacter, Moraxellaceae); Moraxella osloensis (Enhydrobacter, Moraxellaceae); and Empedobacter brevis (Empedobacter, Weeksellaceae). The microbiota in this group are associated with high lipid metabolism. In addition, the beetles' ovaries and testes were more highly developed in the S. nigrum group than in the other two groups. These findings provide valuable information for elucidating the complex roles the gut microbiota play in the fecundity of H. vigintioctopunctata, and may also contribute to developing future novel control strategies involving this economically important pest.

Effects of Pre-Diapause Temperature and Body Weight on the Diapause Intensity of the Overwintering Generation of Bactrocera minax (Diptera: Tephritidae).

The Chinese citrus fruit fly, Bactrocera minax (Enderlein), is an economically important pest of citrus. The fly has an obligatory pupal diapause in soil from November to March. However, techniques for predicting or determining the emergence of the adult have, thus far, not been well documented. In this study, we investigated the effects of different pre-diapause temperatures (8, 12, 16, and 20°C) and pupal body weight (five groups according to pupal weight: G-58, 55.0-61.0 mg; G-68, 65-71 mg; G-78, 75-81 mg; G-88, 85-91 mg; G-95, 92-98 mg) on pupal period (the indicator of diapause intensity). The pupal period of B. minax larvae pupated at 8°C was 193.41 d, which was significantly shorter than that of larvae incubated at higher temperatures, suggesting that there was a lower diapause intensity for larvae pupated at lower pre-diapause temperatures. There were also significant differences in the pupal periods at different pupal body weights. The pupal period of G-58 was significantly shorter than that of the heavier groups (G-88 and G-95), and the pupal period increased with increasing pupal body weight in the five groups. Moreover, the pupal period of B. minax significantly and positively correlated to pupal body weight. These findings demonstrate that the pre-diapause temperature and pupal body weight are suitable indicators for predicting the pupal period of overwintering individuals, and the results of this study will contribute to the development of new and effective strategies for predicting the occurrence and population dynamics of B. minax adult.

A salivary ferritin in the whitefly suppresses plant defenses and facilitates host exploitation.

The whitefly Bemisia tabaci is an important pest of worldwide agriculture. Previous work has shown that B. tabaci actively suppresses host plant defenses, but our knowledge of the specific mechanisms involved remains limited. Here we describe a B. tabaci salivary protein, the ferritin BtFer1, and its role in facilitating exploitation of host plants. We show that BtFer1 exhibits Fe2+ binding ability and ferroxidase activity, and that secretion of BtFer1 during B. tabaci feeding suppresses H2O2-generated oxidative signals in tomato (Solanum lycopersicum). Silencing BtFer1 enhanced the induction of the jasmonic acid (JA)-mediated defense signaling pathway in response to whitefly feeding, and led to increased callose deposition and the production of proteinase inhibitors that prevent whiteflies from continuously ingesting and digesting phloem sap. Consistent with these effects, silencing BtFer1 reduced whitefly survival on tomato but not on artificial diet. Using a JA-deficient spr2 mutant plant further showed that suppression of JA defenses by BtFer1 is sufficient to increase B. tabaci survival. Taken together, these results demonstrate that BtFer1 acts as an effector protein that mediates whitefly-tomato interactions. These findings represent an important step forward in understanding the molecular mechanisms by which whiteflies and other insect herbivores suppress host plant defenses.

Effect of Dietary Protein and Carbohydrates on Survival and Growth in Larvae of the Henosepilachna vigintioctopunctata (F.) (Coleoptera: Coccinellidae).

The ratio of protein and carbohydrate in an insect's nutritional regime can significantly influence its survival, growth, and fecundity. The effects of 11 different artificial diets containing protein (p): carbohydrate (c) ratios were determined in larvae of the phytophagus ladybug, Henosepilachna vigintioctopunctata (F.) (Coleoptera: Coccinellidae). We recorded the developmental times and survival rates of the larvae and weighed their pupae. When the concentration of carbohydrates was kept constant while the concentration of proteins was increased (p29:c20, p31:c20, p33:c20, and p35:c20), H. vigintioctopunctata could successfully complete the larval and pupal stages. The highest survival rate and greatest pupal mass of H. vigintioctopunctata were 72% and 19.5 mg, respectively, when reared on the p33:c20 diet. H. vigintioctopunctata larvae, however, were unable to develop into adults when the concentration of protein remained constant while the level of carbohydrates was increased (p20:c23, p20:c25, p20:c27, and p20:c29), or when the total amount (p + c) was kept at 48% (p22:c26, p 24:c24, p26:c22). Evidently, changing the availability of quality diet, especially the total protein levels, can significantly affect the performance to H. vigintioctopunctata. Our results indicated that the maximum development and survival of H. vigintioctopunctata larvae occurred within a narrow range-when the p:c ratio was (33:20).

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.

Phylogenetic divergences of the true bugs (Insecta: Hemiptera: Heteroptera), with emphasis on the aquatic lineages: the last piece of the aquatic insect jigsaw originated in the Late Permian/Early Triassic.

Heteroptera are among the most diverse hemimetabolous insects. Seven infraorders have been recognized within this suborder of Hemiptera. Apart from the well-established sister-group relationship between Cimicomorpha and Pentatomomorpha (= Terheteroptera), the two terminal lineages, the relationships among the other five infraorders are still controversial, of which three (Gerromorpha, Nepomorpha and Leptopodomorpha) are intimately connected to aquatic environments. However, the various and often conflicting available phylogeny hypotheses do not offer a clear background for a connection between diversification and palaeoenvironments. In this study, a molecular data set representing 79 taxa and 10 149 homologous sites is used to infer the phylogenetic relationships within Heteroptera. Bayesian inference, maximum-likelihood and maximum parsimony analyses were employed. The results of phylogenetic inferences largely confirm the widely accepted phylogenetic context. Estimation of the divergence time based on the phylogenetic results revealed that Gerromorpha, Nepomorpha and Leptopodomorpha originated successively during the period from the Late Permian to Early Triassic (269-246 Ma). This timescale is consistent with the origin and radiation time of various aquatic holometabolans. Our results indicate that the aquatic and semi-aquatic true bugs evolved under environmental conditions of high air temperature and humidity in an evolutionary scenario similar to that of the aquatic holometabolans.

Seasonal migration of Ctenoplusia agnata (Lepidoptera: Noctuidae) over the Bohai Sea in northern China.

Ctenoplusia agnata (Staudinger) (Lepidoptera: Noctuidae) is an important polyphagous pest in East Asia. Previous studies showed that C. agnata moths possesses the potential to undertake long-distance migration; however, knowledge of whether or not the migration of C. agnata moths is a regular ecological behavior and what the pattern of seasonal migrations is in case of regular migration is currently lacking. In the current study, systemic monitoring of population dynamics of C. agnata was conducted by a searchlight trap on an island in the center of Bohai gulf in northern China, during 2003-2013. Our results provided strong evidence for the hypothesis that C. agnata is one of the pest species undertaking regular high altitude long-distance migration and we have depicted the seasonal migration pattern over the Bohai Sea. The first capture of C. agnata generally appeared in late April and early May, then the daily number of catches increased to high levels in late July and formed two waves of migration through August and early September, and finally, the moths disappeared in late October. The mean time from the earliest trapping to the latest trapping within a year was 141.0 +/- 3.0 days. The index of ovarian development of female C. agnata showed seasonal variability and suggested that its migratory flight may be independent of the degree of ovarian development and mating status. In addition, strong migration events took place in 2003, 2004, 2008, and 2010 (annual sum of catches > 10,000). The research result from this work is helpful for understanding the occurrence regularity of C. agnata and developing an integrated pest management strategy.

Adult Feeding Experience Determines the Fecundity and Preference of the Henosepilachna vigintioctopunctata (F.) (Coleoptera: Coccinellidae).

Both larvae and adults of the Henosepilachna vigintioctopunctata feed on leaves of potatoes, tomatoes, and eggplants. Given the variation in planting times of host plants in the Jianghan Plain, host switching between larvae and adults of H. vigintioctopunctata is inevitable to ensure continuous food availability. We evaluated the effect of consistent versus diverse larval and adult host plant feeding experience on growth performance, fecundity, longevity, and feeding preferences of H. vigintioctopunctata through match-mismatch experiments. Host plant quality significantly influences larval development and adult reproduction. Potatoes are identified as the optimal host plant for H. vigintioctopunctata, whereas eggplants significantly negatively affect the adult fecundity. Adult stage host feeding experience determines the fecundity of H. vigintioctopunctata, irrespective of the larval feeding experience. The fecundity of H. vigintioctopunctata adults on eggplant leaves remains significantly lower than that observed on potato leaves. Similarly, adult H. vigintioctopunctata demonstrate a preference for consuming potato leaves, irrespective of the larval feeding experience. Although host switching between larval and adult stages offers lesser benefits for the performance of herbivorous insects compared to a consistent diet with potato leaves, it maintains H. vigintioctopunctata population continuity amidst shortages of high-quality potato hosts.

Current advances in the identification of plant nematode diseases: From lab assays to in-field diagnostics.

Plant parasitic nematodes (PPNs) cause an important class of diseases that occur in almost all types of crops, seriously affecting yield and quality and causing great economic losses. Accurate and rapid diagnosis of nematodes is the basis for their control. PPNs often have interspecific overlays and large intraspecific variations in morphology, therefore identification is difficult based on morphological characters alone. Instead, molecular approaches have been developed to complement morphology-based approaches and/or avoid these issues with various degrees of achievement. A large number of PPNs species have been successfully detected by biochemical and molecular techniques. Newly developed isothermal amplification technologies and remote sensing methods have been recently introduced to diagnose PPNs directly in the field. These methods have been useful because they are fast, accurate, and cost-effective, but the use of integrative diagnosis, which combines remote sensing and molecular methods, is more appropriate in the field. In this paper, we review the latest research advances and the status of diagnostic approaches and techniques for PPNs, with the goal of improving PPNs identification and detection.

CYP314A1-dependent 20-hydroxyecdysone biosynthesis is involved in regulating the development of pupal diapause and energy metabolism in the Chinese citrus fruit fly, Bactrocera minax.

BACKGROUND: Diapause is an environmentally preprogrammed period of arrested development, and characterized by metabolic depression that can occur during any development stage of insect. The insect steroid hormone 20-hydroxyecdysone (20E), is converted from ecdysone by the cytochrome P450 enzyme shade (CYP314A1), and it exerts a potent effect on the induction and maintenance of diapause in obligatory diapause insects. However, the regulatory mechanism of 20E in obligatory diapause development remains unclear. In this study, the function of 20E in the pupal diapause of Bactrocera minax was investigated. RESULTS: We determined the expression pattern of Halloween P450 genes from larval to adult B. minax, and found differential expression of CYP314A1 from other P450 genes, with a high level in larvae and a low level in pupae. Dysfunction of CYP314A1 by dsCYP314A1 microinjection in third-instar larvae caused significant larval mortality or abnormal pupae. Compared with dsGFP and DEPC-water, dsCYP314A1-injected larvae had significantly reduced 20E titer and altered energy metabolism, and many individuals failed to pupate. Exogenous 20E microinjected into late third-instar larvae or 20E fed to early third-instar larvae both caused similar energy metabolism changes. The 20E-treated larvae of B. minax had reduced total lipids and increased amounts of trehalose and glycogen. Furthermore, 20E-treated diapause individuals showed rapid pupal development. CONCLUSION: The 20E biosynthesis was regulated by the expression of CYP314A1, and was involved in the induction and termination phase of obligate diapause by regulating energy metabolism in B. minax. © 2022 Society of Chemical Industry.

Effect of Insulin Receptor on Juvenile Hormone Signal and Fecundity in Spodoptera litura (F.).

Insulin signaling can regulate various physiological functions, such as energy metabolism and reproduction and so on, in many insects, including mosquito and locust. However, the molecular mechanism of this physiological process remains elusive. The tobacco cutworm, Spodoptera litura, is one of the most important pests of agricultural crops around the world. In this study, phosphoinositide 3-kinase (SlPI3K), protein kinase B (SlAKT), target of rapamycin (SlTOR), ribosomal protein S6 kinase (SlS6K) and transcription factor cAMP-response element binding protein (SlCREB) genes, except transcription factor forkhead box class O (SlFoxO), can be activated by bovine insulin injection. Then, we studied the influence of the insulin receptor gene (SlInR) on the reproduction of S. litura using RNA interference technology. qRT-PCR analysis revealed that SlInR was most abundant in the head. The SlPI3K, SlAKT, SlTOR, SlS6K and SlCREB genes were decreased, except SlFoxO, after the SlInR gene knockdown. Further studies revealed that the expression of vitellogenin mRNA and protein, Methoprene-tolerant gene (SlMet), could be down-regulated by the injection of dsRNA of SlInR significantly. Furthermore, a depletion in the insulin receptor by RNAi significantly decreased the content of juvenile hormone III (JH-III), total proteins and triacylgycerol. These changes indicated that a lack of SlInR could impair ovarian development and decrease fecundity in S. litura. Our studies contribute to a comprehensive insight into reproduction, regulated by insulin and the juvenile hormone signaling pathway through nutrition, and a provide theoretical basis for the reproduction process in pest insects.

Comparative transcriptome analysis of different developmental stage of Bactrocera minax (Diptera: Tephritidae): Implication of the molecular basis of its obligatory diapause induction.

The Chinese citrus fruit fly, Bactrocera minax (Enderlein), a devastating pest in citrus, has an obligatory diapause at the pupal stage. Although the physiology of obligatory diapause has been well described, the molecular mechanisms underlying the process remain unknow. Here we investigated the molecular mechanisms of obligatory diapause induction in B. minax using high-throughput RNA-Seq data from second-instar larva (2L), third-instar larva (3L) and pupa (P) stages. A total of 116,402 unigenes were obtained, of which 54,781 unigenes were successfully annotated in public databases, and the differentially expressed genes in the 3L vs 2L, P vs 2L, and P vs 3L comparisons were identified. The cluster co-expression patterns of the differentially expressed genes revealed that significantly differentially-expressed genes in the pupal stage were predicted to be related to diapause induction. All differentially expressed genes were investigated by GO functional and KEGG pathway analysis, and the results showed that genes involved in processes such as 20-hydroxyecdysone (20E) biosynthesis, cell cycle and metabolic pathways are likely related to obligatory diapause induction in B. minax. These results provide important information on the transcriptome of the Chinese citrus fruit fly that can be used for further functional studies as well as contributing to our understanding of the molecular basis of obligatory diapause induction and suggesting potential molecular targets for the control of this pest.

Genetic Diversity of the root-knot nematode Meloidogyne enterolobii in Mulberry Based on the Mitochondrial COI Gene.

This study explores the genetic diversity and structure of Meloidogyne enterolobii in mulberry in China. The COI mitochondrial gene (mtCOI) in M.enterolobii populations in Guangdong, Guangxi, and Hunan Provinces was PCR-amplified, sequenced, and analyzed for genetic diversity. The total number of variations, haplotypes (Hap), the average number of nucleotide differences (k), haplotype diversity (H), and nucleotide diversity (π) of mtCOI were 25, 11, 4.248, 0.900, and 0.00596, respectively. Insignificant differences in Fst value (0.0169) and a high level of gene flow (7.02) were detected among the 19-mulberry root-knot nematode populations, and high genetic variation within each population and a small genetic distance among populations were observed. Both phylogenetic analyses and network mapping of the 11 haplotypes revealed a dispersed distribution pattern of 19 mulberry root-knot nematode populations and an absence of branches strictly corresponding to the 19 range sampling sites. The neutrality test and mismatch analysis indicated that mulberry root-knot nematode populations experienced a population expansion in the past. The analysis of molecular variance (AMOVA) revealed that the genetic differentiation of M. enterolobii was mainly contributed by the variation within each group. No significant correlation was found between the genetic distance and geographical distance of M. enterolobii populations. The findings of this study provide a profound understanding of the M. enterolobii population and will inform the development of strategies to combat and manage root-knot nematodes in mulberry.