Fine-mapping and candidate gene analysis for the foxglove aphid resistance gene Raso2 from wild soybean PI 366121.

KEY MESSAGE: The foxglove aphid resistance gene Raso2 from PI 366121 was fine-mapped to 77 Kb region, and one candidate gene was identified. The foxglove aphid (FA: Aulacorthum solani Kaltenbach) is an important insect pest that causes serious yield losses in soybean. The FA resistance gene Raso2 from wild soybean PI 366121 was previously mapped to a 13 cM interval on soybean chromosome 7. However, fine-mapping of Raso2 was needed to improve the effectiveness of marker-assisted selection (MAS) and to eventually clone it. The objectives of this study were to fine-map Raso2 from PI 366121 using Axiom® 180 K SoyaSNP array, to confirm the resistance and inheritance of Raso2 in a different background, and to identify candidate gene(s). The 105 F4:8 recombinant inbred lines were used to fine-map the gene and to test antibiosis and antixenosis of Raso2 to FA. These efforts resulted in the mapping of Raso2 on 1 cM interval which corresponds to 77 Kb containing eight annotated genes based on the Williams 82 reference genome assembly (Wm82.a2.v1). Interestingly, all nonsynonymous substitutions were in Glyma.07g077700 which encodes the disease resistance protein containing LRR domain and expression of the gene in PI 366121 was significantly higher than that in Williams 82. In addition, distinct SNPs within Glyma.07g077700 that can distinguish PI 366121 and diverse FA-susceptible soybeans were identified. We also confirmed that Raso2 presented the resistance to FA and the Mendelian inheritance for single dominant gene in a different background. The results of this study would provide fundamental information on MAS for development of FA-resistant cultivars as well as functional study and cloning of the candidate gene in soybean.

Insulin signaling mediates previtellogenic development and enhances juvenile hormone-mediated vitellogenesis in a lepidopteran insect, Maruca vitrata.

BACKGROUND: Insulin/insulin-like growth peptide signaling (IIS) down-regulates hemolymph sugar level and facilitates larval growth in the soybean pod borer, Maruca vitrata. The objective of this study is to determine whether IIS of M. vitrata can mediate ovarian development of adult females. RESULTS: A pair of ovaries consists of 8 ovarioles, each of which is separated into distal germarium and proximal vitellarium in M. vitrata. In the germarium, oocyte development occurred with active mitotic activity which was visible by incorporating bromodeoxyribose uridine. Previtellogenic development and subsequent vitellogenesis began soon after adult emergence. They continued with increase of female age. Oocyte development was facilitated by up-regulation of vitellogenin (Vg) and Vg receptor (VgR) gene expression. Larval diets significantly influenced on ovarian development of M. vitrata because oocyte development varied with pupal size derived from larvae treated with different nutritional diets. Its ovarian development was dependent on endocrine signal(s) from the head because decapitation soon after adult emergence prevented oogenesis and subsequent vitellogenesis along with marked reduction of Vg and VgR expression. Topical application of juvenile hormone (JH) significantly recovered its ovarian development whereas farnesoic acid (a precursor of JH biosynthesis) or 20-hydroxyecdysone treatment did not. JH stimulated vitellogenesis and choriogenesis, but not previtellogenic development. In contrast, insulin injection to decapitated females stimulated oocyte differentiation and vitellogenesis along with increase of Vg and VgR expression. To further analyze the effect of insulin on ovarian development, expression of four IIS components (InR, FOXO, Akt, and TOR) genes was manipulated by RNA interference. Hemocoelic injection of gene-specific double stranded RNAs significantly reduced their target gene mRNA levels and interfered with ovarian development. An addition of insulin to JH treatment against decapitated females enhanced the gonadotropic effect of JH by stimulating oogenesis. CONCLUSIONS: IIS plays crucial role in mediating previtellogenic development of M. vitrata in response to nutrient signal. It also enhances the gonadotropic effect of JH II on vitellogenesis.

Insulin-like peptides of the legume pod borer, Maruca vitrata, and their mediation effects on hemolymph trehalose level, larval development, and adult reproduction.

Insulin-like peptides (ILPs) of insects mediate various physiological processes including hemolymph sugar level, immature growth, female reproduction, and lifespan. In target cells of ILPs, insulin/insulin-like growth factor signaling (IIS) is highly conserved in animals. IIS in the legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), is known to be involved in maintaining hemolymph trehalose levels and promoting larval growth. However, ILPs in M. vitrata have not been reported yet. This study predicted two ILP genes of Mv-ILP1 and Mv-ILP2 from transcriptome of M. vitrata. Mv-ILP1 and Mv-ILP2 shared high sequence homologies and domain architecture with Drosophila ILPs. Both ILPs exhibited similar expression patterns in most developmental stages, showing high expression levels in adult stage. In the larval stage, Mv-ILP1 and Mv-IlP2 were expressed mostly in the brain and fat body. However, in the adult stage, both ILP genes were expressed more in the abdomen than those in the head containing the brain. RNA interference (RNAi) of either Mv-ILP1 or Mv-ILP2 during larval stage resulted in significant malfunctioning in regulating hemolymph trehalose titers. RNAi-treated larvae also exhibited significant retardation of larval growth. RNAi treatment in adult stage interfered with the ovarian development of females. These results suggest that Mv-ILP1 and Mv-ILP2 play crucial roles in mediating larval growth and adult reproduction.

Identification and bacterial characteristics of Xenorhabdus hominickii ANU101 from an entomopathogenic nematode, Steinernema monticolum.

An entomopathogenic nematode, Steinernema monticolum, was collected in Korea. Its identity was confirmed by morphological and molecular characters. Its symbiotic bacterium, Xenorhabdus hominickii ANU101, was isolated and assessed in terms of bacterial characteristics. Sixty-eight different carbon sources were utilized by X. hominickii ANU101 out of 95 different sources from a Biolog assay. Compared to other Xenorhabdus species, X. hominickii ANU101 was relatively susceptible to high temperatures and did not grow above 34°C. Furthermore, its growth rate was much slower than other Xenorhabdus species. X. hominickii exhibited insecticidal activities against coleopteran, dipteran, and lepidopteran insect pests. The bacterial virulence was not correlated with its host nematode virulence with respect to relative insecticidal activity against target insects. X. hominickii ANU101 exhibited antibiotics tolerance. The bacterium possesses four different plasmids (Xh-P1 (104,132bp), Xh-P2 (95,975bp), Xh-P3 (88,536bp), and Xh-P4 (11,403bp)) and encodes 332 open reading frames. Subsequent predicted genes include toxin/antitoxins comprising a multidrug export ATP-binding/permease. This study reports bacterial characters of X. hominickii and its entomopathogenicity.

An entomopathogenic bacterium, Xenorhabdus hominickii ANU101, produces oxindole and suppresses host insect immune response by inhibiting eicosanoid biosynthesis.

An entomopathogenic bacterium, Xenorhabdus hominickii ANU101, was isolated from an entomopathogenic nematode, Steinernema monticolum. X. hominickii exhibited significant insecticidal activities at ≥6.6×102 colony-forming units per larva against a lepidopteran insect, Spodoptera exigua with hemocoelic injection. The insecticidal activity of X. hominickii was reduced by an addition of arachidonic acid (AA, a catalytic product of PLA2), but enhanced by an addition by dexamethasone (DEX, a specific inhibitor of PLA2). S. exigua could defend the bacterial infection by forming hemocyte nodules. However, live X. hominickii significantly reduced the hemocytic nodulation compared to similar treatment with heat-killed X. hominickii. An addition of AA to live X. hominickii significantly rescued the immunosuppression. X. hominickii also inhibited phenoloxidase activity in hemolymph of S. exigua larvae. Furthermore, the bacteria suppressed gene expressions of antimicrobial peptides, such as attacin-1, attacin-2, defensin, gallerimycin and transferrin-1 of S. exigua. An organic extract of X. hominickii-cultured broth with ethyl acetate possessed oxindole and significantly suppressed hemocyte nodulation. Again, an addition of AA diminished the inhibitory activity of the organic extract against hemocyte nodulation. Oxindole alone inhibited hemocyte nodulation and PLA2 enzyme activity. These results suggest that the entomopathogenicity of X. hominickii comes from its inhibitory activity against eicosanoid biosynthesis of target insects.

The Biochemical Adaptations of Spotted Wing Drosophila (Diptera: Drosophilidae) to Fresh Fruits Reduced Fructose Concentrations and Glutathione-S Transferase Activities.

Spotted wing drosophila, Drosophila suzukii Matsumura, is an invasive and economically damaging pest in Europe and North America. The females have a serrated ovipositor that enables them to infest almost all ripening small fruits. To understand the physiological and metabolic basis of spotted wing drosophila food preferences for healthy ripening fruits, we investigated the biological and biochemical characteristics of spotted wing drosophila and compared them with those of Drosophila melanogaster Meigen. We found that the susceptibility to oxidative stressors was significantly increased in spotted wing drosophila compared with those of D. melanogaster. In addition, we found that spotted wing drosophila had significantly reduced glutathione-S transferase (GST) activity and gene numbers. Furthermore, fructose concentrations found in spotted wing drosophila were significantly lower than those of D. melanogaster. Our data strongly suggest that the altered food preferences of spotted wing drosophila may stem from evolutionary adaptations to fresh foods accompanied by alterations in carbohydrate metabolism and GST activities.

Detection of novel QTLs for foxglove aphid resistance in soybean.

KEY MESSAGE: The Raso2 , novel QTL for Korea biotype foxglove aphid resistance in soybean from PI 366121 was identified on chromosome 7 using GoldenGate SNP microarray. Foxglove aphid, Aulacorthum solani (Kaltenbach), is a hemipteran insect that infects a wide variety of plants worldwide and causes serious yield losses in crops. The objective of this study was to identify the putative QTL for foxglove aphid resistance in wild soybean, PI 366121, (Glycine soja Sieb. and Zucc.). One hundred and forty-one F4-derived F8 recombinant inbred lines developed from a cross of susceptible Williams 82 and PI 366121 were used. The phenotyping of antibiosis and antixenosis resistance was done through choice and no-choice tests with total plant damage and primary infestation leaf damage; a genome-wide molecular linkage map was constructed with 504 single-nucleotide polymorphism markers utilizing a GoldenGate assay. Using inclusive composite interval mapping analysis for foxglove aphid resistance, one major candidate QTL on chromosome 7 and three minor QTL regions on chromosomes 3, 6 and 18 were identified. The major QTL on chromosome 7 showed both antixenosis and antibiosis resistance responses. However, the minor QTLs showed only antixenosis resistance response. The major QTL mapped to a different chromosome than the previously identified foxglove aphid resistance QTL, Raso1, from the cultivar Adams. Also, the responses to the Korea biotype foxglove aphid were different for Raso1, and the gene from PI 366121 against the Korea biotype foxglove aphid was different. Thus, the foxglove aphid resistance gene from PI 366121 was determined to be an independent gene from Raso1 and was designated as Raso2. This result could be useful in breeding for new foxglove aphid-resistant soybean cultivars.

Assessment of the occurrence of the second generation of Mythimna loreyi Duponchel (Lepidoptera: Noctuidae) using temperature-dependent developmental and oviposition models.

A significant crop pest, Mythimna loreyi, migrates annually to Korea and has been frequently observed in rice and corn fields. However, the phenology of this pest, particularly in relation to its ecological interactions and host crop seasons in Korea, remains poorly understood. This study aims to clarify the timing of the second generation of M. loreyi in Korea to enhance pest management strategies. To achieve this, we developed temperature-dependent models for developmental and ovipositional rates, studying these processes across five constant temperatures (15, 20, 25, 30, and 35°C). Our models, which showed a high correlation with observed data (r2 ≥ 0.93), include a theoretical approach that combines the developmental variation of immatures with the necessary degree-days for 50% egg laying and complete egg development. These predictions allow for the forecasting of the second generation's occurrence, with relatively small deviations (one to three days) observed at two different field sites. The insights from this study are critical for both understanding the ecology of M. loreyi and for informing practical management decisions, such as optimal placement of barriers to prevent immigration and strategies for controlling local populations.

Temperature-dependent development of Helicoverpa armigera (Lepidoptera: Noctuidae) at constant temperatures: instar pathways and stage transition models with semifield validation.

Temperature-dependent development of Helicoverpa armigera (Hüber) fed with an artificial diet was studied at different temperatures. The instar pathway (IPW) defined as the number of instars prior to pupation significantly affected larval development time, with higher IPW leading to longer larval development time. The IPW was determined at the fifth instar to proceed to 6-7 IPW, when the development time of fifth instar was largely shortened. Accordingly, the development time after the fourth instar was combined (i.e., the fifth-seventh instar) as a single stage to simplify the various IPW and applied to develop phenology models. In linear models, the lower threshold temperature (LT) and thermal constant (degree-days, DD) for each stage were estimated. DD based on the common LT of 10.7 °C were 43, 287, and 191 DD for eggs, larvae, and pupae, respectively. DD model (253.6 DD with LT 10.3 °C for larvae and 181.5 DD with 11.6 °C for pupae) showed good performance in predicting the 50% occurrences of pupae and adults. In nonlinear models, stage transition (ST) models were constructed using the development rate and distribution models to simulate the proportion of individuals shifted from one stage to the next stage. The ST model showed good performance, indicating an average discrepancy of 1.74 days at 25%, 50%, 75%, and 90% adult emergence. Our models developed here will be useful to predict the phenology of H. armigera in the field and to construct a deterministic population model in the future.

Chymotrypsin is a molecular target of insect resistance of three corn varieties against the Asian corn borer, Ostrinia furnacalis.

The Asian corn borer, Ostrinia furnacalis, is a serious insect pest that can infest corn leaves and stems. Due to its internal feeding behavior, its larvae are not exposed to insecticides that are usually sprayed for pest control. To minimize crop damage caused by O. furnacalis, improving insect resistance trait of corn has been considered as an optimal control tactic. This study screened 27 corn varieties for their insect resistance trait and selected three varieties of Ilmichal (IM), P3394, and Kwangpyeongok (KP) that showed insect resistance trait. O. furnacalis larvae did not show any significant difference in preference between these three insect-resistant corn varieties and a control susceptible variety. However, these resistant varieties after ingestion significantly interfered with larval development of O. furnacalis. This suggests that the insect resistance trait is induced by antibiosis, but not by antixenosis. Indeed, larvae fed with these varieties suffered from low chymotrypsin (CHY) activities in the midgut juice. To determine the target CHY inhibited by resistant corn varieties, a total of nine CHY genes (Of-CHY1~Of-CHY9) were predicted from the transcriptome of O. furnacalis. Six genes (Of-CHY1~Of-CHY6) were expressed in all developmental stages and tissues. Especially, Of-CHY3 was highly expressed in the midgut of O. furnacalis larvae. RNA interference (RNAi) using double-stranded RNA (dsRNA) specific to Of-CHY3 (2 µg of dsRNA injected to each L5 larva) resulted in significant reduction of Of-CHY3 expression level at 24 h post-treatment. Feeding L3 larvae with this dsRNA also significantly suppressed the expression level of Of-CHY3 and reduced its enzyme activity at 24 h post-treatment. A recombinant Escherichia coli expressing dsRNA specific to Of-CHY3 was constructed using L4440 vector. Feeding such recombinant bacteria suppressed the expression level of Of-CHY3 and prevented larval development of O. furnacalis. These results suggest that the three resistant varieties can produce a resistance factor(s) to inhibit the CHY activity of O. furnacalis and suppress larval growth. This study suggests that CHY might be an inhibition target in O. furnacalis for breeding insect-resistant corns.

Discovery of piperidinyl aminopyrimidine derivatives as IKK-2 inhibitors.

A serine-threonine kinase IKK-2 plays an important role in activation of NF-κB through phosphorylation of the inhibitor of NF-κB (IκB). As NF-κB is a major transcription factor that regulates genes responsible for cell proliferation and inflammation, development of selective IKK-2 inhibitors has been an important area of anti-inflammatory and anti-cancer research. In this study, to obtain active and selective IKK-2 inhibitors, various substituents were introduced to a piperidinyl aminopyrimidine core structure. The structure-activity relationship study indicated that hydrogen, methanesulfonyl, and aminosulfonyl groups substituted at the piperidinylamino functionality provide high inhibitory activity against IKK-2. Also, morpholinosulfonyl and piperazinosulfonyl group substituted at the aromatic ring attached to the aminopyrimidine core significantly increased the inhibitory activity of the resulting derivatives. In particular, compound 17 with the aromatic piperazinosulfonyl substituent showed the most potent (IC(50)=1.30 µM) and selective (over other kinases such as p38α, p38ß, JNK1, JNK2, JNK3, and IKK-1) inhibitory activity against IKK-2.

Sequence information on simple sequence repeats and single nucleotide polymorphisms through transcriptome analysis of mungbean.

Mungbean (Vigna radiata (L.) Wilczek) is a unique species in its ability to fix atmospheric nitrogen, with early maturity, and relatively good drought resistance. We used 454 sequencing technology for transcriptome sequencing. A total of 150 159 and 142 993 reads produced 5 254 and 6 374 large contigs (≥ 500 bp) with an average length of 833 and 853 for Sunhwa and Jangan, respectively. Functional annotation to known sequences yielded 41.34% and 41.74% unigenes for Jangan and Sunhwa. A higher number of simple sequence repeat (SSR) motifs was identified in Jangan (1 630) compared with that of Sunhwa (1 334). A similar SSR distribution pattern was observed in both varieties. A total of 8 249 single nucleotide polymorphisms (SNPs) and indels with 2 098 high-confidence candidates were identified in the two mungbean varieties. The average distance between individual SNPs was approximately 860 bp. Our report demonstrates the utility of transcriptomic data for implementing a functional annotation and development of genetic markers. We also provide large resource sequence data for mungbean improvement programs.

CRISPR/Cas9 mutagenesis against sex pheromone biosynthesis leads to loss of female attractiveness in Spodoptera exigua, an insect pestt.

Virgin female moths are known to release sex pheromones to attract conspecific males. Accurate sex pheromones are required for their chemical communication. Sex pheromones of Spodoptera exigua, a lepidopteran insect, contain unsaturated fatty acid derivatives having a double bond at the 12th carbon position. A desaturase of S. exigua (SexiDES5) was proposed to have dual functions by forming double bonds at the 11th and 12th carbons to synthesize Z9,E12-tetradecedienoic acid, which could be acetylated to be a main sex pheromone component Z9,E12-tetradecenoic acetate (Z9E12-14:Ac). A deletion of SexiDES5 using CRISPR/Cas9 was generated and inbred to obtain homozygotes. Mutant females could not produce Z9E12-14:Ac along with Z9-14:Ac and Z11-14:Ac. Subsequently, pheromone extract of mutant females did not induce a sensory signal in male antennae. They failed to induce male mating behavior including hair pencil erection and orientation. In the field, these mutant females did not attract any males while control females attracted males. These results indicate that SexiDES5 can catalyze the desaturation at the 11th and 12th positions to produce sex pheromone components in S. exigua. This study also suggests an application of the genome editing technology to insect pest control by generating non-attractive female moths.

Alteration of insulin signaling to control insect pest by using transformed bacteria expressing dsRNA.

BACKGROUND: Insulin/insulin-like growth factor signaling (IIS) is known to mediate larval growth and adult reproduction in the legume pod borer, Maruca vitrata (Lepidoptera: Crambidae). Four IIS components (InR, FOXO, Akt, and TOR) play crucial roles in the IIS pathway. RESULTS: RNA interference (RNAi) against any of these four IIS component genes was effective in suppressing each target mRNA level by either hemocoelic injection or oral administration using gene-specific double-stranded RNAs (dsRNAs). These RNAi treatments interfered with larval growth, leading to small pupae or significant larval mortality. For massive production of dsRNA, transformed bacteria expressing dsRNAs of these four IIS components were prepared with L4440 expression vector and HT115 strain of Escherichia coli. The transformed bacteria killed the larvae in a dose-dependent manner by feeding administration. An ultra-sonication pretreatment was performed to impair bacterial membrane and increase dsRNA release from the bacteria in insect intestine. This pretreatment increased the insecticidal activity of these recombinant bacteria. To further increase dsRNA toxicity, its mixture with Bacillus thuringiensis (Bt) was prepared and showed significant increase of Bt insecticidal activity in the laboratory. The bacterial mixture also showed a high control efficacy (83.3%) in an adzuki bean (Vigna angularis) field infested by M. vitrata. Furthermore, such a dsRNA effect was specific for M. vitrata, but not for non-target insects. CONCLUSION: The bacteria expressing dsRNA specific to IIS components can be used to develop dsRNA insecticide. © 2019 Society of Chemical Industry.

Development, Reproduction, and Life Table Parameters of the Foxglove Aphid, Aulacorthum solani Kaltenbach (Hemiptera: Aphididae), on Soybean at Constant Temperatures.

We investigated several characteristics of the development and reproduction of the aphid Aulacorthum solani raised on soybean (Glycine max) at 10 constant temperatures between 2.5 and 30 °C, and described the relationship between temperature and several critical biological characteristics using mathematical models. We found that A. solani could survive and reproduce on soybean at temperatures ranging from 5 to 27.5 °C. High fecundity was observed at temperatures from 12.5 to 20 °C. The lower developmental threshold and thermal constant for this species' nymphal stages were estimated to be 5.02 °C and 131.2 degree-days, respectively, using a linear model. The upper developmental threshold was estimated to be 33.9 °C using the Lactin-2 model. The optimum temperature for nymphal development was estimated to be 26.9 °C. The maximum total fecundity was estimated as ca. 76.9 nymphs per adult at 18.1 °C. The daily fecundity sharply increased at earlier adult ages, and slowly decreased thereafter until final parthenogenesis occurred, over a range of temperatures from 12.5 to 25 °C. The maximum daily fecundity was estimated to be ca. 6.1 nymphs per adult per day for a 5.2 day old of adult at 21.3 °C using an age- and temperature-dependent model of adult fecundity. In terms of life table statistics, the intrinsic rates of increase and the finite rate of increase were both highest at 25 °C, while the net reproductive rate was highest at 20 °C.

Cross-species amplification and polymorphism of microsatellite loci in the soybean aphid, Aphis glycines.

We tested the utility of 18 previously characterized Aphis spp. microsatellite loci for polymorphism and differentiation among populations of the soybean aphid, Aphis glycines. Loci were chosen from a closely related species (Aphis gossypii) and a more distantly related species (Aphis fabae). We found nine loci to be polymorphic among Korean and North American populations. Overall expected heterozygosity was moderate (average: 0.47; range: 0-1), although populations substantially differed in deviations from Hardy-Weinberg equilibrium. These loci will be valuable in characterizing population differentiation, migration and adaptation in an economically important pest of soybeans.

The complete mitochondrial genome of Laodelphax striatellus (Fallén, 1826) (Hemiptera: Delphacidae) collected in a mid-Western part of Korean peninsula.

Laodelphax striatellus (Fallén, 1826) is one of key rice pests in Northeast Asia. We have determined the mitochondrial genome of L. striatellus collected in a mid-western part of Korean peninsula. The circular mitogenome of L. striatellus is 16,359 bp long including 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNAs, and a single large non-coding region of 1,972 bp. The base composition was AT-biased (77.3%). In comparison of the two Chinese L. striatellus mitogenomes with Korean mitogenome, total 140 and 40 single nucleotide polymorphisms and 166 and 118 insertions and deletions were identified, presenting intra-species variations based on geographical distribution.

The complete mitochondrial genome of Laodelphax striatellus (Fallén, 1826) (Hemiptera: Delphacidae) collected in a southern part of Korean peninsula.

A 16,359 bp mitochondrial genome of Laodelphax striatellus collected in a southern part of Korean peninsula was completed and their intraspecies variations were compared with Korean and Chinese L. striatellus mitogenomes. The circular mitogenome of L. striatellus contains 13 protein-coding genes, two rRNA genes, 22 tRNAs, and a single large non-coding region of 1,972 bp. The base composition was AT-biased (77.3%). It has only one single nucleotide polymorphism (SNP) in AT-rich region compared to other Korean mitogenome, but total 41 and 141 SNPs and 118 and 166 insertions and deletions, respectively, compared to two Chinese mitogenomes, suggesting the possibility of tracing migration based on geographic genetic diversity.

Regulation of hemolymph trehalose titers by insulin signaling in the legume pod borer, Maruca vitrata (Lepidoptera: Crambidae).

A disaccharide, trehalose, is a main hemolymph sugar of the legume pod borer, Maruca vitrata larvae, but its titers fluctuated with feeding activity. During diurnal feeding in the photophase, hemolymph trehalose remained at a relatively low level (69 mM) and increased (98 mM) during scotophase. Starvation significantly increased the hemolymph trehalose level, in which the elevation of trehalose titers was dependent on the non-feeding period. The down-regulation of the trehalose level during the active feeding period seemed to result from mediation of the insulin/IGF signal (IIS). Injection of a porcine insulin suppressed the trehalose level in a dose-dependent manner. Genes associated with IIS of M. vitrata were predicted from its larval transcriptome, and their expression was confirmed in different developmental stages and tissues. All seven IIS genes selected were expressed in all developmental stages and different tissues. Silencing of four IIS genes (insulin receptor, Forkhead box O, a serine-threonine protein kinase, target of rapamycin) by RNA interference significantly modulated the hemolymph trehalose level. Starvation treatment changed expression of two trehalose metabolism-associated genes (trehalose phosphate synthase (TPS) and trehalase (TRE)) as well as the IIS genes. Silencing of TPS or TRE expression significantly down- or up-regulated the hemolymph trehalose level, respectively. In addition, silencing of IIS genes altered both TPS and TRE expression, indicating a functional link between IIS and trehalose metabolism. These results suggest that nutrients obtained from feeding activate IIS of M. vitrata, which then down-regulates the hemolymph trehalose level by altering trehalose metabolism.

Application of insulin signaling to predict insect growth rate in Maruca vitrata (Lepidoptera: Crambidae).

Insect growth is influenced by two major environmental factors: temperature and nutrient. These environmental factors are internally mediated by insulin/insulin-like growth factor signal (IIS) to coordinate tissue or organ growth. Maruca vitrata, a subtropical lepidopteran insect, migrates to different climate regions and feeds on various crops. The objective of this study was to determine molecular tools to predict growth rate of M. vitrata using IIS components. Four genes [insulin receptor (InR), Forkhead Box O (FOXO), Target of Rapamycin (TOR), and serine-threonine protein kinase (Akt)] were used to correlate their expression levels with larval growth rates under different environmental conditions. The functional association of IIS and larval growth was confirmed because RNA interference of these genes significantly decreased larval growth rate and pupal weight. Different rearing temperatures altered expression levels of these four IIS genes and changed their growth rate. Different nutrient conditions also significantly changed larval growth and altered expression levels of IIS components. Different local populations of M. vitrata exhibited significantly different larval growth rates under the same nutrient and temperature conditions along with different expression levels of IIS components. Under a constant temperature (25°C), larval growth rates showed significant correlations with IIS gene expression levels. Subsequent regression formulas of expression levels of four IIS components against larval growth rate were applied to predict growth patterns of M. vitrata larvae reared on different natural hosts and natural local populations reared on the same diet. All four formulas well predicted larval growth rates with some deviations. These results indicate that the IIS expression analysis explains the growth variation at the same temperature due to nutrient and genetic background.