Fall Webworm Hyphantria Cunea Research Articles

Chromosome-level genome assembly of Chouioia cunea Yang, the parasitic wasp of the fall webworm

Chouioia cunea Yang 1989 is a parasitic wasp of many lepidopteran insects during their pupal stage, and has been successfully used to control pests such as the fall webworm Hyphantria cunea. Here we reported the chromosome-level genome of C. cunea by using short (MGI-SEQ), long (Oxford Nanopore), chromatin-linked (Hi-C) sequencing reads and transcriptomic data, representing the first chromosome-level genome of parasitic wasps of the family Eulophidae. The total assembly length is 171.99 Mb, containing 6 pesudo-chromosomes with a GC content of 36.89% and the scaffold/contig N50 length of 31.70/26.52 Mb. The BUSCO completeness of the assembly was estimated to be 98.7%. A total of 12,258 protein-coding genes (PCGs), 10,547 3′-UTRs, and 10,671 5′-UTRs were annotated. This high-quality genome is an important step toward a better understanding of the genomes of the Eulophidae (Chalcidoidea), and will serve as a valuable resource for analyses of phylogenetic relationships and the evolution of Hymenoptera.

Mild Winter Causes Increased Mortality in the Fall Webworm Hyphantria cunea (Lepidoptera: Arctiidae).

The fall webworm Hyphantria cunea (Drury) is native to North America and Mexico and has currently expanded its distribution to the temperate areas of the Northern Hemisphere including Japan. According to the data on seasonal fluctuations of this moth for 18 years collected in western-central Japan, the abundance of adults of the overwintered generation showed a negative correlation with winter temperature. We investigated survival, weight loss, and fungal infection of diapausing pupae at 3.0 (an approximate temperature of cold winter) and 7.4 °C (a temperature of mild winter). In the results, mortality was higher and weight loss was larger in pupae exposed to 7.4 °C than in those exposed to 3.0 °C. In addition, pupae that were heavier at the start of cold exposure survived longer than lighter ones. Furthermore, almost all pupae that died at 7.4 °C were infected by fungi. It has been reported that the distribution range of this moth shifts to higher latitudes. According to the experiments conducted, it has been observed that warm winters can lead to a decrease in pupae weight and an increase in fungal deaths; however, the impact of warm winters on populations in the field can be more complicated and multifaceted.

Effects of chlorantraniliprole and chromafenozide on mortality and feeding cessation of the fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae)

The fall webworm (FWW) Hyphantria cunea, native to North America, is a globally invasive pest of a wide range of forest and fruit trees. Spraying of pesticides is the primary method for the control of FWW. In this study, toxicity and feeding cessation of two potential pesticides against the FWW, chlorantraniliprole, and chromafenozide, were evaluated. Both pesticides were slow to affect FWW. For chlorantraniliprole, the highest mortality of third instar larvae occurred at 72 h with an LC50 of 10.34 mg/L, while for chromafenozide, the highest mortality occurred at 72 h with an LC50 value 74.0950 mg/L. Low concentrations of both pesticides led to larvae ceasing to feed after six hours (chlorantraniliprole) and 24 h (chromafenozide). Both pesticides had persistent effects; thirty days after being applied at concentrations of 16, 26.67, and 35.56 mg/L to leaves, 93.33% of newly contacted larvae died after seven days. Our study showed that chlorantraniliprole and chromafenozide could be alternatively used against FWW and form a component of integrated control programs. The results provide information to guide the usage of chlorantraniliprole and chromafenozide in FWW control.

Genome of the webworm Hyphantria cunea unveils genetic adaptations supporting its rapid invasion and spread

BackgroundThe fall webworm Hyphantria cunea is an invasive and polyphagous defoliator pest that feeds on nearly any type of deciduous tree worldwide. The silk web of H. cunea aids its aggregating behavior, provides thermal regulation and is regarded as one of causes for its rapid spread. In addition, both chemosensory and detoxification genes are vital for host adaptation in insects.ResultsHere, a high-quality genome of H. cunea was obtained. Silk-web-related genes were identified from the genome, and successful silencing of the silk protein gene HcunFib-H resulted in a significant decrease in silk web shelter production. The CAFE analysis showed that some chemosensory and detoxification gene families, such as CSPs, CCEs, GSTs and UGTs, were expanded. A transcriptome analysis using the newly sequenced H. cunea genome showed that most chemosensory genes were specifically expressed in the antennae, while most detoxification genes were highly expressed during the feeding peak. Moreover, we found that many nutrient-related genes and one detoxification gene, HcunP450 (CYP306A1), were under significant positive selection, suggesting a crucial role of these genes in host adaptation in H. cunea. At the metagenomic level, several microbial communities in H. cunea gut and their metabolic pathways might be beneficial to H. cunea for nutrient metabolism and detoxification, and might also contribute to its host adaptation.ConclusionsThese findings explain the host and environmental adaptations of H. cunea at the genetic level and provide partial evidence for the cause of its rapid invasion and potential gene targets for innovative pest management strategies.

Influence of invasive pest and natural enemies on entomofauna formation in the Sochi region

The article describes the changes in local entomofauna in the Sochi region. Monitoring for several decades provides information about the spreading and density of invasive species established a long time ago and causing massive damage to agricultural plants during regular outbreaks. This group of species, which includes the Boisduval scale Diaspis boisduvalii, the Japanese planthopper Ricania japonica, and the fall webworm Hyphantria cunea, has had an enormous effect on local insects’ community for the last 50 years. What is most important is that this list of alien species continues to grow. Since the late 1990s, several invaders have been discovered in the Sochi region, including including the sycamore lace bug Corythucha ciliata, the leafhoppers Metcalfa pruinosa and Arboridia kakogawana, the brown marmorated stink bug Halyomorpha halys, the tortoise wax scale Ceroplastes japonicus and the woolly aphid Eriosoma lanigerum. The invasion process causes significant changes in the relationship between newly established populations of invasive pests and their natural enemies, which are partly used as biocontrol agents. So the impact of local and introduced entomophagous species on the fauna is also discussed.

Full-Length Transcriptome Survey and Expression Analysis of Parasitoid Wasp Chouioia cunea upon Exposure to 1-Dodecene

Chouioia cunea (Yang) is an endoparasitic wasp which parasitizes pupae and thus plays an important role in the biological control of the fall webworm (Hyphantria cunea Drury), an important quarantine pest in the entire world and a major invasive pest in China. For the purposes of investigating which proteins are involved in the response of C. cunea to 1-Docecene, one of the chemical compounds of pupae of H. cunea with a significant attracting action to mated female C. cunea, 11.5 Gb transcriptome data was sequenced on the PacBio RS II platform from 1-day old C. cunea adults to generate a reference assembly. Afterwards, 46.88 Gb of clean RNA-Seq data were obtained to assess the transcriptional response of these insects before and after the stimulation with 1-Docecene. After removing redundancy using CD-HIT, a sequence structure analysis predicted 29,105 complete coding sequence (CDS) regions, 51,458 single-sequence repeats (SSRs), and 2,375 long non-coding RNAs. Based on the early transcriptome sequencing in our laboratory, we revealed some new sequences corresponding to chemosensory genes such as odorant binding proteins (OBPs), odorant receptor (OR), gustatory receptors(GRs). Results of quantitative real-time PCR experiments revealed that CcOBP7, CcOBP18, CcCSP4, CcOR2, and CcGR18 were up-regulated after 1-Dodecene stimulation. In addition, the expression of 31 genes, including 1 gene related to phospholipid biosynthesis and 2 genes related to transmembrane transport were up-regulated after 1-Dodecene stimulation; meanwhile, the expression of 22 genes, including 5 genes related to protein phosphorylation and protein serine/threonine kinase activity were significantly down-regulated after 1-Dodecene stimulation. These results suggest that the attraction of adult C. cunea to 1-dodecane is associated with the transmembrane signal transduction and dephosphorylation of some proteins. Our findings will provide useful targets for further studies on the molecular mechanism of host recognition in C. cunea.

CYPs in different families are involved in the divergent regio-specific epoxidation of alkenyl sex pheromone precursors in moths

In moth species that utilize alkenyl sex pheromones, the epoxidation of alkenes confers further diversity on the chemical structures of pheromone components. Hc_epo1 (CYP341B14), the first pheromone gland (PG)-specific epoxidase identified from the fall webworm Hyphantria cunea (Erebidae), specifically epoxidizes the Z9 double bond in the triene precursor, (3Z,6Z,9Z)-3,6,9-henicosatriene (Z3,Z6,Z9-21:H). In the present study, we identified a novel PG-specific epoxidase, As_epo1, from the Japanese giant looper Ascotis selenaria (Geometridae), which secretes cis-3,4-epoxy-(6Z,9Z)-6,9-nonadecadiene (epo3,Z6,Z9-19:H) as the main sex pheromone component. A functional assay using the Sf9 insect cell line–baculovirus expression system showed that As_epo1 specifically epoxidizes the Z3 double bond in the pheromone precursor triene, (3Z,6Z,9Z)-3,6,9-nonadecatriene (Z3,Z6,Z9-19:H). As_epo1 also Z3-specifically epoxidized a triene with a longer carbon chain, Z3,Z6,Z9-21:H, which does not occur in this species. A phylogenetic analysis indicated that As_epo1 belonged to the CYP340 family, not the CYP341 family to which Hc_epo1 belongs. These results suggest that moth PG-specific epoxidases with divergent regio-specificities have evolved independently.

Identification of Venom Proteins of the Indigenous Endoparasitoid Chouioia cunea (Hymenoptera: Eulophidae).

Chouioia cunea (Yang) (Hymenoptera: Eulophidae) is an indigenous pupal endoparasitoid that effectively attacks the exotic fall webworm Hyphantria cunea (Drury) (Lepidoptera: Arctiidae) in China. In this novel association, the parasitoid's venom has played an important role in subduing the immune defense of the host although little is known about the composition and functions of the parasitoid's venom. We therefore first identified the parasitoid's major venom proteins using electrospray ionization-mass spectrometry (ESI-MS). Approximately 207 different proteins were identified from C. cunea's venom; among them 26 types widely existed in other endoparasitoids' venom, including calreticulin and arginine kinase, which inhibited the host immune system.

Haplotype diversity of mtCOI in the fall webworm Hyphantria cunea (Lepidoptera: Arctiidae) in introduced regions in China, Iran, Japan, Korea, and its homeland, the United States

Hyphantria cunea (Drury) has colonized many countries outside its native range of North America and has become a model species for studies of the colonization and subsequent adaptation of agricultural pests. Molecular genetic analyses can clarify the origin and subsequent adaptations to non-native habitats. Using the mitochondrial COI gene, we examined the genetic relationships between invasive populations (China, Iran, Japan, and Korea) and native populations (i.e., the United States). The Jilin (China) and Guilan (Iran) populations showed nine previously unknown haplotypes that differed from those found in the south–central United States, suggesting multiple colonization events and different regions of invasion. A dominant mtDNA haplotype in populations in the United States was shared by all of the populations investigated, suggesting that H. cunea with that haplotype have successfully colonized China, Iran, Japan, and Korea.

Empowering fall webworm surveillance with mobile phone-based community monitoring: a case study in northern China

Recent advances in information and communication technologies, such as mobile Internet and smartphones, have created new paradigms for participatory environment monitoring. The ubiquitous mobile phones with capabilities such as a global positioning system, camera, and network access, offer opportunities to establish distributed monitoring networks that can perform a wide range of measurements for a landscape. This study examined the potential of mobile phone-based community monitoring of fall webworm (Hyphantria cunea Drury). We built a prototype of a participatory fall webworm monitoring system based on mobile devices that streamlined data collection, transmission, and visualization. We also assessed the accuracy and reliability of the data collected by the local community. The system performance was evaluated at the Ziya commune of Tianjin municipality in northern China, where fall webworm infestation has occurred. The local community provided data with accuracy comparable to expert measurements (Willmott’s index of agreement >0.85). Measurements by the local community effectively complemented remote sensing images in both temporal and spatial resolution.

An analysis of the feasibility of incorporating a mobile phone-based participatory field survey into satellite-based fall webworm monitoring

ABSTRACTThis study explores the feasibility of incorporating public participatory field data collection into satellite-based fall webworm (Hyphantria cunea Drury) monitoring. Case studies were conducted through fall webworm monitoring at Jinghai county in the north of China and Lieshan county in the middle-east of China. Sample data generated from participatory monitoring data were used for satellite-based fall webworm defoliation mapping, achieving an average accuracy above 92%. By comparing participatory monitoring data with expert measurements, Willmott’s indices of agreement were greater than 0.88, which demonstrated that infestation data collected by local community could achieve a comparable accuracy to expert value. Meanwhile, the comparison with satellite data confirmed that participatory monitoring data could serve as a valuable, high temporal and spatial resolution data source complementary to satellite data, to help forest managers, planners and scientists to better understand the status of fall webworm infestation.

Cold hardiness characteristic of the overwintering pupae of fall webworm Hyphantria cunea (Drury) (Lepidoptera: Arctiidae) in the northeast of China

The fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae), is an invasive and important pest in China. Understanding the physiological–biochemical characteristics of the overwintering pupae is essential for its cold tolerance and potential distribution in the northeast of China. Results indicated that the supercooling point (SCP) of H. cunea overwintering pupae varied significantly among Jilin city (43°N, 126°E), Jilin Province, Shenyang (41.8°N, 123.4°E), Liaoning Province and Yingkou (40.39°N, 122.13°E), Liaoning Province. The average SCP in Jilin was the lowest (−23.49±0.46°C) in those of three areas. The water content in overwintering pupae had a significant positive correlation with the SCP (R=0.848,P=0.03). Contents of alanine (ALA) (26.00%), lysine (LYS) (30.51%) and arginine (ARG) (22.88%) in the overwintering pupae's hemolymph were higher than others. During the overwintering, the fat content in pupae's bodies decreased gradually. Analysis of variance (ANOVA) indicated that the content of trehalose in the body of H. cunea was remarkably different (P=0.007) month by month, with the maximum in March (2.88mg·mL−1) and the minimum (0.40mg·mL−1) in November. Pearson's correlation test indicated that SCP had a significant negative correlation with the SOD enzyme activity (R=−0.850; P=0.008). Thermal hysteresis activity (THA) of hemolymph was 0.68°C and 0.7–0.8°C for H. cunea pupae and larva, respectively, using differential scanning calorimetry, which suggests that the H. cunea can improve their cold tolerance through producing antifreeze proteins. These results could help us to predict the distribution of the H. cunea in the northeast of China.

CYP341B14: A cytochrome P450 involved in the specific epoxidation of pheromone precursors in the fall webworm Hyphantria cunea

Two of the four sex pheromone components in the fall webworm Hyphantria cunea (Lepidoptera: Arctiidae), cis-9,10-epoxy-(3Z,6Z)-3,6-henicosadiene and cis-9,10-epoxy-(3Z,6Z)-1,3,6-henicosatriene, possess an epoxy ring within their molecules. These compounds have been suggested to be biosynthesized from dietary linolenic acid via the following enzymatic reactions; chain elongation, terminal desaturation (in the case of the latter component), decarboxylation, and epoxidation. The last step of this biosynthesis, epoxidation, is known to occur specifically in the sex pheromone gland of females. We identified the enzyme involved in the epoxidation of pheromone precursors by focusing on cytochromes P450, which are known to catalyze the oxidation of various compounds. Three P450-like sequences (Hc_epo1, Hc_epo2, and Hc_epo3) were identified in the cDNA library prepared from the sex pheromone gland of H. cunea. Among these clones, only Hc_epo1 was specifically expressed in the pheromone gland. The full-length sequence of Hc_epo1 contained an ORF of 1527 bp, which encoded a protein of 509 amino acids with a predicted molecular weight of 57.9 kDa. The deduced Hc_epo1 amino acid sequence possessed the characteristics of P450. A phylogenetic analysis of the sequence indicated that Hc_epo1 belonged to the CYP341B clade in the CYP341 family. Therefore, it was named CYP341B14. A subsequent functional assay using Sf-9 cells transiently expressing CYP341B14 demonstrated that this P450 protein was able to specifically epoxidize a (Z)-double bond at the 9th position in the pheromone precursor, (3Z,6Z,9Z)-3,6,9-henicosatriene.

Characterization and polymorphism analysis of phosphoglucose isomerase gene in the fall webworm (Hyphantria cunea)

Phosphoglucose isomerase (PGI) plays an important role in energy metabolism, and it is documented that PGI exhibits an extensive polymorphism which can affect insects' fitness and adaptation. In this paper, we studied the structural characteristics and polymorphism of pgi gene in the fall webworm (Hyphantria cunea), an important invasive pest in some European and Asian countries. A 2110-bp pgi full-length cDNA encoding a polypeptide of 556 amino acids was obtained from H. cunea. The pgi full-length in the H. cunea genomic DNA was 14,332 bp with 12 exons and 11 introns, similar to the structures of pgi in other Lepidoptera species. We compared the structures of pgi in different insect species. Moreover, thirteen pgi genotypes comprised of five alleles were identified in the Chinese population. Genotypes pgi-cd, pgi-cc and pgi-ce were the most prevalent with over 70% of individuals allocated to them. Four out of five alleles were sequenced the cDNA full-length. Thirty stably variable sites were found among them with five non-synonymous mutation sites. The frequencies of alleles and genotypes were variable in different Chinese geographic subpopulations. Moreover, comparison of pgi mRNA expression levels in each stage of the moth's lifecycle showed that a high expression level was in the 6th instar larval stage, followed by that in the egg and adult stages. The results will provide a basis for further study of the role of different alleles and genotypes of PGI on fitness and adaptation of the moth H. cunea.

Efficacy of Some Biopesticides and Plant Secondary Metabolites Against Fall Webworm Hyphantria Cunea Drury (F. Arctiidae-Lepidoptera) in the Lab Conditions

Efficacy of Some Biopesticides and Plant Secondary Metabolites Against Fall Webworm Hyphantria Cunea Drury (F. Arctiidae-Lepidoptera) in the Lab Conditions The paper presents the efficacy of the some biopesticides used in the experiments to control fall webworm (Hyphantria cunea), comparatively to some plant secondary metabolites from autochthonous flora. From the first category there were used: spinosad, a secondary metabolite produces by the fermentation from Saccharopolyspora spinosa mushroom and is the active principle of the commercial products of the Naturalyte class; azadyrachtines - a group of limonoids, obtained from the seeds of the Neem tree (Azadirachta indica), and milbecmectin, a product obtained from a metabolite of the Streptomyces hygroscopicus subsp. aureolacrimans bacteria. The results revealed the efficacy of all bio insecticides against fall webworm in 2-7 days period after treatment. Spinosad presented a quick action, comparatively to the other bio pesticides. The secondary metabolites, used into fall webworm control, were extracted from autochthonous plants: the common ladyfern (Drioperis filix mas), the perennial sage (Salvia nemorosa), the wormwood (Artemisia dracunculus, A. vulgaris, A. absinthium) the European birthwort (Aristolochia clematidis), Cow parsnip (Heracleum spondylium), the hedge nettle (Stachis sylvatica), the speedwell (Tanacetum vulgare), the nettle (Urtica dioica), the danewort (Sambucus ebulus) and the yew tree (Taxus baccata) to fall webworm. Plants extracts were obtained from dried ground plants, using 25 g/ 1 litter of cold water, stirred for 24 hours. The extracts in ethylic alcohol were made using the same method, 25 g dried plants in 200 ml alcohol and completed up to 1 litter with water. The experiments were carried out under laboratory conditions, treatments being applied on shoots with leaves affected by fall webworm, placed in growth boxes. Each variant had three replications and each replication contained three infested shoots. The treatments were applied with manual small pumps. Efficacy (E%) was calculated after Săvescu-Iacob formula. The majority of alcoholic plant extracts influenced the decrease of leafs consumption as extracts with water. Extracts of metabolites influenced the eating with repellent effects against larvae, no palatable etc. The future experiments must use more chemical analyses to discriminate the main metabolites, which influence the worm activities.

Abundance trumps quality: bi‐trophic performance and parasitism risk fail to explain host use in the fall webworm

Factors including host abundance, quality, and the degree to which hosts provide enemy‐free space (EFS) may drive host plant choice by phytophagous insects. Herbivores may also experience fitness tradeoffs among hosts, promoting polyphagy. The fall webworm Hyphantria cunea is a dietary generalist that feeds on a broad array of trees across its geographic range. Here, we investigate the drivers of host tree use by the fall webworm in Connecticut (CT) and Maryland (MD). Neither caterpillar performance nor EFS was associated with the frequency with which host trees were used, and no tradeoff between host quality and EFS was identified. Vegetation surveys adjacent to host trees showed that at both localities, host use was non‐random with respect to tree species, and that the main predictor of use among suitable host trees was host tree abundance. This suggests that webworms are under selection to reduce search time for oviposition sites. Although we did not detect a tradeoff between host plant quality and availability in MD, we did identify that tradeoff in CT. This disparity amid otherwise similar patterns of host use between CT and MD may be explained by the relative rarity of high quality hosts in CT compared to MD. Our results illustrate that geographic mosaics in patterns of host use may arise in the absence of local adaptation if host use is based upon availability rather than host plant attributes.

Identification and field evaluation of the sex pheromone of an invasive pest, the fall webworm Hyphantria cunea in China

Pheromone gland extracts of the fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae) were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Based on comparison of retention times and mass spectra of synthetic standards, four compounds were identified as (9Z,12Z)-octadecadienal, (9Z,12Z,15Z)-octadecatrienal, (3Z,6Z,9S,10R)-9,10-epoxy-3,6-heneico-sadiene and (3Z,6Z,9S,10R)-9,10-epoxy-1,3,6-heneicosatriene with a ratio of 2:33.6:58.4:6. Trapping tests showed that quaternary blend of these compounds was highly attractive to male H. cunea. Unitraps baited with the quaternary blend loading of 200–300 μg on a gray rubber septum were very effective for monitoring H. cunea populations.

Mass rearing and augmentative releases of the native parasitoid Chouioia cunea for biological control of the introduced fall webworm Hyphantria cunea in China

The fall webworm, Hyphantria cunea Drury, is an economically important introduced pest in China. A native pupal endoparasitoid, Chouioia cunea Yang (Hymenoptera: Eulophidae, Tetrastichinae) causes considerable mortality of H. cunea pupae in some areas of China. Alternative hosts of C. cunea were tested to find potential substitute hosts for mass rearing the parasitoid. Antheraea pernyi Guerin-Meneville (Lepidoptera: Saturniidae) was chosen as a surrogate host as a single pupa produced an average of 6552 wasps and a maximum of 11,256 per pupa. A mass-rearing technique and successful release program of this parasitoid is described. Parasitism by C. cunea in the areas where it was released averaged 67.74% and was usually over 80%. Other native parasitoids, such as Coccygomimus disparis (Viereck), C. parnasae (Viereck) (Hymenoptera: Ichneumonidae) and Exorita japonica Townes (Diptera: Tachnidae), caused the total average parasitism to exceed 90% on average, reaching a maximum of 96.28% in the release areas. These successful releases indicate that an introduced pest species can also be controlled by the mass-rearing and release of native parasitoids in the country of introduction.

Heat retention by webs of the fall webworm Hyphantria cunea (Lepidoptera: Arctiidae): infrared warming and forced convective cooling

1. Webs of the fall webworm (Lepidoptera: Hyphantria cunea Drury) were studied to determine how web temperatures respond to infrared radiation and wind. Webs were individually mounted under an infrared light source at the end of a wind tunnel.2. During infrared heating, the front (outward-facing) surface and central interior of the web were more effective at trapping and holding heat than the back (inner) surface.3. Thermal heterogeneity inside webs represents options to larvae, allowing the possibility of behavioral thermoregulation.4. During windy conditions, webs slowed air movements enough to allow the web interior to retain some captured heat.

Physiology of diapause and cold hardiness in the overwintering pupae of the fall webworm Hyphantria cunea (Lepidoptera: Arctiidae) in Japan

The fall webworm Hyphantria cunea Drury, which was accidentally introduced to Japan in 1945, overwinters on the ground in pupal diapause. Diapause termination, as indicated by the respiration rate and the period required for adult emergence, began in March and ended in April. Cold hardiness (the ability to survive exposure to −15°C) decreased linearly with diapause development from November to the following April under field conditions. Cold hardiness of diapause pupae (DP) decreased as the acclimation temperature decreased from 15 to −10°C, whereas cold hardiness of non-diapause pupae (NDP) remained high as the acclimation temperature decreased from 5 to −5°C. However, H. cunea in Japan can survive exposure to −5°C for two weeks, whether it is in a diapause or non-diapause state. Trehalose was the main sugar detected in the body, but its level was less than 0.8%. Trehalose levels increased in field-collected pupae from January to March. DP accumulated less trehalose than NDP, as the acclimation temperature was decreased from 5 to −5°C. The alanine content in field-collected pupae increased from November to February. Both diapause and low temperature caused an accumulation of alanine. These results suggest that under field conditions, overwintering pupae of H. cunea in Japan do not accumulate high levels of sugars and polyols and do not develop a high level of cold hardiness. Furthermore, DP do not accumulate high levels of sugars and polyols and their ability to survive exposure to −15°C is not greater than that of NDP. The physiological and biochemical bases of diapause in H. cunea from Japan are discussed.