Hisaaki Tsumuki

An ice-nucleating active fungus isolated from the gut of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)

Abstract The existence of a fungus with the ability to nucleate ice formation in supercooled water was revealed. The fungus was isolated from the gut of larvae of the rice stem borer, Chilo suppressalis Walker, and from rice seedlings which were host plants of this insect. The fungus was identified as a Fusarium sp. on the basis of its morphology. Ice-nucleating activity, at around −5°C, was detected in the mycelial suspension of the fungus and also in the culture filtrate. The presence of the exogenous ice-nucleating active fungus in the gut and on the body surface caused an elevation in crystallization temperature of the larvae.

Tissue distribution of the ice-nucleating agents in larvae of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)

Abstract To investigate the tissue distribution of ice-nucleating agents in the rice stem borer, Chilo suppressalis Walker, the crystallization temperatures of the whole bodies and individual tissues of nondiapausing and diapausing larvae were measured. In nondiapausing mature larvae the crystallization temperature of the gut with its contents was the highest, being about − 8 °C, showing that a freezing site is present in the gut. As food particles in the alimentary canal of hibernating larvae were excreted in autumn, the larval supercooling capacity increased with lowering crystallization temperature of the gut. In diapausing larvae the crystallization temperatures of the muscle and epidermis were the highest, being above − 15 °C, which is similar to that of the whole larvae, and the hemolymph crystallization temperature was the lowest, being below − 25 °C. Furthermore, the crystallization temperatures of the nervous system, trachea, silk gland, salivary gland, and ovary were below − 20 °C, which was equivalent to those of 0.9% NaCl solution. Consequently, in diapausing larvae a primary site of freezing is present in the muscle and epidermis, indicating that the potent ice-nucleating agents exist in these tissues. However, since the epidermis could not be completely divided from the muscle, it was not concluded whether the potent ice-nucleating agents existed in the epidermis or not.

Mapping resistance to cereal aphids in barley

Abstract A set of 150 doubled-haploid (DH) barley (Hordeum vulgare L.) lines derived from the cross of Harrington/TR306 was used to determine the number and chromosomal location of quantitative trait loci (QTLs) controlling resistance to cereal aphids. The experiments were conducted under natural infestation in the field during two growing seasons: 1994 and 1995. Aphid resistance was measured by counting the number of aphids per plot. Counts were made on a weekly basis. Each year at the time of maximum aphid density there was an obvious difference in reaction between the parental genotypes. The DH lines showed continuous variation for aphid density. Simple interval mapping and simplified composite interval mapping revealed that the principal QTL determining cereal aphid resistance is on the distal region of the short arm of chromosome 1. This aphid-resistance QTL is linked with a heading-date QTL. At the time of highest aphid infestation, this QTL accounted for 31% and 22% of the total variance of aphid density in 1994 and 1995, respectively. A QTL on chromosome 5 was also detected but only by simplified composite interval mapping. However, the largest consistent effect was due to the QTL on the short arm of chromosome 1. This QTL may be a useful target for marker-assisted selection for adult plant cereal aphid resistance in barley.

Comparison of cold hardiness and sugar content between diapausing and nondiapausing pupae of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract.  To understand overwintering of the cotton boll worm Helicoverpa armigera, cold hardiness and sugar content are compared between diapausing and nondiapausing pupae. Diapausing and nondiapausing pupae reared at 20 °C under short and long photoperiods are acclimatized with a reduction of 5 °C per 5 days to 0 °C. When the acclimation temperature reaches 0 °C, the survival of diapausing pupae is assessed. The survival gradually decreases as the period of treatment progresses and approximately half survive for 112 days. However, nondiapausing pupae survive only 14 days after exposure to 0 °C. The surpercooling points of nondiapausing, diapausing and acclimatized pupae are approximately −17 °C. The major sugars contained in pupae are trehalose and glucose. Even though trehalose contents in diapausing pupae (initial level: 0.6 mg 100 mg−1 fresh weight) increase significantly during cold acclimation and continue increasing until 58 days after exposure to 0 °C (maximum level: 1.8 mg 100 mg−1), glucose is maintained at low levels (0.02 mg 100 mg−1) for 56 days at 0 °C. However, glucose contents increase (maximum level: 0.8 mg 100 mg−1) with decreasing contents of trehalose 84 days after exposure to 0 °C. Glycogen content gradually decreases during cold acclimation. When nondiapausing pupae are acclimatized with a reduction of 5 °C per 5 days to 5 °C from the beginning of pupation until the eyespots move, trehalose content increases (maximum level: 1.0 mg 100 mg−1). Glucose contents in nondiapausing pupae increase before eclosion (0.09 mg 100 mg−1). From these results, diapausing pupae of H. armigera can overwinter in regions where average winter temperatures are higher than 0 °C, but nondiapausing pupae cannot.

Diapause development and acclimation regulating enzymes associated with glycerol synthesis in the Shonai ecotype of the rice stem borer larva, Chilo suppressalis walker.

Overwintering larvae of the Shonai ecotype of the rice stem borer, Chilo suppressalis, enter diapause in early September and terminate diapause at the end of October. Cold acclimation at 0 degrees C did not influence glycerol, trehalose or glycogen content in larvae collected on 22 September. Acclimation at 0 degrees C increased the glycerol content and reduced the glycogen content significantly in larvae collected on 2 October and 22 November compared with acclimation at 15 degrees C. These results indicate that overwintering larvae at different phases of diapause development respond differently to the low temperature stimulus for glycerol synthesis. Thus, we evaluated the metabolic rearrangements associated with glycerol synthesis during diapause development and after temperature acclimation. Larvae collected on 2 October were acclimated at 15 degrees C for 15 and 60 days. Some of those acclimated at 15 degrees C were then moved to 0 degrees C for 15 days. The larvae acclimated at 15 degrees C for 15 days were in deep diapause and accumulated little glycerol, while larvae acclimated at 15 degrees C for 60 days were nearly ready to emerge from diapause and accumulated glycerol at 155.5 &mgr;mol/g. When larvae acclimated to 15 degrees C for 15 days were transferred to 0 degrees C, glycerol accumulation was stimulated to the same extent (ca 140 &mgr;mol/g) as it was in larvae that were acclimated to 15 degrees C for 60 days and then transferred to 0 degrees C. These results indicate that low temperature has a cumulative effect on glycerol production in larvae at different phases of diapause development. Glycerol accumulation was accomplished by activation of glycogen phosphorylase and inhibition of fructose-1,6-bisphosphatase, and activation of enzymes associated with glycerol synthesis, mainly glyceraldehyde-3-phosphatase and polyol dehydrogenase with glyceraldehyde activity.

Changes in chemical components in the freshwater apple snail, Pomacea canaliculata (Gastropoda: Ampullariidae), in relation to the development of its cold hardiness.

The apple snail, Pomacea canaliculata, is an invasive freshwater snail. It increases its cold hardiness before winter. However, the physiological mechanism of cold hardiness in molluscs is poorly understood, especially in freshwater molluscs. In this study, we examined the changes in low molecular weight compounds, glycogen and lipids, in the body of P. canaliculata in association with the development of cold hardiness. When snails without cold hardiness were experimentally cold-acclimated, the amount of glycerol, glutamine, and carnosine increased, while glycogen and phenylalanine decreased. Overwintering cold-tolerant snails collected from a drained paddy field in November also showed increased glycerol in their bodies with decreasing glycogen concentration, compared to summer snails collected from a submerged field. Water content also decreased during the cold acclimation, although the water loss was minimal. These results indicate that the freshwater snail, P. canaliculata enhances cold hardiness by accumulation of some kinds of low molecular weight compounds in its body as some insects do. However, the actual function of each low molecular compound is still unknown.

Genomic organization of the para-sodium channel α-subunit genes from the pyrethroid-resistant and -susceptible strains of the diamondback moth

We examined the genomic organization of the para-sodium channel alpha-subunit gene of the diamondback moth, Plutella xylostella (L.). The nucleotide sequence contained 34 putative exons, which covered almost the entire coding region of the gene producing 1,889 amino acid residues. Deduced amino acid identity to the hscp locus of Heliothis virescens was 84%. Comparison of deduced amino acid sequences of the permethrin-resistant and -susceptible strains showed two substitutions other than kdr and super-kdr-like substitutions. They were Ala to Thr (A1060T) and Pro to Ser (P1836S) at the linker region of the domains II-III and the carboxyl terminus, respectively. Furthermore, we developed PCR amplification protocols for the rapid detection of both substitutions.

Alternatively spliced sodium channel transcripts expressed in field strains of the diamondback moth

The frequencies of the L1014F and T929I mutations, both of which are involved in nerve insensitive resistance to pyrethroids, were examined in field and laboratory strains of the diamondback moth, Plutella xylostella at DNA and RNA levels. Results showed that the resistance allele frequencies at the L1014F and T929I sites in the field strains were respectively, 82.8-100% and 72.9-94.4%. No posttranscriptional regulation of the L1014F mutation was observed. The examined insects were classifiable into four groups according to the expression patterns of mutually exclusive exons 18a and 18b. Most insects in the field strains expressed transcripts containing exon 18b more abundantly than those containing exon 18a, although both transcripts were expressed with similar proportions in all insects of the laboratory strains. Some other insects expressed a chimeric transcript comprising parts of exons 18a and 18b. Deduced amino acid sequences of the chimeric transcript encoded amino substitution from Met to Ile at the site corresponding to the super-kdr mutation (M918T) in Musca domestica. The frequencies of the M918I mutation in the field strains were 5.0-19.4%. Analyses of the genomic organization revealed that the chimeric sequences are encoded in the genome.

Pupal diapause of Helicoverpa armigera (Lepidoptera: Noctuidae): sensitive stage for thermal induction in the Okayama (western Japan) population

Helicoverpa armigera (Hübner) exhibits a facultative pupal diapause, which depends on temperature and photoperiod. Pupal diapause is induced at 20 degrees C by short photoperiods and inhibited by long photoperiods during the larval stage. However, in some pupae (35% of males and 57% of females) of a non-selected field population from Okayama Prefecture (34.6 degrees N), diapause is not induced by short photoperiods. In the present experiment, the importance of temperature for diapause induction was studied in the non-diapausing strain, which was selected from such individuals reared at 20 degrees C under a short photoperiod of 10L:14D. Furthermore, the sensitive stage for thermal determination of pupal diapause was determined by transferring larvae of various instars and pupae between 20 degrees C and 15 degrees C. Diapause was induced by 15 degrees C without respect to photoperiod. When larvae or pupae reared from eggs at 20 degrees C under a short or a long photoperiod were transferred to 15 degrees C in the periods of the middle fifth instar to the first three days after pupation, the diapause induction rate was significantly reduced in both males and females, especially in females. In contrast, when larvae or pupae reared at 15 degrees C were transferred to 20 degrees C in the same periods, diapause was induced in males, but not in females. However, the diapause induction rate of pupae transferred to 20 degrees C on the fourth day after pupation was significantly increased in females. The results show that temperature is the major diapause cue in the photoperiod-insensitive strain and the periods of middle fifth larval instar to early pupal stage are the thermal sensitive stages for pupal diapause induction with some different responses to temperatures between males and females in H. armigera.

Identification of tissues showing the lowest tolerance to freezing in larvae of the rice stem borer, Chilo suppressalis

Abstract.  Even though overwintering larvae of the rice stem borer, Chilo suppressalis, are freeze‐tolerant, they cannot survive below −30 °C. Furthermore, nondiapausing larvae cannot survive freezing. However, the cause of death due to freezing is unclear. To identify the cause of death by freezing in larvae, those tissues most injured by low temperatures are identified using the vital stain trypan blue. In overwintering larvae, the midgut of dead larvae stains blue, and remarkable colour density differences between dead and surviving larvae are observed in the midgut. In nondiapausing larvae incubated at −10 °C for several hours, the fat body of dead larvae is strongly stained. Furthermore, increases in mortality with treatment time correspond with increases in the area of the fat body stained. Sterile nondiapausing larvae with lower supercooling points, below −20 °C, do not freeze at −10 °C and survive the treatment. However, all the larvae die when subjected to inoculative freezing at −10 °C, and the fat body stains blue. These results suggest that the midgut in overwintering larvae and the fat body in nondiapausing larvae have the lowest tolerance to freezing.