Extremely high relative growth rate makes the cabbage white, Pieris rapae, a global pest with highly abundant and migratory nature.

The small cabbage white butterfly, Pieris rapae, is an extraordinarily abundant migratory pest of cabbage that causes severe damage worldwide without known reasons. I here show that the average relative growth rate (RGR: the ratio of the daily increase of biomass to total biomass) of herbivore (Gh; an indicator of the growth speed of herbivore) of P. rapae on cabbage during the larval period is larger by far than those of all other insect-plant pairs tested. It exceeds 1.15 (/day),-meaning that the biomass more than doubles each day-compared to 0.1-0.7 for most insect-plant pairs, including that of Pieris melete, a sibling of P. rapae which never becomes a pest of cabbage. My data further showed the RGR in the larval stage (larval Gh), positively correlates with abundance and/or migratoriness of insect herbivores. These results together with my mathematical food web model suggest that the extraordinarily high larval Gh of P. rapae is the primary reason for its ubiquitously severe pest status accompanied with its abundance and migratoriness, and that the RGR of herbivores, Gh, characterizing the plant-herbivore interface at the bottom of the food webs is an important factor affecting whole ecosystems, including animal abundance, fauna size, plant damage levels, competitiveness among herbivorous species, determination of hostplant, invasiveness, and the evolution of animal traits involved in the so-called r/K strategy, such as migratoriness. Knowledge about Gh will be crucial to controlling pests and improving the negative effects of human activity on ecosystems including faunal decline (or defaunation).

Neurexins in serotonergic neurons regulate neuronal survival, serotonin transmission, and complex mouse behaviors.

Extensive serotonin (5-hydroxytryptamine, 5-HT) innervation throughout the brain corroborates 5-HT's modulatory role in numerous cognitive activities. Volume transmission is the major mode for 5-HT transmission but mechanisms underlying 5-HT signaling are still largely unknown. Abnormal brain 5-HT levels and function have been implicated in autism spectrum disorder (ASD). Neurexin (Nrxn) genes encode presynaptic cell adhesion molecules important for the regulation of synaptic neurotransmitter release, notably glutamatergic and GABAergic transmission. Mutations in Nrxn genes are associated with neurodevelopmental disorders including ASD. However, the role of Nrxn genes in the 5-HT system is poorly understood. Here, we generated a mouse model with all three Nrxn genes disrupted specifically in 5-HT neurons to study how Nrxns affect 5-HT transmission. Loss of Nrxns in 5-HT neurons reduced the number of serotonin neurons in the early postnatal stage, impaired 5-HT release, and decreased 5-HT release sites and serotonin transporter expression. Furthermore, 5-HT neuron-specific Nrxn knockout reduced sociability and increased depressive-like behavior. Our results highlight functional roles for Nrxns in 5-HT neurotransmission, 5-HT neuron survival, and the execution of complex behaviors.

Glia transmit negative valence information during aversive learning in Drosophila.

During Drosophila aversive olfactory conditioning, aversive shock information needs to be transmitted to the mushroom bodies (MBs) to associate with odor information. We report that aversive information is transmitted by ensheathing glia (EG) that surround the MBs. Shock induces vesicular exocytosis of glutamate from EG. Blocking exocytosis impairs aversive learning, whereas activation of EG can replace aversive stimuli during conditioning. Glutamate released from EG binds to N-methyl-d-aspartate receptors in the MBs, but because of Mg2+ block, Ca2+ influx occurs only when flies are simultaneously exposed to an odor. Vesicular exocytosis from EG also induces shock-associated dopamine release, which plays a role in preventing formation of inappropriate associations. These results demonstrate that vesicular glutamate released from EG transmits negative valence information required for associative learning.

Intravenous transplantation of amnion-derived mesenchymal stem cells promotes functional recovery and alleviates intestinal dysfunction after spinal cord injury.

Spinal cord injury (SCI) is often accompanied by gastrointestinal dysfunction due to the disconnection of the spinal autonomic nervous system. Gastrointestinal dysfunction reportedly upregulates intestinal permeability, leading to bacterial translocation of the gut microbiome to the systemic circulation, which further activates systemic inflammation, exacerbating neuronal damage. Mesenchymal stem cells (MSC) reportedly ameliorate SCI. Here, we aimed to investigate their effect on the associated gastrointestinal dysfunction. Human amnion-derived MSC (AMSCs) were intravenously transplanted one day after a rat model of midthoracic SCI. Biodistribution of transplanted cells, behavioral assessment, and histological evaluations of the spinal cord and intestine were conducted to elucidate the therapeutic effect of AMSCs. Bacterial translocation of the gut microbiome was examined by in situ hybridization and bacterial culture of the liver. Systemic inflammations were examined by blood cytokines, infiltrating immune cells in the spinal cord, and the size of the peripheral immune tissue. AMSCs released various neurotrophic factors and were mainly distributed in the liver and lung after transplantation. AMSC-transplanted animals showed smaller spinal damage and better neurological recovery with preserved neuronal tract. AMSCs transplantation ameliorated intestinal dysfunction both morphologically and functionally, which prevented translocation of the gut microbiome to the systemic circulation. Systemic inflammations were decreased in animals receiving AMSCs in the chronic phase. Intravenous AMSC administration during the acute phase of SCI rescues both spinal damage and intestinal dysfunction. Reducing bacterial translocation may contribute to decreasing systemic inflammation.

Retrieval of conditioned fear activates the basolateral and intercalated nucleus of amygdala.

The amygdala is one of the crucial brain structures for conditioned fear, in which conditioned stimuli are received by the basolateral nucleus of the amygdala (BLA), inducing a fear reaction via the central nucleus of the amygdala (CeA). Whereas BLA sends glutamatergic projections into CeA, the intercalated nucleus of the amygdala (ITC) sends GABAergic projections into CeA, which is doubly regulated by BLA and ITC. In the present study, we investigated the characteristics of the neural cells activated by retrieval of conditioned fear in BLA and ITC using immunohistochemistry, in situ hybridization, and Western blot analysis of transcription factors and neural cell markers. Because most conditioned fear-induced c-Fos-positive cells in BLA were glutaminase positive and 67-kDa isomer of glutamic acid decarboxylase (GAD67) negative, these cells are speculated to be glutamatergic. Seventy-eight percent of the phosphorylated CREB (pCREB)-positive cells were glutaminase double positive and 13% of the pCREB-positive cells were GAD67 double positive, indicating that many of the conditioned fear-induced pCREB-positive cells in BLA were glutamatergic, but at least some of the pCREB-positive cells were GABAergic. These results suggested that CREB phosphorylation was increased both in glutamatergic and in GABAergic neurons, but c-Fos expression was increased mainly in glutamatergic neurons in BLA. CREB phosphorylation but not c-Fos expression in ITC was specifically increased by retrieval of conditioned fear. It is therefore speculated that ITC GABAergic neurons were activated by retrieval of conditioned fear and that transcription factors other than c-Fos were relevant to the activation.

Expression of a gene for an MLX56 defense protein derived from mulberry latex confers strong resistance against a broad range of insect pests on transgenic tomato lines.

Insect pests cause serious damage in crop production, and various attempts have been made to produce insect-resistant crops, including the expression of genes for proteins with anti-herbivory activity, such as Bt (Bacillus thuringiensis) toxins. However, the number of available genes with sufficient anti-herbivory activity is limited. MLX56 is an anti-herbivory protein isolated from the latex of mulberry plants, and has been shown to have strong growth-suppressing activity against the larvae of a variety of lepidopteran species. As a model of herbivore-resistant plants, we produced transgenic tomato lines expressing the gene for MLX56. The transgenic tomato lines showed strong anti-herbivory activities against the larvae of the common cutworm, Spodoptera litura. Surprisingly, the transgenic tomato lines also exhibited strong activity against the attack of western flower thrips, Frankliniera occidentalis. Further, growth of the hadda beetle, Henosepilachna vigintioctopunctata, fed on leaves of transgenic tomato was significantly retarded. The levels of damage caused by both western flower thrips and hadda beetles were negligible in the high-MLX56-expressing tomato line. These results indicate that introduction of the gene for MLX56 into crops can enhance crop resistance against a wide range of pest insects, and that MLX56 can be utilized in developing genetically modified (GM) pest-resistant crops.

Excitatory and inhibitory receptors utilize distinct post- and trans-synaptic mechanisms in vivo.

Ionotropic neurotransmitter receptors at postsynapses mediate fast synaptic transmission upon binding of the neurotransmitter. Post- and trans-synaptic mechanisms through cytosolic, membrane, and secreted proteins have been proposed to localize neurotransmitter receptors at postsynapses. However, it remains unknown which mechanism is crucial to maintain neurotransmitter receptors at postsynapses. In this study, we ablated excitatory or inhibitory neurons in adult mouse brains in a cell-autonomous manner. Unexpectedly, we found that excitatory AMPA receptors remain at the postsynaptic density upon ablation of excitatory presynaptic terminals. In contrast, inhibitory GABAA receptors required inhibitory presynaptic terminals for their postsynaptic localization. Consistent with this finding, ectopic expression at excitatory presynapses of neurexin-3 alpha, a putative trans-synaptic interactor with the native GABAA receptor complex, could recruit GABAA receptors to contacted postsynaptic sites. These results establish distinct mechanisms for the maintenance of excitatory and inhibitory postsynaptic receptors in the mature mammalian brain.

Mesenchymal Stem Cell Sheet Promotes Functional Recovery and Palliates Neuropathic Pain in a Subacute Spinal Cord Injury Model.

Stem cell therapy has been shown to reverse the sequelae of spinal cord injury (SCI). Although the ideal treatment route remains unknown, providing a large number of stem cells to the injured site using less invasive techniques is critical to achieving maximal recovery. This study was conducted to determine whether administration of bone marrow stem cell (BMSC) sheet made on its own without a scaffold is superior to intramedullary cell transplantation in a rat subacute SCI model. Adult female Sprague-Dawley rats were subjected to SCI by 30 g clip compression at the level of Th6 and Th7 and were administered BMSC cell sheet (7 × 104 cells, subdural), cell suspension (7 × 104 cells, intramedullary), or control seven days after the injury. Motor and sensory assessments, as well as histological evaluation, were performed to determine the efficacy of the different cell transplantation procedures. While both the cell sheet and cell intramedullary injection groups showed significant motor recovery compared to the control group, the cell sheet group showed better results. Furthermore, the cell sheet group displayed a significant sensory recovery compared to the other groups. A histological evaluation revealed that the cell sheet group showed smaller injury lesion volume, less inflammation, and gliosis compared to other groups. Sensory-related fibers of µ-opioid receptors (MOR, interneuron) and hydroxytryptamine transporters (HTT, descending pain inhibitory pathway), located around the dorsal horn of the spinal cord at the caudal side of the SCI, were preserved only in the cell sheet group. Stem cells could also be found inside the peri-injured spinal cord in the cell sheet group. BMSC cell sheets were able to promote functional recovery and palliate neuropathic pain more effectively than intramedullary injections, thus serving as a good treatment option for SCI.

GABA, ß-alanine and glycine in the digestive juice of privet-specialist insects: convergent adaptive traits against plant iridoids.

The privet tree, Ligustrum obtusifolium (Oleaceae), defends its leaves against insects with a strong lysine-decreasing activity that make proteins non-nutritive. This is caused by oleuropein, an iridoid glycoside. We previously found that some privet-specialist caterpillars adapt by secreting glycine in the digestive juice as a neutralizer that prevents the loss of lysine. Here, we extended the survey into 42 lepidopteran and hymenopteran species. The average concentration of glycine in digestive juice for 11 privet-feeding species (40.396 mM) was higher than that for 32 non-privet-feeding species (2.198 mM). The glycine concentrations exceeded 10 mM in 7 out of 11 privet-feeding species. In Macrophya timida (Hymenoptera), it reached 164.8 mM. Three out of the four remaining privet-feeding species had other amino acids instead. Larvae of a privet-specialist butterfly, Artopoetes pryeri (Lycaenidae), had a high concentration (60.812 mM) of GABA. In two other specialists, ß-alanine was found. GABA, ß-alanine, and glycine as well as alanine, amines, and ammonium ion inhibited the lysine decrease, indicating that amino residues are responsible for the inhibition. However, the three amino acids found in the specialists were far more effective (20 mM showed 80% inhibition) than the rest (>140 mM was required for 80% inhibition). Our results show a clear and rare case of the apparent convergent evolution of herbivores' molecular adaptations of feeding on a plant with a chemical defense in a manner that minimizes the cost of adaptation. The novel role of GABA in plant-herbivore interactions shown here is probably the first reported non-neuronal role of animal-derived GABA.

FTY720 Attenuates Neuropathic Pain after Spinal Cord Injury by Decreasing Systemic and Local Inflammation in a Rat Spinal Cord Compression Model.

Neuropathic pain severely impairs rehabilitation and quality of life after spinal cord injury (SCI). The sphingosine-1-phosphate receptor agonist, FTY720, plays an important protective role in neuronal injury. This study aims to examine the effects of FTY720 in a rat acute SCI model, focusing on neuropathic pain. Female rats with SCI induced by 1-min clip compression were administered vehicle or 1.5 mg/kg of FTY720 24 h after the injury. Using the mechanical nociceptive threshold test, we monitored neuropathic pain and performed histological analysis of the pain pathway, including the µ opioid receptor (MOR), hydroxytryptamine transporter (HTT), and calcitonin gene-related peptide (CGRP). Motor score, SCI lesion volume, residual motor axons, inflammatory response, glial scar, and microvascular endothelial dysfunction were also compared between the two groups. FTY720 treatment resulted in significant attenuation of post-traumatic neuropathic pain. It also decreased systemic and local inflammation, thereby reducing the damaged areas and astrogliosis and resulting in motor functional recovery. Whereas there was no difference in the CGRP expression between the two groups, FTY720 significantly preserved the MOR in both the caudal and rostral areas of the spinal dorsal horn. Whereas HTT was preserved in the FTY720 group, it was significantly increased in the rostral side and decreased in the caudal side of the injury in the vehicle group. These results suggest that FTY720 ameliorates post-traumatic allodynia through regulation of neuroinflammation, maintenance of the blood-brain barrier, and inhibition of glial scar formation, thereby preserving the connectivity of the descending inhibitory pathway and reducing neuropathic pain.

The peritrophic membrane as a target of proteins that play important roles in plant defense and microbial attack.

The peritrophic membrane (or peritrophic matrix: PM) is a thin membranous structure that lies along the midgut epithelium in the midgut lumen and consists of chitin and proteins. PM exists between ingested food material and midgut epithelium cells and it is on the frontline of insect-plant and insect-microbe interactions. Therefore, proteins that play major roles in plant defense against herbivorous insects and in microbial attack on insects should penetrate, destroy or modify the PM to accomplish their roles. Recently, it has become clear that some proteins crucial to plant defense or microbial attack have the PM as their primary target. In addition, several plant defense proteins have been reported to affect the PM, although it is still unclear whether the PM is their primary target. This review introduces several of these proteins: fusolin and enhancin, two proteins produced by insect viruses that greatly enhance infection of the viruses by disrupting the PM; the MLX56 family proteins found in mulberry latex as defense proteins against insect herbivores, which modify the PM to a thick structure that inhibits digestive processes; Mir1-CP, a defense cysteine protease from maize that inhibits the growth of insects at very low concentrations and degrades the PM structures; and chitinases and lectins. The importance, necessary characteristics, and modes of action of PM-targeting proteins are then discussed from a strategic point of view, by spotlighting the importance of selective permeability of the PM. Finally, the review discusses the possibility of applying PM-targeting proteins for the control of pest insects.

Effects of ozone and ammonium sulfate on cauliflower: Emphasis on the interaction between plants and insect herbivores.

Ammonium sulfate [(NH4)2SO4] deposition and elevated ozone (O3) concentrations may negatively affect plants and trophic interactions. This study aimed to evaluate for the first time the interactive effects of high (NH4)2SO4 load and elevated O3 levels on cauliflower (Brassica oleracea L.) under field conditions. Cauliflower seedlings were treated with 0 (AS0) or 50 (AS50) kg ha-1 (NH4)2SO4 and exposed to ambient (AOZ, ≈20 ppb) or elevated (EOZ, ≈55 ppb) O3 for about one month, in a Free Air O3 Concentration Enrichment (FACE) system. The oligophagous diamondback moth (Plutella xylostella Linnaeus, 1758) showed a clear preference towards the seedlings treated with AS50, which intensively grazed. Plant-herbivore interactions were driven by (NH4)2SO4 availability, rather than O3, via increased nitrogen content in the leaves. Further laboratory bioassays were followed to confirm the validity of these observations using polyphagous Eri silkmoth larvae (Samia ricini) as a biological model in a standardized experimental setup. Choice assays, where larvae could select leaves among leaf samples from the different experimental conditions, and no-choice assays, where larvae could graze leaves from just one experimental condition, were conducted. In the choice assay, the larvae preferred AS50-treated leaves, in agreement with the field observations with diamondback moth. In the no-choice assay, larval body mass growth was inhibited when fed with leaves treated with EOZ and/or AS50. Larvae fed with AS50-treated leaves displayed increased mortality. These observations coincide with higher NO3 and Zn content in AS50-treated leaves. This study shows that plant-herbivore interactions can be driven by (NH4)2SO4 availability, independently of O3, and suggests that high N deposition may have severe health implications in animals consuming such plant tissues. Key message: Plant-herbivore interactions are driven by high (NH4)2SO4 availability, independently of O3.

Regioselective formation of quercetin 5-O-glucoside from orally administered quercetin in the silkworm, Bombyx mori.

The cocoons of some races of the silkworm, Bombyx mori, have been shown to contain 5-O-glucosylated flavonoids, which do not occur naturally in the leaves of their host plant, mulberry (Morus alba). Thus, dietary flavonoids could be biotransformed in this insect. In this study, we found that after feeding silkworms a diet rich in the flavonol quercetin, quercetin 5-O-glucoside was the predominant metabolite in the midgut tissue, while quercetin 5,4'-di-O-glucoside was the major constituent in the hemolymph and silk glands. UDP-glucosyltransferase (UGT) in the midgut could transfer glucose to each of the hydroxyl groups of quercetin, with a preference for formation of 5-O-glucoside, while quercetin 5,4'-di-O-glucoside was predominantly produced if the enzyme extracts of either the fat body or silk glands were incubated with quercetin 5-O-glucoside and UDP-glucose. These results suggest that dietary quercetin was glucosylated at the 5-O position in the midgut as the first-pass metabolite of quercetin after oral absorption, then glucosylated at the 4'-O position in the fat body or silk glands. The 5-O-glucosylated flavonoids retained biological activity in the insect, since the total free radical scavenging capacity of several tissues increased after oral administration of quercetin.

Abnormal swelling of the peritrophic membrane in Eri silkworm gut caused by MLX56 family defense proteins with chitin-binding and extensin domains.

MLX56 family defense proteins, MLX56 and its close homolog LA-b, are chitin-binding defense proteins found in mulberry latex that show strong growth-inhibitions against caterpillars when fed at concentrations as low as 0.01%. MLX56 family proteins contain a unique structure with an extensin domain surrounded by two hevein-like chitin-binding domains, but their defensive modes of action remain unclear. Here, we analyzed the effects of MLX56 family proteins on the peritrophic membrane (PM), a thin and soft membrane consisting of chitin that lines the midgut lumen of insects. We observed an abnormally thick (>1/5 the diameter of midgut) hard gel-like membrane consisted of chitin and MLX56 family proteins, MLX56 and LA-b, in the midgut of the Eri silkworms, Samia ricini, fed a diet containing MLX56 family proteins, MLX56 and LA-b. When polyoxin AL, a chitin-synthesis-inhibitor, was added to the diet containing MLX56 family proteins, the toxicity of MLX56 family proteins disappeared and PM became thinner and fragmented. These results suggest that MLX56 family proteins, through their chitin-binding domains, bind to the chitin framework of PM, then through their extensin-domain (gum arabic-like structure), which functions as swelling agent, expands PM into an abnormally thick membrane that inhibits the growth of insects. This study shows that MLX56 family proteins are plant defense lectins with a totally unique mode of action, and reveals the functions of extensin domains and arabinogalactan proteins as swelling (gel-forming) agents of plants.

Differential effects of sugar-mimic alkaloids in mulberry latex on sugar metabolism and disaccharidases of Eri and domesticated silkworms: enzymatic adaptation of Bombyx mori to mulberry defense.

Mulberry leaves (Morus spp.) exude latex rich in sugar-mimic alkaloids, 1,4-dideoxy-1,4-imino-d-arabinitol (d-AB1) and 1-deoxynojirimycin (DNJ), as a defense against herbivorous insects. Sugar-mimic alkaloids are inhibitors of sugar-metabolizing enzymes, and are toxic to the Eri silkworm, Samia ricini, a generalist herbivore, but not at all to the domesticated silkworm, Bombyx mori, a mulberry specialist. To address the phenomena, we fed both larvae diets containing different sugar sources (sucrose, glucose or none) with or without sugar-mimic alkaloids from mulberry latex. In S. ricini, addition of sugar-mimic alkaloids to the sucrose (the major sugar in mulberry leaves) diet reduced both growth and the absorption ratio of sugar, but it reduced neither in B. mori. The midgut soluble sucrase activity of S. ricini was low and inhibited by very low concentrations of sugar-mimic alkaloids (IC(50)=0.9-8.2microM), but that of B. mori was high and not inhibited even by very high concentrations (IC(50)>1000microM) of sugar-mimic alkaloids. In S. ricini, the addition of sugar-mimic alkaloids to the glucose diet still had considerable negative effects on growth, although it did not reduce the absorption ratio of glucose. The hemolymph of S. ricini fed sugar-mimic alkaloids contained sugar-mimic alkaloids. The trehalose concentration in the hemolymph increased significantly in S. ricini fed sugar-mimic alkaloids, but not in B. mori. The trehalase activities of S. ricini were lower and inhibited by lower concentrations of sugar-mimic alkaloids than those of B. mori. These results suggest that sugar-mimic alkaloids in mulberry latex exert toxicity to S. ricini larvae first by inhibiting midgut sucrase and digestion of sucrose, and secondly, after being absorbed into hemolymph, by inhibiting trehalase and utilization of trehalose, the major blood sugar. Further, our results reveal that B. mori larvae evolved enzymatic adaptation to mulberry defense by developing sucrase and trehalase that are insensitive to sugar-mimic alkaloids.

Late postnatal shifts of parvalbumin and nitric oxide synthase expression within the GABAergic and glutamatergic phenotypes of inferior colliculus neurons.

The inferior colliculus (IC) is partitioned into three subdivisions: the dorsal and lateral cortices (DC and LC) and the central nucleus (ICC), and serves as an integration center of auditory information. Recent studies indicate that a certain population of IC neurons may represent the non-GABAergic phenotype, while they express well-established cortical/hippocampal GABAergic neuron markers. In this study we used the optical disector to investigate the phenotype of IC neurons expressing parvalbumin (PV) and/or nitric oxide synthase (NOS) in C57BL/6J mice during the late postnatal period. Four major types of IC neurons were defined by the presence (+) or absence (-) of PV, NOS, and glutamic acid decarboxylase 67 (GAD67): PV+ /NOS- /GAD67+ , PV+ /NOS+ /GAD67+ , PV+ /NOS- /GAD67- , and PV- /NOS+ /GAD67- . Fluorescent in situ hybridization for vesicular glutamate transporter 2 mRNA indicated that almost all GAD67- IC neurons represented the glutamatergic phenotype. The numerical densities (NDs) of total GAD67+ IC neurons remained unchanged in all subdivisions. The NDs of PV+ /NOS- /GAD67+ neurons and PV- /NOS+ /GAD67- neurons were reduced with age in the ICC, while they remained unchanged in the DC and LC. By contrast, the NDs of PV+ /NOS+ /GAD67+ neurons and PV+ /NOS- /GAD67- neurons were increased with age in the ICC, although there were no changes in the DC and LC. The cell body size of GAD67+ IC neurons did not vary according to the expression of PV with or without NOS. The present findings indicate that the expression of PV and NOS may shift with age within the GABAergic and glutamatergic phenotypes of IC neurons during the late postnatal period. J. Comp. Neurol. 525:868-884, 2017. © 2016 Wiley Periodicals, Inc.

Involvement of hormones in olfactory imprinting and homing in chum salmon.

The olfactory hypothesis for salmon imprinting and homing to their natal stream is well known, but the endocrine hormonal control mechanisms of olfactory memory formation in juveniles and retrieval in adults remain unclear. In brains of hatchery-reared underyearling juvenile chum salmon (Oncorhynchus keta), thyrotropin-releasing hormone gene expression increased immediately after release from a hatchery into the natal stream, and the expression of the essential NR1 subunit of the N-methyl-D-aspartate receptor increased during downstream migration. Gene expression of salmon gonadotropin-releasing hormone (sGnRH) and NR1 increased in the adult chum salmon brain during homing from the Bering Sea to the natal hatchery. Thyroid hormone treatment in juveniles enhanced NR1 gene activation, and GnRHa treatment in adults improved stream odour discrimination. Olfactory memory formation during juvenile downstream migration and retrieval during adult homing migration of chum salmon might be controlled by endocrine hormones and could be clarified using NR1 as a molecular marker.

Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps.

The activity and composition of leafhopper saliva are important in interactions with the host rice plant, and it may play a physiological role in detoxifying toxic plant substances or ingesting sap. We have characterized diphenoloxidase in the salivary glands of Nephotettix cincticeps, its activity as a laccase, and its presence in the watery saliva with the objective of understanding its function in feeding on rice plants. Nonreducing SDS-PAGE of salivary gland homogenates with staining by the typical laccase substrate 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydroquinone or syringaldazine revealed a band at a molecular mass of approximately 85 kDa at pH 5. A band also appeared at a molecular mass of approximately 200 kDa when the gels were treated with dopamine, L-3,4-dihydroxyphenylalanine (DOPA) or catechol at pH 7. The ABTS-oxidizing activity of the homogenates was drastically inhibited by N-hydroxyglycine, a specific inhibitor of laccase. However, the dopamine-oxidizing activity was not inhibited by N-hydroxyglycine, while it was inhibited by phenylthiourea (PTU). Thus, the salivary glands of N. cincticeps contain two types of phenoloxidases: a laccase (85 kDa) and a phenoloxidase (200 kDa). Laccase activity was detected in a holidic sucrose diet that was fed on for 16 h by two females, but only a trace of catechol oxidase activity was observed, suggesting that the laccase-type phenoloxidase was the predominant phenoloxidase secreted in watery saliva. The laccase exhibited an optimum pH of 4.75-5 in McIlvaine buffer and had a PI of 4.8. Enzyme activity was histochemically localized in V cells of the posterior lobe of the salivary glands. It remained at the same level throughout the adult stage from 2 days after eclosion. A possible function of N. cincticeps salivary laccase may be rapid oxidization of potentially toxic monolignols to nontoxic polymers during feeding on the rice plant. This is the first report proving that laccase occurs in the salivary glands of Hemiptera species and is secreted in the watery saliva.

Synergistic defensive function of raphides and protease through the needle effect.

Raphides, needle-shaped calcium oxalate crystals in tissues of many plants, have been thought to play defensive roles against herbivores without detailed bioassays for their defensive roles and modes of function using purified raphides. In order to examine the defensive roles and modes of function of raphides in a clear experimental system, we performed bioassays giving the larvae of the Eri silkmoth, Samia ricini (Saturniidae), leaves of their host plant, the castor oil plant, Ricinus communis (Euphorbiaceae), painted with the raphides purified from kiwifruits, Actinidia deliciosa (Actinidiaceae), in presence or absence of cysteine protease, which often coincide with raphides in plant tissues. Raphides alone or cysteine protease alone showed only weak defensive activities around experimental concentrations. However, when raphides and cysteine protease coexisted, they synergistically showed very strong growth-reducing activities, and the mortality of caterpillars was very high. In contrast, amorphous calcium oxalate did not show synergism with cysteine protease on defensive activities, indicating that the needle-shape of raphides is essential for the synergism. The present study provides the first clear experimental evidence for the synergism between raphides and other defensive factors. Further, the study suggests that "the needle effect", which intensify the bioactivities of other bioactive factors by making holes to the barriers (cell membrane, cuticle, epithelium, the nuclear membrane, etc.) and facilitate the bioactive factors to go through them and reach the targets, is important in the defensive activities of raphides, and possibly in the allergy caused by raphides, and in the carcinogenic activities of other needle-shaped components including asbestos and plant derived silica needles.

Purification, cDNA cloning and recombinant protein expression of a phloem lectin-like anti-insect defense protein BPLP from the phloem exudate of the wax gourd, Benincasa hispida.

Latex and other exudates in plants contain various proteins that are thought to play important defensive roles against herbivorous insects and pathogens. Herein, the defensive effects of phloem exudates against the Eri silkworm, Samia ricini (Saturniidae, Lepidoptera) in several cucurbitaceous plants were investigated. It was found that phloem exudates are responsible for the defensive activities of cucurbitaceous plants, such as the wax gourd Benincasa hispida and Cucumis melo, especially in B. hispida, whose leaves showed the strongest growth-inhibitory activity of all the cucurbitaceous plants tested. A 35 kDa proteinaceous growth-inhibitory factor against insects designated BPLP (B. hispida Phloem Lectin-like Protein) was next isolated and purified from the B. hispida exudate, using anion exchange and gel filtration chromatography. A very low concentration (70 µg/g) of BPLP significantly inhibited growth of S. ricini larvae. The full-length cDNA (1076 bp) encoding BPLP was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of BPLP had 51% identity with a cucurbitaceous phloem lectin (phloem protein 2, PP2), and showed binding specificity to oligomers of N-acetylglucosamine. Some features of BPLP indicated that it does not have a cysteine residue and it is composed of two repeats of similar sequences, suggesting that BPLP is distinct from PP2. Recombinant BPLP, obtained by expressing the cDNA in Escherichia coli, showed both chitin-binding lectin activity and growth-inhibitory activity against S. ricini larvae. The present study thus provides experimental evidence that phloem exudates of Cucurbitaceae plants, analogous to plant latex, play defensive roles against insect herbivores, especially against chewing insects, and contain defensive substances toxic to them.