Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

Electrophysiological and Morphological Characterization of Contact Chemosensilla in Adults and Larvae of the Butterfly, Atrophaneura alcinous.

Distribution and electrophysiological responses of contact chemosensilla were examined in the Aristolochiaceae-feeding butterfly Atrophaneuraalcinous. In adult butterflies, tarsal contact chemosensilla of the foreleg were classified into two groups based on length: long- and short-type sensilla. Long-type sensilla were distributed much more widely in females than in males, whereas short-type sensilla were found at the edge of the tarsi in a similar manner in both sexes. Taste responses of the long- and short-type sensilla to methanol extracts of Aristolochia debilis and Citrus spp. were recorded. Aristolochia debilis extracts evoked spikes with different amplitudes, whereas Citrus spp. extracts evoked spikes with a single amplitude in the long-type sensilla. Short-type sensilla did not respond to either extract. Moreover, we recorded responses to different concentrations of sucrose and NaCl. Results suggest that adult butterflies can discriminate the taste of host plant components from other chemicals using long-type sensilla during oviposition and may recognize diets containing sugar and salts during feeding using short-type sensilla. In the larval mouthparts, there were lateral and medial styloconic sensilla on the maxillary galea and epipharyngeal sensillum on the epipharynx. Electrophysiological responses of these sensilla suggest that larvae can discriminate between host plant compounds.

Long horns protect Hestina japonica butterfly larvae from their natural enemies.

Animals sometimes have prominent projections on or near their heads serving diverse functions such as male combat, mate attraction, digging, capturing prey, sensing or defence against predators. Some butterfly larvae possess a pair of long frontal projections; however, the function of those projections is not well known. Hestina japonica butterfly larvae have a pair of long hard projections on their heads (i.e., horns). Here we hypothesized that they use these horns to protect themselves from natural enemies (i.e., predators and parasitoids). Field surveys revealed that the primary natural enemies of H. japonica larvae were Polistes wasps. Cage experiments revealed that larvae with horns intact and larvae with horns removed and fitted with horns of other individuals succeeded in defending themselves against attacks of Polistes wasps significantly more often than larvae with horns removed. We discuss that the horns counter the paper wasps' hunting strategy of first biting the larvae's 'necks' and note that horns evolved repeatedly only within the Nymphalidae in a phylogeny of the Lepidoptera. This is the first demonstration that arthropods use head projections for physical defence against predators.

Long Frontal Projections Help Battus philenor (Lepidoptera: Papilionidae) Larvae Find Host Plants.

Animals sometimes develop conspicuous projections on or near their heads as, e.g., weaponry, burrowing or digging tools, and probes to search for resources. The frontal projections that insects generally use to locate and assess resources are segmented appendages, including antennae, maxillary palps, and labial palps. There is no evidence to date that arthropods, including insects, use projections other than true segmental appendages to locate food. In this regard, it is noteworthy that some butterfly larvae possess a pair of long antenna-like projections on or near their heads. To date, the function of these projections has not been established. Larvae of pipevine swallowtail butterflies Battus philenor (Papilionidae) have a pair of long frontal fleshy projections that, like insect antennae generally, can be actively moved. In this study, we evaluated the possible function of this pair of long moveable frontal projections. In laboratory assays, both frontal projections and lateral ocelli were shown to increase the frequency with which search larvae found plants. The frontal projections increased finding of host and non-host plants equally, suggesting that frontal projections do not detect host-specific chemical cues. Detailed SEM study showed that putative mechanosensillae are distributed all around the frontal as well as other projections. Taken together, our findings suggest that the frontal projections and associated mechanosensillae act as vertical object detectors to obtain tactile information that, together with visual information from lateral ocelli and presumably chemical information from antennae and mouthparts, help larvae to find host plants. Field observations indicate that host plants are small and scattered in southern Arizona locations. Larvae must therefore find multiple host plants to complete development and face significant challenges in doing so. The frontal projections may thus be an adaptation for finding a scarce resource before starving to death. This is the first evidence that arthropods use projections other than true segmental appendages such as antennae, mouthparts and legs, to locate food resources.

Molecular cloning of a novel calcium-binding protein in the secreted saliva of the green rice leafhopper Nephotettix cincticeps.

Green rice leafhoppers (Nephotettix cincticeps) secrete watery and coagulable saliva in the feeding process. In our study, the watery salivary secretion was concentrated by ultrafiltration from "fed diet" and subjected to SDS-PAGE. The N-terminal amino acid sequence of the most predominant band at 84 kDa (designated NcSP84) was analyzed by Edman degradation. This sequence was completely consistent with the most abundant protein in the salivary gland extracts, which was separated by two-dimensional gel electrophoresis. Based on the N-terminal amino acid sequence, the complete cDNA of this protein was cloned by 5'- and 3'-RACE using degenerate primers. The deduced NcSP84 contained an open reading frame of 2061 bp encoding a putative 687 amino acids with a putative signal sequence composed of 19 amino acids. The nucleotide and amino acid sequences of NcSP84 did not share statistically significant homology with any sequences in public databases. Motif search predicted that this protein had EF-hands, the most common motif found in Ca(2+) -binding proteins. As predicted, NcSP84 exhibited Ca(2+)-binding activity. The SDS-PAGE mobility of purified NcSP84 bound to Ca(2+) tended to decline discretely, depending on the concentration of CaCl(2) with which it was mixed for 1h before adding SDS buffer. In situ hybridization and immunohistochemistry showed that the NcSP84 gene and gene product were expressed and stored in type III cells, which are the largest lobes in the primary salivary glands. The NcSP84 protein was detected in the phloem sap of rice exposed to leafhoppers, verifying that the NcSP84 protein was injected into the sieve tubes. These results suggest that NcSP84 could be secreted into the sieve tubes during feeding, which might bind Ca(2+) ions that flow into sieve tubes in response to stylet puncturing. This might suppress sieve-element clogging and facilitate continuous ingestion from sieve tubes.

Ligand carrier protein genes expressed in larval chemosensory organs of Bombyx mori.

Expressed sequence tags (ESTs) of the maxillary galea of the silkworm were analyzed to identify proteins involved in food selection systems. From the 1251 redundant genes of the ESTs, we identified 7 odorant-binding protein-like genes (bmObpL), 6 takeout-like genes (bmToL), and 6 chemosensory protein genes (bmCsp). Quantitative RT-PCR analysis indicated that bmObpL1, bmObpL2, bmObpL3, bmObpL5, bmToL1, bmToL3, and bmorCsp15 were predominantly expressed in the larval oral appendages, such as the maxilla, labrum, labium and antenna. Immunocytochemical analysis indicated that the proteins of bmObpL1, bmObpL3, and bmToL1 were localized in the gustatory chemosensilla on the maxillary galea and olfactory sensilla in the antenna. The proteins encoded by bmObpL1 and bmObpL3 were detected in the gustatory chemosensilla of the epipharynx. However, bmObpL1 and bmToL1 were also detected in tactile hairs and in the epidermis of several chemosensory organs. The bmObpL2 protein was localized inside and in the epidermis around the chemosensilla, tactile hairs, and wide surface of the epipharynx. From these results, bmObpL3 is the most likely to have a dedicated role in chemoreception in the silkworm, Bombyx mori.

Molecular characterization and expression of laccase genes in the salivary glands of the green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae).

The green rice leafhopper, Nephotettix cincticeps has a laccase (EC 1.10.3.2) in its salivary glands and saliva, possibly playing an important role in detoxifying plant phenolics and in salivary sheath coagulation during feeding. We aimed to clarify the function of saliva-specific laccase in a vascular-feeding insect, N. cincticeps, for which we cloned 2 cDNAs (NcLac1S and NcLac1G) from the salivary glands and 1 cDNA (NcLac2) from the epidermis. The NcLac1S, NcLac1G, and NcLac2 transcripts encoded 701-, 792-, and 729-amino-acid proteins, respectively. The putative proteins encoded by NcLac1S and NcLac2 were predicted to be soluble, whereas that encoded by NcLac1G was hydrophobic and predicted to have a C-terminal transmembrane domain. Real-time reverse transcriptase polymerase chain reaction analysis revealed that NcLac1S was expressed exclusively, and at a much higher level than NcLac1G and NcLac2 in the salivary glands. NcLac1G was also expressed in the epidermis, midgut, and Malpighian tubules. NcLac2 expression was highest in the epidermis. In situ hybridization revealed NcLac1S expression in the V-cells of the salivary glands, having proven laccase activity. Expression of NcLac1G and NcLac2 were not detected clearly in all cells in the salivary glands. Therefore, NcLac1S is responsible for the laccase activity detected in the salivary glands and saliva of this insect. This is the first report on gene cloning of salivary laccase from a vascular-feeding insect.

An oviposition stimulant binding protein in a butterfly: Immunohistochemical localization and electrophysiological responses to plant compounds.

Oviposition is evoked by plant compounds, which are recognized by chemoreceptive organs of insects. The swallowtail butterfly, Atrophaneura alcinous, oviposits its eggs on the host plant, Aristolochia debilis, in the presence of only two stimulating compounds: an alkaloid, aristolochic acid, and a monosaccharide, sequoyitol. In our previous study, a unique protein of 23 kDa [Oviposition stimulant(s) binding protein (OSBP)] was found in the forelegs of female, but not male A. alcinous. The electrophysiological response of A. alcinous to an extract of A. debilis was depressed by the presence of OSBP antiserum, suggesting that OSBP presumably binds to oviposition stimulant(s). We show here, using a highly sensitive fluorescence micro-binding assay that native OSBP binds to a main oviposition stimulant, aristolochic acid, from its host plant, A. debilis. Three-dimensional molecular modeling studies also gave a reasonable structure for the OSBP/aristolochic acid complex. This is the first report of a native chemoreceptive protein binding to an oviposition stimulant ligand in insects.

Comparative pharmacology of two D1-like dopamine receptors cloned from the silkworm Bombyx mori.

Dopamine (DA) is a physiologically important biogenic amine in insect peripheral and nervous tissues.We recently cloned two DA receptors (BmDopR1 and BmDopR2) from the silkworm Bombyx mori and identified them as D1-like receptors, which activate adenylate cyclase to increase intracellular cAMP levels. In this study, these two receptors were stably expressed in HEK-293 cells, and the dose-responsiveness to DA and their pharmacological properties were examined using cAMP assays. BmDopR1 showed a dose-dependent increase in cAMP levels at DA concentrations up to 10(-7) M with EC(50) of 3.30 nM, while BmDopR2 required 10(-6) M DA for activation. In BmDopR1-expressing cells, DA at 10(-6)-10(-4) M induced 30-50% lower cAMP production than 10(-7) MDA. BmDopR2-expressing cells showed a standard sigmoidal dose-response, with maximum cAMP levels attained with 10(-5)-10(-4) M DA and EC(50) of 1.30 microM. Both receptors had similar agonist profiles, and the typical vertebrate D1-like receptor agonist SKF-38393 was ineffective. Experiments with antagonists revealed that BmDopR1 exhibits D1-like features. However, the pharmacology of BmDopR2 was distinct from D1-like receptors; the typical vertebrate D1-like receptor antagonist SCH-23390 was less potent than the nonselective antagonist flupenthixol and the D2-like receptor antagonist chlorpromazine. The rank order of activities of several antagonists for BmDopR1 and BmDopR2 was more similar to that of Drosophila melanogaster DA receptors than Apis mellifera DA receptors. These data suggest that DA receptors could be potential targets for specific insecticides or insectistatics.

Characterization of chemoreceptive protein binding to an oviposition stimulant using a fluorescent micro-binding assay in a butterfly.

A native female-specific chemoreceptive protein of a swallowtail butterfly [oviposition stimulant binding protein (OSBP)] was shown to specifically bind to aristolochic acid, a main stimulant for oviposition from its host plant. Oviposition stimulants are recognized by chemoreceptive organs of insects. OSBP isolated previously from the chemoreceptive organs was assumed to bind to an oviposition stimulant. Using a highly sensitive fluorescent micro-binding assay, we clarified OSBP bound to aristolochic acid. Three-dimensional molecular modeling revealed the structure of the OSBP-aristolochic acid complex. This is the first report of a native chemoreceptive protein binding to an oviposition stimulant as a ligand in insects.

An odorant-binding protein facilitates odorant transfer from air to hydrophilic surroundings in the blowfly.

Chemical sense-related lipophilic ligand-binding protein (CRLBP) is an insect odorant-binding protein (OBP) found abundantly in the taste and olfactory organs of the blowfly, Phormia regina. Through computational construction, a three-dimensional molecular model of a CRLBP indicated good fitting to a fluorescent ligand, 7-hydroxycoumarin (7-HC), in its ligand-binding pocket. By showing that the fluorescence of 7-HC bound to CRLBP migrated in a native electrophoresis gel, we confirmed that CRLBP formed a stable complex with 7-HC. In an odorant-binding experiment, 7-HC vapor odor was introduced by aeration to the aquatic solution containing CRLBP and its binding to CRLBP fluorospectrometrically quantified. Because olfactory organs as well as taste organs of flies respond to vapors, we suggest that CRLBP effectively transfers odorants from the air into aquatic surroundings by forming stable complexes with airborne molecules in both chemosensory organs.