Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT₇ genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT_1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.     

Knockdown of specific cuticular proteins analogous to peritrophin 3 genes disrupt larval and ovarian development in Bactrocera dorsalis (Diptera: Tephritidae)

Cuticular proteins (CPs) are critical components of the insect cuticle and play important roles in maintaining normal insect development and defense against various environmental stresses. The oriental fruit fly (Bactrocera dorsalis) is one of the most destructive pests worldwide, and its eight CPs analogous to peritrophin 3 (BdCPAP3) family genes have been identified in our previous study. In the present study, we further explored the possible roles of CPAP3 genes in B. dorsalis development. Each sequence of BdCPAP3 genes contained three conserved ChtBD2 (chitin-binding) domains. Spatial and temporal expression patterns revealed that the four BdCPAP3 genes (BdCPAP3-A1, B, E, and E2) might play important roles in larval pupariation of B. dorsalis. Moreover, treatment with a juvenile hormone analog (methoprene) significantly restricted expression of these four CPAP3 genes, whereas treatment with 20-hydroxy-ecdysone induced expression. The RNA interference (RNAi) results revealed that down-regulated CPAP3 genes led to significant delay of pupariation, and injection of dsBdCPAP3-E into 5-d-old B. dorsalis larvae caused approximately 40% mortality. Interestingly, we also confirmed that BdCPAP3-D2 was involved in B. dorsalis ovarian development. This study showed that some specific CPAP3 genes had crucial roles in B. dorsalis development, and these CP genes could be used as potential targets to control this pest via RNAi.     

基于转录组学的管纹艳虎天牛表皮蛋白基因家族的鉴定及特征分析

表皮蛋白(cuticular protein,CP)在昆虫生长发育、抵御外界不良环境等方面具有重要作用,但在管纹艳虎天牛Rhaphuma horsfieldi中关于CP基因的研究尚属空白.本研究基于已测序的转录组,通过生物信息学技术从管纹艳虎天牛中一共鉴定到108个CP基因,包括41个CP-RR1、30个CP-RR2、...     

A chromosome‐level genome assembly of the parasitoid wasp Pteromalus puparum

Parasitoid wasps represent a large proportion of hymenopteran species. They have complex evolutionary histories and are important biocontrol agents. To advance parasitoid research, a combination of Illumina short‐read, PacBio long‐read and Hi‐C scaffolding technologies was used to develop a high‐quality chromosome‐level genome assembly for Pteromalus puparum, which is an important pupal endoparasitoid of caterpillar pests. The chromosome‐level assembly has aided in studies of venom and detoxification genes. The assembled genome size is 338 Mb with a contig N50 of 38.7 kb and a scaffold N50 of 1.16 Mb. Hi‐C analysis assembled scaffolds onto five chromosomes and raised the scaffold N50 to 65.8 Mb, with more than 96% of assembled bases located on chromosomes. Gene annotation was assisted by RNA sequencing for the two sexes and four different life stages. Analysis detected 98% of the BUSCO (Benchmarking Universal Single‐Copy Orthologs) gene set, supporting a high‐quality assembly and annotation. In total, 40.1% (135.6 Mb) of the assembly is composed of repetitive sequences, and 14,946 protein‐coding genes were identified. Although venom genes play important roles in parasitoid biology, their spatial distribution on chromosomes was poorly understood. Mapping has revealed venom gene tandem arrays for serine proteases, pancreatic lipase‐related proteins and kynurenine–oxoglutarate transaminases, which have amplified in the P. puparum lineage after divergence from its common ancestor with Nasonia vitripennis. In addition, there is a large expansion of P450 genes in P. puparum. These examples illustrate how chromosome‐level genome assembly can provide a valuable resource for molecular, evolutionary and biocontrol studies of parasitoid wasps.     

Quantitative changes and synthesis of cyanoprotein in whole body and tissues during development of the bean bug,Riptortus clavatus

Changes in biliverdin-binding cyanoprotein content in whole body and tissue extracts during development of nymphal and adult (non-diapause) bean bugs, Riptortus clavatus were analyzed by rocket immunoelectrophoresis (RIE). RIE using anti-CPegg serum can be used to determine the content of CP-A (Cp-1, 2 and 3) and CP-B (CP-4) separately. During the nymphal stage CP content of whole body changes cyclically in each instar. In the first nymphal instar, CPegg is the main CP which disappears during the first-second instar ecdysis. In nymphal bugs from the 2nd to 4th instars only CP-B (CP-4) is detected, and at the beginning of each instar the CP content is very low but increases toward the next ecdysis, after which CP decreases and disappears very rapidly. In the 5th nymphal instar, CP-B is the major CP but CP-A (CP-1, 2 and 3) is also detected. These changes in whole body CP content of 5th instar nymphs are observed in both females and males. The content of total CPs in the 5th instar nymph reaches about 1000 μg in the whole insect. During nymphal-adult ecdysis, nymphal CPs decrease and disappear at day 2 after emergence. In female adults CP-A (CP-1 only) increases rapidly after day 4 of adult emergence, while no CP is detected in male adults. In females CPs were detected only in the fat body, hemolymph and ovary. In the mid-5th-instar nymphs, CPs (CP-A and B) are mainly distributed in the hemolymph. CPs in the Hemolymph decrease during nymphal-adult ecdysis, whereas they increase in the fat body. CPs disappear from both the hemolymph and fat body by 2 days after ecdysis. Subsequently in the adult stage only CP-A increases again in the fat body and ovary. By tracer experiments using [³⁵S]-methionine, the fat body was shown to be the site of CP synthesis. CP-A and B synthetic activity was detected in nymphal females whereas, only CP-A synthesis was observed in adult females, while no CP synthesis was seen in adult males.     

Monoclonal antibodies recognizing larval- and pupal-specific cuticular proteins of Tenebrio molitor (Insecta,Coleoptera)

To study the sequential expression of insect epidermal cells during metamorphosis, a library of monoclonal antibodies (MABs) was prepared against the water-soluble proteins from preecdysial pupal cuticle of Tenebrio molitor. Six selected MABs recognizing only larval and pupal cuticular proteins (CPs) in immunoblot analysis were classified into three types. Type 1 recognized a 21.5 and a 22 kDa polypeptide, type 2, a 26 kDa polypeptide, and type 3, three polypeptides of 18.5, 19.5 and 21.5 kDa. They did not immunoreact with any protein of fat bodies or haemolymph from pharate pupae, suggesting that the antigens originate from the epidermis. The stage-specificity was confirmed by electron microscopic immunogold labelling. Type 1 and 3 MABs recognized antigens characterizing larval and pupal preecdysial sclerotized cuticles, while the antigens recognized by type 2 were localized in the first few lamellae of unsclerotized postecdysial cuticle. When the expression of the adult programme was inhibited by application of a juvenile hormone analogue, the larval-/pupal-specific CPs were detected in the supernumerary pupal cuticle. These results suggest that the genes encoding these proteins are juvenile hormone dependent. These MABs should be useful tools to isolate pupal-specific genes whose regulation sems to be different from that of the adult-specific ones.     

CLONING,EXPRESSION PATTERNS,AND PRELIMINARY CHARACTERIZATION OF AccCPR24, A NOVEL RR‐1 TYPE CUTICLE PROTEIN GENE FROM Apis cerana cerana

Cuticular proteins (CPs) are key components of insect cuticle, a structure that plays a pivotal role in insect development and defense. In this study, we cloned the full‐length cDNA of a CP gene from Apis cerana cerana (AccCPR24). An amino acid sequence alignment indicated that AccCPR24 contains the conserved Rebers and Riddiford consensus sequence and shares high similarity with the genes from other hymenopteran insects. We then isolated the genomic DNA and found that the first intron, which is present in other CP genes, is absent in AccCPR24. Real‐time quantitative polymerase chain reaction (qPCR) analysis revealed that AccCPR24 is highly expressed in the late pupal stage and midgut. Expression was inhibited by an exogenous ecdysteroid in vitro but was enhanced by this hormone in vivo; environmental stressors, such as heavy metals and pesticides, also influenced gene expression. In addition, a disc diffusion assay showed that AccCPR24 enhanced the ability of bacterial cells to resist multiple stresses. We infer from our results that AccCPR24 acts in honeybee development and in protecting these insects from abiotic stresses.     

Conserved roles of Osiris genes in insect development,polymorphism and protection

Much of the variation among insects is derived from the different ways that chitin has been moulded to form rigid structures, both internal and external. In this study, we identify a highly conserved expression pattern in an insect‐only gene family, the Osiris genes, that is essential for development, but also plays a significant role in phenotypic plasticity and in immunity/toxicity responses. The majority of Osiris genes exist in a highly syntenic cluster, and the cluster itself appears to have arisen very early in the evolution of insects. We used developmental gene expression in the fruit fly, Drosophila melanogaster, the bumble bee, Bombus terrestris, the harvester ant, Pogonomyrmex barbatus, and the wood ant, Formica exsecta, to compare patterns of Osiris gene expression both during development and between alternate caste phenotypes in the polymorphic social insects. Developmental gene expression of Osiris genes is highly conserved across species and correlated with gene location and evolutionary history. The social insect castes are highly divergent in pupal Osiris gene expression. Sets of co‐expressed genes that include Osiris genes are enriched in gene ontology terms related to chitin/cuticle and peptidase activity. Osiris genes are essential for cuticle formation in both embryos and pupae, and genes co‐expressed with Osiris genes affect wing development. Additionally, Osiris genes and those co‐expressed seem to play a conserved role in insect toxicology defences and digestion. Given their role in development, plasticity, and protection, we propose that the Osiris genes play a central role in insect adaptive evolution.     

Genes acting in longevity-related pathways in the endoparasitoid,Pteromalus puparum

Among insects, lifespans vary over a broad range, from the short-lived mayflies to the 17-year periodical cicadas. Generally, lifespans are determined by a phase in life, the reproductive lifespan, which varies among species. Numerous pathways, such as the insulin/insulin-like growth factor signaling pathway, the target of rapamycin pathway and the mitogen-activated protein kinase/extracellular signal-regulated kinases pathways, influence aging and lifespan. Components of these pathways were identified as lifespan-related genes, including genes mediating growth, metabolism, development, resistance, and other processes. Many age-related genes have been discovered in fruit flies, honeybees, and ants among other insect species. Studies of insect aging and longevity can help understand insect biology and develop new pest management technologies. In this paper, we interrogated the new Pteromalus puparum genome, from which we predicted 133 putative lifespan-related genes based on their homology with known lifespan-related genes of Drosophila melanogaster. These genes function in five signaling pathways and three physiological processes. The conserved domain structures of these genes were predicted and their expression patterns were analyzed. Amino acid sequence alignments and domain structure analysis indicate that most components remain conserved across at least six insect orders. The data in this paper will facilitate future work on parasitoid lifespans, which may have economic value in biocontrol programs.     

The biosynthesis of Caryophyllaceae-like cyclic peptides in Saponaria vaccaria L. from DNA-encoded precursors

Cyclic peptides (CPs) are produced in a very wide range of taxa. Their biosynthesis generally involves either non‐ribosomal peptide synthases or ribosome‐dependent production of precursor peptides. Plants within the Caryophyllaceae and certain other families produce CPs which generally consist of 5–9 proteinogenic amino acids. The biological roles for these CPs in the plant are not very clear, but many of them have activity in mammalian systems. There is currently very little known about the biosynthesis of CPs in the Caryophyllaceae. A collection of expressed sequence tags from developing seeds of Saponaria vaccaria was investigated for information about CP biosynthesis. This revealed genes that appeared to encode CP precursors which are subsequently cyclized to mature CPs. This was tested and confirmed by the expression of a cDNA encoding a putative precursor of the CP segetalin A in transformed S. vaccaria roots. Similarly, extracts of developing S. vaccaria seeds were shown to catalyze the production of segetalin A from the same putative (synthetic) precursor. Moreover, the presence in S. vaccaria seeds of two segetalins, J [cyclo(FGTHGLPAP)] and K [cyclo(GRVKA)], which was predicted by sequence analysis, was confirmed by liquid chromatography/mass spectrometry. Sequence analysis also predicts the presence of similar CP precursor genes in Dianthus caryophyllus and Citrus spp. The data support the ribosome‐dependent biosynthesis of Caryophyllaceae‐like CPs in the Caryophyllaceae and Rutaceae.     

Annotation and expression analysis of cuticular proteins from the tobacco hornworm,Manduca sexta

The insect cuticle is a unique material that covers the exterior of the animal as well as lining the foregut, hindgut, and tracheae. It offers protection from predators and desiccation, defines body shape, and serves as an attachment site for internal organs and muscle. It has demonstrated remarkable variations in hardness, flexibility and elasticity, all the while being light weight, which allows for ease of movement and flight. It is composed primarily of chitin, proteins, catecholamines, and lipids. Proteomic analyses of cuticle from different life stages and species of insects has allowed for a more detailed examination of the protein content and how it relates to cuticle mechanical properties. It is now recognized that several groups of cuticular proteins exist and that they can be classified according to conserved amino acid sequence motifs. We have annotated the genome of the tobacco hornworm, Manduca sexta, for genes that encode putative cuticular proteins that belong to seven different groups: proteins with a Rebers and Riddiford motif (CPR), proteins analogous to peritrophins (CPAP), proteins with a tweedle motif (CPT), proteins with a 44 amino acid motif (CPF), proteins that are CPF-like (CPFL), proteins with an 18 amino acid motif (18 aa), and proteins with two to three copies of a C-X₅-C motif (CPCFC). In total we annotated 248 genes, of which 207 belong to the CPR family, the most for any insect genome annotated to date. Additionally, we discovered new members of the CPAP family and determined that orthologous genes are present in other insects. We established orthology between the M. sexta and Bombyx mori genes and identified duplication events that occurred after separation of the two species. Finally, we utilized 52 RNAseq libraries to ascertain gene expression profiles that revealed commonalities and differences between different tissues and developmental stages.     

Cyanoprotein: Developmental stage,sex and diapause-dependent expression,and synthesis regulation by juvenile hormone in the bean bug,Riptortus clavatus

Cyanoprotein (CP) synthesis was studied in nymphal and nondiapause adult, diapause adult, and juvenile hormone (JH) treated adult bean bugs, Riptortus clavatus. Hemolymph collected from bugs injected with [³⁵S]-methionine was analyzed by native polyacrylamide gel electrophoresis (PAGE) and fluorography, and CP synthesis was also determined quantitatively by rocket immunoelectrophoresis of hemolymph and counting. In the nymphal stages synthesis of CP-1, 2, 3, and 4 (with CP-4 synthesis predominant) reached a maximum at mid-instar and was not detected at each ecdysis, so that the synthetic activity of CPs changed cyclically in each instar. During the nymphal stages CP synthesis showed the same pattern and level in both females and males, and both diapause and nondiapause oriented bugs. In the adult stage, however, CP synthesis differed in the two sexes and nondiapause or diapause conditions. In nondiapause males CP synthesis was not detected in the adult, but in nondiapause females CP (only CP-1) was synthesized through the reproductive stages. CP-1 accumulated in the egg yolk together with vitellogenin. In diapause adults (both females and males) CP-1 to 4 were synthesized at a very low rate for over 2 months and accumulated in the hemolymph. JH or JH analog (JHA) methoprene and also long day condition, which terminate diapause, switched the main CP synthesis CP-4 to CP-1 in females. CP synthesis in diapause males stopped after JH(A) treatment. The activities of CP synthesis, thus, changed in developmental stages, sexes, and diapause. This is an excellent system for study of specific gene expression and switching controlled by insect hormones and sex. © 1992 Wiley-Liss, Inc.     

Conjugation in Archaea: Frequent Occurrence of Conjugative Plasmids inSulfolobus

We describe five novel conjugative plasmids (CPs) and two subfamilies, each comprising several closely related variants of CPs isolated from colony-cloned strains of the extremely thermophilic, heterotrophic archaeonSulfolobus islandicus,which were obtained by plating of samples from Icelandic solfataras after liquid enrichment. They are related to each other and to the previously described CP pNOB8 from a JapaneseSulfolobusstrain in that they share essential functions and limited similarity of genomes as demonstrated by DNA cross-hybridization and sequences. All these plasmids thus form a family of highly efficient self-spreading elements directly transferred from donor into recipient cells. Conjugation is initiated by pair formation, followed by selective transfer of the plasmids into the recipient and expression of transfer functions. Some of these CPs exclude superconjugation of the transcipients with closely related CPs. The novel CPs are stable upon conjugative transfer, but vary upon growth of transcipients. The stability of the CPs is higher in their original hosts or in relatedS. islandicusstrains, than inSulfolobus solfataricusstrain PH1 as recipient. The deletion variant pING3 has lost the ability to transfer itself but is still subject to being transferred by the transfer apparatus of its complete relative, pING6. The dissection of genes and functions has been initiated by characterizing this incomplete variant.