半翅目昆虫卵黄原蛋白及其合成调控的研究进展

昆虫卵黄原蛋白（Vitellogenins, Vg）是一种多功能的生殖发育关键调控蛋白,在不同昆虫体内的结构、合成调控及功能不尽相同。随着基因编辑技术的成熟,运用分子手段调控Vg的合成,可减少卵黄发生,降低昆虫的繁殖力,成为有效防治害虫的优势方法之一。因此,Vg及其合成调控的研究受到广泛关注。半翅目害虫是农林业的重点防治对象之一,除直接刺吸为害寄主外,其常传播植物病原体,对农业生产造成了严重危害。半翅目昆虫Vg除在生殖发育中的关键作用外,还与病原菌的传播、寄主免疫等密切相关,可成为分子水平防治半翅目害虫及其继发病害的优势靶标。因此,本文总结了半翅目昆虫Vg的合成方式、合成场所,指明了其结构上蛋白亚基数目的差异,概述了其与昆虫免疫反应、植物防御、病毒传播等有关的研究进展,总结了其合成的保幼激素（包括保幼激素受体Methoprene-tolerant和转录因子Krüppel homolog 1等关键调控因子等）、蜕皮激素和胰岛素信号通路等主要的内分泌激素调控通路,以及以营养信号调控为主的非激素调控通路,为探索半翅目害虫的分子防控手段提供理论依据。     

Comparative peptidomics of four related hemipteran species: pyrokinins, myosuppressin, corazonin, adipokinetic hormone, sNPF, and periviscerokinins

We performed the first comprehensive peptidomic analysis of neurohormones from hemipteran insects by analyzing the neuropeptides of two major neurohemal organs, namely the corpora cardiaca and abdominal perisympathetic organs. For the experiments we selected four related species of polyphagous stinkbugs (Pentatomidae), three of which are known to attack several important food crops. Peptide sequences were identified by MALDI-TOF mass spectrometry; tandem fragmentation of myosuppressin, sNPF, CAPA-periviscerokinins and pyrokinins revealed novel sequences not known from other insects so far. Most Leu/Ile and Glu/Lys ambiguities could be solved by either specific side-chain fragmentations or on-plate acetylation experiments. The identification of the specific sequences provides a solid basis for forthcoming pharmacological tests to study the neuroendocrine system of these pest insects. However, it should be mentioned in this context that the sequences of the peptides from different stinkbugs are likely not representative of Hemiptera in general. The forthcoming release of the genome from the reduviid Rhodnius prolixus will provide sufficient data to clear this point.     

Phylogenetic congruence of armored scale insects (Hemiptera: Diaspididae) and their primary endosymbionts from the phylum Bacteroidetes

Insects in the sap-sucking hemipteran suborder Sternorrhyncha typically harbor maternally transmitted bacteria housed in a specialized organ, the bacteriome. In three of the four superfamilies of Sternorrhyncha (Aphidoidea, Aleyrodoidea, Psylloidea), the bacteriome-associated (primary) bacterial lineage is from the class Gammaproteobacteria (phylum Proteobacteria). The fourth superfamily, Coccoidea (scale insects), has a diverse array of bacterial endosymbionts whose affinities are largely unexplored. We have amplified fragments of two bacterial ribosomal genes from each of 68 species of armored scale insects (Diaspididae). In spite of initially using primers designed for Gammaproteobacteria, we consistently amplified sequences from a different bacterial phylum: Bacteroidetes. We use these sequences (16S and 23S, 2105 total base pairs), along with previously published sequences from the armored scale hosts (elongation factor 1alpha and 28S rDNA) to investigate phylogenetic congruence between the two clades. The Bayesian tree for the bacteria is roughly congruent with that of the hosts, with 67% of nodes identical. Partition homogeneity tests found no significant difference between the host and bacterial data sets. Of thirteen Shimodaira-Hasegawa tests, comparing the original Bayesian bacterial tree to bacterial trees with incongruent clades forced to match the host tree, 12 found no significant difference. A significant difference in topology was found only when the entire host tree was compared with the entire bacterial tree. For the bacterial data set, the treelengths of the most parsimonious host trees are only 1.8-2.4% longer than that of the most parsimonious bacterial trees. The high level of congruence between the topologies indicates that these Bacteroidetes are the primary endosymbionts of armored scale insects. To investigate the phylogenetic affinities of these endosymbionts, we aligned some of their 16S rDNA sequences with other known Bacteroidetes endosymbionts and with other similar sequences identified by BLAST searches. Although the endosymbionts of armored scales are only distantly related to the endosymbionts of the other sternorrhynchan insects, they are closely related to bacteria associated with eriococcid and margarodid scale insects, to cockroach and auchenorrynchan endosymbionts (Blattabacterium and Sulcia), and to male-killing endosymbionts of ladybird beetles. We propose the name "Candidatus Uzinura diaspidicola" for the primary endosymbionts of armored scale insects.     

Occurrence of midgut perimicrovillar membranes in paraneopteran insect orders with comments on their function and evolutionary significance

Hemipterans are characterized by the absence of the peritrophic membrane, an anatomical structure that envelopes the food bolus in the majority of insects. However, the microvillar membranes of many hemipteran midgut cells are not in direct contact with the food bolus, due to the existence of the so-called perimicrovillar membrane (PMM), which covers the microvilli extending into the gut lumen with dead ends. alpha-Glucosidase is a biochemical marker for PMM in the seed sucker bug Dysdercus peruvianus (Heteroptera: Pyrrhocoridae). In this article, we report that adults of the major hemipteran infra-orders (Sternorrhyncha, Auchenorrhyncha, and Heteroptera) have PMM and a major membrane bound alpha-glucosidase, which has properties similar to those of the D. peruvianus enzyme. A polyclonal antibody raised against the enzyme of D. peruvianus recognized the enzymes present in PMM from the above-mentioned hemipteran groups. The same antibody was also able of recognizing perimicrovillar alpha-glucosidase from thrips. No PMM nor membrane-bound alpha-glucosidase were found in Psocoptera and Phthiraptera midguts. This suggests that PMM and PMM-bound-alpha-glucosidase are widespread among insects of the order Hemiptera and of the sister order Thysanoptera. The data support the hypothesis that PMM may have originated in the Condylognatha (Paraneopteran taxon including Hemiptera and Thysanoptera) ancestral stock and are associated with plant sap feeding.     

Host‐associated differentiation in a highly polyphagous,sexually reproducing insect herbivore

Insect herbivores may undergo genetic divergence on their host plants through host‐associated differentiation (HAD). Much of what we know about HAD involves insect species with narrow host ranges (i.e., specialists) that spend part or all their life cycle inside their hosts, and/or reproduce asexually (e.g., parthenogenetic insects), all of which are thought to facilitate HAD. However, sexually reproducing polyphagous insects can also exhibit HAD. Few sexually reproducing insects have been tested for HAD, and when they have insects from only a handful of potential host‐plant populations have been tested, making it difficult to predict how common HAD is when one considers the entire species' host range. This question is particularly relevant when considering insect pests, as host‐associated populations may differ in traits relevant to their control. Here, we tested for HAD in a cotton (Gossypium hirsutum) pest, the cotton fleahopper (CFH) (Pseudatomoscelis seriatus), a sexually reproducing, highly polyphagous hemipteran insect. A previous study detected one incidence of HAD among three of its host plants. We used Amplified fragment length polymorphism (AFLP) markers to assess HAD in CFH collected from an expanded array of 13 host‐plant species belonging to seven families. Overall, four genetically distinct populations were found. One genetically distinct genotype was exclusively associated with one of the host‐plant species while the other three were observed across more than one host‐plant species. The relatively low degree of HAD in CFH compared to the pea aphid, another hemipteran insect, stresses the likely importance of sexual recombination as a factor increasing the likelihood of HAD.     

Phosphorylation of microtubule-associated proteins from the ovaries of hemipteran insects by MPF and MAP kinase: Possible roles in the regulation of microtubules during oogenesis

Nutritive tubes that link the developing oocytes to the nurse cells in ovarioles of hemipteran insects contain extensive arrays of microtubules. These are established, then later depolymerised, by developmentally regulated processes. Breakdown of the microtubules corresponds with the activation of M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase), late in oogenesis, as the oocytes proceed to arrest at the first meiotic metaphase [Lane and Stebbings, Roux's Arch Dev Biol 205:150–159 (1995)]. The mechanisms that lead to the breakdown of nutritive tube microtubules are unknown. Here, we have investigated the possibility that the insect ovarian microtubules are regulated by MPF- or MAP kinase-dependent phosphorylation, focusing upon the prominent high molecular weight microtubule-associated protein (HMW MAP) enriched in this system, which is a potential target for protein kinase activity in vivo. We have purified the prominent HMW MAPs from the ovaries of two species of hemipterans, and have shown them to be substrates in vitro for the activities of MPF and MAP kinase. However, although the catalytic component of MPF (p34^cdc2) is present within microtubule-rich portions of hemipteran ovarioles, we have found that neither this protein nor its regulatory partner (cyclin B) co-purify with microtubules during taxol-mediated microtubule isolation. Arch. Insect Biochem. Physiol. 39:81–90, 1998.© 1998 Wiley-Liss, Inc.     

Stolbur phytoplasma genome survey achieved using a suppression subtractive hybridization approach with high specificity

Phytoplasmas are unculturable bacterial plant pathogens transmitted by phloem-feeding hemipteran insects. DNA of phytoplasmas is difficult to purify because of their exclusive phloem location and low abundance in plants. To overcome this constraint, suppression subtractive hybridization (SSH) was modified and used to selectively amplify DNA of the stolbur phytoplasma infecting a periwinkle plant. Plasmid libraries were constructed, and the origins of the DNA inserts were verified by hybridization and PCR screenings. After a single round of SSH, there was still a significant level of contamination with plant DNA (around 50%). However, the modified SSH, which included a second round of subtraction (double SSH), resulted in an increased phytoplasma DNA purity (97%). Results validated double SSH as an efficient way to produce a genome survey for microbial agents unavailable in culture. Assembly of 266 insert sequences revealed 181 phytoplasma genetic loci which were annotated. Comparative analysis of 113 kbp indicated that among 217 protein coding sequences, 83% were homologous to "Candidatus Phytoplasma asteris" (OY-M strain) genes, with hits widely distributed along the chromosome. Most of the stolbur-specific SSH sequences were orphan genes, with the exception of two partial coding sequences encoding proteins homologous to a mycoplasma surface protein and riboflavin kinase.     

An analysis of mutualistic interactions between exotic ants and honeydew producers in the Yanbaru district of Okinawa Island,Japan

Invasive exotic ants often have a mutualistic relationship with other insects excreting honeydew, and this is considered to play a key role in their invasion success. We investigated the multispecies association patterns between ants and hemipteran insects in the Yanbaru forests, Okinawa, Japan, an Asian biodiversity hotspot. We especially focused on roadside environments, which are the frontlines of invasion for exotic ants. We found that only a small number of herbaceous and pioneer plants were predominant on the roadsides. Four honeydew producers, Melanaphis formosana, Dysmicoccus sp. A, Heteropsylla cubana, and Sogata hakonensis, living on these roadside plants accounted for 94.9% of the total honeydew-producer aggregations observed. Only a few exotic ants, such as Technomyrmex brunneus and Anoplolepis gracilipes, were observed with these honeydew-producer aggregations, and densities of these ants and honeydew producers were often positively correlated. An ant exclusion experiment showed that exotic ant occurrence improved the survival of some of the hemipteran colonies. Interestingly, the abundance of native ants was not correlated with the abundance of honeydew producers, and the local density of Pheidole noda was negatively correlated with that of M. formosana. These findings, i.e., only a few ants, all exotic, tended to hemipteran honeydew producers despite the existence of many native ants, and the abundances of those exotic ants and those hemipteran insects had positive correlations, provide some insights into the mechanism of biological invasion and provide information for the management of exotic ants.     

Ecological effects of multi‐species,ant–hemipteran mutualisms in citrus

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Allergenicity assessment of Allium sativum leaf agglutinin, a potential candidate protein for developing sap sucking insect resistant food crops

Background

Mannose-binding Allium sativum leaf agglutinin (ASAL) is highly antinutritional and toxic to various phloem-feeding hemipteran insects. ASAL has been expressed in a number of agriculturally important crops to develop resistance against those insects. Awareness of the safety aspect of ASAL is absolutely essential for developing ASAL transgenic plants.

Methodology/Principal Findings

Following the guidelines framed by the Food and Agriculture Organization/World Health Organization, the source of the gene, its sequence homology with potent allergens, clinical tests on mammalian systems, and the pepsin resistance and thermostability of the protein were considered to address the issue. No significant homology to the ASAL sequence was detected when compared to known allergenic proteins. The ELISA of blood sera collected from known allergy patients also failed to show significant evidence of cross-reactivity. In vitro and in vivo assays both indicated the digestibility of ASAL in the presence of pepsin in a minimum time period.

Conclusions/Significance

With these experiments, we concluded that ASAL does not possess any apparent features of an allergen. This is the first report regarding the monitoring of the allergenicity of any mannose-binding monocot lectin having insecticidal efficacy against hemipteran insects.     

Relationship between intraspecific variations and host insects of Ophiocordyceps nutans collected in Japan

To investigate the host specificity of Ophiocordyceps nutans against hemipteran insects in the wild, we determined the relationship between host species and rDNA-internal transcribed spacer (ITS) variation in O. nutans. The analyzed fungal specimens infected 16 host species belonging to four families of Hemiptera. The molecular phylogenetic analysis revealed that O. nutans can be classified into two types corresponding to their host families. The genetic distance values between the two types were very remote (>0.084), and the strains of O. nutans that parasitized Halyomorpha halys and Plautia crossota stali, well-known insect pests, formed a subclade. The results suggest that O. nutans should have host specificity which can be valuable for developing biological control agents against specific hemipteran insects.     

18S rRNA secondary structure and phylogenetic position of Peloridiidae (Insecta, hemiptera)

A secondary structure model for 18S rRNA of peloridiids, relict insects with a present-day circumantarctic distribution, is constructed using comparative sequence analysis, thermodynamic folding, a consensus method using 18S rRNA models of other taxa, and support of helices based on compensatory substitutions. Results show that probable in vivo configuration of 18S rRNA is not predictable using current free-energy models to fold the entire molecule concurrently. This suggests that refinements in free-energy minimization algorithms are needed. Molecular phylogenetic datasets were created using 18S rRNA nucleotide alignments produced by CLUSTAL and rigorous interpretation of homologous position based on certain secondary substructures. Phylogenetic analysis of a hemipteran data matrix of 18S rDNA sequences placed peloridiids sister to Heteroptera. Resolution of affiliations between the three main euhemipteran lineages was unresolved. The peloridiid 18S RNA model presented here provides the most accurate template to date for aligning homologous nucleotides of hemipteran taxa. Using folded 18S rRNA to infer homology of character as morpho-molecular structures or nucleotides and scoring particular sites or substructures is discussed.     

Stolbur Phytoplasma Genome Survey Achieved Using a Suppression Subtractive Hybridization Approach with High Specificity

Phytoplasmas are unculturable bacterial plant pathogens transmitted by phloem-feeding hemipteran insects. DNA of phytoplasmas is difficult to purify because of their exclusive phloem location and low abundance in plants. To overcome this constraint, suppression subtractive hybridization (SSH) was modified and used to selectively amplify DNA of the stolbur phytoplasma infecting a periwinkle plant. Plasmid libraries were constructed, and the origins of the DNA inserts were verified by hybridization and PCR screenings. After a single round of SSH, there was still a significant level of contamination with plant DNA (around 50%). However, the modified SSH, which included a second round of subtraction (double SSH), resulted in an increased phytoplasma DNA purity (97%). Results validated double SSH as an efficient way to produce a genome survey for microbial agents unavailable in culture. Assembly of 266 insert sequences revealed 181 phytoplasma genetic loci which were annotated. Comparative analysis of 113 kbp indicated that among 217 protein coding sequences, 83% were homologous to “Candidatus Phytoplasma asteris” (OY-M strain) genes, with hits widely distributed along the chromosome. Most of the stolbur-specific SSH sequences were orphan genes, with the exception of two partial coding sequences encoding proteins homologous to a mycoplasma surface protein and riboflavin kinase.     

甜菜夜蛾几丁质合成酶基因A和B启动子的克隆

几丁质不仅是昆虫的表皮和围食膜的主要成分,也是一个非常关键的害虫控制靶标,主要通过几丁质合成酶（chitin synthase,CHS）基因合成。本文在克隆甜菜夜蛾Spodoptera exigua的两个几丁质合成酶基因（SeCHSA和SeCHSB）cDNA和基因组序列的基础上,从基因的5′末端设计特异性引物和构建特定的基因组文库, 采用PCR的方法获得了5′端侧翼序列。通过5′RACE的方法确定SeCHSA和SeCHSB基因的转录起始位点后,获到了启动子序列。这为研究昆虫几丁质合成和转录调控奠定了基础。     

Two different farnesyl diphosphate synthase genes exist in the genome of the green peach aphid, Myzus persicae.

Farnesyl diphosphate synthase (FPS; EC 2.5.1.1, 2.5.1.10) catalyzes biosynthesis of farnesyl diphosphate, which is important to insects as the precursor of juvenile hormone and the substrate for (E)-beta-farnesene synthase. Here, two FPS cDNAs were isolated from the green peach aphid, Myzus persicae (EU334430 and EU334431). Their shared identity within the coding region is approximately 82%. The deduced amino acid sequences of the two M. persicae FPS cDNAs have the highly conserved motifs characteristic of most known FPSs. Phylogenetic analyses showed that they are closely related to other insect FPSs. Homology modeling of structures suggested a very good fit between the three-dimensional structures of the two putative M. persicae FPSs (designated as MpFPS1 and MpFPS2) and the avian FPS crystal structure. The corresponding genomic DNA sequences were subsequently determined (EU429295 and EU429296). Sequence comparisons revealed a different splicing pattern between the two MpFPS genes. Furthermore, the two MpFPS genes exhibited a seemingly very primitive gene-splicing pattern at 5' ends but a gene-splicing style similar to mammalian FPS genes at 3' ends. These data, combined with results of Southern blotting, suggest that M. persicae contains two different FPS genes. This is the first report that two different FPS genes exist in a hemipteran insect.