意大利蝗卵发育过程中血蓝蛋白基因表达分析

【目的】意大利蝗Calliptamus italicus是新疆草原的主要优势危害种,以卵在土壤中越冬。呼吸代谢可反映蝗卵的生理状态,呼吸蛋白对于呼吸系统不完善的蝗卵尤为重要。本研究旨在明确意大利蝗卵发育过程中血蓝蛋白基因的表达情况。【方法】采用实时荧光定量PCR方法检测不同发育阶段的蝗卵以及1龄蝗蝻的血蓝蛋白2个亚基基因Hc1和Hc2的表达量。【结果】根据解剖形态观察,将意大利蝗越冬卵的整个发育过程分为10个阶段,包括9个卵发育阶段(C-Ⅰ-C-Ⅹ)和1龄蝗蝻阶段(C-Ⅹ)。Hc1和Hc2在越冬蝗卵各发育阶段以及1龄蝗蝻中均有表达。其中,在蝗卵早期发育阶段(C-Ⅰ, C-Ⅱ和C-Ⅲ),Hc1表达量逐渐增加,C-Ⅲ阶段表达量显著高于C-Ⅰ和C-Ⅱ阶段;滞育阶段(C-Ⅳ, C-Ⅴ和C-Ⅵ),胚胎发育停滞,Hc1表达量较C-Ⅲ,C-Ⅶ和C-Ⅷ阶段低;滞育后发育阶段(C-Ⅶ和C-Ⅷ),蝗卵解除滞育,快速发育,Hc1表达量较早期发育阶段和滞育阶段高,其中,C-Ⅷ阶段Hc1表达量最高(212.3156±10.5470),显著高于其他所有阶段;1龄蝗蝻(C-Ⅹ)的Hc1表达量最低,为0.4017±0.1010。Hc2表达量在C-Ⅴ阶段最高(679.7511±54.5719),显著高于其他所有阶段;除C-Ⅴ阶段外,其他各阶段之间Hc2表达量差异均不显著。Hc1在蝗卵滞育后阶段高表达,而Hc2在蝗卵滞育阶段高表达。【结论】血蓝蛋白亚基基因Hc1和Hc2在整个意大利蝗卵发育过程均有表达,且具有阶段特异性。Hc1与Hc2协同作用为蝗卵发育供氧,其中,Hc1主要负责蝗卵滞育后发育期间的氧气运载,而Hc2主要维持滞育期间的氧气运载,且载氧效率较低。研究结果可为进一步探讨意大利蝗卵的抗逆机制提供科学依据。     

Hemocyanin gene family evolution in spiders (Araneae), with implications for phylogenetic relationships and divergence times in the infraorder Mygalomorphae

Hemocyanins are multimeric copper-containing hemolymph proteins involved in oxygen binding and transport in all major arthropod lineages. Most arachnids have seven primary subunits (encoded by paralogous genes a–g), which combine to form a 24-mer (4 × 6) quaternary structure. Within some spider lineages, however, hemocyanin evolution has been a dynamic process with extensive paralog duplication and loss. We have obtained hemocyanin gene sequences from numerous representatives of the spider infraorders Mygalomorphae and Araneomorphae in order to infer the evolution of the hemocyanin gene family and estimate spider relationships using these conserved loci. Our hemocyanin gene tree is largely consistent with the previous hypotheses of paralog relationships based on immunological studies, but reveals some discrepancies in which paralog types have been lost or duplicated in specific spider lineages. Analyses of concatenated hemocyanin sequences resolved deep nodes in the spider phylogeny and recovered a number of clades that are supported by other molecular studies, particularly for mygalomorph taxa. The concatenated data set is also used to estimate dates of higher-level spider divergences and suggests that the diversification of extant mygalomorphs preceded that of extant araneomorphs. Spiders are diverse in behavior and respiratory morphology, and our results are beneficial for comparative analyses of spider respiration. Lastly, the conserved hemocyanin sequences allow for the inference of spider relationships and ancient divergence dates.     

The respiratory proteins of insects

For a long time, respiratory proteins have been considered unnecessary in most insects because the tracheal system was thought to be sufficient for oxygen supply. Only a few species that survive under hypoxic conditions were known exceptions. However, recently it has become evident that (1) intracellular hemoglobins belong to the standard repertoire of insects and (2) that hemocyanin is present in many "lower" insects. Intracellular hemoglobins have been identified in Drosophila, Anopheles, Apis and many other insects. In all investigated species, hemoglobin is mainly expressed in the fat body and the tracheal system. The major Drosophila hemoglobin binds oxygen with high affinity. This hemoglobin type possibly functions as a buffer system for oxygen supply at low partial pressures and/or for the protection from an excess of oxygen. Similar hemoglobins, present in much higher concentrations, store oxygen in specialized tracheal organs of the botfly and some backswimmers. The extracellular hemoglobins in the hemolymph of chironomid midges are evolutionary derivatives of the intracellular insect hemoglobins, which emerged in response to the hypoxic environment of the larvae. In addition, several hemoglobin variants of unknown functions have been discovered in insect genomes. Hemocyanins transport oxygen in the hemolymph of stoneflies, but also in the Entognatha and most hemimetabolan taxa. Apparently, hemocyanin has been lost in Holometabola. At present, no physiological or morphological character is known that could explain the presence or loss of hemocyanins in distinct taxa. Nevertheless, the occurrence of respiratory proteins in insects adds further complexity to our view on insect respiration.     

RNA interference unveils functions of the hypertrehalosemic hormone on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blattella germanica

Hypertrehalosemic hormone (HTH) is a neuropeptide within the adipokinetic hormone (AKH) family that induces a release of trehalose from fat body into hemolymph in a number of insects. In this study, we first showed that female adult German cockroach, Blattella germanica, displayed a cyclic fluctuation of hemolymph trehalose levels correlated to the maturation of oocytes in the reproductive cycle. After cloning the HTH cDNA from the German cockroach (Blage-HTH), expression studies indicated that Blage-HTH mRNA showed the cyclic changes during the first reproductive cycle, where peak values occurred in 8-day-old virgin female cockroaches, which were going to produce oothecae. The functions of Blage-HTH were studied using RNA interference (RNAi) to knockdown its expression. Adult virgin females of B. germanica injected with Blage-HTH dsRNA increased hemolymph trehalose levels in the late period of vitellogenesis more slowly than control. Furthermore, RNAi of Blage-HTH delayed oviposition time and some (10%) individuals did not produce the first ootheca until 15 days after eclosion, whereas the control group produced ootheca before 9 days in all cases.     

Fluorescence and circular dichroism studies with hemocyanin from taiwan snails

Several derivatives of hemocyanin from Taiwan snails (Achatina fulica) have been prepared. The reconstituted protein (R-HcO₂) has lower Cu content, lower circular dichroism intensity, and higher fluorescence intensity than native oxyhemocyanin (HcO₂). The Co(II) derivative (CoHc) does not take up molecular oxygen and only 50% of the total sites for Cu in native hemocyanin is taken up by Co. The half-apo derivative (half-apo-Hc) contains a single Cu per active site. Divalent cations quench the tryptophan fluorescence in the hemocyanin species and also quench the fluorescence from Tb³⁺ bound to the protein. The collisional quenching constants decrease in the order Co²⁺ > Mn²⁺ > Ca²⁺. The static component is negligible. For carboxy hemocyanin (HcCO), fluorescence originates from a Cu(I) CO complex and was used to study reaction of Hc CO with CN⁻.     

Hemolymph Proteins and Molting in Crustaceans and Insects

The exoskeleton of crustaceans and insects is formed by cellsof the hypodermis, but several hemolymph proteins contributeto the synthesis of the new exoskeleton. These hemolymph proteinsshare a surprising degree of sequence similarity and are membersof the hemocyanin gene family. Copper-containing prophenoloxidasesof crustaceans and insects are directly involved in cross-linkingand hardening of the exoskeleton during molting and repair.Crustacean cryptocyanin and insect hexamerins lack copper andhave probably evolved from a copper-free product of an earlyhemocyanin gene duplication. These proteins have been implicatedin transport of hormones and phenols, and may be used directlyas structural components of the new exoskeleton. They are synthesizedelsewhere in the body, transported in the hemolymph, and probablytaken up by the hypodermis via specific receptors. Hemocyaninshave some residual phenoloxidase activity, in addition to theirprimary role of supplying oxygen to the metabolizing tissues.Thus multiple members of the hemocyanin gene family play vitalroles during molting, and a molecular phytogeny of these proteinswill contribute to our understanding of the evolution of formand function of these molecules from oxygen transport to molt-relatedactivities. Further studies on the expression of prophenoloxidase,cryptocyanin, hexamerins and hemocyanin, potential marker proteins,may extend our understanding of the relationship between othermolting animals in the proposed clade, Ecdysozoa.     

Co(II)-substituted Octopus vulgaris hemocyanin

Co(II)-substituted hemocyanin (Co(II)Hc) of the octopus, Octopus vulgaris, has been prepared by dialysis of apohemocyanin against Co(II·) ion and subsequent Chelex-treatment. The blue 50%-Co(II)Hc (half-apo Co(II)Hc), in which binuclear coppers are replaced in the hemocyanin by a single Co(II), exhibits two absorption maxima at 560 (?_Co=250) and 594 nm (?_Co=320 M^?1 cm^?1) and a shoulder near 610 nm, all of which are attributed to a dd transition of high spin Co(II) (S=3/2) with a tetrahedral geometry. The magnetic circular dichroism (MCD) spectrum in this region also suggests the existence of a tetrahedral Co(II) species in the protein. The visible absorption and MCD spectra of octopus 50%-Co(II)Hc are quite similar to those of squid 50%-Co(II)Hc described in the previous paper (S. Suzuki, J. Kino, M. Kimura, W. Mori and A. Nakahara, Inorg. Chim. Acta, 66, 41 (1982)). The formation of half-apo Co(II)Hc demonstrates that the binuclear copper sites in native octopus hemocyanin may differ from each other in coordination geometry, as in other molluscan hemocyanins, squid and snail hemocyanins. The coordination environment of the active-site Co(II) substituted for Cu in the octopus hemocyanin is the same as that of the corresponding active site of the squid hemocyanin.     

Unusual oxygen binding behavior of a 24-meric crustacean hemocyanin

Hemocyanins from Crustacea usually are found as 1 × 6 or 2 × 6-meric assemblies. An exception is the hemocyanin isolated from thalassinidean shrimps where the main component is a 24-meric structure. Our analysis of oxygen binding data of the thalassinidean shrimp Upogebia pusilla based on a three-state MWC-model revealed that despite the 24-meric structure the functional properties can be described very well based on the hexamer as allosteric unit. In contrast to the hemocyanins from other thalassinidean shrimps the oxygen affinity of hemocyanin from U. pusilla is increased upon addition of l-lactate. A particular feature of this hemocyanin seems to be that l-lactate already enhances oxygen affinity under resting conditions which possibly compensates the rather low intrinsic affinity observed in absence of l-lactate. The fast rate of oxygen dissociation might indicate that in this hemocyanin a higher cooperativity is less important than a fast response of saturation level to changes in oxygen concentration.     

Identification and characterization of a hemocyanin-derived phenoloxidase from the crab Charybdis japonica

To determine effective activators of crab hemocyanin (Hc) and the properties of Hc-derived phenoloxidase (HdPO), Hc, for the first time, was purified from hemolymph of Charybdis japonica, and the properties of activated HdPO were studied by using L-DOPA as a substrate. Three distinct subunits were isolated, and each had a molecular mass of about 80, 75 and 70 kDa, respectively. SDS and HLS were much effective in conversion of Hc into HdPO whose PO activity was optimal at pH 7.0 and temperature of 40 °C. The Km value of the HdPO was 2.90 mM for L-DOPA and 7.33 mM for tyrosine. The PO activity of HdPO was most sensitive to 1-phenyl-2-thiourea, cysteine and ascorbic acid, and much sensitive to thio urea and sodium sulfite. Based on its inhibition characteristics and the substrate specificity, this HdPO could be classified as a kind of tyrosinase-type phenoloxidase. The PO activity of HdPO was also strongly inhibited by Cu²⁺, Zn²⁺, ethylenediaminetetraacetic acid (EDTA) and diethyldithiocarbamate (DETC). The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu²⁺ on DETC-inhibited PO activity, indicate that the HdPO is a kind of copper-containing metalloenzyme. All these imply that the Hc, as an oxygen carrier, can be activated to have PO activities by SDS or HLS, and the activated HdPO has the properties of a tyrosinase-type copper-containing phenoloxidase. This study makes us to understand more easily the multifunctions of crustacean Hc in oxygen carrier and melaninization at certain stresses in host defence as well.     

Neurophysiological changes associated with paralysis arising from body stress in the cockroach, Periplaneta americana

Two hours after physical stress, Periplaneta americana could be separated into three behavioural categories: normal to hyperactive; torpid with ataxia; and paralysed. At 2 hr, 68 per cent were either torpid or paralysed, at 20 hr, 83 per cent were paralysed. Weight loss was a distinct physiological symptom of stress paralysis: The calculated mean loss was 9·5 mg/hr for torpid insects and 12·4 mg/hr for paralysed cockroaches, losses were four to six times larger than those occurring in starved cockroaches. However, the haemolymph osrnolarities of the three categories showed no appreciable differences. Only starved and paralysed cockroaches showed a noticeable reduction in muscle fibre membrane potentials of the flexor tibia—a mean value below 40 mV for starved insects and a mean value below 50 mV for paralysed insects. Both of these categories consistently showed a lower amplitude for junctional potentials, but paralysed cockroaches showed a much higher incidence of complete failure to neural stimulation. Most muscle fibres of completely paralysed insects lost their sensitivity to direct extracellular stimulation while the loss in sensitivity was less evident in starved cockroaches. Axonal conduction on the crural nerve was not changed by stress, and the spontaneous efferent activity of completely paralysed insects was similar to the pattern of activity for normal cockroaches. Stress seemed to alter the volume and content of the intermyofibral spaces of muscles.     

The social biology of domiciliary cockroaches: colony structure, kin recognition and collective decisions

A substantial body of research on eusocial insects seen in the last decades has gone hand-in-hand with the development of social evolution theory. In contrast, little attention has been given to the non-eusocial insect species that nevertheless exhibit a rich spectrum of social behaviours, thus effectively skewing our vision of insect sociality. Recent studies on the behaviour, ecology and genetic of “gregarious” cockroaches (Blattodea) have revealed a diversity of social structures and group dynamics unique among insects, providing an important comparative model for the broader understanding of insect social evolution. Here, we present an overview of the social biology of the domiciliary cockroaches (ca. 25 species adapted to human habitats) based on research on two model species, Blattella germanica and Periplaneta americana. We discuss the evolution of these domiciliary cockroaches, considering them in the context of “social herds” within the insect sociality framework.     

Evaluation of Aprostocetus hagenowii (Hymenoptera: Eulophidae) for the control of American cockroaches (Dictyoptera: Blattidae) in sewers and crevices around buildings

The objective of this study was to evaluate the potential of Aprostocetus hagenowii (Ratzeburg) (Hymenoptera: Eulophidae) to control American cockroaches, Periplaneta americana (L.) (Dictyoptera: Blattidae), in sewer manholes and in crevices around buildings. Parasitoids were released weekly for 12 wk from laboratory parasitized heat-killed oothecae, and parasitism monitored using sentinel oothecae of American cockroaches. In addition, preference of A. hagenowii for 1- to 4-wk-old oothecae was evaluated in the laboratory. A. hagenowii females showed no preference for any ootheca age. Twenty of the 30 tested females parasitized one ootheca, whereas the other 10 parasitized two oothecae. The total progeny (males, females, and total) that emerged from a single ootheca parasitized by a female was not significantly different to the total progeny that emerged from two oothecae parasitized by a female. The number of males, females, and total progeny that emerged from the second parasitized ootheca was significantly less than the number that emerged from the first parasitized ootheca. The weekly mean sentinel oothecal parasitism rate in wall crevices was 18.1 +/- 3.2% and in sewer manholes was 13.3 +/- 2.0%. The mean number of released A. hagenowii females per number of parasitized sentinel oothecae recorded in crevices was 189 +/- 18, whereas it was 428 +/- 50 in sewers. A. hagenowii females were more effective at parasitizing sentinel oothecae placed at high and middle levels in manholes than at a low level when releases were made at the midpoint of the manhole shaft.     

Self-association and oxygen-binding characteristics of the isolated subunits of Limulus polyphemus hemocyanin

The hemocyanin of the horseshoe crab Limulus polyphemus is characteristic of arthropod hemocyanins in that it is a high-molecular-weight oligomer composed of functionally and structurally distinct subunits. The protein forms a 48-subunit complex, the largest form of arthropod hemocyanin, whose oxygen-binding characteristics are modulated by subunit interaction within the oligomer. It has previously been shown that a number of electrophoretic isozymes, which are identical immunochemically, are present in dissociated Limulus hemocyanin. In this study it is demonstrated that the electrophoretic differences in the antigenically identical subunits are not reflected in their oxygen-binding and self-assembly properties or in the roles they play in reassembly and function of the 48-subunit native molecule. The chloride-dependent modulation of the oxygen-binding properties of those Limulus subunits which do not self-assemble, as documented here, illustrates that this allosteric effect may be operable at the tertiary level. For each of the purified subunits the effects of pH and calcium ions on oxygen-binding characteristics and self-assembly reactions are reported, and the roles of specific subunits in reassembly of distinct aggregation states are further documented.     

Hemocyte components in crustaceans convert hemocyanin into a phenoloxidase-like enzyme

The functional conversion of hemocyanin (Hc), an oxygen transporter, into an enzyme was investigated in crustaceans. Hc is converted into a phenoloxidase-like enzyme by hemocyte components, which is triggered by beta-1,3-glucan. This activation is severely hampered with leupeptin and E-64 treatment, indicating that the serine/cysteine proteases in the hemocytes are involved in the activation. In a SDS-PAGE-analysis, no change was observed between normal and activated Hc under reduced conditions. However, under non-reduced condition of normal Hc, several minor bands were observed at oligomeric position of Hc subunit, which disappeared upon activation. These results indicate that a split of the reductive bond, such as the disulfide bond between subunits, is essential for Hc activation. This is the first report to show the enzymatic conversion of Hc and the presence of the covalent bond in the Hc subunit of crustaceans.     

Dietary effects of allopurinol and sulfinpyrazone on development, survival, and reproduction of German cockroaches (Dictyoptera: Blattellidae).

Diets containing two gout medications, allopurinol and sulfinpyrazone, were fed ad libitum to first- or second-instar German cockroaches for 15 wk or until 100% mortality was reached. Cockroaches fed greater than or equal to 0.10% allopurinol diets weighed significantly less than those fed the control diet. Mortality of cockroaches fed diets containing greater than or equal to 0.05% allopurinol was significantly greater than those fed the control diet. The LT50 (6.1 wk) of cockroaches fed diets containing 0.10% allopurinol was significantly less than those fed any other diet containing allopurinol. LT50s and slopes were proportional and inversely related, respectively, to percentage of allopurinol in the diet. The addition of sulfinpyrazone to allopurinol diets minimally enhanced the blatticidal nature of the diets. Nymphs fed diets containing greater than or equal to 0.05% allopurinol experienced significant delays in adult emergence. Cockroaches fed greater than or equal to 0.01% allopurinol diets aborted a significantly greater percentage of their oothecae than those fed the 0.001% allopurinol or control diets. Hatched oothecae from cockroaches fed the 0.01% allopurinol diet had significantly fewer nymphs than those fed the 0.001% allopurinol or control diets. Percentage of oothecae aborted and number of nymphs per hatched ootheca from cockroaches fed a 2% sulfinpyrazone diet did not differ significantly from the control.     

Reproduction and development of Blattella asahinai (Dictyoptera: Blattellidae).

The development of the Asian cockroach, Blattella asahinai Mizukubo, was studied under laboratory conditions. Immature development and adult longevity were 67.8 and 103.5 d for females and 65.7 and 48.5 d for males. Females produced an average of 3.7 oothecae, 64.6% of which were viable. Viable oothecae contained an average of 37.5 eggs with an 88% rate of hatch. Females required 13 d after eclosion to produce the first ootheca and 7.9 d between dropping one ootheca and producing the next. Viable oothecae were incubated an average of 19.2 d. Nonviable oothecae were either dropped within 4 d or carried for a period similar to the incubation period of viable oothecae. The estimated lifetime reproductive potential of female Asian cockroaches was 79.6 hatched eggs. This estimate is similar to that of another outdoor species, Blattella vaga Hebard, but approximately one half that of the closely related German cockroach, B. germanica (L.), under similar conditions. Tests in the field and laboratory indicated that chemical control is feasible with application of residual pesticides to lawns and low vegetation where Asian cockroaches are found.     

Oxygen Affects Gut Bacterial Colonization and Metabolic Activities in a Gnotobiotic Cockroach Model

The gut microbiota of termites and cockroaches represents complex metabolic networks of many diverse microbial populations. The distinct microenvironmental conditions within the gut and possible interactions among the microorganisms make it essential to investigate how far the metabolic properties of pure cultures reflect their activities in their natural environment. We established the cockroach Shelfordella lateralis as a gnotobiotic model and inoculated germfree nymphs with two bacterial strains isolated from the guts of conventional cockroaches. Fluorescence microscopy revealed that both strains specifically colonized the germfree hindgut. In diassociated cockroaches, the facultatively anaerobic strain EbSL (a new species of Enterobacteriaceae) always outnumbered the obligately anaerobic strain FuSL (a close relative of Fusobacterium varium), irrespective of the sequence of inoculation, which showed that precolonization by facultatively anaerobic bacteria does not necessarily favor colonization by obligate anaerobes. Comparison of the fermentation products of the cultures formed in vitro with those accumulated in situ indicated that the gut environment strongly affected the metabolic activities of both strains. The pure cultures formed the typical products of mixed-acid or butyrate fermentation, whereas the guts of gnotobiotic cockroaches accumulated mostly lactate and acetate. Similar shifts toward more-oxidized products were observed when the pure cultures were exposed to oxygen, which corroborated the strong effects of oxygen on the metabolic fluxes previously observed in termite guts. Oxygen microsensor profiles of the guts of germfree, gnotobiotic, and conventional cockroaches indicated that both gut tissue and microbiota contribute to oxygen consumption and suggest that the oxygen status influences the colonization success.     

Initial analysis of the hemocyanin subunit type 1 (Hc1 gene) from <Emphasis Type="Italic">Locusta migratoria manilensis</Emphasis>

Hemocyanins are copper-containing (Cu⁺) proteins that transport oxygen in many arthropods hemolymph. We characterized Hc1 gene from the grasshopper species Locusta migratoria manilensis. In particular, we cloned and sequenced the corresponding cDNAs and studied their expression at different developmental stages. The cDNA of Hc1 gene (GenBank accession no.:HQ213937) is 2271 bp in length and the open reading frame is 2016 bp, which encodes a 672 amino acids protein with a calculated molecular mass of 77.9 kD and the isoelectric point of 6.06. Sequence alignment analysis result showed that this gene shares 94.7% identity with Schistocerca americana EHP. In addition, analysis of quantitative RT-PCR indicated that, LmiHc1 was expressed in the embyro (24, 39, 62, 86, 144, and 193 h after hatch), nymphs (1st instar, 2nd instar, 3rd instar, 4th instar and 5th instar) and in adult. These results showed that Hc1 plays an important role in grasshopper, which may be related to an enhanced oxygen supply. Phylogenetic analysis of insecta based on Hc1 are basically consistent with the morphology.