A target protease activity of serpins in insect hemolymph

Two zymogens of the serine enzymes (prophenoloxidase activating enzyme and BAEEase, an enzyme hydrolyzing ethyl ester), which are thought to be components of prophenoloxidase cascade in silkworm (Bombyx mori) plasma, were activated through the action of microbial cell wall components. The two active enzymes of the zymogens were studied with regard to the regulation of their activities by two endogenous serpins (silkworm anti-trypsin and silkworm anti-chymotrypsin).

BAEEase activity was shown to be inactivated by silkworm antitrypsin, whereas the inactivation of prophenoloxidase activating enzyme by either of silkworm antitrypsin and silkworm antichymotrypsin could not be demonstrated under the experimental conditions.     

Flight-correlated activity changes in neurons of the lateral accessory lobes in the brain of the locust Schistocerca gregaria

The study investigates activity changes in neurons of the lateral accessory lobes in the brain of the locust Schistocerca gregaria during wind-elicited tethered flight. Neurons with ascending projections from the ventral nerve cord to the lateral accessory lobes showed flight-associated excitations which were modulated in the flight motor rhythm. Descending neurons with ramifications in the lateral accessory lobes were tonically excited corresponding to flight duration. The onset of wind-elicited responses in the descending neurons preceded the onset of flight motor activity by 22–60 milliseconds. Neurons connecting the lateral accessory lobes with the central body, the anterior optic tubercles, or other brain areas showed a variety of responses including activity changes during flight initiation and flight termination. Activity in many of these neurons was less tightly coupled to the flight situation and often returned to background levels before flight was terminated. Most of the recorded neurons responded, in addition, to stationary visual stimuli. The results suggest that the lateral accessory lobes in the locust brain are integrative links between the central body, visual pathways, and the ventral nerve cord. The possible involvement of these brain areas in flight control is discussed.     

Pollination success in a population of dioecious rain forest trees

Pollination success in female trees was determined for a population of Neolitsea dealbata (R. Br.) Merr., a locally abundant dioecious tree pollinated by small, unspecialized insects in northern Queensland rain forest, Australia. The population consisted of a clustered group of trees with a mean male-to-female distance of 4.5 m and more isolated individuals, including females more than 90 m away from the nearest pollen source. A map of all reproductive trees was produced to determine accurate male-to-female distances. The size of the pollen source available to females was defined as a function of the distance to the nearest ten male trees and their sizes (male neighbourhood index). The rate of pollen movement to females was measured by counting pollen tubes (and the number of tubes per style) in female trees 6 days after the commencement of population flowering. The pollination rate decreased steeply to less than half when the nearest male was only 6.5 m away. Although pollen reached a female 330 m away from the nearest pollen source, only 10% of receptive flowers had been pollinated. The short flowering period (2–3 weeks) combined with the the slow rate of pollen movement means that a large proportion of flowers in isolated trees are unpollinated, confirming an earlier finding that isolated females set fewer fruits than gregarious females. The reliability of pollen transfer to females was determined by quantifying insects and their pollen loads trapped at female trees with a range of male neighbourhood indices. Quantities of insects and pollen were significantly correlated with the size of the male neighbourhood index, indicating a strong density-dependent response by vectors to flowering. Pollen was also collected from insect visitors to non-flowering trees. Females with large male neighbourhood indices received more pollen than non-flowering trees with equivalent male neighbourhood indices. However, when the male neighbourhood indices were small for both female and non-flowering trees, the changces of pollinators encountering female and non-flowering trees were similar, suggesting random movements of pollinators in sparse-flowering sub-populations. The dioecious breeding system, brief, synchronous flowering period, clustered population structure and random, opportunistic foraging behaviour of vectors interacted in a way that reduced reproduction in relatively isolated trees. These results demonstrate a mechanism for differential breeding success between trees in natural populations and emphasize the possible impact of logging regimes on pollen flow between trees. Large interconspecific distances in species-rich environments may have been a factor in the selection for synchronous flowering between trees in outcrossing tree species with generalist insect pollinators.     

Flight metabolism in the African fruit beetle,Pachnoda sinuata

Metabolite concentrations in flight muscles and in abdomen of beetles (Pachnoda sinuata) were measured after various periods of tethered flight and subsequent rest. Three distinct phases of energy metabolism are found in active flight muscles: (1) during the first minutes of flight proline is used as main substrate and concomitantly alanine accumulated as an end product; (2) the second phase is characterized by a large-scale degradation of glycogen; (3) after about 8 min of flight the metabolite levels stabilize, while flight performance appears unchanged. After the termination of flight the preflight proline concentration (70 mol·g^-1 fw) is re-established in less than 60 min, whereas restoration of resting levels of other metabolites requires longer. The pattern of maximal enzyme activities and the respiratory rates of mitochondria with different substrates confirm the significance of proline and carbohydrates as the main fuels of working flight muscles.Abbreviations CS citrate synthetase - Cytox cytochrome c oxidase - EDTA ethylenediaminetetra-acetate - fw fresh weight - GluDH glutamate dehydrogenase - GPT alanine aminotransferase - HOAD hydroxyacyl-coenzyme A dehydrogenase - HPLC high pressure liquid chromatography - ME malic enzyme - PCA perchloric acid - RQ repiratory quotient - TRA triethanolamine     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.     

Two bacteria causing farmer's lung: Fine structure ofThermoactinomyces vulgaris andSaccharopolyspora rectivirgula

Larval cuticle ofHelicoverpa (Heliothis)zea and yeast extract added to a minimal medium (MM) induced germination of conidia ofNomuraea rileyi whereas sterile distilled water or MM alone did not. Yeast extract increased mycelial yield, but when cuticle was added, mycelial yield significantly decreased. Proteases and chitinases ofN. rileyi were only expressed when cuticle was added to the MM.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by US Department of Agriculture.     

东方粉蝶幼虫马氏管的超微构造分化

东方粉蝶幼虫有6条马氏管,每条管可以简易地分为四个部分:直肠导,迴肠纲,黄色段和白色段,所有这四个部分首要细胞的顶面都折叠形成微绒毛(特别是迴肠纲,其顶面进一步折叠形成微道),线粒体几乎延伸到微绒毛顶端。在基部,大量基膜折叠形成细胞内管道,向细胞顶部延伸。在细胞质中,有许多线粒体,糙面内质纲和空泡。每一个细胞的细胞核中有一些散的染色质物质,并且外形呈不规则形。在黄色段首要细胞中,有大量证明是由矿物质沉积形成的电子密集颗粒,及猜疑为微孢子原虫的细胞质囊球。上述每段可能具有的功能会在这报告中讨论。     

Two Clip Domain Family of Serine Proteases and a Serine Protease Homolog from the Fall Webworm, Hyphantria cunea; cDNA Cloning and Characterization

Two types of serine proteases and a serine protease homologue cDNAs were isolated from Hyphantria cunea larvae induced immune response due to an injection of a microorganism through RT‐PCR and cDNA library screening, and their characteristics were examined. The isolated cDNAs are composed 2.1 kb, 2.2 kb, and 2.5 kb nucleotide each, which encoded 388, 390, 580 amino acid residues, and were designated as HcPE‐1, HcPE‐2 and HcPE‐3, respectively. They were revealed as serine proteases or a serine protease homologue with the clip domain through a database search. The deduced amino acid sequence comparison showed high homology of 72‐78% among them. Six Cys residues of the N‐terminal clip domain forming the disulfide bond, Cys residues of the catalytic domain, and Cys residues forming inter‐bridge between clip domain and catalytic domain were also well preserved. Three amino acid residues, His, Asp, and Ser, within the active site were perfectly conserved in HcPE‐2 and HcPE‐3, however, His was replaced with Gln¹⁷⁸ in HcPE‐1. The Arg residues (HcPE‐1, Arg¹³²; HcPE‐2, Arg¹³⁴; HcPE‐3, Arg³²⁵) known as the activation sites by proteolytic cleavage were preserved well in all three types of protein. In case of HcPE‐3, three continuous clip‐like domains existed in the N terminal. As the result of phylogenetic analysis, three clip domain family of protein from H. cunea make groups with arthropod proclotting enzyme precursor. Northern blot analysis showed all three genes were induced through an injection of Escherichia coli, but expression patterns were varied.