Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae)

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites (His109, Asp156, Ser257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp251, Gly274, Gly284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).     

谷蠹生态学特性的研究

谷蠹Rhyzoperthadominica（F．）生态学特性的试验结果表明,温度、湿度、粮食含水量对其各发育历期影响非常显著;食物影响不显著。     

Outdoor flight activity and immigration of Rhyzopertha dominica into seed wheat warehouses

The flight activity of lesser grain borer, Rhyzopertha dominica F. (Coleoptera: Bostrichidae), was monitored at two Foundation seed wheat warehouses during the 2003 and 2004 field seasons, using pheromone‐baited Lindgren funnel traps positioned indoors and outdoors. General stored‐product insect activity was also monitored using unbaited sticky traps positioned inside the warehouses around overhead doors. Pheromone‐baited traps were useful for monitoring R. dominica activity, however insect captures decreased when lures were not changed weekly. Flight peaks were documented in early May and again from September through October, and insect captures inside warehouses correlated with timing of outdoor captures. Multiple regression analyses showed that slightly more than half of the variability in R. dominica captures could be explained by mean ambient air temperature and wind speed during the 2 h preceding sunset. Stored‐product Coleoptera captured on unbaited glue boards around overhead doors included Ahasverus advena, Cryptolestes ferrugineus, R. dominica, Sitophilus oryzae, Tribolium castaneum, Trogoderma variabile, and Typhaea stercorea. Door gaskets significantly reduced the number of insect captures on glue boards placed around the overhead doors, and generally restricted their entry to ground level. These studies demonstrated that outdoor pheromone‐baited traps are effective monitoring tools for determining when grain‐handling facilities are most susceptible to infestation and that exclusion may be an effective component of a pest management program.     

玉米面水分含量对谷蠹生长发育和繁殖的影响

本文研究了玉米面的水分含量(21.33%、19.35%、18.67%、13.64%、10.98%)对谷蠹Rhyzopertha dominica Fabricius生长发育及产卵的影响。结果表明:玉米面的水分含量对谷蠹幼虫的发育历期影响显著,对卵和蛹的发育历期影响不显著;谷蠹卵的孵化率、幼虫和蛹的存活率在玉米面水分10.98%～19.35%的条件下增大,在玉米面水分21.33%条件下下降;谷蠹幼虫龄数也受到玉米面水分含量的影响,玉米面水分19.35%条件下幼虫龄数在3～5间变化,10.98%条件下幼虫龄数在3～8间变化;在试验的5种玉米面水分中,谷蠹只在19.35%和18.67%条件下才产卵,19.35%条件下日均产卵量和产卵期均大于18.67%条件下的相应值。     

Field evaluation of spinosad as a grain protectant for stored wheat in Australia: efficacy against Rhyzopertha dominica (F.) and fate of residues in whole wheat and milling fractions

Abstract  The potential of spinosad as a grain protectant for the lesser grain borer, Rhyzopertha dominica , was investigated in a silo-scale trial on wheat stored in Victoria, Australia. Rhyzopertha dominica is a serious pest of stored grain, and its resistance to protectants and the fumigant phosphine is becoming more common. This trial follows earlier laboratory research showing that spinosad may be a useful pest management option for this species. Wheat (300 t) from the 2005 harvest was treated with spinosad 0.96 mg/kg plus chlorpyrifos-methyl 10 mg/kg in March 2006, and samples were collected at intervals during 7.5 month storage to determine efficacy and residues in wheat and milling fractions. Chlorpyrifos-methyl is already registered in Australia for control of several other pest species, and its low potency against R. dominica was confirmed in laboratory-treated wheat. Grain moisture content was stable at about 10%, but grain temperature ranged from 29.3°C in March to 14.0°C in August. Bioassays of all treated wheat samples over 7.5 months resulted in 100% adult mortality after 2 weeks exposure and no live progeny were produced. In addition, no live grain insects were detected during outload sampling after a 9 month storage. Spinosad and chlorpyrifos-methyl residues tended to decline during storage, and residues were higher in the bran layer than in either wholemeal or white flour. This field trial confirmed that spinosad was effective as a grain protectant targeting R. dominica .     

Molecular cloning and characterization of a midgut chymotrypsin-like enzyme from the lesser grain borer, Rhyzopertha dominica

A cDNA encoding a chymotrypsinogen-like protein in midguts of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) was cloned and sequenced. The 901 bp cDNA contains an 816-nucleotide open reading frame encoding 272-amino acids. The predicted molecular mass and pI of the mature enzyme are 23.7 kDa and 4.64, respectively. The encoded protein includes amino acid sequence motifs that are conserved with 5 homologous chymotrypsinogen proteins from other insects. Features of the putative chymotrypsin-like protein from R. dominica include the serine proteinase active site (His(90), Asp(133), Ser(226)), conserved cysteine residues for disulfide bridges, the residues (Gly(220), Gly(243), Asp(252)) that determine chymotrypsin specificity, and both zymogen activation and signal peptides. A TPCK-sensitive caseinolytic protein (P6) with an estimated molecular mass of 24 kDa is present in midgut extracts of R. dominica and can be resolved by electrophoresis on 4-16% polyacrylamide gels. The molecular mass of this caseinolytic enzyme is similar to that of the chymotrypsin deduced from cDNA. Midgut extracts of R. dominica readily hydrolyzed azocasein and N-succinyl-alanine-alanine-proline-phenylalanine-p- nitroanilide (SAAPFpNA), a chymotrypsin-specific substrate. Properties of the enzymes responsible for these activities were partially characterized with respect to distribution in the gut, optimum pH, and sensitivity toward selected proteinase inhibitors. Optimal activity against both azocasein and SAAPFpNA occurs in a broad pH range from about 7 to 10. Both azocasein and SAAPFpNA activities, located primarily in the anterior midgut region, are inhibited by aprotinin, phenylmethyl sulphonylfluoride (PMSF), and soybean trypsin inhibitor (STI). TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) and chymostatin inhibited more than 60% of SAAPFpNA but only about 10-20% of azocasein activity. These results provide additional evidence for the presence of serine proteinases, including chymotrypsin, in midguts of R. dominica. Arch. Insect Biochem. Physiol. 43:173-184, 2000.Published 2000 Wiley-Liss, Inc.     

Effect of five diatomaceous earth formulations against Tribofium castaneum （Coleoptera： Tenebrionidae）, Oryzaephflus surinamensis （Coleoptera： Silvanidae） and Rhyzopertha dominica （Coleoptera： Bostrychidae）

Laboratory bioassays were conducted to determine the effect of food source on the survival of Tribolium castaneum Herbst, Oryzaephilus surinamensis L. and Rhyzopertha dominica F., after exposure to five diatomaceous earth （DE） formulations： Protect-It, Insecto, Perma-GuardTM, Dryacide and SilicoSec. Adults of these species were exposed to DEs at the rate of 0.5 mg/cm^2 for 1 day on filter paper inside plastic Petri dishes. After exposure, the initial mortality was counted and live individuals of the three species were held for a week in glass vials containing 50 mg wheat flour, rice and whole wheat, respectively. In the second experiment, after 1 day exposure to DEs, beetles were transferred to Petri dishes without food and held for a week to determine if the presence of food source would decrease the mortality of beetles. Experiments were carried out at 27℃ and 55% RH in the dark. The initial mortality in both of the experiments reached 100% for the three species exposed to Protect-It and in the case ofR. dominica and O. surinamensis exposed to Dryacide. In contrast, low level of mortality （〈 10%） was observed for T. castaneum exposed to Perma-GuardTM and Insecto. The mortality after the post-treatment period on food was decreased for the three species exposed to Perma-GuardTM and in the case of T. castaneum and R. dominica exposed to Insecto and SilicoSec. Adults of O. surinamensis were the most susceptible followed by R. dominica and 100% adult mortality was obtained, whereas T. castaneum were the least susceptible beetles to DEs. Protect-It and Dryacide were the most efficient DE formulations and can be used effectively in a stored grain integrated pest management program.     

Potential host affinities for the lesser grain borer, Rhyzopertha dominica: behavioral responses to host odors and pheromones and reproductive ability on non-grain hosts

Behavioral responses of male and female Rhyzopertha dominica L. (Coleoptera: Bostrichidae) to odors from pulverized wheat seeds, peanuts, cowpeas, potato tubers, acorns, and twigs from cedar and pine were compared in dual‐choice, still‐air bioassays. We investigated the reproductive success of R. dominica on five of the seven plant tissues (wheat, peanuts, cowpeas, dried potato tubers, and acorns). A field experiment was also conducted to investigate responses of dispersing R. dominica to semiochemicals emitted by live males placed on different plant species. Results showed that both sexes of R. dominica responded to plant volatiles, but responses were strongest to seeds of wheat, a plant species judged to be most suitable for beetle development due to the number of progeny produced on the plant species. Similarly, insects reared on wheat were heavier than those reared on less suitable materials. In general, behavioral responses by males to plant volatiles were faster than responses by females. Responses of conspecifics to aggregation pheromones produced by males feeding on different host materials were skewed toward females although both sexes were attracted. Male R. dominica feeding on wheat recruited more conspecifics than beetles feeding on less suitable hosts (acorns, cowpeas, peanut, and potato tubers).     

Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae)

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-α-benzoyl- -arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl- -leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23–24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK.Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77–81% sequence identity. The three encoded trypsinogens shared 54–62% identity in their amino acid sequences and had 16–18 residues of signal peptides and 12–15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG^62–65, the catalytic amino acid triad of serine proteinase active sites (His¹⁰⁹, Asp¹⁵⁶, Ser²⁵⁷), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp²⁵¹, Gly²⁷⁴, Gly²⁸⁴) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys¹⁵⁸ near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).     

A comparison of some effects of phosphine, hydrogen cyanide and anoxia in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae)

The effects of phosphine, hydrogen cyanide and anoxia on levels of ATP, pyruvate and lactate in Rhyzopertha dominica are compared. The effect of phosphine on anaerobiosis is not directly comparable either with HCN or anoxia. Reduction of catalase by feeding 3 amino 1,2,4 triazole does not enhance the toxicity of phosphine in treated insects.     

Effect of plant lectins on larval development of the legume pod borer, Maruca vitrata

The legume pod-borer Maruca vitrata (Fabricius), [Lepidoptera: Pyralidae] is a major constraint restricting increased cowpea production in tropical Africa and Asia. Since lectins are known to have insecticidal properties against several pests, a survey was undertaken to screen for the effects of 25 lectins from 15 plant families on the development of Maruca pod borer (MPB) larvae. The list included 8 galactose/N-acetylgalactosamine-, 7 mannose-, 5 complex glycan-, 2 sialic acid- and 3, N-acetylglucosamine-specific lectins. Feeding bioassays using artificial diet were carried out at 2% (w/w) topical levels. Although a total of 16 lectins had detrimental effects pertaining either to larval survival, weight, feeding inhibition, pupation, adult emergence and/or fecundity, only the Listera ovata agglutinin (LOA) (Orchidaceae) and Galanthus nivalis (Amaryllidaceae) agglutinin were effective against MPB larvae for all six parameters examined. Larval mortality and feeding inhibition caused by the most active lectin (LOA) was above 60%.     

Free flight capacity determination in a sustained flight tunnel: effects of age and sexual state on the flight duration of Prostephanus truncatus

Abstract. . Adult Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) steering anemotactically upwind to a pheromone source in a wind tunnel, respond normally to, and alter their ground speed according to the direction and speed of, the movement of ground patterns beneath them. By manipulating this optomotor reaction component of their behaviour, studies were conducted on their flight duration. The results show a great deal of individual variation in flight duration, and the general data on single flights were skewed towards short flights. Pre-reproductive and inter-reproductive male and female beetles flew significantly longer than older beetles that had passed their peak of reproduction. There was no significant difference in the flight duration of male and female beetles at any age. The overall results suggest mat younger P. truncatus are capable of long-distance flights, and support the proposition that flight could be important in the spread of the beetle.     

寄主植物与温度对大豆食心虫滞育期间糖类和脂质含量的影响

【目的】本文探究了大豆食心虫Leguminirora glycinioorella(Mats.)Obraztsov进入滞育以后体内糖类、脂质和水分含量的变化,以及不同寄主植物和滞育诱导期温度对大豆食心虫体内糖类、脂质和水分含量的影响。【方法】通过蒽酮硫酸法和氯仿甲醇分离法测定了滞育前后、以大豆Glycine max(L.)Merr和野大豆Glycine soja Sieb.et Zucc.两种植物为寄主以及不同滞育诱导温度下的大豆食心虫体内脂质、水分、总糖、糖原和海藻糖的含量,结果利用SPSS17.0数据分析软件进行分析。【结果】进入滞育阶段的大豆食心虫体内脂质、总糖、海藻糖和糖原含量显著增加,含水量显著下降。以大豆为寄主的大豆食心虫在滞育阶段体内水分含量、总糖和糖原含量较野大豆寄主的高,海藻糖含量较野大豆的低,差异均显著。不同滞育诱导温度下的大豆食心虫体内生化物质含量基本一致。【结论】滞育前后大豆食心虫体内各种生化物质变化明显,以栽培大豆为寄主和以野大豆为寄主的大豆食心虫体内生化物质含量不同,而滞育诱导温度对大豆食心虫体内生化物质含量影响不大。     

The effects of prolonged food deprivation on the covering behavior of the sea urchins Glyptocidaris crenularis and Strongylocentrotus intermedius

In this study, we investigated effects of 24 weeks of food deprivation on covering behavior of Glyptocidaris crenularis and Strongylocentrotus intermedius. Neither G. crenularis nor S. intermedius significantly reduced covering behavior during both short- and long-term food deprivation. However, G. crenularis and S. intermedius had significantly different behavioral patterns over the 24 weeks. Covering behavior of G. crenularis greatly increased from the first to the second week, with decreasing reaction time and increasing covering ability, while the covering behavior of S. intermedius generally remained unchanged during the experiment of 24 weeks. As prolonged food deprivation does not significantly affect covering behavior of G. crenularis and S. intermedius, the present study highlights the innate nature and biological significance of covering behavior in sea urchins, providing new insights into its behavioral mechanism.     

Effects of Schistocephalus solidus (Cestoda) on the food intake and diet of the three-spined stickleback, Gasterosteus aculeatus

Seasonal differences were detected between uninfected sticklebacks and those infected with Schistocephalus solidus in their stomach fullness and diet composition.     

Effects of food deprivation and high fat diet on immunoreactive dynorphin A(1–8) levels in brain regions of Zucker rats

The levels of immunoreactive dynorphin A(1–8) (ir-DYN8) were measured in discrete brain regions of lean Zucker rats subjected to food deprivation for 72 hr and to a high fat diet, and in fatty Zucker rats after food deprivation for 72 hr. Fatty rats showed higher concentrations of ir-DYN8 in the cortex and midbrain, when compared to lean rats fed a stock diet ad lib. Food deprivation increased ir-DYN8 levels in the cortex of lean rats and fatty rats and in the hippocampus of fatty rats, but decreased its content in the striatum of lean rats and in the midbrain of fatty rats. The high fat diet increased ir-DYN8 levels in the cortex and midbrain of lean rats. These results suggest that ir-DYN8 levels in extrahypothalamic structures of Zucker rats could be differentially modified under conditions of hereditary obesity and dietary manipulations.     

Effect of diet and feeding history on flight of Colorado potato beetle, Leptinotarsa decemlineata

We evaluated the hypothesis that Colorado potato beetle (Leptinotarsa decemlineata Say) (CPB) flight frequency is related to diet, and that it changes with duration of food unavailability or exposure to poor quality food by exposing adult overwintered and summer CPB populations to an acceptable host plant (conventional foliage), a poor host (insect resistant transgenic foliage expressing Bacillus thuringiensis tenebrionis[Btt] Cry3a toxin) and no host. Exposure to poor host and no host treatments (with or without water) decreased mean daily flight frequencies and the overall number of overwintered CPB flying, but increased the mean daily flight frequency and number of summer population CPB that flew. Overwintered CPB did not react to an absence of plants at emergence whereas summer CPB increased mean daily flight frequencies when plants and water were not available. The flight response to insect resistant foliage was similar to that for starvation treatments in both populations indicating that flight may not be triggered by Btt toxins but by starvation brought on by feeding on poor quality food. Flight was observed in all treatments for the duration of the test with two exceptions; overwintered beetles fed insect resistant foliage ceased flying after day 17 and summer beetles starved without water ceased after day 8 of a 29‐day study.     

The effects of long-term food deprivation on respiration and haematology of the neotropical fish Hoplias malabaricus

Growth of adult traíras Hoplias malabaricus ceased and body mass ( M ) decreased during starvation periods of 30, 60, 90, 150, 180 and 240 days. Hepatic reserves were mobilized in fish starved for 30 days, but liver mass of fish starved for longer periods was not significantly different from those starved for 30 days. Perivisceral fat bodies were consumed gradually, being completely exhausted after 240 days of food deprivation. Length of starvation was associated with a significant decrease in the oxygen uptake ( V o₂). In spite of this reduction, the respiratory frequency ( f _R) was kept nearly constant during the starvation periods. The haematocrit and the number of red blood cells decreased after 150 and 240 days of starvation, respectively. These parameters did not recover after refeeding (after 90 and 240 days of starvation). This hypometabolic state in response to food deprivation contributed to energy conservation during these periods. Traíras can survive food deprivation for periods of up to 180 days without reductions in metabolism and when they do become hypometabolic, normal metabolic rates are rapidly restored upon refeeding.