Evaluation of hydrolytic enzyme activities from digestive fluids of Anthonomus grandis (Coleoptera: Curculionidae).

The cotton boll weevil, Anthonomus grandis, is a major pest of cotton crops in South America. In this work, partial biochemical characterizations of (hemi) cellulases and pectinases activities in the digestive system (head- and gut- extracts) of A. grandis were evaluated. Gut extract section from third instar larvae exhibited endoglucanase, xylanase, ß-glucosidase, and pectinase activities. The endoglucanase and xylanase activities were localized in the foregut, whereas ß-glucosidase activity was mainly detected in the hindgut. In addition, no difference in pectinase activity was observed across the gut sections. Thus, A. grandis digestive system is a potentially interesting reservoir for further lignocellulolytic enzymes research.

A differential transcriptional profile by Culex quinquefasciatus larvae resistant to Lysinibacillus sphaericus IAB59 highlights genes and pathways associated with the resistance phenotype.

BACKGROUND: The study of the mechanisms by which larvae of the Culex quinquefasciatus mosquito survive exposure to the entomopathogen Lysinibacillus sphaericus has benefited substantially from the generation of laboratory-selected colonies resistant to this bacterium. One such colony, RIAB59, was selected after regular long-term exposure of larvae to the L. sphaericus IAB59 strain. This strain is characterized by its ability to produce the well known Binary (Bin) toxin, and the recently characterized Cry48Aa/Cry49Aa toxin, able to kill Bin-resistant larvae. Resistance to Bin is associated with the depletion of its receptor, Cqm1 α-glucosidase, from the larvae midgut. This study aimed to identify novel molecules and pathways associated with survival of the RIAB59 larvae and the resistance phenotype. METHODS: A transcriptomic approach and bioinformatic tools were used to compare the profiles derived from the midguts of larvae resistant and susceptible to L. sphaericus IAB59. RESULTS: The RNA-seq profiles identified 1355 differentially expressed genes (DEGs), with 673 down- and 682 upregulated transcripts. One of the most downregulated DEGs was cqm1, which validates the approach. Other strongly downregulated mRNAs encode the enzyme pantetheinase, apolipoprotein D, lipases, heat-shock proteins and a number of lesser known and hypothetical polypeptides. Among the upregulated DEGs, the top most encodes a peroxisomal enzyme involved in lipid metabolism, while others encode enzymes associated with juvenile hormone synthesis, ion channels, DNA binding proteins and defense polypeptides. Further analyses confirmed a strong downregulation of several enzymes involved in lipid catabolism while the assignment of DEGs into metabolic pathways highlighted the upregulation of those related to DNA synthesis and maintenance, confirmed by their clustering into related protein networks. Several other pathways were also identified with mixed profiles of down- and upregulated transcripts. Quantitative RT-PCR confirmed the changes in levels seen for selected mRNAs. CONCLUSIONS: Our transcriptome-wide dataset revealed that the RIAB59 colony, found to be substantially more resistant to Bin than to the Cry48Aa/Cry49Aa toxin, developed a differential expression profile as well as metabolic features co-selected during the long-term adaptation to IAB59 and that are most likely linked to Bin resistance.

Recruited lysosomal enzymes as major digestive enzymes in insects.

The mass recruitment to the midgut contents of lysosomal proteolytic enzymes occurred in insects under three major selective pressures. Hemipteran (true bugs, aphids, and cicadas) ancestors lost their serine peptidases (SP) on adapting to feed on protein-free plant sap. When they returned to protein diets, their cathepsins L and B were recruited to replace their lost SP. Among beetles of the series Cucujiformia, cathepsins L were recruited to hydrolyze ingested plant inhibitors that affect their major SP and/or to deal with special seed proteins, such as prolamins. Larval flies have a very acid middle midgut and use cathepsin D to digest bacteria from their infected food. All the recruited enzymes originated from duplicated genes. The recruited digestive enzymes differ from their lysosomal counterparts in critical regions of their amino acid sequences that resulted in changes in substrate specificities and other kinetic properties. The discharge of digestive cathepsins in the midgut contents, instead of lysosomes, seems to be a consequence of their overexpression or the existence of new targeting signals. Their activation at the midgut contents occurs by an autoactivation mechanism or with the help of other enzymes or by a combination of both. The targeting to lysosomes of the insect lysosomal enzymes does not follow the mammalian mannose 6-phosphate route, but an incompletely known mechanism.

Novel pseudo-aspartic peptidase from the midgut of the tick Rhipicephalus microplus.

The characterization of Rhipicephalus microplus tick physiology can support efforts to develop and improve the efficiency of control methods. A sequence containing a domain with similarity to one derived from the aspartic peptidase family was isolated from the midgut of engorged female R. microplus. The lack of the second catalytic aspartic acid residue suggest that it may be a pseudo-aspartic peptidase, and it was named RmPAP. In this work we confirm the lack of proteolytic activity of RmPAP and investigate it's non-proteolytic interaction with bovine hemoglobin by Surface Plasmon Resonance and phage display. Moreover we carried out RNAi interference and artificial feeding of ticks with anti-RmPAP antibodies to assess it's possible biological role, although no changes were observed in the biological parameters evaluated. Overall, we hypothesize that RmPAP may act as a carrier of hemoglobin/heme between the tick midgut and the ovaries.

Molecular machinery of starch digestion and glucose absorption along the midgut of Musca domestica.

Until now there is no molecular model of starch digestion and absorption of the resulting glucose molecules along the larval midgut of Musca domestica. For addressing to this, we used RNA-seq analyses from seven sections of the midgut and carcass to evaluate the expression level of the genes coding for amylases, maltases and sugar transporters (SP). An amylase related protein (Amyrel) and two amylase sequences, one soluble and one with a predicted GPI-anchor, were identified. Three highly expressed maltase genes were correlated with biochemically characterized maltases: one soluble, other glycocalyx-associated, and another membrane-bound. SPs were checked as being apical or basal by proteomics of microvillar preparations and those up-regulated by starch were identified by real time PCR. From the 9 SP sequences with high expression in midgut, two are putative sugar sensors (MdSP4 and MdSP5), one is probably a trehalose transporter (MdSP8), whereas MdSP1-3, MdSP6, and MdSP9 are supposed to transport glucose into cells, and MdSP7 from cells to hemolymph. MdSP1, MdSP7, and MdSP9 are up-regulated by starch. Based on the data, starch is at first digested by amylase and maltases at anterior midgut, with the resulting glucose units absorbed at middle midgut. At this region, low pH, lysozyme, and cathepsin D open the ingested bacteria and fungi cells, freeing sugars and glycogen. This and the remaining dietary starch are digested by amylase and maltases at the end of middle midgut and up to the middle part of the posterior midgut, with resulting sugars being absorbed along the posterior midgut.

The role of the peritrophic matrix and red blood cell concentration in Plasmodium vivax infection of Anopheles aquasalis.

BACKGROUND: Plasmodium vivax is predominant in the Amazon region, and enhanced knowledge of its development inside a natural vector, Anopheles aquasalis, is critical for future strategies aimed at blocking parasite development. The peritrophic matrix (PM), a chitinous layer produced by the mosquito midgut in response to blood ingestion, is a protective barrier against pathogens. Plasmodium can only complete its life-cycle, and consequently be transmitted to a new host, after successfully passing this barrier. Interestingly, fully engorged mosquitoes that had a complete blood meal form a thicker, well-developed PM than ones that feed in small amounts. The amount of red blood cells (RBC) in the blood meal directly influences the production of digestive enzymes and can protect parasites from being killed during the meal digestion. A specific study interrupting the development of the PM associated with the proteolytic activity inhibition, and distinct RBC concentrations, during the P. vivax infection of the New World malaria vector An. aquasalis is expected to clarify whether these factors affect the parasite development. RESULTS: Absence of PM in the vector caused a significant reduction in P. vivax infection. However, the association of chitinase with trypsin inhibitor restored infection rates to those of mosquitoes with a structured PM. Also, only the ingestion of trypsin inhibitor by non-chitinase treated mosquitoes increased the infection intensity. Moreover, the RBC concentration in the infected P. vivax blood meal directly influenced the infection rate and its intensity. A straight correlation was observed between RBC concentrations and infection intensity. CONCLUSIONS: This study established that there is a balance between the PM role, RBC concentration and digestive enzyme activity influencing the establishment and development of P. vivax infection inside An. aquasalis. Our results indicate that the absence of PM in the midgut facilitates digestive enzyme dispersion throughout the blood meal, causing direct damage to P. vivax. On the other hand, high RBC concentrations support a better and thick, well-developed PM and protect P. vivax from being killed. Further studies of this complex system may provide insights into other details of the malaria vector response to P. vivax infection.

Biochemical Properties of α-Amylase from Midgut of Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) Larvae.

The lesser mealworm, Alphitobius diaperinus (Panzer), is the main insect pest in the poultry industry, thus causing serious damage to production. In this work, the properties of midgut α-amylase from larvae of A. diaperinus were characterized, and its in vitro activity to proteinaceous preparations from different cultivars of common bean (Phaseolus vulgaris) was determined, as well as the amylolitic activity of insects reared on different types of poultry diet. In order to establish some assay conditions, time course and enzyme concentration upon the reaction rate were determined. Product proceeded linearly with time, and the activity was directly proportional to the enzyme concentration. Banding patterns in mildly denaturing electrophoresis showed a single band with apparent molecular weight of 42 kDa. α-Amylase reached optimal temperature at 45°C and pH 5.0 as the optimal one. It maintained 34.6% of the activity after being kept at 60°C for 5 min, and 23%, after 60 min. However, at 80°C, only 14 and 6% remained after 5 and 60 min, respectively. The presence of Ca2+ and Na+ ions decreased the enzyme activity at concentrations higher than 2 and 100 mM, respectively. The activity was significantly inhibited by some proteinaceous extracts from common bean cultivars, and it declined with increasing proteinaceous concentration. No significant difference was observed when the amylolytic activity was determined in A. diaperinus reared on different poultry diets, offered to broilers in the starter, grower, finisher, and layer phases.

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Binding targets of termiticidal lectins from the bark and leaf of Myracrodruon urundeuva in the gut of Nasutitermes corniger workers.

BACKGROUND: Lectins, carbohydrate-binding proteins, from the bark (MuBL) and leaf (MuLL) of Myracrodruon urundeuva are termiticidal agents against Nasutitermes corniger workers and have been shown to induce oxidative stress and cell death in the midgut of these insects. In this study, we investigated the binding targets of MuBL and MuLL in the gut of N. corniger workers by determining the effects of these lectins on the activity of digestive enzymes. In addition, we used mass spectrometry to identify peptides from gut proteins that adsorbed to MuBL-Sepharose and MuLL-Sepharose columns. RESULTS: Exoglucanase activity was neutralized in the presence of MuBL and stimulated by MuLL. α-l-Arabinofuranosidase activity was not affected by MuBL but was inhibited by MuLL. Both lectins stimulated α-amylase activity and inhibited protease and trypsin-like activities. Peptides with homology to apolipophorin, trypsin-like enzyme, and ABC transporter substrate-binding protein were detected from proteins that adsorbed to MuBL-Sepharose, while peptides from proteins that bound to MuLL-Sepharose shared homology with apolipophorin. CONCLUSION: This study revealed that digestive enzymes and transport proteins found in worker guts can be recognized by MuBL and MuLL. Thus, the mechanism of their termiticidal activity may involve changes in the digestion and absorption of nutrients. © 2018 Society of Chemical Industry.

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases.

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Propolis residue inclusion in the diet affects digestive enzyme activity in broiler chickens / A inclusão do resíduo da própolis na dieta afeta a atividade de enzimas digestivas em frangos de corte

This study investigated the effects of the inclusion of solid propolis residue (PR) from alcoholic propolis extraction in the diets of broiler chickens from 1 to 21-d of age on their performance, intestinal morphology, and digestive enzyme activity. 405 male chicks were distributed in a completely randomized design with five treatments (inclusion of 0, 1, 2, 3 and 4% PR in the diets), and three replications with 27 birds each. The birds were fed experimental diets from 1 to 21-d of age and basal diet from 22 to 42-d of age. Feed intake (from 1 to 21-d and 1 to 42-d of age) decreased linearly with increasing levels of PR inclusion (P < 0.05). Dietary inclusion of PR did not affect intestinal morphology at 7 and 21-d of age (P > 0.05). Pancreatic amylase activity presented a quadratic response at 7 and 21-d of age, with its lowest activity estimated at 2.45 and 1.91% PR inclusion, respectively. At 7-d of age, trypsin activity decreased linearly, whereas chymotrypsin activity varied quadratically, with its activity estimated at 2.06% PR inclusion. Intestinal maltase activity varied quadratically with lowest activity predicted at 1.57% PR inclusion at 21-d of age. The dietary inclusion of solid propolis residue of propolis decreases feed intake in broilers and modulates their intestinal and pancreatic enzyme activity.(AU)

Este estudo investigou os efeitos da inclusão do resíduo sólido da extração alcoólica da própolis (PR) em dietas de frangos de corte de 1 a 21 dias de idade no desempenho, morfologia intestinal e atividade de enzimas digestivas. 405 frangos machos foram distribuídos em um delineamento inteiramente casualizado com cinco tratamentos (inclusão de 0, 1, 2, 3 e 4% de PR na ração), e três repetições com 27 aves cada. As aves receberam as dietas experimentais de 1 a 21 dias de idade e dieta basal de 22 a 42 dias de idade. O consumo de ração (1 a 21 dias e 1 a 42 dias) diminuiu linearmente com o aumento de inclusão de PR (P < 0,05). A inclusão de PR não afetou a morfometria intestinal aos 7 e 21 dias de idade (P > 0.05). A atividade da amilase pancreática apresentou resposta quadrática aos 7 e 21 dias de idade (P < 0,05), com menores atividades estimadas ao nível de 2,45 e 1,91% de inclusão de PR, respectivamente. Aos 7 dias de idade, a atividade da tripsina diminuiu linearmente, enquanto a atividade da quimiotripsina variou quadraticamente, com menor atividade estimada com inclusão de 2,06% de PR. A atividade da maltase intestinal variou quadraticamente com menor atividade estimada com inclusão de 1,57% de inclusão de PR aos 21 dias. A inclusão do resíduo sólido da própolis na dieta de frangos de corte diminui o consumo de ração e modula a atividade das enzimas digestivas intestinais e pancreáticas.(AU)

Synchronization to light and mealtime of daily rhythms of locomotor activity, plasma glucose and digestive enzymes in the Nile tilapia (Oreochromis niloticus).

The light-dark cycle and feeding can be the most important factors acting as synchronizers of biological rhythms. In this research we aimed to evaluate synchronization to feeding schedule of daily rhythms of locomotor activity and digestive enzymes of tilapia. For that purpose, 120 tilapias (65.0±0.6g) were distributed in 12 tanks (10 fish per tank) and divided into two groups. One group was fed once a day at 11:00h (zeitgeber time, ZT6) (ML group) and the other group was fed at 23:00h (ZT18) (MD group). The fish were anesthetized to collect samples of blood, stomach and midgut at 4-hour intervals over a period of 24h. Fish fed at ML showed a diurnal locomotor activity (74% of the total daily activity occurring during the light phase) and synchronization to the feeding schedule, as this group showed anticipation to the feeding time. Fish fed at MD showed a disruption in the pattern of locomotor activity and became less diurnal (59%). Alkaline protease activity in the midgut showed daily rhythm with the achrophase at the beginning of the dark phase in both ML and MD groups. Acid protease and amylase did not show significant daily rhythms. Plasma glucose showed a daily rhythm with the achrophase shifted by 12h in the ML and MD groups. These results revealed that the feeding time and light cycle synchronize differently the daily rhythms of behavior, digestive physiology and plasma metabolites in the Nile tilapia, which indicate the plasticity of the circadian system and its synchronizers.

Role of cathepsins D in the midgut of Dysdercus peruvianus.

Hemipteran ancestors probably lost their digestive serine peptidases on adapting to a plant sap diet. On returning to protein ingestion, these insects start using cathepsin (lysosomal) peptidases as digestive enzymes, from which the less known is cathepsin D. Nine of the ten cathepsin D transcribing genes found in Dysdercus peruvianus midgut are expressed exclusively in this tissue and only DpCatD10 is also expressed in other tissues. The main action of cathepsins D is in the first (V1) (from three, V1-3) midgut regions, where 40% of the total proteolytic activity was assigned to aspartic peptidases with an optimum pH of 3.5. The most expressed cathepsins D were identified in the midgut luminal contents by proteomics. The data indicate that D. peruvianus have kept a lysosomal gene expressed in all tissues and evolved another set of genes with a digestive function restricted to midgut. Digestive cathepsins D apparently complement the action of digestive cathepsin L and they are arguably responsible for the hydrolysis of cysteine peptidase inhibitors known to be present in the cotton seeds eaten by the insect, before they meet cathepsin L.

Propolis residue inclusion in the diet affects digestive enzyme activity in broiler chickens / A inclusão do resíduo da própolis na dieta afeta a atividade de enzimas digestivas em frangos de corte

This study investigated the effects of the inclusion of solid propolis residue (PR) from alcoholic propolis extraction in the diets of broiler chickens from 1 to 21-d of age on their performance, intestinal morphology, and digestive enzyme activity. 405 male chicks were distributed in a completely randomized design with five treatments (inclusion of 0, 1, 2, 3 and 4% PR in the diets), and three replications with 27 birds each. The birds were fed experimental diets from 1 to 21-d of age and basal diet from 22 to 42-d of age. Feed intake (from 1 to 21-d and 1 to 42-d of age) decreased linearly with increasing levels of PR inclusion (P 0.05). Pancreatic amylase activity presented a quadratic response at 7 and 21-d of age, with its lowest activity estimated at 2.45 and 1.91% PR inclusion, respectively. At 7-d of age, trypsin activity decreased linearly, whereas chymotrypsin activity varied quadratically, with its activity estimated at 2.06% PR inclusion. Intestinal maltase activity varied quadratically with lowest activity predicted at 1.57% PR inclusion at 21-d of age. The dietary inclusion of solid propolis residue of propolis decreases feed intake in broilers and modulates their intestinal and pancreatic enzyme activity.

Este estudo investigou os efeitos da inclusão do resíduo sólido da extração alcoólica da própolis (PR) em dietas de frangos de corte de 1 a 21 dias de idade no desempenho, morfologia intestinal e atividade de enzimas digestivas. 405 frangos machos foram distribuídos em um delineamento inteiramente casualizado com cinco tratamentos (inclusão de 0, 1, 2, 3 e 4% de PR na ração), e três repetições com 27 aves cada. As aves receberam as dietas experimentais de 1 a 21 dias de idade e dieta basal de 22 a 42 dias de idade. O consumo de ração (1 a 21 dias e 1 a 42 dias) diminuiu linearmente com o aumento de inclusão de PR (P 0.05). A atividade da amilase pancreática apresentou resposta quadrática aos 7 e 21 dias de idade (P < 0,05), com menores atividades estimadas ao nível de 2,45 e 1,91% de inclusão de PR, respectivamente. Aos 7 dias de idade, a atividade da tripsina diminuiu linearmente, enquanto a atividade da quimiotripsina variou quadraticamente, com menor atividade estimada com inclusão de 2,06% de PR. A atividade da maltase intestinal variou quadraticamente com menor atividade estimada com inclusão de 1,57% de inclusão de PR aos 21 dias. A inclusão do resíduo sólido da própolis na dieta de frangos de corte diminui o consumo de ração e modula a atividade das enzimas digestivas intestinais e pancreáticas.

Integrated assessment of biomarker responses and microbiological analysis of oysters from São Luís Island, Brazil.

This study was conducted to evaluate the use of biochemical biomarkers and microbiological analysis to identify levels of oyster contamination at different ports in São Luís Island (Maranhão), Brazil. Oysters were analyzed for total coliforms, thermotolerant coliforms, Escherichia coli and Aeromonas spp. In addition, tissue was removed from the digestive gland to determine the glutathione-S-transferase (GST) and catalase (CAT) activity. The highest percentage of microbiological contamination of oyster samples occurred during the rainy season. The activity of GST and catalase in oysters was also higher in the rainy season, coinciding with the greatest abundance of total and thermotolerant coliforms. Among the prospective biomarkers, GST showed the best results for identification of areas with higher levels of contamination.

Effects of specific antisera targeting peritrophic matrix-associated proteins in the sand fly vector Phlebotomus papatasi.

In many hematophagous insects, the peritrophic matrix (PM) is formed soon after a blood meal (PBM) to compartmentalize the food bolus. The PM is an important component of vector competence, functioning as a barrier to the development of many pathogens including parasites of the genus Leishmania transmitted by sand flies. PM morphology and permeability are associated with the proteins that are part of the PM scaffolding, including several peritrophins, and chitin fibers. Here, we assessed the effects of specific antisera targeting proteins thought to be an integral part of the PM scaffolding and its process of maturation and degradation. Phlebotomus papatasi sand flies were fed with red blood cells reconstituted with antisera targeting the chitinase PpChit1, and the peritrophin PpPer2. Sand fly midguts were dissected at different time points and processed for light microscopy (LM), confocal and transmission electron (TEM) microscopies (24, 42-46, 48 and 72h PBM), scanning electron (SEM) (48h PBM) and atomic force (AFM) (30h PBM) microscopies. TEM and WGA-FITC staining indicate PM degradation was significantly delayed following feeding of flies on anti-PpChit1. AFM analysis at 30h PBM point to an increase in roughness' amplitude of the PM of flies that fed on either anti-PpChit1 or anti-PpPer2. Collective, our data suggest that antibodies targeting PM-associated proteins affects the kinetics of PM maturation, delaying its degradation and disruption and are potential targets on transmission-blocking vaccines strategies.

Degradabilidade ruminal e digestibilidade intestinal dos grãos de soja crus e tostados em bovinos leiteiros / Ruminal degradability and intestinal digestibility of raw and roasted soy beans in dairy cows

Objetivou-se estudar o efeito de diferentes tratamentos térmicos, tempos e procedimentos na degradação ruminal de grãos de soja crus e tostados e sua ação na digestão intestinal da proteína não degradada no rúmen (PNDR) pelo método dos três estágios. Para a degradação ruminal in situ foram pesados cinco gramas de matéria natural em sacos de náilon incubados durante 2; 4; 8; 16; 24 e 48 horas. No tempo zero foi efetuado o mesmo procedimento, excetuando a incubação ruminal. Os resíduos de cada tratamento formaram uma amostra composta para determinar a matéria seca (MS) e proteína bruta (PB). Para a digestibilidade intestinal realizou-se a incubação in situ por 16 horas, usando a técnica dos três estágios. A degradabilidade efetiva da MS com taxa de passagem de 5%/hora para a soja crua (SC) foi de 71,94% e tostada entre 52,23% a 68,78%. Após 16 horas de incubação a PNDR variou de 32,12 a 67,72% e a digestibilidade intestinal de 73,21% a 86,02%. A menor degradação da MS e PB foi da soja tostada a 145oC durante um minuto com steeping (STC1). A digestibilidade intestinal in vitro dos grãos crus foi superior e diferiu dos tostados, exceto a soja tostada a 115oC durante quatro minutos com steeping. A menor degradação proteica foi obtida da STC1de 67,72% da PNDR,52,33% a mais do que à SC. A tostagem dos grãos de soja a 145oC(STC1)contribuiu para uma menor degradabilidade ruminal

This study analyse ruminal degradation the technique of the nylon bags of dry matter ( DM) and crude protein (CP ) and the intestinal digestibility of rumen undegraded protein (RUP) by the method of the three stages of raw and roasted soybeans at different temperatures with and without The in situ ruminal degradation were weighed five grams of natural matter in nylon bags incubated 2; 4;8; 16; 24 and 48 hours. Zero time was made the same procedure, except ruminal incubation. The residue of treatment formed a composite sample to determine the DM and CP. The intestinal digestibility was in situ incubation for 16 hours, using the technique of the three stages. The degradability of DM at a passage rate of 5 % / hour, for raw soybean (RS), was 71.94 % and roasted between 52.23 % and 68.78 %. After 16 hours of incubation(RUP) ranged from 32.12 to 67.72% and the intestinal digestibility of 73.21% to 86.02 %. The lowest degradation of DM and CP was in the roasted soybeans at 145ºC for one minute with steeping (STC1). The in vitro intestinal digestibility of raw grains was higher and differed from the toasted, except for the ones toasted at 115 ºC for four minutes with steeping. For STC1, was obtained the lowest protein degradation of 67.72 % RUP, which 52.33 %, more when compare to RS. The toast of soybeans at 145 ºC (STC1) contributed to a lower ruminal degradability of crude protein.

Degradabilidade ruminal e digestibilidade intestinal dos grãos de soja crus e tostados em bovinos leiteiros / Ruminal degradability and intestinal digestibility of raw and roasted soy beans in dairy cows

Objetivou-se estudar o efeito de diferentes tratamentos térmicos, tempos e procedimentos na degradação ruminal de grãos de soja crus e tostados e sua ação na digestão intestinal da proteína não degradada no rúmen (PNDR) pelo método dos três estágios. Para a degradação ruminal in situ foram pesados cinco gramas de matéria natural em sacos de náilon incubados durante 2; 4; 8; 16; 24 e 48 horas. No tempo zero foi efetuado o mesmo procedimento, excetuando a incubação ruminal. Os resíduos de cada tratamento formaram uma amostra composta para determinar a matéria seca (MS) e proteína bruta (PB). Para a digestibilidade intestinal realizou-se a incubação in situ por 16 horas, usando a técnica dos três estágios. A degradabilidade efetiva da MS com taxa de passagem de 5%/hora para a soja crua (SC) foi de 71,94% e tostada entre 52,23% a 68,78%. Após 16 horas de incubação a PNDR variou de 32,12 a 67,72% e a digestibilidade intestinal de 73,21% a 86,02%. A menor degradação da MS e PB foi da soja tostada a 145oC durante um minuto com steeping (STC1). A digestibilidade intestinal in vitro dos grãos crus foi superior e diferiu dos tostados, exceto a soja tostada a 115oC durante quatro minutos com steeping. A menor degradação proteica foi obtida da STC1de 67,72% da PNDR,52,33% a mais do que à SC. A tostagem dos grãos de soja a 145oC(STC1)contribuiu para uma menor degradabilidade ruminal(AU)

This study analyse ruminal degradation the technique of the nylon bags of dry matter ( DM) and crude protein (CP ) and the intestinal digestibility of rumen undegraded protein (RUP) by the method of the three stages of raw and roasted soybeans at different temperatures with and without The in situ ruminal degradation were weighed five grams of natural matter in nylon bags incubated 2; 4;8; 16; 24 and 48 hours. Zero time was made the same procedure, except ruminal incubation. The residue of treatment formed a composite sample to determine the DM and CP. The intestinal digestibility was in situ incubation for 16 hours, using the technique of the three stages. The degradability of DM at a passage rate of 5 % / hour, for raw soybean (RS), was 71.94 % and roasted between 52.23 % and 68.78 %. After 16 hours of incubation(RUP) ranged from 32.12 to 67.72% and the intestinal digestibility of 73.21% to 86.02 %. The lowest degradation of DM and CP was in the roasted soybeans at 145ºC for one minute with steeping (STC1). The in vitro intestinal digestibility of raw grains was higher and differed from the toasted, except for the ones toasted at 115 ºC for four minutes with steeping. For STC1, was obtained the lowest protein degradation of 67.72 % RUP, which 52.33 %, more when compare to RS. The toast of soybeans at 145 ºC (STC1) contributed to a lower ruminal degradability of crude protein.(AU)

Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites.

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus and Cortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatants of termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensional gel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.

The Effect of Protease Inhibitors on Digestive Proteolytic Activity in the Raspberry Weevil, Aegorhinus superciliosus (Guérin) (Coleoptera: Curculionidae).

The raspberry weevil, Aegorhinus superciliosus (Guérin) (Coleoptera: Curculionidae), is an economically important pest of blueberry in southern Chile. The digestive protease activity of adult insects was investigated using general and specific substrates and inhibitors. Enzymatic assays demonstrated the presence of trypsin- and chymotrypsin-like serine proteinases. Furthermore, in vitro assays using phenylmethylsulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) at 0.01 and 0.1 mM showed percentages of enzymatic inhibition between 0 and 16% for PMSF and 67 to 76% for SBTI, whereas in vivo assays indicated that SBTI caused between 50 and 90% mortality in males and between 80 and 100% in females. Our data indicate the presence of serine proteases and suggest that digestive proteases could be a target for the design and development of strategies to control the raspberry weevil.