Inga vera trypsin inhibitor interferes in the proteolytic activity and nutritional physiology of Ephestia kuehniella larvae

Plant peptidase inhibitors provide plants with a defense strategy to inhibit insect digestive enzymes and have been studied as an alternative strategy for pest control as they interfere in normal insect physiology. We evaluated the effects of ingestion of the trypsin inhibitor from Inga vera Willd. (Fabaceae) seeds on the nutritional and digestive physiology of Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) larvae. Inga vera trypsin inhibitor (IVTI) reduced the efficiency of the conversion of ingested and digested food in these larvae and increased the metabolic cost, causing an anti‐nutritional effect. In both short‐ and long‐term bioassays, the ingestion of IVTI inactivated most of the insect's trypsin activity, but increased chymotrypsin activity as a compensatory response by the insect; however, protein digestion continued to be partially blocked. Consequently, chymotrypsin‐like enzymes, which were over‐produced in the gut, were excreted more into the frass of IVTI‐fed larvae. As such, the resistance of IVTI to hydrolysis by insect midgut proteases resulted in detrimental effects to larvae. These data provide support for the use of IVTI as a biotechnological tool for pest control.     

建鲤和异育银鲫摄食不同质量饲料时的氮收支和能量收支比较

实验探讨了建鲤和异育银鲫摄食低质和高质饲料时氮和能量的收支情况.低质饲料以豆粕为主要蛋白源,饲料蛋白含量为33.91%,高质饲料以鱼粉为主要蛋白源,饲料蛋白含量为45.59%.55d的生长结果显示,氮收支和能量收支受到饲料质量和鱼类种类的显著影响:摄食低质饲料时,建鲤的生长氮和生长能比例显著低于异育银鲫,排泄氮、排泄能和代谢能比例显著高于异育银鲫;摄食高质饲料时,两种鱼的氮收支和能量收支无显著差异;建鲤的氮收支和能量收支受饲料质量的显著影响,摄食低质饲料时,其生长氮和生长能比例均显著低于摄食高质饲料时,而排泄氮、粪能和代谢能比例均显著高于摄食高质饲料时;异育银鲫的氮收支、生长能和代谢能比例不受饲料质量的显著影响.结果表明,在低质饲料条件下,建鲤利用氮和能量的能力弱于异育银鲫,在高质饲料条件下,两种鱼没有显著差异.与异育银鲫相比,建鲤利用氮和能量的能力受饲料质量的影响更为显著.       

Lipid and fatty acid composition of eggs producing larvae with high survival rate in the Japanese eel

Eggs of Japanese eel Anguilla japonica were obtained by artificial induction of maturation. Fertilization and hatching rates, and survival rates of normal larvae were examined. The levels of total lipids and polar lipids (PL) of high quality (HQ; >80% survival rate at 8 days post-hatch) eggs were significantly lower than low quality (LQ; hatching and survival rates were zero) eggs. The docosahexaenoic acid (DHA) level in PL of HQ eggs, however, was significantly higher than in LQ eggs. In contrast to DHA, arachidonic acid level was negatively correlated with egg quality variables and it was lower in HQ than in LQ eggs. Most of the egg lipid and fatty acid components showed larger variation in LQ than in HQ eggs.     

Substrate specificity in the control of digestive enzymes in larvae of the black carpet beetle

When starved larvae of the black carpet beetle, Attagenus megatoma, were fed selected diets, increases in proteolytic, trypsin, and chymotrypsin activity were correlated with total midgut protein and not with the amount of food consumed. Although larvae initially consumed more of a starch diet than of 2 diets that contained added protein, total protease activity in these larvae was minimal. Starch-fed larvae and larvae fed a casein-sucrose diet had a consistently higher level of sucrase activity than larvae fed an all-casein diet. These total results support a secretagogue mechanism for control of digestive enzyme synthesis in insects. In addition, the absence of parallel stimulation of different digestive enzymes by a single substrate (starch) indicated nutrient class specificity in the control of inducible midgut enzymes in this species.     

Trypsin activity and physiological aspects in larval rearing of European sea bass (Dicentrarchus labrax) using live prey and compound diets

The aim of this study was to evaluate the potential of a compound diet as a live prey substitute for feeding European sea bass larvae ( Dicentrarchus labrax L.). The effect of a commercial diet (Nippai ML feed) and live prey ( Artemia nauplii) on tryptic enzyme activity, protein content, growth (standard length) and survival rates of sea bass larvae were tested during a 27-day rearing experiment. Sea bass larvae were divided into two groups. The live food group (control group) was fed exclusively on newly hatched Artemia nauplii (Inve AF grade), the test group was fed exclusively with the compound diet from day 15 onwards. As trypsin has been demonstrated to be a useful indicator for evaluating digestibility of food and the nutritional condition of fish larvae, individual tryptic enzyme activity was determined in both feeding groups. Larvae older than 14 days after hatching and fed on live food showed a significantly higher tryptic enzyme activity than larvae fed the compound diet. A similar relationship between tryptic activity and standard length in both test groups was detected only in small larvae (standard length < 7 mm). The usefulness of proteolytic enzyme activity measurements in larval fish research, as well as its use in aquaculture nutrition research, was confirmed. Protein content, increase in length and survival rates of the sea bass larvae were additionally determined in order to evaluate an influence on the diet. The protein content of larvae fed the Artemia nauplii was higher and the growth of larvae fed the compound diet was reduced. Larval mortality was not affected by the diet given.     

Prey-mediated effects of the protease inhibitor aprotinin on the predatory carabid beetle Nebria brevicollis

To investigate the potential non-target impacts of transgenic pest-resistant plants, prey-mediated impacts of a protease inhibitor (PI) on the predatory carabid, Nebria brevicollis, were investigated. The PI used was aprotinin, a serine PI of mammalian origin with insecticidal properties when incorporated in artificial diet or expressed in transgenic plants. Field-collected N. brevicollis adults, kept at 23 °C, 16:8 L:D, were fed, over their pre-aestivation activity period of 24 days, with Helicoverpa armigera larvae reared on an artificial diet containing 0.5% (w:w, fresh mass) aprotinin. These larvae contained 22.62 μg aprotinin/g insect. Control prey was reared on diet without aprotinin. Beetle survival and body mass were unaffected by prey type. Beetles consuming PI-fed prey lost significantly more mass than the control beetles during two periods of mass loss, but gained significantly more mass during the final period of mass gain. This was not due to differences in amounts of prey supplied or consumed. The final mass gain coincided with increased consumption of PI-prey. Female beetles were significantly heavier than males, but we found no consistent gender-based differences in response to PI-prey. At the end of the experiment, body mass of all beetles was similar to field-collected ones (approximately 55 mg). All experimental beetles had significantly lower activities of digestive cysteine proteases and the serine proteases chymotrypsin and trypsin than field-collected ones. Beetles consuming PI-fed prey had significantly lower levels of trypsin and higher levels of chymotrypsin and elastase than the control beetles.     

Compensatory responses of Samea multiplicalis larvae when fed leaves of different fertilization levels of the aquatic weed Pistia stratiotes

Compensatory responses of caterpillars fed low quality food include increased consumption and utilization of essential nutrients. Information about an insect's responses to nutritional challenges from their host plants could benefit weed biological control efforts in the selection and establishment of new agents. The target weed, Pistia stratiotes L. (Araceae) is a floating aquatic plant that has relatively low nitrogen levels which are further diluted with high water content. Efforts to establish the insect Spodoptera pectinicornis (Hampson) (Lepidoptera: Noctuidae) for biological control of P. stratiotes could benefit by examining the nutritional responses of a similar widely established lepidopteran species, Samea multiplicalis (Guenèe) (Lepidoptera: Pyralidae). Larvae of this species were fed leaves of P. stratiotes plants that had been fertilized (NPK) at high and low rates. The leaves of the fertilized plants had a 4.3-fold increase in nitrogen (dry weight) and a 1.6-fold increase in water content. The results suggest that no compensatory increases occurred in larvae fed leaves from the low fertilized plants as no changes were found in fresh mass consumption or nitrogen utilization efficiency. Consequently, development time from second-third instars to pupation was delayed about 3 days compared with larvae fed the high nitrogen leaves. Furthermore, consumption of nitrogen was only 30% and its accumulation into larval tissues was only 60% compared with the larvae fed the high fertilized leaves. The resulting larvae had both a final biomass and a growth rate that were reduced by 40%. Regardless of plant fertilizer level, the larvae fed at a rate 5–10 times greater than that of similar lepidopteran species consuming either low or high quality diets, suggesting that the S. multiplicalis larvae may be functioning at their biological limit for ingesting food.     

Partial characterization of the proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus, and effects of soybean Kunitz trypsin inhibitor on larval performance

The proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), have been characterized. Both larvae and adults rely on a complex proteolytic system based on at least cathepsin D‐, cathepsin B‐, trypsin‐, chymotrypsin‐, leucine aminopeptidase‐, carboxypeptidase A‐, and carboxypeptidase B‐like activities. All endoproteolytic activities were higher in the anterior section of the gut, whereas the exopeptidases were evenly distributed in the anterior and middle sections, and almost no activity was detected in the posterior section. Gelatin‐containing gels confirmed the spatial organization of the proteolytic digestive process. According to this proteolytic profile, the STI (soybean Kunitz trypsin inhibitor) was tested in vivo to establish its potential as a resistance factor against C. sordidus. Newly hatched larvae fed on diets containing 0.2% (w/w) STI experience lower survival rates and display significant reductions in larval growth. Biochemical analysis carried out on guts of larvae reared on STI‐treated diet showed a reduction of trypsin‐like activity compared to that from larvae fed on control diet. This decrease was compensated with an induction of cathepsin B, whereas cathepsin D, chymotrypsin, and leucine aminopeptidase were not affected. These results are discussed as a basis for selecting appropriate inhibitors to obtain transgenic banana and plantain plants with enhanced resistance to this pest.     

Effects of food quality on trade‐offs among growth,immunity and survival in the greater wax moth Galleria mellonella

The resources available to an individual in any given environment are finite, and variation in life history traits reflect differential allocation of these resources to competing life functions. Nutritional quality of food is of particular importance in these life history decisions. In this study, we tested trade‐offs among growth, immunity and survival in 3 groups of greater wax moth (Galleria mellonella) larvae fed on diets of high and average nutritional quality. We found rapid growth and weak immunity (as measured by encapsulation response) in the larvae of the high‐energy food group. It took longer to develop on food of average nutritional quality. However, encapsulation response was stronger in this group. The larvae grew longer in the low‐energy food group, and had the strongest encapsulation response. We observed the highest survival rates in larvae of the low‐energy food group, while the highest mortality rates were observed in the high‐energy food group. A significant negative correlation between body mass and the strength of encapsulation response was found only in the high‐energy food group revealing significant competition between growth and immunity only at the highest rates of growth. The results of this study help to establish relationships between types of food, its nutritional value and life history traits of G. mellonella larvae.     

Purification and characterization of the western spruce budworm larval midgut proteinases and comparison of gut activities of laboratory-reared and field-collected insects.

Three proteolytic enzymes, trypsin, chymotrypsin, and aminopeptidase-N (APN), were purified from laboratory-reared western spruce budworm, Choristoneura occidentalis [Freeman], larvae. Budworm trypsin exhibited a high degree of substrate specificity, was inactivated by DFP and TLCK, and was inhibited by trypsin inhibitors. The western spruce budworm chymotrypsin hydrolyzed SAAPFpNA and SAAPLpNA, but not SFpNA, SGGFpNA, SGGLpNA or BTpNA. The chymotrypsin was inactivated by DFP, and was inhibited by chymostatin and the chymotrypsin inhibitor, POT-1. Purified budworm chymotrypsin exhibited little BTEE esterolytic activity and was insensitive to inhibition with TPCK. The N-terminal sequence of budworm trypsin, chymotrypsin, and APN were obtained. Similar levels of trypsin and APN gut activities were found in laboratory-reared and field-collected larvae. However, in comparison to laboratory-reared insects, considerably less chymotrypsin activity, and a much higher level of gut carboxypeptidase activity were found in field-collected western spruce budworm larvae.     

Increased food utilization indices and decreased proteolytic activity in Helicoverpa armigera larvae fed sublethal Bacillus thuringiensis‐treated diet

The cotton bollworm Helicoverpa armigera is one of the most devastating insect pests. A set of protease enzymes allows this species to feed on different host plant species. Control measures in agriculture often involve the application of the pathogenic bacterium Bacillus thuringiensis subsp. kurstaki (Btk). In the present study, the effects of sublethal Btk doses are evaluated with respect to the food utilization indices and proteolytic activities of Helicoverpa armigera. Accordingly, the H. armigera larvae are fed with artificial diet containing sublethal Btk doses (LC₅, LC₁₀, LC₁₅, LC₂₀ and LC₂₅) and a Btk‐free diet as control. All but one of the food utilization indices we measured is observed to increase significantly with increasing Btk doses. By contrast, the specific activity of total protease, chymotrypsin and elastase enzymes decrease significantly with an increasing Btk concentration. We conclude that Btk was not toxic to H. armigera larvae and any damage that it causes can be compensated for by H. armigera larvae via various mechanisms. In conclusion, increased nutritional indices in the larvae fed with Btk diet represent an important issue that needs to be considered to avoid the pest establishing Bt resistant populations. Meanwhile, the lack of effect of Btk sublethal concentrations on trypsin enzyme specific activity can bolster this challenge.     

Maternal effects are long‐lasting and influence female offspring's reproductive strategy in the swordtail fish Xiphophorus multilineatus

The adaptive benefits of maternal investment into individual offspring (inherited environmental effects) will be shaped by selection on mothers as well as their offspring, often across variable environments. We examined how a mother's nutritional environment interacted with her offspring's nutritional and social environment in Xiphophorus multilineatus, a live‐bearing fish. Fry from mothers reared on two different nutritional diets (HQ = high quality and LQ = low quality) were all reared on a LQ diet in addition to being split between two social treatments: exposed to a large adult male during development and not exposed. Mothers raised on a HQ diet produce offspring that were not only initially larger (at 14 days of age), but grew faster, and were larger at sexual maturity. Male offspring from mothers raised on both diets responded to the exposure to courter males by growing faster; however, the response of their sisters varied with mother's diet; females from HQ diet mothers reduced growth if exposed to a courter male, whereas females from LQ diet mothers increased growth. Therefore, we detected variation in maternal investment depending on female size and diet, and the effects of this variation on offspring were long‐lasting and sex specific. Our results support the maternal stress hypothesis, with selection on mothers to reduce investment in low‐quality environments. In addition, the interaction we detected between the mother's nutritional environment and the female offspring's social environment suggests that female offspring adopted different reproductive strategies depending on maternal investment.     

Changes in midgut endopeptidase activity of Spodoptera frugiperda (Lepidoptera: Noctuidae) are responsible for adaptation to soybean proteinase inhibitors

The development of transgenic maize plants expressing soybean proteinase inhibitors could reduce the economic damage of one of the major maize pests in Brazil, the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797). We examined the influence of soybean proteinase inhibitors on digestive enzyme properties and development of S. frugiperda larvae. The inhibition of trypsin and chymotrypsin activities in vitro by soybean proteinase inhibitors suggested that either Kunitz (SBTI) or Bowman-Birk (SBBI) would have a potential antimetabolic effect when ingested by insect larvae. However, chronic ingestion of semipurified soybean inhibitors did not result in a significant reduction of growth and development of fall armyworm. Therefore, digestive serine proteinase activities (trypsin and chymotrypsin) of fall armyworm larvae were characterized. The results suggest that S. frugiperda was able to physiologically adapt to dietary proteinase inhibitors by altering the complement of proteolytic enzymes in the insect midguts.     

Protein digestion in larvae of the red oak borer Enaphalodes rufulus

Abstract In the Ozark Mountains of the U.S.A., the red oak borer Enaphalodes rufulus contributes to the destruction of red oaks. To understand nutrient digestion in E. rufulus larvae, digestive proteinases are compared in both larvae fed heartwood phloem and those transferred to artificial diet. The pH of gut extracts is approximately 6.3 in the midgut and foregut and decreases to 5.5 in the hindgut region. The hydrolysis of casein by midgut extracts from E. rufulus larvae fed either artificial diet or phloem from tree sections increases in buffers greater than pH 6.19, with maximum hydrolysis being observed at pH 10.1. Casein zymogram analysis reveals two major proteinase activities in larval midgut extracts of diet‐fed larvae, with molecular masses of approximately 25 and 40–60 kDa, whereas phloem‐fed larvae have proteinase activities corresponding to 40, 45, 60, 80 and >100 kDa. Substrate analysis indicates at least one major trypsin‐like activity in both gut extracts with a molecular mass of >100 kDa, but two chymotrypsin‐like activities of approximately 25 and >200 kDa are found only in diet‐fed larvae. Inhibitors of serine proteinases are most effective in reducing the general proteolytic activity of midgut extracts from larvae fed either food source. The data indicate that serine proteinase inhibitors have the potential to reduce E. rufulus larval damage to oaks. In particular, transgenic technologies incoporating trypsin inhibitors may be effective in reducing protein digestion in phloem‐feeding larvae.     

The suitability of the free-living nematode Panagrellus redivivus as live food for first-feeding fish larvae

The free‐living nematode Panagrellus redivivus has been recommended as a suitable food source for first‐feeding fish. A new technology for mass production of P. redivivus enables fish hatchery operators to rely on an inexpensive, standardized and permanently available live food for first‐feeding fish larvae. The proximate composition, and the fatty acid and amino acid profiles of nematodes mass produced on oat‐based and purified ingredient media were determined. The quality of nematodes was significantly influenced by the culture medium used. The lipid content and fatty acid composition of nematodes could be modified by using lipid‐enriched media. Mass‐produced nematodes were tested on first‐feeding common carp (Cyprinus carpio L.) and whitefish (Coregonus lavaretus) larvae. Carp larvae, grown on nematodes cultured on oat medium enriched with sunflower oil, showed a higher survival rate (87.1%) than the control group fed frozen zooplankton (82.9%) at the end of the 1‐week feeding experiment. Differences in larval mass between the treatments disappeared after subsequent feeding of a dry diet for 2 weeks. Whitefish larvae can be reared exclusively on a dry diet; here, the initial feeding of nematodes had no effect on final biomass and survival of larvae.     

西江鲤仔稚鱼生长及消化酶活性变化

为了探究了鲤(Cyprinus carpio)仔稚鱼的生长过程中主要消化酶活性变化规律, 实验测定了鲤从孵化出膜到40 日龄(日龄, Day after hatching)仔稚鱼期间的生长、可溶性蛋白含量和几种消化酶活性变化。结果显示: 仔稚鱼全长以及体重在15 日龄后增速加快, 特定生长率为14.81%。淀粉酶、脂肪酶、胰蛋白酶, 糜乳蛋白酶、碱性磷酸酶以及氨基肽酶在1 日龄仔鱼体内均能检测到活性。在仔稚鱼发育过程中, 其可溶性蛋白含量先下降后上升。仔鱼摄食前消化酶的活性出现一定的上升, 随着仔稚鱼消化系统发育以及营养方式的转变, 其消化酶活性从3到25 日龄处于不断的变化状态, 而25 日龄到实验结束鲤仔稚鱼的消化酶处于一种相对稳定的状态, 标志着其消化功能趋于完善。根据不同发育时期鲤消化酶活性的变化, 设计有机可腐化的材料制备人工鱼巢以提高人工鱼巢的生态修复功能, 降低饥饿对仔稚鱼成活率的影响。     

Malnutrition may affect common sole (Solea solea L.) growth, pigmentation and stress response: molecular, biochemical and histological implications

In the last decades there have been several evidences that traditionally used live preys like rotifers and Artemia salina have nutritional deficiencies that result in a general decrease of fish health, causing anomalies in the development, in growth and in pigmentation. In this study a partial of total replacement of traditional live preys with preserved copepods that represent the natural food of the larvae was evaluated during Solea solea culture. In this study a positive effect of co-feeding preserved copepods in sole larviculture was observed since larvae fed this diet growth and survived better, showed a better tolerance to captive conditions and had a better response to the final thermal/density stress-test with respect to larvae fed a traditional diet. Morphometric data were fully supported by molecular and biochemical ones. Moreover, liver histological investigations, revealed that the inclusion of preserved copepods in the larval diet was able to improve lipid assimilation. In conclusion, preserved copepods may be considered a suitable food for sole when used as a supplement to the traditional diet based on rotifers and Artemia nauplii.     

Phylloplane bacteria increase the negative impact of food limitation on insect fitness

1. When populations of herbivorous insects increase in density, they can alter the quantity or quality of their food. The impacts of diet‐related stressors on insect fitness have been investigated singly, but not simultaneously. 2. Foliage quantity and quality of red alder, Alnus rubra, were manipulated together with the presence of non‐entomopathogenic phylloplane bacteria to investigate their impacts, singly and in combination, on survival, pupal mass, growth rate, fecundity and egg quality of a cyclic forest insect, the western tent caterpillar, Malacosoma californicum pluviale. 3. Food limitation (half food) had strong negative impacts on all life‐history traits. When the larvae were fed continuously, however, neither ingesting phylloplane bacteria nor eating leaves from damaged branches (induced foliage) affected survival. In the half‐food treatment, ingesting bacteria further increased mortality, while feeding on induced foliage improved survival. 4. Growth rate and pupal mass of both sexes were reduced for larvae with food limitation compared with continuously fed insects and this was exacerbated when the larvae also ate bacteria‐treated leaves. A combination of bacteria and induced foliage also reduced larval growth rate by 5% in the full‐food treatment. 5. Fecundity (eggs per egg mass) was 2.7 times greater in full‐food than in food‐limited treatments but neither phylloplane bacteria nor plant induction had an effect. Insects fed induced foliage produced smaller eggs. Overall, there was no evidence of a three‐way interaction between the three stressors, although there were negative synergistic effects, primarily between food limitation and the ingestion of phylloplane bacteria.