Transgenic microalgae expressing <Emphasis Type="Italic">Escherichia coli</Emphasis> AppA phytase as feed additive to reduce phytate excretion in the manure of young broiler chicks

Microbial phytases are widely used as feed additive to increase phytate phosphorus utilization and to reduce fecal phytates and inorganic phosphate (iP) outputs. To facilitate the process of application, we engineered an Escherichia coli appA phytase gene into the chloroplast genome of the model microalga, Chlamydomonas reinhardtii, and isolated homoplasmic plastid transformants. The catalytic activity of the recombinant E. coli AppA can be directly detected in the whole-cell lysate, termed Chlasate, prepared by freeze-drying the transgenic cell paste with liquid nitrogen. The E. coli AppA in the Chlasate has a pH and temperature optima of 4.5 and 60°C, respectively, which are similar to those described in the literature. The phytase-expressed Chlasate contains 10 phytase units per gram dry matter at pH 4.5 and 37°C. Using this transgenic Chlasate at 500 U/kg of diet for young broiler chicks, the fecal phytate excretion was reduced, and the iP was increased by 43% and 41%, respectively, as compared to those of the chicks fed with only the basal diet. The effectiveness of the Chlasate to break down the dietary phytates is compatible with the commercial Natuphos fungal phytase. Our data provide the first evidence of functional expression of microbial phytase in microalgae and demonstrate the proof of concept of using transgenic microalgae as a food additive to deliver dietary enzymes with no need of protein purification.     

Fitness consequences of artificial diets with different macronutrient composition for the predatory bug Orius majusculus

The commercial production of Orius spp. (Hemiptera: Anthocoridae), including Orius majusculus (Reuter), relies on the use of eggs of Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) as rearing diet. However, E. kuehniella eggs have become an expensive fodder thus increasing the price of these key biological control agents. The use of artificial diets potentially decreases the production costs. In this regard, establishing a link between dietary composition and fitness could advance the development of an optimum alternative artificial diet for these predatory insects. The aim of the current study was to test the effect of six artificial diets with different macronutrient composition on the development and reproductive fitness of O. majusculus when compared with the effect of E. kuehniella eggs. In general, nymphal survival was not affected by diet, whereas development was slightly delayed on artificial diets. However, female body mass and fecundity were significantly lower on all of the artificial diets compared with the E. kuehniella eggs diet, suggesting that artificial diets were of inferior quality. Within artificial diets, females fed the viable diet with highest content in lipid laid more eggs than those raised on the most protein-rich diets. We found there was some variation in carcass composition between the O. majusculus fed the various diets, but these variations did not match the differences found in the fitness parameters measured.     

Colour intensity in angelfish (Pterophyllum scalare) as influenced by dietary microalgae addition

A study was conducted to determine the effects of increasing the levels of dietary carotenoid‐rich microalgae biomass on the skin colouration of angelfish (Pterophyllum scalare). A natural microalgae product characterised by a high content (approximately 5% w/w) of the carotenoid astaxanthin was included in a basal diet at 0.25, 0.50, 1.00 and 2.00 g per 100 g as a substitute for wheat flour. Each diet was fed at 3% of live weight to three replicates of 10 fish each for a period of 28 days. Final weights of replicates were determined as indications of growth. Further, non‐invasive techniques were applied to evaluate skin colour. Whole body images of individual fish were processed using specialised computer software to measure the different components of the RGB, HSV and CIE L*a*b* colour evaluation systems, which were also assessed by three independent judges familiar with the rearing and trade of ornamental fishes. No significant influence of the microalgae addition was detected (P > 0.05) on the final live weight of the fish. The colour components Red, Hue, Value and L* were not linearly related (P < 0.05) to dietary microalgae inclusion levels. Where related (Green, Blue, Saturation, a*, b*), correlation coefficients were 0.66 at most. Second‐order polynomial regression models showed that maximum or minimum values for colour measurements were frequently outside the range (0.00–2.00 g per 100 g) of the microalgae inclusion levels applied. When visual evaluation (the most common assessment approach in practice) was used, the judges clearly confirmed that the colour changes were directly related to increasing levels of microalgae inclusion.     

Bio‐safety evaluation of Cry1Ac,Cry2Ab,Cry1Ca,Cry1F and Vip3Aa on Harmonia axyridis larvae

Dietary exposure studies are initial steps in environmental risk assessments of genetically engineered plants on non‐target organisms. These studies are conducted in the laboratory where surrogate species are exposed to purified and biologically active insecticidal compounds at higher concentrations than those expected to occur in transgenic crops foliage. Thus, dietary exposure (early tier) tests provide robust data needed to make general conclusions about the susceptibility of the surrogate species to the test substance. For this, we developed suitable artificial diet and used it to establish a dietary exposure test for assessing the toxicity of midgut‐active insecticidal compounds to the larvae of the Asian ladybird beetle Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). Using boric acid as a model compound, we validated the bioassay established for H. axyridis larvae. An artificial diet containing boric acid which negatively affected survival, development and adult weights was offered to larvae and indicated that the bioassay was able to detect toxic effects of insecticidal substances incorporated in diets. Using this dietary exposure test, environmental risk assessment of Cry1Ac, Cry2Ab, Cry1Ca, Cry1F and the non‐Cry protein Vip3Aa was evaluated by analysing pupation rates, adult emergence rates, 7‐day larval weights, and freshly emerged male and female weights among the toxin treatments and a pure artificial diet. These life‐table parameters did not vary among artificial diets containing 200 μg/g Bt proteins or pure artificial diet. In contrast, boric acid adversely affected all life‐table parameters. Thus on these bases, we concluded H. axyridis larvae are not sensitive to these Bt proteins expressed in genetically engineered crops.     

Diatom artificial medium (DAM): a new artificial medium for the diatom <Emphasis Type="Italic">Haslea ostrearia</Emphasis> and other marine microalgae

Artificial media are used in physiological studies of microalgae to maintain consistent conditions from one experiment to another and these media must be adapted to the needs of the organism studied. The artificial medium, in this case named diatom artificial medium (DAM), was designed to maintain long-term cultures of Haslea ostrearia and 19 other planktonic microalgae, and to allow physiological studies related to metal metabolism. The biomass and biochemical composition of H. ostrearia grown in the DAM and in a modified Provasoli medium were compared to assess the suitability of this new artificial medium for the culture of this diatom. The DAM provided sufficient nutrients to allow H. ostrearia to grow as efficiently as in the enriched seawater medium, without negative impact on metabolism. The DAM was tested with 19 other microalgae in order to widen its potential use, and 18 of the 19 showed a good adaptation to this medium. The chemical speciation of metals (Cd, Cu, Pb, Zn) was assessed using a speciation mathematical model. The presence of EDTA resulted in the total complexation of the trace metals implying that they were present in a sole chemical species in the DAM.     

Utilization of α-starch in ayu, Plecoglossus altivelis, relating to growth and body composition

To examine the possibility of dietary α‐starch in reducing feed costs in a practical diet, α‐starch was supplemented at 10, 20, 30 and 40% in a composed diet having the same protein level. The four diets were fed to ayu, Plecoglossus altivelis (initial weight 9.1 g) for 43 days. Growth and feed efficiency increased with the supplement, with values highest in the 30–40%α‐starch diet. The level of dietary α‐starch did not affect the proximate muscle composition; although the hepatosomatic index was not affected, liver glycogen increased with increasing dietary α‐starch. The dietary α‐starch did not influence evacuation time from the gut, and was well digested through passage in the gut, mainly between the stomach and the anterior part of the intestine. Ayu have an ability to adapt their metabolism to high dietary α‐starch, and can digest 40% or more in a composed diet. Although the muscle lipid content did not change, the fatty acid composition was influenced by dietary starch. With the elevation of dietary starch, a decrease of C18:2n‐6 and an increase of C22:6n‐3 occurred. These results indicate that at least 40%α‐starch can be used in practical diets for ayu.     

Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci

In insect reverse genetics, dietary delivery of interfering RNAs is a practical approach in nonmodel species, such as thrips, whose small size, and feeding behavior restricts the use of other delivery methods. In a laboratory context, an unsuitable diet could confound the interpretation of an RNA interference (RNAi) phenotype, however well-formulated artificial diets can minimize experimental variability, reduce the need for insect handling, and can further be used for roles, such as delivering double-strand RNA (dsRNA)-expressing recombinant bacteria. In this study, artificial diets for oral delivery of dsRNA were developed for two important pest thrips species, western flower thrips (Frankliniella occidentalis) and onion thrips (Thrips tabaci), with the goal of (a) stimulating feeding behavior, (b) supporting optimal growth rates of dsRNA-expressing symbiotic bacteria, and (c) nutritionally supporting the thrips for sufficient periods to observe RNAi phenotypes. The efficacy of artificial diets for ingesting “naked” dsRNA or dsRNA-expressing symbionts and dsRNA delivery via host plant uptake was evaluated. Compared with previously published diet formulations, new combinations based on tryptone, yeast, and soy were superior for enhancing feeding and longevity. However, simply adding “naked” dsRNA to an artificial diet was an unreliable form of RNAi delivery in our hands due to dsRNA degradation. Delivery via host plants was more successful, and the new diet formulation was suitable for symbiont-mediated dsRNA delivery, which we believe is the most convenient approach for large-scale knockdown experiments. This study, therefore, provides alternative methodologies for thrips rearing, dietary RNAi delivery, and insights into the challenges of performing dietary RNAi in nonmodel insects.     

Comparison of different artificial neural network architectures in modeling of Chlorella sp. flocculation

Biodiesel production from microalgae feedstock should be performed after growth and harvesting of the cells, and the most feasible method for harvesting and dewatering of microalgae is flocculation. Flocculation modeling can be used for evaluation and prediction of its performance under different affective parameters. However, the modeling of flocculation in microalgae is not simple and has not performed yet, under all experimental conditions, mostly due to different behaviors of microalgae cells during the process under different flocculation conditions. In the current study, the modeling of microalgae flocculation is studied with different neural network architectures. Microalgae species, Chlorella sp., was flocculated with ferric chloride under different conditions and then the experimental data modeled using artificial neural network. Neural network architectures of multilayer perceptron (MLP) and radial basis function architectures, failed to predict the targets successfully, though, modeling was effective with ensemble architecture of MLP networks. Comparison between the performances of the ensemble and each individual network explains the ability of the ensemble architecture in microalgae flocculation modeling.     

Seasonal variation in prey preference,diet partitioning and niche breadth in a rich large carnivore guild

Large carnivore community structure is affected by direct and indirect interactions between intra-guild members. Co-existence between different species within a carnivore guild may occur through diet, habitat or temporal partitioning. Since carnivore species are highly dependent on availability and accessibility of prey, diet partitioning is potentially one of the most important mechanisms in allowing carnivores to co-exist. Intra-guild interactions may vary over time as carnivore prey preference and diet overlap can change due to seasonal changes in resource availability. We conducted scat analysis to compare the seasonal changes in prey preference, diet partitioning and niche breadth of four large carnivore species, namely leopard Panthera pardus, spotted hyena Crocuta crocuta, brown hyena Parahyaena brunnea and wild dog Lycaon pictus in central Tuli, Botswana. Large carnivores in central Tuli display a high dietary overlap, with spotted hyena and brown hyena displaying almost complete dietary overlap and the other carnivore species displaying slightly lower but still significant dietary overlap. Dietary niche breadth for both hyena species was high possibly due to their flexible foraging strategies, including scavenging, while leopard and wild dog showed a relatively low niche breadth, suggesting a more specialised diet. High dietary overlap in central Tuli is possibly explained by the high abundance of prey species in the area thereby reducing competition pressure between carnivore species. Our research highlights the need to assess the influence of diet partitioning in structuring large carnivore communities across multiple study sites, by demonstrating that in prey rich environments, the need for diet partitioning by carnivores to avoid competition may be limited.     

Similarities,differences, and seasonal patterns in the diets ofEulemur rubriventer andEulemur fulvus rufus in the Ranomafana National Park,Madagascar

I studied the diets of two sympatric species ofEulemur (E. fulvus rufus andE. rubriventer) in the Ranomafana National Part in southeastern Madagascar from July 1988 through August 1989. Both species were highly frugivorous throughout the year and devoted similar amounts of time to feeding daily; the composition of their diets were similar. Three aspects of both lemur species' diets were correlated with seasonal food availability: changes in interspecific dietary overlap, the length of feeding bouts, and the amount of time devoted to feeding daily. Throughout the year neither species had a more diverse or more folivorous diet than its sympatric congener. However, there were several consistent year-round differences between their diets.E. f. rufus initiated more feeding bouts daily that were shorter in duration than those ofE. rubriventer. E. f. rufus also consistently exploited more unripe fruits and mature leaves thanE. rubriventer did throughout the study.E. f. rufus migrated from the study site during a period of fruit scarcity. These dietary patterns are discussed here in regard to feeding patterns observed in other sympatric congeneric primate species, whether these dietary differences can contribute to these two species' ability to coexist, and how differences in diet may be influenced by differences in social structure. I suggest that subtle, consistent differences in diet and seasonal dietary differences are sufficient to allow these two species to coexist.     

Mitochondrial activity,hemocyte parameters and lipid composition modulation by dietary conditioning in the Pacific oyster Crassostrea gigas

Several parameters can affect membrane lipid composition in bivalves, including diet. Although two fatty acids (FA) 22:6n-3 and 20:5n-3 are essential membrane components, they are sparingly synthesized by bivalves and must be obtained from their diet. Here, effects of dietary modifications of membrane lipid composition were studied at both cellular and subcellular levels in the oyster Crassostrea gigas. To this end, we compared oysters fed two monoalgal diets that differed markedly in their FA composition and a mix of both. As expected, algae impacted phospholipids, in particular 22:6n-3 and 20:5n-3, reflecting differences of dietary microalgae FA composition. Meantime, total saturated FA, total monounsaturated FA, total polyunsaturated FA and total non-methylene-interrupted FA varied little and phospholipid class composition was only slightly affected by diets. Measures made in hemocytes indicated that only mitochondrial membrane potential was affected by diets. Total ROS production as well as mitochondrial superoxide production did not differ with diet. There was no difference in phosphorylating (state 3) and non-phosphorylating (state 4) rates of oxygen consumption rates or in cytochrome c oxidase activity of mitochondria isolated from gills between the three diets. Similarly, neither cytochromes a, b, c or c ₁ content nor citrate synthase activities were changed, suggesting that number and morphology of mitochondria were not affected by dietary treatment. These results suggest that oysters could possess high homeostatic capabilities, at both cellular and subcellular levels, to minimize the effect of dietary FA and related membrane lipid FA modifications on mitochondrial functions. These capabilities could be a means to face variations in diet composition in their natural environment and to preserve important oyster physiological functions such as growth and reproduction.     

Potassium chloride deters Lygus hesperus feeding behavior

A series of bioassays were conducted to determine the response of adult western tarnished plant bugs, Lygus hesperus Knight (Heteroptera: Miridae), to artificial diets containing potassium chloride (KCl). We first examined the feeding behavior of L. hesperus by direct observation in a no‐choice diet feeding arena. We observed a total of 22 Lygus feeding events lasting an average of 411 ± 64 s on the control artificial diet and only three feeding events lasting an average of 11 ± 3 s on the KCl‐treated diet. We then conducted several multiple diet choice bioassays to determine the feeding response of L. hesperus when exposed simultaneously to five artificial diet treatments containing different amounts of KCl. For the first bioassay, we used standard clear parafilm diet packets and for the second bioassay we used dark green parafilm diet packets to hold the various diet treatments. Regardless of the diet packet color, L. hesperus overwhelmingly selected the 0% KCl diet treatment over diets containing 3, 6, 9, or 12% KCl. The third and fourth multiple diet choice bioassays were identical to the first bioassay, except that concentrations of the KCl‐treated diets were reduced. Lygus hesperus consistently selected the control diet over all diets containing more than 0.5% KCl. However, when the concentration of KCl in the diet was reduced to ≤0.4%, there were no significant differences in feeding activity exhibited by L. hesperus. Finally, to determine if the addition of KCl to the diet influenced their upwind response, we examined the responses of L. hesperus that were simultaneously exposed to a control artificial diet and a diet containing 12% KCl in a Y‐tube olfactometer bioassay. Of the 95 adults tested, 47 selected the arm containing the normal diet and 48 selected the arm containing KCl‐treated diet, indicating that dietary constituents did not preferentially attract or repel L. hesperus. The results from these studies strongly suggest that KCl negatively affects L. hesperus feeding behavior by functioning as a strong gustatory deterrent when concentrations exceed 0.5%. Visual and volatile cues appeared to have no role in mediating orientation or feeding behavior under these test conditions.     

The relative contributions of diet and associated microbiota to the accumulation of UV‐absorbing mycosporine‐like amino acids in the freshwater copepod Boeckella antiqua

1. Mycosporine‐like amino acids (MAAs) are ubiquitous compounds in aquatic organisms that are usually considered sunscreens that protect them from harmful ultraviolet radiation. Given that virtually all animals lack the metabolic pathways to synthesise MAAs de novo, they must acquire them either from their diet or from microorganisms living in close association. In freshwater copepods, accumulation of MAAs is stimulated by exposure to ultraviolet and/or visible radiation. 2. A 2 × 2 factorial experiment was performed to assess the contributions of dietary and microbial sources of MAAs in the freshwater copepod Boeckella antiqua. The treatments consisted of two different diets: an MAA‐free diet, including only Chlamydomonas reinhardtii, and an MAA‐rich diet, including both C. reinhardtii and Peridinium inconspicuum, crossed with two antibiotic treatments, with and without chloramphenicol. Treatment with chloramphenicol was intended to inhibit the development of bacteria associated with the copepods. 3. MAA concentration in B. antiqua was affected by the experimental conditions: (i) exposure to artificial PAR + UVR stimulated the accumulation of several MAAs (up to 62% increase in total MAA concentration with respect to the initial concentration); (ii) the presence of chloramphenicol in the culture media reduced the MAA concentration in copepods fed an MAA‐free diet; (iii) in the absence of chloramphenicol, copepods fed the MAA‐rich diet had significantly higher total MAA concentration than those fed the MAA‐deficient diet; but (iv) dietary supplementation with an MAA‐rich algae in the presence of chloramphenicol failed to significantly increase total MAA concentration. 4. Analysis of profiles from denaturing gradient gel electrophoresis (DGGE) showed that the prokaryotic community associated with the copepods was affected by chloramphenicol. Dendograms constructed from digitalised DGGE images consistently grouped the antibiotics treatments separately from the initial samples and the treatments without antibiotics. Two band positions were exclusive to treatments without antibiotics. 5. We conclude that when offered an MAA‐rich diet, B. antiqua may accumulate a proportion of MAAs from diet. However, we suspect that in the absence of an MAA‐rich dietary source (as in its natural habitat), virtually all MAAs present in B. antiqua are produced by copepod‐associated prokaryotes.     

Chemical and structural defenses in the sea fan Gorgonia ventalina: effects against generalist and specialist predators

Cyphoma gibbosum is an ovulid predatory gastropod that specializes on gorgonians, many of which contain secondary metabolites and calcified sclerites that serve as antipredator defenses. In field and shipboard feeding assays, we examined the role of gorgonian crude extracts and sclerites as feeding deterrents to generalist predators and to C. gibbosum. Crude extracts and sclerites were isolated from Gorgonia ventalina, a Caribbean sea fan on which C. gibbosum feeds, and incorporated into a carrageenan-based artificial diet. In shipboard feeding experiments, artificial diet containing G. ventalina crude extracts was consumed 49% less by C. gibbosum, than artificial diet lacking extracts. The addition of G. ventalina sclerites to the diet also reduced feeding by Cyphoma by about one half. The addition of extracts to the artificial diet reduced feeding by natural assemblages of tropical fishes at Cross Harbor, Great Abaco Island, Bahamas by 87%; sclerites reduced feeding by fishes by 95%. Gorgonia ventalina extracts were composed of at least a dozen nonpolar terpenoids. Fractions containing these compounds were feeding deterrents towards fishes in the field. Unlike many terrestrial oligophagous specialists, C. gibbosum is not immune to the defenses produced by its prey.     

Growth response of African catfish,Clarias gariepinus (B.), larvae and fingerlings fed protease‐incorporated diets

African catfish, Clarias gariepinus (B.), is one of the promising freshwater fish species in African aquaculture but the expansion of its farming needs more production of its larvae. The use of live food organisms at first feeding for larvae is still obligatory. That increases the cost of larvae production. Hence, the incorporating of exogenous enzymes especially protease in artificial microdiets may provide affordable alternatives for enhancing the larvae performance. The present study was carried out to evaluate the growth and survival of larvae or fingerlings of African catfish fed artificial diets incorporated with different protease levels. Four artificial diets were formulated and enriched with protease enzyme at levels of 0.0, 750, 1,000, and 1,250 unit/kg diet; after that diets were made into crumbles (100–200 µm diameter). After absorption of the yolk sac, diets were offered to fish larvae (3.6 ± 0.2 mg) in triplicates as a starter feed up to apparent satiation every two hours for 30 days. In another treatment, fish larvae were fed on newly hatched Artemia nauplii (2,500 Artemia/L) as a starter food. In another experiment, African catfish fingerlings (10.1 ± 1.6 g) were fed on the same diets up to satiation twice a day for 2 months. It was noticed that the dietary protease improved larval growth and survival but not as Artemia nauplii did where fish larvae fed on Artemia nauplii showed highest growth and survival followed by those fed a diet enriched with 1,250 unit/kg diet of protease. The mortality of larvae fed protease‐enriched diets as well as the control diet was occurred mostly at the first week reaching its maximum at the third week. The poor growth was observed with fish larvae fed the control diet. Meanwhile, catfish fingerlings fed protease‐enriched diets showed higher growth over those fed the control diet. The larvae survival (11.0%–41.7%) was enhanced by increasing protease levels and it was lower than that of fingerlings (95.6%–100.0%). Furthermore, protein retention and digestibility were significantly improved with protease supplementation over the control diet especially at a level of 1,000 unit/kg diet. As compared with the previous studies, live food should be used in larvae rearing for the first week after that a starter diet enriched with protease at levels of 1,250 unit/kg diet should be used. In case of fish fingerlings, the dry diets should be enriched with 1,100 unit/kg diet to improve diet digestibility and subsequently enhance their growth.     

25-Azasteroid inhibition of development and conversion of C28 and C29 phytosterols to cholesterol in Spodoptera litura (F.)

Larvae of Spodoptera litura (F.) grown on an artificial diet completed larval development in 19.2 days and attained a maximum weight of 873.2 mg. When fed dietary concentrations of 50 ppm of 25-azacholesterol or 10 ppm of 25-azacholestane, the larval developmental period increased to 28.4 and 23.4 days, and the larval weights were 447.5 and 542.3 mg, respectively. Both compounds induced distinct melanization effects and caused production of larval-pupal intermediates and severe mortality. Treatments with concentrations of 50 ppm or more of either azasteroid caused a decline in pupal period and earlier eclosion and emergence of abnormal adults. Egg laying and hatchability decreased with increasing concentrations of azasteroids in the larval diets. When 1 ppm or more of 25-azasteroid is added to the artificial diet, the insect larvae contain identifiable amounts of desmosterol, in addition to cholesterol, campesterol, and sitosterol, which are present in Spodoptera grown on artificial diet alone. Desmosterol accumulation in the insect body is due to an inhibition of the Δ²⁴-sterol reductase by 25-azasteroids. An increase in the concentration of these azasteroids in the diet results in an increase in sitosterol concentration and simultaneous reduction in the cholesterol levels due to inhibition of conversion of sitosterol. This inhibition appears to be more pronounced with 25-azacholestane treatment than with 25-azacholesterol.     

Use of an artificial diet system to study the toxicity of gut-active insecticidal compounds on larvae of the green lacewing Chrysoperla sinica

A semi-liquid artificial diet was established and found to be a suitable food source for Chrysoperla sinica larvae, comparable to aphid prey. Using the artificial diet, we established and validated a dietary exposure assay by using the insecticidal potassium arsenate (PA) as positive control. Dose-dependent responses were documented for all observed life-table parameters of C. sinica larvae such as survival rate, pupation rate, larval weight, and larval development time. Thus, the dietary assay can detect the effects of insecticidal compounds on the survival and development of C. sinica larvae. Using the established dietary assay, we subsequently tested the toxicity of Cry1Ab, Cry1Ac, and Cry2Aa proteins (which are produced by transgenic maize, cotton or rice plants) to C. sinica larvae. Artificial diets containing Galanthus nivalis agglutinin (GNA) or PA were included as positive controls. Survival and development of C. sinica larvae were not affected when the artificial diet contained purified Cry1Ab, Cry1Ac, or Cry2Aa at 200 μg/g diet. In contrast, C. sinica larvae were adversely affected when the diet contained PA and GNA. The stability and bioactivity of the Cry proteins in the diet and Cry protein uptake by the lacewing larvae were confirmed by bioassay with a Cry-sensitive insect species and by ELISA. The current study describes a suitable experimental system for assessing the potential effects of gut-active insecticidal compounds on green lacewing larvae. The experiments with the Cry proteins demonstrate that C. sinica larvae are not sensitive to Cry1Ab, Cry1Ac, and Cry2Aa.     

Beneficial effect of supplementing an artificial diet for Amblyseius swirskii with Hermetia illucens haemolymph

Artificial diets have been developed to sustain the mass rearing of a wide range of arthropod natural enemies, with varying success. In some cases, such diets can be optimized using insect‐derived materials, such as haemolymph. In this study, we examined the effect of supplementing haemolymph of the black soldier fly, Hermetia illucens, to a basic artificial diet for the phytoseiid mite Amblyseius swirskii. The survival, development and reproduction of the predatory mite were assessed when fed on artificial diets composed of honey, sucrose, tryptone, yeast extract and egg yolk, supplemented with 5%, 10%, or 20% of H. illucens pre‐pupal haemolymph. Developmental time from larva to adult was shorter for males and females offered artificial diets supplemented with 20% haemolymph vs. the basic diet. The oviposition rate and total fecundity of females reared on the basic diet were substantially lower than those of females supplied with the enriched diets. The intrinsic rate of increase was highest on the diet containing 20% haemolymph, followed by those containing 10% and 5% haemolymph. In a subsequent diet‐switching experiment, mites fed on the basic diet in their juvenile stages were switched upon adulthood to diet enriched with different concentrations of H. illucens haemolymph. The females that were fed with the enriched diets from the adult stage on had higher oviposition rates and fecundities than those maintained on the basic diet, but their reproductive parameters were not significantly affected by the concentration of the haemolymph in the artificial diet. In conclusion, supplementing artificial diets with black soldier fly haemolymph significantly improved their nutritional value for A. swirskii. Our findings indicate the potential of using H. illucens as a cheap source for haemolymph in artificial diets, as the fly can be cost‐effectively produced at a large scale on organic waste materials.     

Microalgae aquaculture feeds

Microalgae feeds are currently used in relatively small amounts in aquaculture, mainly for the production of larvae and juvenile shell- and finfish, as well as for raising the zooplankton required for feeding of juvenile animals. The blue-green algaSpirulina is used in substantial amounts (over 100 t y^–1) as a fish and shrimp feed, and even larger markets can be projected if production costs could be reduced. Another potential large-scale application of microalgae is the cultivation ofHaematococcus for the production of the carotenoid astaxanthin, which gives salmon flesh its reddish color. In the long-term microalgae biomass high in lipids (omega-3 fatty acids) may be developed as substitutes for fish oil-based aquaculture feeds. In shrimp ponds the indigenous algal blooms supply a part of the dietary requirements of the animals, but it is difficult to maximize algal productivities. A separate algal production system could feed the shrimps and minimize the need for added feed. Bivalves feed essentially exclusively on marine microalgae throughout their life cycle. The development of cultivation technologies for such microalgae would allow the onshore production of these animals, with greatly improved product quality and safety.This paper was presented at the Symposium on Applied Phycology at the Fourth International Phycological Congress, Duke University.     

Artificial leaf aids analysis of chlorophyll fluorescence and P700 absorbance in studies involving microalgae

Measuring chlorophyll fluorescence and P700 absorbance has been widely used to study photosynthesis in both terrestrial plants and algae. However, in order to apply these measurement techniques to study microalgae, a concentrated suspension of algae, which is usually prepared by centrifugation, is required. In this study, instead of using centrifugation, we concentrated microalgae on a nitrocellulose membrane using filtration to create an ‘artificial leaf’ before analysis. Overall, we were able to generate values of the appropriate photosynthetic parameters that were comparable to those obtained when chlorophyll fluorescence and P700 absorbance were measured following centrifugation. There were no statistically significant differences (P > 0.05) between the artificial leaf method and the traditional cuvette method for determining chlorophyll fluorescence or P700 absorbance at appropriate chlorophyll concentrations. We were also able to reduce background noise by using a filter membrane as a carrier. Therefore, an artificial leaf has the potential to be a valuable tool for phycologists interested in studying microalgal photosynthesis by enabling them to eliminate tedious centrifugation steps. In addition, fluorometers commonly used for studying the leaves of higher plants will also be suitable for studying microalgae.