The association of Western flower thrips, Frankliniella occidentalis, with a near Erwinia species gut bacteria: transient or permanent?

Associations between insects and gut bacteria are ubiquitous. It is possible to make a distinction between permanent associations (called symbiosis), in which the same type of bacteria is present in more than one generation of the insect, and transient associations. Transient bacteria are ingested together with food but do not settle in the insect gut in such a way that they will be passed on to the next generation. In this study, we describe the permanent association between Western flower thrips (Frankliniella occidentalis), a polyphagous insect species that is a major pest worldwide, and one type of gut bacteria. On the basis of direct microscopic observations and counts of bacteria, it was found that thrips from the populations studied contained large numbers of bacteria in their hindgut. Bacteria were isolated from their host and grown on 10 different agar media. The number of bacteria isolated on agar media equaled the number of direct counts. All isolates had the same colony morphology. On the basis of their 16S rDNA sequence these bacteria were identified as Enterobacteriaceae, closely related to Escherichia coli. Isolates tested with API 20E biochemical tests were Erwinia species. This was the only type of bacteria found in all thrips individuals on any of the 10 different agar media. Universal primers, which would potentially pick up DNA from any bacterium present in the insect, were applied on crude DNA extracts from thrips with bacteria. We only found 16S rDNA sequences similar to those of the isolated thrips gut bacteria. The same type of bacteria was present in all life stages of the thrips and was found to persist in the thrips populations for at least 2 years (more than 50 generations).     

Growth and transmission of gut bacteria in the Western flower thrips, Frankliniella occidentalis

The Western flower thrips (Frankliniella occidentalis), a polyphagous insect with global distribution, has a permanent association with a near Erwinia species TAC bacterium in its hindgut. Since this bacterium is able to grow outside the thrips, it is a facultative symbiont that is not completely dependent on the host. In this study we address the question of how the association is maintained and how bacteria are transmitted to newly hatched thrips larvae. Bacteria are passed on to new thrips via the food source. No evidence was found for vertical transmission from mother to offspring via the egg. Gut bacteria show unlimited growth during the larval (feeding) stages, and in the second instar stage 100% of the larvae become infected with high numbers of bacteria. In the prepupal and pupal stage, the number of bacteria declines, but increases again during the adult phase. A method to rear aposymbiotic (bacteria-free) thrips is described which enables studies on the impact of bacteria on the fitness of thrips.     

Characterization of bacterial symbionts in Frankliniella occidentalis (Pergande), Western flower thrips

Many insects have associations with bacteria, although it is often difficult to determine the intricacies of the relationships. In one such case, facultative bacteria have been discovered in a major crop pest and virus vector, the Western flower thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Several bacterial isolates have been studied in Netherlands greenhouse thrips populations, with molecular data indicating that these bacteria were similar to Escherichia coli, although biochemical properties suggested these microbes might actually be most similar to plant pathogenic bacteria in the genus Erwinia. We focused on the bacterial flora of the Hawaiian Islands thrips population where these gut bacteria were first reported in 1989. We also analyzed a German population and a 1965 California population preserved in ethanol. Culture and culture-independent techniques revealed a consistent microflora that was similar to the Netherlands isolates studied. The similarity among thrips microbes from multiple populations and environments suggested these bacteria and their hosts share a widespread association. Molecular phylogeny based on the 16S rRNA gene and biochemical analysis of thrips bacteria suggested two distinctive groups of microbes are present in thrips. Phylogenetic analysis also revealed support for one thrips bacterial group having a shared ancestry with Erwinia, whereas the second group of thrips bacteria fell out with E. coli, but without support. Although species-specific relationships were indeterminable due to the conservative nature of 16S, there is strong indication that thrips symbionts belong to two different genera and originated from environmental microbes.     

Intraguild predation among plant pests: western flower thrips larvae feed on whitefly crawlers

Omnivores obtain resources from more than one trophic level, and choose their food based on quantity and quality of these resources. For example, omnivores may switch to feeding on plants when prey are scarce. Larvae of the western flower thrips Frankiniella occidentalis Pergande (Thysanoptera: Thripidae) are an example of omnivores that become predatory when the quality of their host plant is low. Western flower thrips larvae usually feed on leaf tissue and on plant pollen, but may also attack eggs of predatory mites, their natural enemies, and eggs of the two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae), one of their competitors. Here, we present evidence that western flower thrips larvae prey on Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae), another competitor for plant tissue. We tested this on two host plant species, cucumber (Cucumis sativa L.), considered a host plant of high quality for western flower thrips, and sweet pepper (Capsicum annuum L.), a relatively poor quality host. We found that western flower thrips killed and fed especially on whitefly crawlers and that the incidence of feeding did not depend on host-plant species. The developmental rate and oviposition rate of western flower thrips was higher on a diet of cucumber leaves with whitefly crawlers than on cucumber leaves without whitefly crawlers, suggesting that thrips do not just kill whiteflies to reduce competition, but utilize whitefly crawlers as food.     

Host-plant species modifies the diet of an omnivore feeding on three trophic levels

The diet choice of omnivores feeding on two adjacent trophic levels (either plants and herbivores or herbivores and predators) has been studied extensively. However, omnivores usually feed on more than two trophic levels, and this diet choice and its consequences for population dynamics have hardly been studied. We report how host-plant quality affects the diet choice of western flower thrips feeding on three trophic levels: plants (cucumber or sweet pepper), eggs of spider mites and eggs of a predatory mite that attacks spider mites. Spider mites feed on the same host plants as thrips and produce a web that hampers predator mobility. To assess the indirect effects of spider mites on predation by thrips, the thrips were offered spider-mite eggs and predatory-mite eggs on cucumber or sweet pepper leaf discs that were either clean, damaged by spider mites but without spider-mite web, or damaged and webbed. We show that, overall, thrips consumed more eggs on sweet pepper, a plant of low quality, than on cucumber, a high quality host plant. On damaged and webbed leaf discs (mimicking the natural situation), thrips killed more predator eggs than spider-mite eggs on sweet pepper, but they killed equal numbers of eggs of each species on cucumber. This is because web hampered predation on spider-mite eggs by thrips on sweet pepper, but not on cucumber, whereas it did not affect predation on predatory-mite eggs. We used the data obtained to parameterize a model of the local dynamics of this system. The model predicts that total predation by the omnivore has little effects on population dynamics, whereas differential attack of predator eggs and spider-mite eggs by the omnivore has large effects on the dynamics of both mite species on the two host plants.     

Oviposition preference of western flower thrips for cucumber leaves from different positions along the plant stem

While the distribution of herbivorous insects over leaves along the stem often shows a peak at some distance from the apex this does not necessarily reflect an innate preference as alternative explanations can be provided such as impact of predators and inter- or intraspecific competitors. It is of interest to determine which factors shape the distribution of insects over the leaves of a plant. Do leaves from different positions differ in suitability for insects and is that reflected in the insect's preference, or are other factors involved? In this paper we assess how the herbivorous insect western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), chooses among leaves from different positions relative to the apex of cucumber, Cucumis sativus (L.) plants. On leaf discs of a susceptible and three partially resistant cucumber accessions, thrips reproduction was highest on apical leaves and lowest on basal leaves. In dual-choice essays thrips females preferred younger leaves over older leaves for oviposition in all cucumber accessions tested, as was predicted from the no-choice assay. This indicates that differences in leaf suitability are an important factor in determining thrips distribution on cucumber plants.     

Symbiotic bacteria enable olive flies (Bactrocera oleae) to exploit intractable sources of nitrogen

Insects are often associated with symbiotic micro‐organisms, which allow them to utilize nutritionally marginal diets. Adult fruit flies (Diptera: Tephritidae) associate with extracellular bacteria (Enterobacteriaceae) that inhabit their digestive tract. These flies obtain nutrients by foraging for plant exudates, honeydew and bird droppings scattered on leaves and fruit – a nutritional niche which offers ample amounts of carbohydrates, but low quantities of available nitrogen. We identified the bacteria resident in the gut of the olive fly (Bactrocera oleae) – a worldwide pest of olives and examined their contribution to nitrogen metabolism in the adult insect. By suppressing bacteria in the gut and monitoring female fecundity, we demonstrate that bacteria contribute essential amino acids and metabolize urea into an available nitrogen source for the fly, thus significantly elevating egg production. In an ecological context, bacteria were found to be beneficial to females subsisting on bird droppings, but not on honeydew – two natural food sources. We suggest that a main gut bacterium (Candidatus Erwinia dacicola) forms an inseparable, essential part of this fly's nutritional ecology. The evolution of this symbiosis has allowed adult flies to utilize food substrates which are low or imbalanced in assimilable nitrogen and thereby to overcome the nitrogen limitations of their natural diet.     

Influence of salicylaldehyde and methyl salicylate on post-landing behaviour of Frankliniella occidentalis Pergande

This study investigates behavioural responses of adult western flower thrips (Frankliniella occidentalis Pergande; Thysan., Thripidae) females to direct contact with repellent phenylpropanoid plant compounds (salicylaldehyde and methyl salicylate) applied on bean and cucumber leaves. The residence time of F. occidentalis females until take off was significantly shorter on bean or cucumber leaf discs treated with salicylaldehyde at 1% concentration compared with control leaf discs. A methyl salicylate (1%) treatment of cucumber resulted in shorter time periods until thrips took off the treated leaf discs compared with the control leaf discs. In a choice experiment thrips avoided to settle on a 1% salicylaldehyde treatment of bean and cucumber leaf discs for a maximum of 3 h, on a 1% methyl salicylate treatment for a 5‐h period. Within a 24‐h period neither the egg‐laying nor the feeding activity of F. occidentalis was affected after salicylaldehyde application (0.1%, 1%) on bean or cucumber. In contrast, methyl salicylate (1%) applied on bean and cucumber significantly prevented thrips females from oviposition and reduced the percentage of damaged area caused by their feeding activity for 24 h. As olfactory repellent plant volatiles applied on crop plants may elicit diverse post‐landing responses of F. occidentalis, short‐ and long‐term effects should be considered when evaluating the factual applicability of secondary plant compounds in a successful thrips management strategy.     

Effect of bacteria on nutritional status and reproductive success of the Mediterranean fruit fly Ceratitis capitata

Abstract.  Mediterranean fruit flies ( Ceratitis capitata Wiedemann, Diptera: Tephritidae) harbor a community of diazotrophic bacteria in their digestive system. The present study aims to test the hypothesis that bacteria contribute to fly fitness by enhancing copulatory success and egg production in males and females, respectively. After eclosion, flies were fed a full diet containing peptides, sugar and minerals, or a sugar diet, lacking peptides. Subgroups from each diet were fed a mixture of the antibiotics ciprofloxacin and piperacillin. The presence of bacteria, food consumption, weight gain, lipid and protein levels, oviposition in females and copulatory success of males were quantified in the four groups. The antibiotic treatment effectively cleared the gut of bacteria. The relative amounts of food consumed (with or without antibiotics) are similar in all groups. The antibiotics do not inhibit feeding, and their ingestion does not affect dry weight or the amount of protein stored, yet females feeding on the full diet without antibiotics have increased lipid levels. Females fed the full diet produce significantly more eggs than females on the sugar diet, but the presence of bacteria does not affect numbers of eggs produced. However, in the absence of bacteria, the oviposition rate of nutritionally stressed females is significantly accelerated. The presence of bacteria in sugar fed males does not provide them with a mating advantage. Conversely, in males fed a full diet, the presence of bacteria is associated significantly with a shorter latency to mate. It is concluded that, because the bacterial community is present at all stages of the fly's life cycle, at least some species are effectively transmitted from parents to offspring, and removal of bacteria affects measurable physiological and behavioural parameters related to fitness, the association between bacteria and the medfly is mutualistic.     

The use of floral homeotic mutants as a novel way to obtain durable resistance to insect pests

We have developed a novel strategy for the introduction of durable insect resistance in crops. This strategy was based on intervention in the natural relationship between plants and insects. For many insects, including pests such as thrips (Frankliniella occidentalis), the flower is an important factor in their life cycle, serving either as a food source or as a place for mating. The insects are attracted to the flower by scent, which is mainly produced by the petals, and by the bright colour of these floral organs. We therefore anticipated that removal or changing the identity of the petals would significantly reduce the attractiveness of the flower to thrips. To test this hypothesis, we used cucumber as a model species because most modern varieties are parthenocarpic, in which the fruit develops without fertilization. The cucumber mutant green petals, in which the petals are homeotically transformed into green sepals, was particularly useful for this study. The susceptibility of the cucumber plants to damage by thrips was determined by recording thrip numbers and by measuring leaf damage. Large differences were observed when greenhouse compartments with either wild-type or green petal mutant plants were compared. The rate of population growth of the insects on the mutant plants was significantly reduced and the leaves were almost undamaged. These results demonstrate that alterations in the structure of flowers may interfere with the life cycle of insects, providing the means for a novel and natural strategy for obtaining insect resistance.     

西花蓟马在黄瓜和架豆上的空间分布型及理论抽样数

西花蓟马Frankliniella occidentalis（Pergande）已在北京部分蔬菜园区成功定居,对蔬菜生产造成危害。为了解该虫在蔬菜田的种群空间分布型,指导田间取样,本文应用几种聚集度指标的计算公式以及Taylor、Iwao的回归方程式,分析和测定了西花蓟马的种群空间分布格局。结果表明：在黄瓜和架豆上西花蓟马的空间分布型一致,均为聚集分布型;该虫的空间分布型不受种群密度的影响,并且也不随取样时间的变化而变化。种群数量动态研究显示西花蓟马成虫和若虫在黄瓜植株的中部叶片分布较多（从顶部向下数第4叶至第17叶片上成、若虫的分布数量多于其他叶片上的数量）,未展开的叶片、嫩叶和下部老叶危害较轻。用Iwao回归法中α、β参数计算出在允许误差范围内的理论抽样数。     

Erwinia carotovora Evf antagonizes the elimination of bacteria in the gut of Drosophila larvae

Erwinia Virulence Factor (Evf) has been identified in Erwinia carotovora carotovora 15 (Ecc15) as a virulence factor that promotes colonization of the Drosophila larval gut and provokes the triggering of a systemic immune response. Here we have analysed how Evf promotes persistence and colonization of bacteria inside the larval gut. Erwinia evf mutants do not persist in immune-deficient Drosophila, indicating that Evf does not act by counteracting immunity. The results indicated that Evf is not a toxin because various gram-negative bacteria expressing evf can persist without affecting viability of Drosophila larvae. Evf did not appear to be a factor antagonizing a host-specific reaction because in vitro assays failed to reveal detoxifying enzymatic activities against various compounds thought to contribute to the hostile environment of the gut. These findings were corroborated by the observation that Evf is not required for survival in midgut organ cultures. By contrast, bacteria expressing evf allow persistence in trans of bacteria lacking evf indicating that Evf promotes the accumulation of gram-negative bacteria in the anterior midgut by affecting gut physiology.     

抗生素和肠道细菌对小菜蛾适合度的影响

【目的】小菜蛾是危害十字花科蔬菜的世界性重要害虫。通过分析抗生素对小菜蛾的毒性效应,了解肠道细菌对小菜蛾适合度的影响,有助于更好地阐明小菜蛾肠道细菌的功能。【方法】利用抗生素处理含有高丰度肠道细菌的萝卜苗饲养品系(FZss)小菜蛾幼虫,同时利用抗生素处理饲料饲养的无肠道细菌(SLss)小菜蛾幼虫,分析抗生素及肠道细菌对小菜蛾适合度的影响。【结果】抗生素处理FZss品系小菜蛾导致了小菜蛾发育历期延长,虫重、蛹重、化蛹率、产卵量和成虫寿命降低。利用抗生素处理无肠道菌的SLss品系小菜蛾幼虫,小菜蛾化蛹率和单雌产卵量均显著降低,而对发育历期、虫重和蛹重则无影响。【结论】综合两个研究的结果发现抗生素处理后宿主适合度的降低一方面是由于抗生素的毒性效应导致,另一方面是由于小菜蛾肠道细菌的缺失引起。抗生素的毒性效应主要表现为对化蛹率和单雌产卵量的影响,而肠道细菌则对小菜蛾的发育历期、虫重、蛹重以及成虫寿命具有重要的促进作用。     

Flower visitation and oviposition behavior of Frankliniella occidentalis (Tysan., Thripidae) on cucumber plants

Oviposition behaviour of western flower thrips, Frankliniella occidentalis (Pergande) on greenhouse cucumber, Cucumis satifus (L.) was investigated. Most eggs were laid in the leaves, along veins and under leaf hairs, with only a few on plant stems and flowers. Oviposition rate was higher during the day than during the night. During the day, more adult thrips were found in the flowers than during the night. The number of adult thrips per flower increased rapidly after sunrise with the highest densities occurring around noon and thereafter the number of thrips in flowers decreased during the afternoon. No differences were found in the number of larvae (first and second instars) in flowers during the same period. The number of adult thrips on male and female cucumber flowers was not different, indicating that pollen is not the only attraction in flowers for thrips.     

Effect of Test Leaf and Temperature on Mortality of Frankliniella occidentalis in Leaf Dip Bioassay of Insecticides

Effects of the kind of test leaf and temperature on leaf dip bioassay to the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), were examined with nine different insecticides. For the test of different leaves, cucumber, pepper and potato leaves were evaluated. The test leaves significantly influenced thrips mortalities on leaf dip bioassay. Generally the mortalities on pepper leaf were higher than on cucumber leaf or filter paper. Temperature effects were determined at 15, 20, 25 and 30°C. Regardless of the test leaf, the mortality was increased with increasing temperature except for the case of imidacloprid. Local variations in mortality of F. occidentalis populations were not observed in this study.     

Apple pollen as a supplemental food for the western flower thrips, Frankliniella occidentalis: response of individuals and populations

Experiments were performed to investigate the influence of apple pollen, plant diet, and relative humidity on the individual life‐history traits and on the population growth of the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). In experiments with individual thrips, availability of bean pods plus a mixture of apple pollen and lycopodium shortened larval development time, and hence time from egg to adult, compared to bean pods alone. Similarly, the total number of eggs laid and the mean number of eggs laid per female per day were greater in the presence of pollen plus bean pods than on bean pods alone. Diet did not affect survival or length of the pre‐oviposition and oviposition periods. A diet including bean pods plus pollen shortened the generation time and population‐doubling time, and increased the net reproductive rate and the intrinsic rate of increase, compared to bean pods alone. In experiments on populations, growth was measured with different amounts of pollen on bean pods under two relative humidities, ca. 38% and 66%, and separately on two bean substrates, pods or leaves, at ca. 45% r.h. The presence or amount of pollen had no effect on population growth regardless of the other variables. Greater population growth occurred under the higher relative humidity regardless of substrate. Population growth was greater on bean pods than on bean leaves. Despite reports on the nutritional benefit of pollen for F. occidentalis, this benefit may be affected by pollen type, host plant, and other factors. In situations in which pollen has little influence on the growth of F. occidentalis populations, pollen that benefits a thrips predator could be used in an integrated management plan for controlling thrips.     

No evidence that gut microbiota impose a net cost on their butterfly host

Gut microbes are believed to play a critical role in most animal life, yet fitness effects and cost–benefit trade‐offs incurred by the host are poorly understood. Unlike most hosts studied to date, butterflies largely acquire their nutrients from larval feeding, leaving relatively little opportunity for nutritive contributions by the adult's microbiota. This provides an opportunity to measure whether hosting gut microbiota comes at a net nutritional price. Because host and bacteria may compete for sugars, we hypothesized that gut flora would be nutritionally neutral to adult butterflies with plentiful food, but detrimental to semistarved hosts, especially when at high density. We held field‐caught adult Speyeria mormonia under abundant or restricted food conditions. Because antibiotic treatments did not generate consistent variation in their gut microbiota, we used interindividual variability in bacterial loads and operational taxonomic unit abundances to examine correlations between host fitness and the abdominal microbiota present upon natural death. We detected strikingly few relationships between microbial flora and host fitness. Neither total bacterial load nor the abundances of dominant bacterial taxa were related to butterfly fecundity, egg mass or egg chemical content. Increased abundance of a Commensalibacter species did correlate with longer host life span, while increased abundance of a Rhodococcus species correlated with shorter life span. Contrary to our expectations, these relationships were unchanged by food availability to the host and were unrelated to reproductive output. Our results suggest the butterfly microbiota comprises parasitic, commensal and beneficial taxa that together do not impose a net reproductive cost, even under caloric stress.     

饲料类型对茶尺蠖幼虫肠道细菌多样性及幼虫存活率的影响

【目的】茶尺蠖是茶园中的重要害虫。研究茶尺蠖寄主食物-肠道菌群-茶尺蠖生长发育三者之间的关系对于茶尺蠖的防治具有重要的理论指导价值。【方法】分析不添加茶叶因子的纯人工饲料和茶树鲜叶对茶尺蠖幼虫的存活影响;用高通量测序技术分析不同饲料饲喂的茶尺蠖幼虫的肠道菌群异同。【结果】取食人工饲料的幼虫死亡率远远高于取食茶树鲜叶的幼虫;取食人工饲料的幼虫肠道细菌多样性和丰富度高于取食茶树鲜叶的幼虫;茶尺蠖幼虫肠道中存在很多促进宿主生长的细菌。【结论】饲料类型影响茶尺蠖幼虫的存活;饲料类型影响茶尺蠖幼虫肠道菌群结构。     

Life history characteristics of Frankliniella occidentalis on cucumber leaves with and without supplemental food

The development time, fecundity, longevity, and resultant intrinsic growth rate of the western flower thrips Frankliniella occidentalis (Pergande) [Thysanoptera: Thripidae] encaged on a cucumber leaf were compared among seven types of food supplied: six pollen species and a mixture of milk powder and yeast. The rationale was to find a food source that offers the least benefit for thrips and could therefore be considered as a food source for the preventative introduction of thrips predators. With the exception of the mixture of milk powder and yeast, all the food sources tested offered a nutritional benefit for the thrips. The addition of pollen increased fecundity and reduced development time, mainly during the larval stage. Betula pubescens and Corylus avellana pollen also increased adult longevity. The nutritional benefit of Pinus sylvestris pollen was greater than that of the other five pollen species, as manifested by its significantly greater positive effect on fecundity. The other pollen species could not be ranked in terms of nutritional benefit to F. occidentalis. The negative effect of the milk powder plus yeast mix on the life‐table parameters of F. occidentalis probably only occurs in an encaged situation where thrips cannot escape from the unfavorable environment. The crude estimate of the intrinsic growth rate of F. occidentalis increased from 0.163 on the plain cucumber leaf to 0.240 when P. sylvestris pollen was added to the leaf. The differences in intrinsic growth rate mainly reflect the differences in fecundity among the food sources. Thus, the peak oviposition rate may be used as a measure of the nutritional benefit F. occidentalis can obtain by feeding on supplemental food sources. The positive effect of a supplemental food source on thrips does not necessarily mean it is unsuitable for the preventative introduction of thrips predators, because the supplemental food can also affect the population dynamics of the predator and the predator–prey interaction and, hence, the outcome of biological control.     

Gut microbiota of the pine weevil degrades conifer diterpenes and increases insect fitness

The pine weevil (Hylobius abietis), a major pest of conifer forests throughout Europe, feeds on the bark and cambium, tissues rich in terpenoid resins that are toxic to many insect herbivores. Here, we report the ability of the pine weevil gut microbiota to degrade the diterpene acids of Norway spruce. The diterpene acid levels present in ingested bark were substantially reduced on passage through the pine weevil gut. This reduction was significantly less upon antibiotic treatment, and supplementing the diet with gut suspensions from untreated insects restored the ability to degrade diterpenes. In addition, cultured bacteria isolated from pine weevil guts were shown to degrade a Norway spruce diterpene acid. In a metagenomic survey of the insect's bacterial community, we were able to annotate several genes of a previously described diterpene degradation (dit) gene cluster. Antibiotic treatment disrupted the core bacterial community of H. abietis guts and eliminated nearly all dit genes concordant with its reduction in diterpene degradation. Pine weevils reared on an artificial diet spiked with diterpenes, but without antibiotics, were found to lay more eggs with a higher hatching rate than weevils raised on diets with antibiotics or without diterpenes. These results suggest that gut symbionts contribute towards host fitness, but not by detoxification of diterpenes, as these compounds do not show toxic effects with or without antibiotics. Rather the ability to thrive in a terpene‐rich environment appears to allow gut microbes to benefit the weevil in other ways, such as increasing the nutritional properties of their diet.