The role of larvae of black soldier fly and house fly and of feed substrate microbes in biotransformation of aflatoxin B1.

Over the past few years, there has been growing interest in the ability of insect larvae to convert various organic side-streams containing mycotoxins into insect biomass that can be used as animal feed. Various studies have examined the effects of exposure to aflatoxin B1 (AFB1) on a variety of insect species, including the larvae of the black soldier fly (BSFL; Hermetia illucens L.; Diptera: Stratiomyidae) and the housefly (HFL; Musca domestica L.; Diptera: Muscidae). Most of these studies demonstrated that AFB1 degradation takes place, either enzymatic and/or non-enzymatic. The possible role of feed substrate microorganisms (MOs) in this process has thus far not been investigated. The main objective of this study was therefore to investigate whether biotransformation of AFB1 occurred and whether it is caused by insect-enzymes and/or by microbial enzymes of MOs in the feed substrate. In order to investigate this, sterile and non-sterile feed substrates were spiked with AFB1 and incubated either with or without insect larvae (BSFL or HFL). The AFB1 concentration was determined via LC-MS/MS analyses and recorded over time. Approximately 50% of the initially present AFB1 was recovered in the treatment involving BSFL, which was comparable to the treatment without BSFL (60%). Similar patterns were observed for HFL. The molar mass balance of AFB1 for the sterile feed substrates with BSFL and HFL was 73% and 78%, respectively. We could not establish whether non-enzymatic degradation of AFB1 in the feed substrates occurred. The results showed that both BSFL and substrate-specific MOs play a role in the biotransformation of AFB1 as well as in conversion of AFB1 into aflatoxin P1 and aflatoxicol, respectively. In contrast, HFL did not seem to contribute to AFB1 degradation. The obtained results contribute to our understanding of aflatoxin metabolism by different insect species. This information is crucial for assessing the safety of feeding fly larvae with feed substrates contaminated with AFB1 with the purpose of subsequent use as animal feed.

A systematic literature review on the effects of mycotoxin exposure on insects and on mycotoxin accumulation and biotransformation.

Novel protein sources for animal feed are needed, and the use of insects as feed ingredient is explored. The insect production sector offers opportunities for a circular and sustainable approach to feed production by upgrading waste or side streams into high-quality proteins. However, potential food or feed safety issues should be studied in advance. Mycotoxins, such as aflatoxin B1, are natural contaminants commonly found in agricultural crops and have proven to be detrimental to the agricultural industry, livestock, and human health. This systematic review aims to provide a comprehensive overview of the published evidence on effects of mycotoxin exposure on insect growth and survival, mycotoxin accumulation within the insect body, and metabolization of various mycotoxins by insects. The review includes 54 scientific articles published in the past 55 years, in total covering 32 insect species. The main findings are the following: (1) Insects of the order Coleoptera show lower mortality after exposure to aflatoxin B1 when compared to Lepidoptera and Diptera; (2) effects of mycotoxins on larval growth and survival are less detrimental in later larval stages; (3) accumulation of mycotoxins was low in most insect species; (4) mycotoxins are metabolized within the insect body, the degree of which depends on the particular mycotoxin and insect species; (5) cytochrome P450s are the main family of enzymes involved in biotransformation of mycotoxins in some insect species. Results of this review support an optimistic outlook for the use of mycotoxin-contaminated waste streams as substrate for insect rearing.

LEDs Make It Resilient: Effects on Plant Growth and Defense.

Light spectral composition influences plant growth and metabolism, and has important consequences for interactions with plant-feeding arthropods and their natural enemies. In greenhouse horticulture, light spectral composition can be precisely manipulated by light-emitting diodes (LEDs), and LEDs are already used to optimize crop production and quality. However, because light quality also modulates plant secondary metabolism and defense, it is important to understand the underlying mechanisms in the context of the growth-defense trade-off. We review the effects of the spectral composition of supplemental light currently used, or potentially used, in greenhouse horticulture on the mechanisms underlying plant growth and defense. This information is important for exploring opportunities to optimize crop performance and pest management, and thus for developing resilient crop-production systems.

Photoreceptor spectral sensitivity of the compound eyes of black soldier fly (Hermetia illucens) informing the design of LED-based illumination to enhance indoor reproduction.

Mating in the black soldier fly (BSF) is a visually mediated behaviour that under natural conditions occurs in full sunlight. Artificial light conditions promoting mating by BSF were designed based on the spectral characteristics of the compound eye retina. Electrophysiological measurements revealed that BSF ommatidia contained UV-, blue- and green-sensitive photoreceptor cells, allowing trichromatic vision. An illumination system for indoor breeding based on UV, blue and green LEDs was designed and its efficiency was compared with illumination by fluorescent tubes which have been successfully used to sustain a BSF colony for five years. Illumination by LEDs and the fluorescent tubes yielded equal numbers of egg clutches, however, the LED illumination resulted in significantly more larvae. The possibilities to optimize the current LED illumination system to better approximate the skylight illuminant and potentially optimize the larval yield are discussed.

Plant-mediated interactions between two herbivores differentially affect a subsequently arriving third herbivore in populations of wild cabbage.

Plants are part of biodiverse communities and frequently suffer from attack by multiple herbivorous insects. Plant responses to these herbivores are specific for insect feeding guilds: aphids and caterpillars induce different plant phenotypes. Moreover, plants respond differentially to single or dual herbivory, which may cascade into a chain of interactions in terms of resistance to other community members. Whether differential responses to single or dual herbivory have consequences for plant resistance to yet a third herbivore is unknown. We assessed the effects of single or dual herbivory by Brevicoryne brassicae aphids and/or Plutella xylostella caterpillars on resistance of plants from three natural populations of wild cabbage to feeding by caterpillars of Mamestra brassicae. We measured plant gene expression and phytohormone concentrations to illustrate mechanisms involved in induced responses. Performance of both B. brassicae and P. xylostella was reduced when feeding simultaneously with the other herbivore, compared to feeding alone. Gene expression and phytohormone concentrations in plants exposed to dual herbivory were different from those found in plants exposed to herbivory by either insect alone. Plants previously induced by both P. xylostella and B. brassicae negatively affected growth of the subsequently arriving M. brassicae. Furthermore, induced responses varied between wild cabbage populations. Feeding by multiple herbivores differentially activates plant defences, which has plant-mediated negative consequences for a subsequently arriving herbivore. Plant population-specific responses suggest that plant populations adapt to the specific communities of insect herbivores. Our study contributes to the understanding of plant defence plasticity in response to multiple insect attacks.

Variation in plant-mediated interactions between rhizobacteria and caterpillars: potential role of soil composition.

Selected strains of non-pathogenic rhizobacteria can trigger induced systemic resistance (ISR) in plants against aboveground insect herbivores. However, the underlying mechanisms of plant-mediated interactions between rhizobacteria and herbivorous insects are still poorly understood. Using Arabidopsis thaliana Col-0-Pseudomonas fluorescens WCS417r as a model system, we investigated the performance and the molecular mechanisms underlying plant-mediated effects of rhizobacteria on the generalist caterpillar Mamestra brassicae and the specialist Pieris brassicae. Rhizobacteria colonisation of Arabidopsis roots resulted in decreased larval weight of M. brassicae, whereas no effect was observed on larval weight of P. brassicae. Using a jasmonic acid (JA)-impaired mutant (dde2-2), we confirmed the importance of JA in rhizobacteria-mediated ISR against M. brassicae. Interestingly, in some experiments we also observed rhizobacteria-induced systemic susceptibility to M. brassicae. The role of soil composition in the variable outcomes of microbe-plant-insect interactions was then assessed by comparing M. brassicae performance and gene transcription in plants grown in potting soil or a mixture of potting soil and sand in a 1:1 ratio. In a mixture of potting soil and sand, rhizobacteria treatment had a consistent negative effect on M. brassicae, whereas the effect was more variable in potting soil. Interestingly, at 24 h post-infestation (hpi) rhizobacteria treatment primed plants grown in a mixture of potting soil and sand for stronger expression of the JA- and ethylene-regulated genes PDF1.2 and HEL, respectively. Our study shows that soil composition can modulate rhizobacteria-plant-insect interactions, and is a factor that should be considered when studying these belowground-aboveground interactions.

Assessing the efficacy of candidate mosquito repellents against the background of an attractive source that mimics a human host.

Mosquito repellents are used around the globe to protect against nuisance biting and disease-transmitting mosquitoes. Recently, there has been renewed interest in the development of repellents as tools to control the transmission of mosquito-borne diseases. We present a new bioassay for the accurate assessment of candidate repellent compounds, using a synthetic odour that mimics the odour blend released by human skin. Using DEET (N,N-diethyl-meta-toluamide) and PMD (p-menthane-3,8-diol) as reference compounds, nine candidate repellents were tested, of which five showed significant repellency to the malaria mosquito Anopheles gambiae sensu stricto (Diptera: Culicidae). These included: 2-nonanone; 6-methyl-5-hepten-2-one; linalool; δ-decalactone, and δ-undecalactone. The lactones were also tested on the yellow fever mosquito Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae), against which they showed similar degrees of repellency. We conclude that the lactones are highly promising repellents, particularly because these compounds are pleasant-smelling, natural products that are also present in human food sources.

Rhizobacteria modify plant-aphid interactions: a case of induced systemic susceptibility.

Beneficial microbes, such as plant growth-promoting rhizobacteria and mycorrhizal fungi, may have a plant-mediated effect on insects aboveground. The plant growth-promoting rhizobacterium Pseudomonas fluorescens can induce systemic resistance in Arabidopsis thaliana against several microbial pathogens and chewing insects. However, the plant-mediated effect of these beneficial microbes on phloem-feeding insects is not well understood. Using Arabidopsis as a model, we here report that P. fluorescens has a positive effect on the performance (weight gain and intrinsic rate of increase) of the generalist aphid Myzus persicae, while no effect was recorded on the crucifer specialist aphid Brevicoryne brassicae. Additionally, transcriptional analyses of selected marker genes revealed that in the plant-microbe interaction with M. persicae, rhizobacteria (i) prime the plant for enhanced expression of LOX2, a gene involved in the jasmonic acid (JA)-regulated defence pathway, and (ii) suppress the expression of ABA1, a gene involved in the abscisic acid (ABA) signalling pathway, at several time points. In contrast, almost no effect of the plant-microbe interaction with B. brassicae was found at the transcriptional level. This study presents the first data on rhizobacteria-induced systemic susceptibility to an herbivorous insect, supporting the pattern proposed for other belowground beneficial microbes and aboveground phloem feeders. Moreover, we provide further evidence that at the transcript level, soil-borne microbes modify plant-aphid interactions.

Herbivore-induced volatiles of cabbage (Brassica oleracea) prime defence responses in neighbouring intact plants.

When attacked by herbivores, plants release herbivore-induced plant volatiles (HIPV) that may function in direct defence by repelling herbivores or reducing their growth. Emission of HIPV may also contribute to indirect defence by attracting natural enemies of the herbivore. Here, cabbage (Brassica oleracea L.) plants (receiver plants) previously exposed to HIPV and subsequently induced through feeding by five Pieris brassicae L. caterpillars attracted more Cotesia glomerata L. parasitoids than control plants. HIPVs to which receiver plants had been exposed were emitted by B. oleracea infested with 50 P. brassicae caterpillars. Control plants had been exposed to volatiles from undamaged plants. In contrast, there were no differences in the attraction of wasps to receiver plants induced through feeding of one or ten larvae of P. brassicae compared to control plants. In addition, RT-PCR demonstrated higher levels of LIPOXYGENASE (BoLOX) transcripts in HIPV-exposed receiver plants. Exposure to HIPV from emitter plants significantly inhibited the growth rate of both P. brassicae and Mamestra brassicae caterpillars compared to growth rates of caterpillars feeding on control receiver plants. Our results demonstrate plant-plant signalling leading to priming of both indirect and direct defence in HIPV-exposed B. oleracea plants.

Behavioural responses of Anopheles gambiae sensu stricto to components of human breath, sweat and urine depend on mixture composition and concentration.

Host-seeking behaviour of the anthropophilic malaria vector Anopheles gambiae sensu stricto (Diptera: Culicidae) is mediated predominantly by olfactory cues. Several hundreds of odour components have been identified from human emanations, but only a few have been proven to act as attractants or synergists in the host-seeking behaviour of female An. gambiae. In previous work, aromatics, alcohols and ketones in human odours were found to elicit electrophysiological activity in antennal olfactory neurons of female An. gambiae. However, the behavioural effects of these compounds have not been investigated. In this study, behavioural responses of female An. gambiae to components of human breath, urine and sweat at a series of concentrations, or a single concentration in the case of acetone, were examined in combination with ammonia and L-lactic acid in a dual-choice olfactometer. The results showed that at specific concentrations 4-ethylphenol, indole, 3-methyl-1-butanol and two ketones inhibited the attractive effect of a mixture of ammonia and lactic acid. Acetone on its own was not attractive; however, when combined with lactic acid, the binary mixture was attractive. When combined with ammonia, acetone inhibited the attractiveness exerted by ammonia alone. Dodecanol and dimethyldisulphide did not affect the attraction exerted by ammonia and lactic acid at any of the concentrations tested. By contrast, a human-specific armpit odour, 7-octenoic acid, augmented the attraction exerted by the combination of ammonia and lactic acid at a specific dosage.

Chemosensory basis of behavioural plasticity in response to deterrent plant chemicals in the larva of the Small Cabbage White butterfly Pieris rapae.

Behavioural and electrophysiological responsiveness to three chemically different secondary plant substances was studied in larvae of Pieris rapae L. (Lepidoptera: Pieridae). Three groups of caterpillars were studied that during their larval development were exposed to different rearing diets: an artificial diet or one of two host-plants, cabbage, Brassica oleracea, or nasturtium, Tropaeolum majus. In dual-choice leaf disc assays, caterpillars reared on cabbage were strongly deterred by the phenolic chlorogenic acid, the flavonol glycoside naringin and the alkaloid strychnine. However, behavioural plasticity was found in caterpillars reared on nasturtium or artificial diet in that these did not discriminate against chlorogenic acid. Caterpillars reared on the artificial diet were also significantly less sensitive to naringin and strychnine in the behavioural assay. Electrophysiological studies of the maxillary sensilla styloconica revealed that the deterrent neuron in the medial sensillum, but not in the lateral sensillum, of cabbage-reared caterpillars was more sensitive than the same neuron type of caterpillars reared on nasturtium or artificial diet. We conclude that (1) the diet-induced behavioural habituation to deterrents can at least partly be explained by chemosensory desensitisation of a generalist type of maxillary deterrent neuron; (2) behavioural cross-habituation to the three structurally diverse deterrent compounds can be traced back to cross-sensitivity for these compounds in the same gustatory neuron.

Flower vs. leaf feeding by Pieris brassicae: glucosinolate-rich flower tissues are preferred and sustain higher growth rate.

Interactions between butterflies and caterpillars in the genus Pieris and plants in the family Brassicaceae are among the best explored in the field of insect-plant biology. However, we report here for the first time that Pieris brassicae, commonly assumed to be a typical folivore, actually prefers to feed on flowers of three Brassica nigra genotypes rather than on their leaves. First- and second-instar caterpillars were observed to feed primarily on leaves, whereas late second and early third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed. We investigated growth rates of caterpillars having access exclusively to either leaves of flowering plants or flowers. In addition, we analyzed glucosinolate concentrations in leaves and flowers. Late-second- and early-third-instar P. brassicae caterpillars moved upward into the inflorescences of B. nigra and fed on buds and flowers until the end of the final (fifth) instar, after which they entered into the wandering stage, leaving the plant in search of a pupation site. Flower feeding sustained a significantly higher growth rate than leaf feeding. Flowers contained levels of glucosinolates up to five times higher than those of leaves. Five glucosinolates were identified: the aliphatic sinigrin, the aromatic phenylethylglucosinolate, and three indole glucosinolates: glucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Tissue type and genotype were the most important factors affecting levels of identified glucosinolates. Sinigrin was by far the most abundant compound in all three genotypes. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in both leaves and flowers were significantly higher than in undamaged plants, whereas 4-hydroxyglucobrassicin leaf levels were lower. Our results show that feeding on flower tissues, containing higher concentrations of glucosinolates, provides P. brassicae with a nutritional benefit in terms of higher growth rate. This preference appears to be in contrast to published negative effects of volatile glucosinolate breakdown products on the closely related Pieris rapae.

Interindividual variation in the attractiveness of human odours to the malaria mosquito Anopheles gambiae s. s.

Differences between human individuals in their attractiveness to female mosquitoes have been reported repeatedly, but the underlying mechanisms are not well understood. Skin emanations from 27 human individuals, collected on glass marbles, were tested against ammonia in a dual-choice olfactometer to establish their degrees of attractiveness to anthropophilic Anopheles gambiae s.s. Giles (Diptera: Culicidae) mosquitoes. Ammonia was used as a standard odour source because of its proven attractiveness to An. gambiae s.s. Skin emanations from most volunteers attracted significantly more mosquitoes than ammonia. There were clear differences in the attractiveness of skin emanations from different volunteers relative to that of ammonia, as well as in the strength of the trap entry response. Consistent differences were observed when emanations from the three most and the three least attractive volunteers were tested pairwise. No gender or age effect was found for relative attractiveness or trap entry response. Emanations from volunteers with higher behavioural attractiveness elicited higher electroantennogram response amplitudes in two pairs, but in a third pair a higher electroantennogram response was found for the less attractive volunteer. These results confirm that odour contributes to the differences in attractiveness of humans to mosquitoes.

Hybridization between Helicoverpa armigera and Helicoverpa assulta (Lepidoptera: Noctuidae): development and morphological characterization of F1 hybrids.

Reciprocal hybridizations between Helicoverpa armigera (Hübner) and Helicoverpa assulta (Guenée) were studied. The cross between females of H. armigera and males of H. assulta yielded only fertile males and sterile individuals lacking an aedeagus, valva or ostium bursae. A total of 492 larvae of the F1 generation were obtained and 374 of these completed larval development and pupated. Only 203 pupae were morphologically normal males, the remaining 171 pupae were malformed. Larvae and pupae that gave rise to morphologically abnormal adults exhibited longer development times. Sterility was not only associated with malformed external sex organs, but also a range of abnormalities of the internal reproductive system: (i) loss of internal reproductive organs, (ii) with one to three copies of an undeveloped bursa copulatrix; or (iii) with one or two undeveloped testes. Normal male hybrid adults showed higher flight activity in comparison with males of both species. In contrast, the cross between females of H. assulta and males of H. armigera yielded morphologically normal offspring (80 males and 83 females). The interaction of the Z-chromosome from H. assulta with autosomes from H. armigera might result in morphological abnormalities found in hybrids and backcrosses, and maternal-zygotic incompatibilities might contribute to sex bias attributed to hybrid inviability.

Behavioural and electrophysiological responses of the malaria mosquito Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) to human skin emanations.

Behavioural and electrophysiological responses of Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) to human skin emanations collected on glass beads were studied using a dual-port olfactometer and electroantannography. Glass beads to which skin emanations from human hands had been transferred elicited a level of attraction similar to a human hand. The attractiveness of these handled glass beads faded away 4 h after transfer onto the beads. Storage at -20 degrees C for up to 8 weeks showed a decreased but still attractive effect of the beads. In a choice test between one individual and four others, the emanations from the reference individual were significantly more attractive in three out of four cases. The headspace of handled glass beads elicited a dose-dependent EAG response. The substances causing EAG activity could be removed partially by dichloromethane, ethanol and pentane-ether. Glass beads provide a suitable neutral substrate for the transfer of human odour to enable chemical analysis of the human skin emanations for identification of kairomones of anthropophilic mosquitoes.

Sensitivities of antennal olfactory neurons of the malaria mosquito, Anopheles gambiae, to carboxylic acids.

Single sensillum recordings on the antennae of female Anopheles gambiae s.s. mosquitoes revealed neurons sensitive to aliphatic carboxylic acids within (a) subtype(s) sensilla trichodea. The aliphatic acids, acetic acid, propionic acid, butyric acid, iso-butyric acid and iso-valeric acid evoked an inhibition reaction in one of the cell types recorded from. A different cell type was excited in response to the former aliphatic acids, but showed a broader range of sensitivity, as acids with a longer carbon chain length, like caproic acid, elicited excitations as well. In addition, occasionally 1-octen-3-ol elicited an excitation reaction. This article focuses on the carboxylic acid inhibited cell type and its temporal pattern of response to different doses of the compounds. Furthermore, in order to compare the stimulatory effectiveness of the compounds on a per molecule basis, corrections were made for differences in volatility by determining the absolute number of molecules in the stimulus puff.