Safety, regulatory and environmental issues related to breeding and international trade of edible insects in Africa.

Insect breeding or farming for food and feed is an emerging enterprise that can address the ever-growing demand for protein and curb high unemployment rates in Africa and beyond. However, for the sector to prosper, its value chain needs to be regulated to ensure sustainability and safety for consumers and the environment. Although a few African countries, such as Kenya, Uganda and Rwanda, have promulgated standards on the use of insects as food and feed, greater efforts are needed in other countries, and relevant policies governing the sector need to be formulated. All over the globe, attention to the regulation of the edible insect sector is increasing, and more investment in the industry is foreseen. Safety issues such as identifying which species should be reared, substrate quality and traceability imposed by importing countries will be critical for expansion of the sector. This paper analyses safety, regulatory and environmental issues related to breeding and international trade of edible insects in Africa and provides case studies and recommendations for sustainable use of insects for food and feed.

Les élevages et les fermes d'insectes destinés à l'alimentation humaine et animale sont de nouvelles entreprises qui pourraient répondre à la hausse continue de la demande en protéines tout en réduisant les taux élevés du chômage en Afrique et ailleurs. Néanmoins, pour que ce secteur puisse prospérer, sa chaîne de création de valeur doit être réglementée afin de garantir sa durabilité et son innocuité pour les consommateurs et l'environnement. Si un petit nombre de pays africains dont le Kenya, l'Ouganda et le Rwanda ont élaboré des normes applicables à l'utilisation des insectes pour l'alimentation humaine et animale, dans d'autres pays les efforts doivent se poursuivre et des politiques appropriées doivent être mises en place pour régir ce secteur. Partout dans le monde, la réglementation du secteur des insectes comestibles fait désormais l'objet d'une attention considérable et des investissements accrus dans la filière sont attendus. Les questions de sécurité telles que l'identification des insectes à élever, la qualité du substrat et la traçabilité exigée par les pays importateurs seront cruciales pour le développement du secteur. Les auteurs analysent les questions de sécurité et les enjeux réglementaires et environnementaux liés à l'élevage et au commerce international d'insectes comestibles en Afrique et présentent des études de cas et des recommandations pour une utilisation durable des insectes destinés à l'alimentation humaine et animale.

La cría o producción de insectos con fines de alimentación humana o animal es una actividad incipiente que puede ayudar a responder a la siempre creciente demanda de proteínas y a contener las elevadas tasas de desempleo de África y otras regiones. Para que el sector prospere, no obstante, es preciso reglamentar su cadena de valor a fin de asegurar su sostenibilidad y su inocuidad para el consumidor y el medio ambiente. Aunque unos pocos países africanos, como Kenia, Uganda o Ruanda, tienen promulgadas normas sobre el uso de insectos para la alimentación humana o la producción de piensos, aún hay que redoblar esfuerzos en otros países y formular políticas que ordenen el sector. En todo el planeta se presta hoy una atención sin precedentes a la regulación del sector de los insectos comestibles, un sector que previsiblemente va a recibir cada vez más inversiones. Las cuestiones ligadas a la inocuidad, como la identidad de los insectos que se van a producir, la calidad del substrato o la rastreabilidad impuesta por los países importadores, serán fundamentales para el desarrollo del sector. Los autores analizan las cuestiones reglamentarias, ambientales y de inocuidad que se plantean en relación con la cría y el comercio internacional de insectos comestibles en África, presentan ejemplos concretos y formulan recomendaciones para un uso sostenible de los insectos con fines de alimentación humana o animal.

Utilizing Genomics to Study Entomopathogenicity in the Fungal Phylum Entomophthoromycota: A Review of Current Genetic Resources.

The order Entomophthorales, which formerly contained c.280 species, has recently been recognized as a separate phylum, Entomophthoromycota, consisting of three recognized classes and six families. Many genera in this group contain obligate insect-pathogenic species with narrow host ranges, capable of producing epizootics in natural insect populations. Available sequence information from the phylum Entomophthoromycota can be classified into three main categories: first, partial gene regions (exons+introns) used for phylogenetic inference; second, protein coding gene regions obtained using degenerate primers, expressed sequence tag methodology or de novo transcriptome sequencing with molecular function inferred by homology analysis; and third, primarily forthcoming whole-genome sequencing data sets. Here we summarize the current genetic resources for Entomophthoromycota and identify research areas that are likely to be significantly advanced from the availability of new whole-genome resources.

Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis

Abstract The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host–pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis.

Temperature dependent virulence of obligate and facultative fungal pathogens of honeybee brood.

Chalkbrood (Ascosphaera apis) and stonebrood (Aspergillus flavus) are well known fungal brood diseases of honeybees (Apis mellifera), but they have hardly been systematically studied because the difficulty of rearing larvae in vitro has precluded controlled experimentation. Chalkbrood is a chronic honeybee-specific disease that can persist in colonies for years, reducing both brood and honey production, whereas stonebrood is a rare facultative pathogen that also affects hosts other than honeybees and can likely survive outside insect hosts. Hive infection trials have indicated that accidental drops in comb temperature increase the prevalence of chalkbrood, but it has remained unclear whether virulence is directly temperature-dependent. We used a newly established in vitro rearing technique for honeybee larvae to test whether there are systematic temperature effects on mortality induced by controlled infections, and whether such effects differed between the two fungal pathogens. We found that increasing spore dosage at infection had a more dramatic effect on mortality from stonebrood compared to chalkbrood. In addition, a 24h cooling period after inoculation increased larval mortality from chalkbrood infection, whereas such a cooling period decreased mortality after stonebrood infection. These results raise interesting questions about honeybee defenses against obligate and facultative pathogens and about the extent to which stress factors in the host (dis)favor pathogens with lesser degrees of specialization.

Long-term storage of Ascosphaera aggregata and Ascosphaera apis, pathogens of the leafcutting bee (Megachile rotundata) and the honey bee (Apis mellifera).

Survival rates of Ascosphaera aggregata and Ascosphaera apis over the course of a year were tested using different storage treatments. For spores, the storage methods tested were freeze-drying and ultra-low temperatures, and for hyphae, freeze-drying, agar slants, and two methods of ultra-low temperatures. Spores of A. aggregata and A. apis stored well at -80 degrees C and after freeze-drying. A. aggregata hyphae did not store well under any of the methods tested while A. apis hyphae survived well using cryopreservation. Spores produced from cryopreserved A. apis hyphae were infective. Long-term storage of these two important fungal bee diseases is thus possible.

Bizarre interactions and endgames: entomopathogenic fungi and their arthropod hosts.

Invertebrate pathogens and their hosts are taxonomically diverse. Despite this, there is one unifying concept relevant to all such parasitic associations: Both pathogen and host adapt to maximize their own reproductive output and ultimate fitness. The strategies adopted by pathogens and hosts to achieve this goal are almost as diverse as the organisms themselves, but studies examining such relationships have traditionally concentrated only on aspects of host physiology. Here we review examples of host-altered behavior and consider these within a broad ecological and evolutionary context. Research on pathogen-induced and host-mediated behavioral changes demonstrates the range of altered behaviors exhibited by invertebrates including behaviorally induced fever, elevation seeking, reduced or increased activity, reduced response to semiochemicals, and changes in reproductive behavior. These interactions are sometimes quite bizarre, intricate, and of great scientific interest.

Diversity of entomopathogenic fungi near leaf-cutting ant nests in a neotropical forest, with particular reference to Metarhizium anisopliae var. anisopliae.

We investigated the prevalence of entomopathogenic fungi associated with leaf-cutting ant colonies in a small area of tropical forest in Panama. There was a high abundance of Metarhizium anisopliae var. anisopliae near the colonies. Beauveria bassiana was also detected in the soil, Aspergillus flavus in dump material, and six Camponotus atriceps ants were found infected with Cordyceps sp. Based on a partial sequence of the IGS region, almost all of the M. anisopliae var. anisopliae isolates fell within one of the three main clades of M. anisopliae var. anisopliae, but with there still being considerable diversity within this clade. The vast majority of leaf-cutting ants collected were not infected by any entomopathogenic fungi. While leaf-cutting ants at this site must, therefore, regularly come into contact with a diversity of entomopathogenic fungi, they do not appear to be normally infected by them.

The hidden lifestyles of Bacillus cereus and relatives.

Bacillus cereus sensu lato, the species group comprising Bacillus anthracis, Bacillus thuringiensis and B. cereus (sensu stricto), has previously been scrutinized regarding interspecies genetic correlation and pathogenic characteristics. So far, little attention has been paid to analysing the biological and ecological properties of the three species in their natural environments. In this review, we describe the B. cereus sensu lato living in a world on its own; all B. cereus sensu lato can grow saprophytically under nutrient-rich conditions, which are only occasionally found in the environment, except where nutrients are actively collected. As such, members of the B. cereus group have recently been discovered as common inhabitants of the invertebrate gut. We speculate that all members disclose symbiotic relationships with appropriate invertebrate hosts and only occasionally enter a pathogenic life cycle in which the individual species infects suitable hosts and multiplies almost unrestrained.

[Detection of enterotoxin genes in Bacillus thuringiensis by PCR].

The presence of hemolysin HblA (hblA), enterotoxin BceT (bceT) and enterotoxin S (entS) genes from 45 strains of B. thuringiensis, 4 strains of B. cereus and B. sphaericus have been detected respectively by multiple PCR. The results showed that 95.6% B. thuringiensis strains contain the B component of hblA gene, 91.1% and 93.3% of them contain bceT and entS genes sequences respectively. The enterotoxin productions in all strains have also been analysis using two commercial immunoassay kits(TECRA and RPLA) and it has proved that most of B. thuringiensis stains and the positive B. cereus strain can produce entero toxins during their growth. However, the two hblA sequence positive stains, DBT007 was negative when tested both by RPLA and Tecra, T24 001 was negative when assayed by Tecra and positive by RPLA. One hblA sequence negative strain Dmu39 was negative when tested by RPLA but positive by Tecra. No enterotoxin and enterotoxin gene could be detected in B. thuringiensis DBT248 and the B. sphaericus strains. The results suggest that the potential risk of using B. thuringiensis, as biopesticide needs to be further evaluated.

Natural occurrence of entomophthoralean fungi pathogenic to collembolans.

The aim of this study was to assess the natural occurrence of fungi from the order Entomophthorales (Zygomycetes) pathogenic to a range of collembolan species. A total of 11,709 collembolans representing a total of 15 species from all three subclasses within Collembola were collected and incubated. Infection with Neozygites-like spp. (Entomophthorales) was recorded in the two subclasses, Arthropleona and Symphypleona. The morphology of the primary conidia of the Neozygites-like fungi infecting collembolans showed a high degree of variation in shape and size, and the fungi were divisible into five groups. One group consisted of Neozygites sminthuri, while the other four groups, while still closely resembling the genus Neozygites, did not fit within the description of any known species and were preliminarily designated Neozygites sp. nov. 1 to 4. Neozygites sp. nov. 1, infecting Sphaeridia pumilis, was observed to form globular secondary conidia, which have not been observed before within the genus Neozygites. A formal species description needs, however, yet to be performed. The infection was mainly found in species from the subclass Symphypleona, where two new host genera for Neozygites, Deuterosminthurus and Sphaeridia, were recorded. However, the prevalence of Neozygites spp. was low, up to 10% in S. pumilis, 6% in Sminthurus viridis, and below 1% in Deuterosminthurus sulphureus. Infection with an obligatory entomopathogenic fungus was for the first time well documented within the subclass Arthropleona, where specimens of Orchesella cincta and Orchesella villosa sporulated with a N. sminthuri-like fungus. Additionally, infection with the facultative pathogen Conidiobolus coronatus was recorded from several hosts representing both the subclass Arthropleona (Isotoma anglicana and O. cincta) and the subclass Symphypleona (Dicyrtoma sp. and S. viridis). For most species the prevalence of C. coronatus resembled that of Neozygites spp. and did not exceed 8.8%. The only exception was Dicyrtoma sp., for which the only associated entomophthoralean fungus was C. coronatus, where the prevalence reached 17.8% infection.

Intraspecific variation and host specificity of Entomophthora muscae sensu stricto isolates revealed by random amplified polymorphic DNA, universal primed PCR, PCR-restriction fragment length polymorphism, and conidial morphology.

The intraspecific variations of Entomophthora muscae s. str. associated with particular host species, Musca domestica and Delia radicum, sampled from different localities and different years in Denmark and the variation of E. muscae s. str. originating from different host taxa were investigated. The isolates were compared both by primary spore morphology and by three molecular methods: random amplified polymorphic DNA, universal primed PCR, and PCR-restriction fragment length polymorphism. Analyses of the different molecular data showed the same overall picture and separated E. muscae s. str. into two main groups with all the M. domestica isolates in one group and isolates from D. radicum, Coenosia tigrina, and Pegoplata infirma in the second group. E. muscae s. str. isolates from M. domestica also differ significantly from the rest of the E. muscae s. str. isolates with regard to the morphology of the primary conidia, which were bigger and contained significantly more nuclei per conidium. Several different E. muscae s. str. genotypes were documented and each type was restricted to a single host species, indicating a very high degree of host specificity at or below the level of the subfamily.

Geographical distribution and host range of entomophthorales infecting the green spruce aphid Elatobium abietinum Walker in Iceland.

Entomophthora planchoniana and Neozygites fresenii caused infection in populations of the green spruce aphid, Elatobium abietinum, in Iceland. On this aphid species En. planchoniana was exclusively found in the western part of Iceland, while N. fresenii was exclusively found in the eastern part of Iceland. This discrete and nearly nonoverlapping geographical distribution correlates with the distribution of two different populations of El. abietinum found in Iceland. On other aphid species N. fresenii, En. planchoniana, Pandora neoaphidis, and Conidiobolus obscurus were documented throughout the country. Transmission experiments showed that Pa. neoaphidis and En. planchoniana could infect the eastern population of El. abietinum, although they have never been found on this population in nature. This strongly indicates that there is little or no interaction among El. abietinum, other aphids, and their respective entomophthoralean fungi in the field. Furthermore, this study is the first to record epizootics caused by N. fresenii and En. planchoniana in the subpolar region.

Isolation and in vitro cultivation of the aphid pathogenic fungus Entomophthora planchoniana.

Entomophthora planchoniana is an important fungal pathogen of aphids. Although Entomophthora chromaphidis has been considered a synonym for E. planchoniana, the two species are now separated, and E. planchoniana is reported not to grow in vitro. In this paper, we describe for the first time the isolation and cultivation of this species. Entomophthora planchoniana was isolated from a population of Ovatus crataegarius (Homoptera, Aphididae), which was infected by E. planchoniana only. The isolates did not sporulate, but the sequence of the small subunit rDNA and the restriction fragment length polymorphism patterns of the first part of the large subunit rDNA and the ITS II region confirm that the isolates were E. planchoniana. The isolated fungus grew in a medium consisting of Grace's insect cell culture medium supplemented with lactalbumin hydrolysate, yeastolate, and 10% fetal bovine serum or in GLEN medium with 10% fetal bovine serum. Vegetative cells of E. planchoniana were long and club-shaped and did not stain with Calcofluor, thus suggesting that they were protoplasts.

Entomophthora muscae resting spore formation in vivo in the host Delia radicum.

The formation in vivo of Entomophthora muscae resting spores was investigated in the host, Delia radicum (cabbage root fly), by analysis of field data on the seasonal occurrence of E. muscae resting spores over 4 years. E. muscae resting spores in D. radicum were spherical with an average diameter of 39.4 microm, and the average numbers produced were estimated at 5.7 x 10(4) resting spores/female cadaver. Resting spores were found only after midsummer in D. radicum and almost exclusively in females. The proportion of infected females with resting spores was negatively correlated with average weekly day length after midsummer. We did not detect any significant year effect; thus, the results support the hypothesis that the photoperiod is the most important abiotic factor controlling E. muscae resting spore formation in D. radicum.

Who's in charge here anyway? Managing the management split in mental health organizations.

Most mental health organizations are run by chief executive officers (CEOs) who are not physicians, with medical directors reporting to the CEOs. In this article the historical and organizational origins of this arrangement are reviewed. The well known disadvantages of shared management are discussed, as are the less obvious advantages. Through case vignettes the authors illustrate how bifurcated leadership can promote productive and creative administrative decisions. Guidelines are offered for strengthening collaborations between non-medical and medical mental health program directors.

Natural coprevalence of Strongwellsea castrans, Cystosporogenes deliaradicae, and Bacillus thuringiensis in the host, Delia radicum.

Adult cabbage root flies (Delia radicum) from three Danish localities were diagnosed microscopically for the natural prevalence of Strongwellsea castrans, Cystosporogenes deliaradicae, and Bacillus thuringiensis. C. deliaradicae was significantly coprevalent with S. castrans. B. thuringiensis sporangia were diagnosed in the hemolymph in two D. radicum which were also infected with S. castrans and proved to belong to serovar aizawai and serovar balearica. The biological characterization of S. castrans proved that at 17.5 degrees C flies developed an abdominal hole 7.9 days (mean) after infection and that 5.7 days (mean) passed from the emergence of the hole to the death of the infected host. No mortality effect among D. radicum subjected to B. thuringiensis serovar aizawai, balearica, and kurstaki isolates was detected. RAPD with DNA proved that six B. thuringiensis serovar balearica isolates (all from the same fly) were indistinguisable. This indicates that proliferation of B. thuringiensis in the abdomen of an S. castrans-infected D. radicum may be due to just one genotype. The profiles of one isolated aizawai strain did not correspond to the profiles of other serovar aizawai strains used for comparison. The biological significance of the interaction between the involved pathogens is discussed.

Natural host range and prevalence of the genus strongwellsea (Zygomycota: entomophthorales) in denmark

The natural occurrence and host range of species of the insect pathogenic fungal genus Strongwellsea [Zygomycota: Entomophthorales] were studied by extensive sampling and examination of adult Diptera [Cyclorrhapha]. The host range for Strongwellsea spp. was significantly enlarged. Three families were documented as new hosts: Muscidae (three species), Calliphoridae (one species), and Sarcophagidae (one species). Further, within the family Anthomyiidae six new host species were recorded and three new host species were documented in the Fanniidae. Strongwellsea castrans was identified as the pathogen in the Anthomyiids, while records from Fanniidae belonged to S. magna. The records of S. magna were the first outside the type locality (California). Primary conidia morphology indicated that muscid and calliphorid species were infected by three undescribed species of Strongwellsea. For the sarcophagid fly, no conidia were encountered, so the Strongwellsea species could not be identified. The tested sampling methods had each different advantages. Sweep netting and diagnosis in situ gave the best opportunity to sample a high number of infected dipterans per time unit spend, while sweep netting followed by incubation in the laboratory was the only method for the documentation of resting spores. The prevalence of S. castrans in the cabbage root fly Delia radicum was obtained by two methods: Samples collected by sweep net and incubated and water trap samples. Water trap captures gave higher prevalences of conidial infections than sweep-net captures. Measured prevalences of Strongwellsea spp. infections are therefore highly dependent on sampling method. The occurrence of resting spores of S. castrans in D. radicum was almost exclusively restricted to females and varied during the season. In samples from 1988 through 1993, no infected females in June contained resting spores, while 43.0% of the S. castrans-infected females from samples in August contained resting spores. During September and October, a decreasing proportion of S. castrans-infected D. radicum contained resting spores. The results document that species from the genus Strongwellsea are common fungal pathogens of adult flies from different families, occasionally with high prevalences. It also appears that the two described species of Strongwellsea, S. castrans and S. magna, have a range of dipterous host species that may always belong to a single family, Anthomyiidae and Fanniidae, respectively. Our data shows also that the host family Muscidae may be exploited by two new species of Strongwellsea. Copyright 1999 Academic Press.

Light and electron microscopy studies of the infection of the western flower thrips frankliniella occidentalis (Thysanoptera: thripidae) by the entomopathogenic fungus metarhizium anisopliae

The infection of Frankliniella occidentalis by two isolates of the entomopathogenic fungus Metarhizium anisopliae was studied using fluorescence, scanning, transmission, and confocal scanning laser microscopy techniques. Conidia of M. anisopliae adhered mostly to the wings of adult F. occidentalis but the number declined from 73 to 40% within 72 h postinoculation at 23 degreesC, presumably due to preening. Conidia germinated and produced appressoria on adult, larval, and pupal stages within 12 h post inoculation. However, penetration pegs were not observed until 30 h postinoculation. Light microscopy and transmission electron microscopy provided further information on fungal development inside the insect. The fungus colonized the insect hemocoel from day 3 and sporulated approximately 6 days postinoculation. Copyright 1999 Academic Press.

Relationships of the insect-pathogenic order Entomophthorales (Zygomycota, Fungi) based on phylogenetic analyses of nuclear small subunit ribosomal DNA sequences (SSU rDNA).

We sequenced the nuclear small subunit of ribosomal DNA (SSU rDNA) from seven species within the insect-pathogenic order Entomophthorales. These sequences were aligned with other published SSU rDNA sequences and phylogenies were inferred using phenetic and cladistic methods. Based on three different phylogenetic methods the Entomophthorales (excluding Basidiobolus ranarum) is monophyletic; B. ranarum was more closely related to chytrids from Chytridiales and Neocallimasticales than to Entomophthorales, as was proposed by Nagahama et al. (Mycologia 87: 203-209, 1995). Nuclear characters (large nuclei containing conspicuous condensed chromatin and lack of a prominent nucleolus) were of predictive value for the monophyly of the family Entomophthoraceae. Conidial characters separate the Entomophthoraceae, which only includes obligate pathogens, into at least two lineages: one lineage with uninucleate conidia and another with multinucleate conidia. The two species of Conidiobolus studied were paraphyletic in our analyses and only distantly related to each other. This information may prove to be important in the use of these fungi as biocontrol agents.

Molecular and phenotypic characterization of bacillus thuringiensis isolated from leaves and insects

Bacillus thuringiensis isolates from the phylloplane of organically cultivated cabbage were characterized using molecular and phenotypic methods. Of the 58 isolates under study, 31 belonged to serovar kurstaki, 16 did not react with any of the currently recognized antisera, 7 reacted with known antisera, and 4 could not be serotyped as they were nonmotile. Round crystals were found in 26 isolates, while bipyramidal crystals were found in the remaining 32 isolates, all of which had activity to lepidopteran larvae. Further, one isolate with unknown serotype and round crystals had lepidopteran activity. Colony hybridization was found to be a useful tool for screening the isolates for specific gene homologies and showed good correlation with the phenotypic observations. Polymerase chain reaction (PCR) was used for confirmation of the colony hybridization data, in most cases with concordant results. However, in one case some of the colony hybridization data could not be confirmed by PCR, due to DNA sequence variations in the binding area of one of the primers. The random amplified polymorphic DNA (RAPD) analysis showed that isolates otherwise indistinguishable could be distinguished by this method. However, the method was not able to distinguish the 31 kurstaki isolates. Further, the kurstaki isolates could not be distinguished from the B. thuringiensis serovar kurstaki HD-1 strain used in commercial products for lepidopteran control. One of the isolates was a serovar israelensis, but no genes encoding dipteran activity could be detected, and the RAPD analysis revealed that the DNA fingerprint of this israelensis isolate deviated from the israelensis ONR60A isolate used in commercial products. In conclusion we find that a molecular method like colony hybridization is suitable for screening large collections of bacteria. When colony hybridization data are combined with RAPD analyses isolates can be grouped based on genetic potential and DNA fingerprint, whereby further characterizations by PCR and the more labourious phenotypic methods can be performed more effectively. Copyright 1998 Academic Press.