Aurélie Babin

Marie-Paule Nawrot-Esposito

Armel Gallet

Jean-Luc Gatti
email: jean-luc.gatti@inrae.fr

Marylène Poirié

Differential side-effects of Bacillus thuringiensis bioinsecticide on non-target Drosophila flies

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

www.nature.com/scientificreports/ ingested by insect larvae, the toxin crystals are dissolved by the midgut alkaline pH, releasing ~ 130 kDa protoxins that are then processed by digestive proteases into smaller, soluble, active toxin fragments of ~ 60-70 kDa [START_REF] Bravo | Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control[END_REF][START_REF] Wei | Activation of Bt protoxin Cry1Ac in resistant and susceptible cotton bollworm[END_REF] . Active toxins bind to specific receptors of midgut epithelial cells, eliciting pores formation in the cell membrane, cell lysis and gut epithelium disorganization [START_REF] Bravo | Bacillus thuringiensis: A story of a successful bioinsecticide[END_REF] . This allows gut bacteria, including Bt, to colonize the hemocoel, and leads to rapid septicaemia and death [START_REF] Caccia | Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism[END_REF] .

Numerous impact studies of field application rates and acute intoxications have shown that Bt bioinsecticides are safe or have a limited impact on non-target vertebrates and invertebrates, and associated species communities [START_REF] Glare | Bacillus thuringiensis: Biology, Ecology and Safety[END_REF][START_REF] Rubio-Infante | An overview of the safety and biological effects of Bacillus thuringiensis Cry toxins in mammals[END_REF] . However, the increasing use of bioinsecticides based on Bt spores and toxins has recently raised concern [START_REF] Efsa Panel On | Risks for public health related to the presence of Bacillus cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs[END_REF] and led to the assessment of their potential effects on non-target species, such as auxiliary insects of biological control [START_REF] Amichot | Side effects of Bacillus thuringiensis var. kurstaki on the hymenopterous parasitic wasp Trichogramma chilonis[END_REF] , pollinators [START_REF] Renzi | Chronic toxicity and physiological changes induced in the honey bee by the exposure to fipronil and Bacillus thuringiensis spores alone or combined[END_REF] and species communities which share their habitat with Bt-targeted insect pests [START_REF] Caquet | Effects of repeated field applications of two formulations of Bacillus thuringiensis var. israelensis on non-target saltmarsh invertebrates in Atlantic coastal wetlands[END_REF][START_REF] Duguma | Microbial communities and nutrient dynamics in experimental microcosms are altered after the application of a high dose of Bti[END_REF][START_REF] Venter | Interactions between Bt crops and aquatic ecosystems: A review[END_REF] . There is growing evidence of direct and indirect cross-effects of Bt bioinsecticide formulations and Bt-Cry and Cyt toxins across insect species and orders, or even across phyla, suggesting that Bt targeting is only partly specific [START_REF] Palma | Bacillus thuringiensis toxins: An overview of their biocidal activity[END_REF][START_REF] Venter | Interactions between Bt crops and aquatic ecosystems: A review[END_REF][START_REF] Van Frankenhuyzen | Specificity and cross-order activity of Bacillus thuringiensis pesticidal proteins[END_REF] . Data also showed that almost all of the applied Btk formulation dose was still present on the leaves surface 72 h after spraying [START_REF] Bizzarri | The ecology of Bacillus thuringiensis on the phylloplane: Colonization from soil, plasmid transfer, and interaction with larvae of Pieris brassicae[END_REF] , its amount returning close to environmental levels only 28 days after treatment [START_REF] Raymond | Environmental factors determining the epidemiology and population genetic structure of the Bacillus cereus group in the field[END_REF] . Finally, Bt spores can survive in the soil and on different supports for months and even years after application [START_REF] Hendriksen | Long-term survival and germination of Bacillus thuringiensis var. kurstaki in a field trial[END_REF][START_REF] Hung | Persistence of detectable insecticidal proteins from Bacillus thuringiensis (Cry) and toxicity after adsorption on contrasting soils[END_REF][START_REF] Hung | Fate of insecticidal Bacillus thuringiensis Cry protein in soil: Differences between purified toxin and biopesticide formulation[END_REF][START_REF] Enger | Evaluating the long-term persistence of Bacillus spores on common surfaces[END_REF] . Bt formulations contain also different compounds to protect spores and crystals and aggregate them into a wettable form, surfactants to facilitate spraying and dispersion on plants, and phagostimulants [START_REF] Couch | Industrial fermentation and formulation of entomopathogenic bacteria[END_REF][START_REF] Brar | Recent advances in downstream processing and formulations of Bacillus thuringiensis based biopesticides[END_REF] . Nevertheless, since toxin crystals, and to a much lesser extent spores [START_REF] Setlow | Spore resistance properties[END_REF] , are somewhat sensitive to abiotic conditions (e.g. UV, pH, rainfall), repeated spraying with a minimum delay of 3 to 8 days is often recommended over the period of pest occurrence to achieve the required pest control level [START_REF] Brar | Recent advances in downstream processing and formulations of Bacillus thuringiensis based biopesticides[END_REF][START_REF] Food | Conclusion on the peer review of the pesticide risk assessment of the active substance Bacillus thuringiensis subsp. Kurstaki (strains ABTS 351, PB 54, SA 11, SA 12[END_REF] (https ://www.certi seuro pe.fr; https ://www.certi susa.com). All these can potentially lead to Bt accumulation in the environment, thus raising the rarely addressed issue of potential side-effects of chronic exposure (i.e. continuous and increasing exposure dose for an extended period) of non-target species to doses unexpectedly above those that are recommended.

Diptera are worldwide distributed insects, most of which are not targets of commercial Bt formulations. This is the case of the genus Drosophila, represented by ~ 1500 described species [START_REF] Bächli | The database on Taxonomy of Drosophilidae: Database 2020/1[END_REF] , including the model organism Drosophila melanogaster. In the field, most of these flies feed and reproduce mainly on ripening or rotting/fermenting fruits and are therefore naturally present in areas treated with Bt such as orchards, vineyards and gardening areas. Unable to disperse between food patches, early developmental stages of Drosophila eat intensively and grow exponentially [START_REF] Tennessen | Coordinating growth and maturation-Insights from Drosophila[END_REF] and may thus ingest high doses of Bt bioinsecticides that have accumulated during the treatment periods. Surprisingly, despite the presence of many Drosophila species in Bt-treated areas, their role in the decomposition of organic matter, and the ease of study of some species, only a few studies have focused on these flies. However, most of them suggested susceptibility to Btk, but they used mainly late 3rd instar larvae preparing for pupation, which do not feed much. In addition, these studies used Bt preparations, especially field isolates, that possibly contained highly toxic β-exotoxins, which are not authorized in commercial Bt formulations [START_REF] Benz | The toxic action of Bacillus thuringiensis "exotoxin" on Drosophila reared in yeast-containing and yeastfree media[END_REF][START_REF] Saadoun | Assessment of toxic potential of local Jordanian Bacillus thuringiensis strains on Drosophila melanogaster and Culex sp. (Diptera)[END_REF][START_REF] Khyami-Horani | Toxicity of Bacillus thuringiensis and B. sphaericus to laboratory populations of Drosophila melanogaster (Diptera: Drosophilidae)[END_REF][START_REF] Obeidat | Toxicity of local Bacillus thuringiensis isolates against Drosophila melanogaster[END_REF][START_REF] Obeidat | Toxicity of Bacillus thuringiensis β-exotoxins and δ-endotoxins to Drosophila melanogaster, Ephestia kuhniella and human erythrocytes[END_REF][START_REF] Cossentine | Biological activity of Bacillus thuringiensis in Drosophila suzukii (Diptera: Drosophilidae)[END_REF][START_REF] Biganski | Bacillus thuringiensis serovar israelensis has no effect on Drosophila suzukii Matsumura[END_REF][START_REF] Haller | Effects of purified or plant-produced Cry proteins on Drosophila melanogaster (Diptera: Drosophilidae) larvae[END_REF] . So far, no study addressed the effects of chronic exposure to commercial Bt formulations on developing stages of these Dipterans that are present in Bt-treated areas.

Here, we tested the chronic side-effects of commercial formulations of Btk and, to a lesser extent of Bti, on non-target Drosophila flies (D. melanogaster and seven other Drosophila species), with doses starting from mean recommended spray doses up to ~ 1000 times this dose (i.e. below acute intoxication doses used in most studies). We mainly focused on developmental traits (developmental time, emergence rate), but also on two fitnessrelated traits (longevity and offspring number) of adult flies that developed from the egg under Btk formulation exposure. Our study would be a first-step in the exploration of potential implications of chronic exposure to Btk formulation on Drosophila flies.

Results

Btk formulations adversely impact the development of D. melanogaster. In a dose-response assay, emergence rates (ER) and developmental times (DT) of wild-type D. melanogaster Canton S flies exposed to doses up to 10 7 CFU/g of DELFIN A in a standard low-protein/high-sugar fly medium were similar to those of the control unexposed group (Fig. 1a,b; Table 1). At higher doses, both ER and DT were affected in a dosedependent manner: ER was reduced by 17% at 5 × 10 7 CFU/g (although not statistically significant), up to 100% at 10 9 CFU/g, at which no individual reached the pupal stage. The lethal dose 50 (LD50) was estimated between 5 × 10 7 and 10 8 CFU/g (Fig. 1a). DT was increased of about 0.5 day at 5 × 10 7 CFU/g (+ 4% versus controls) to up to 1.5 days (+ 14%) at 10 8 CFU/g (Fig. 1b; Table 1). The sex-ratio at emergence (SR, proportion of males) was strongly biased towards males at 10 8 CFU/g, with 58% more males compared to the control (Supplementary information S2).

We observed no change in ER using the same dose range of the Btk Cry-free strain 4D22 (Fig. 1a,e; Table 1) and the non-pathogenic Bacillus subtilis (Fig. 1a, Table 1), two controls for the effect of ingestion of high loads of spores. In contrast, addition of the formulation of Bt var. israelensis VectoBac WG reduced ER by 89% only at 10 9 CFU/g (~ 2000 times the recommended dose; Fig. 1a; Table 1; Supplementary information S1). DT varied with the dose of Btk 4D22, the differences being mainly between doses but not with the control. DT increased by ~ 1.5 days at the highest dose of VectoBac WG (Fig. 1b; Table 1) and showed a similar trend with B. subtilis (P = 0.06; Fig. 1b; Table 1). None of these treatments influenced dramatically the SR (Supplementary information S2).

To test whether these effects are generic to Btk formulations, the fly development was evaluated on two other formulations, DELFIN B (same brand) and Scutello DF (brand Dipel), at the critical doses 10 8 and 10 9 CFU/g. As DELFIN A, these formulations contain spores and Cry toxins such as Cry-1A as pro-toxins of ~ 130 kDa, activated toxins of ~ 60-70 kDa, but also as smaller fragments [START_REF] Rubio-Infante | An overview of the safety and biological effects of Bacillus thuringiensis Cry toxins in mammals[END_REF] (Fig. 1e, red asterisks). ER remained unchanged at 10 8 CFU/g whereas no individual reached pupation at 10 9 CFU/g on DELFIN B and very few individuals reached the adult stage on Scutello DF, DT being increased by more than 2 days (Fig. 1c,d; Table 1). No significant bias in SR was observed for either formulation (Supplementary information S2).

Btk formulation strongly impacts survival during the larval stages. Cumulative exposure to DELFIN A from the egg to late stages of the 1st and 2nd instars did not influence larval survival at 10 7 CFU/g but reduced it for both instar larvae above this dose to reach up to 37% mortality at 10 9 CFU/g (Fig. 2a). Reduced survival tended to occur at a lower dose when cumulative survival was measured later in the development, i.e. 10 9 CFU/g for late 1st instar larvae and 10 8 CFU/g for late 2nd instar larvae (Fig. 2a; Table 1). For both instars, larvae surviving at 10 9 CFU/g were noticeably smaller and less active than those surviving at lower doses, and none of these individuals reached the pupal stage (see results above). A 24-h exposure of the 1st or 2nd instar larvae resulted in a 36% decrease in survival of 1st instar larvae at 10 9 CFU/g, whereas survival of 2nd instar larvae was unchanged (Fig. 2b, Table 1).

Total numbers of offspring

Continued

Developmental exposure to Btk formulation does not strongly influence fitness-related traits in adults. Despite a large variation between the two independent sets of experimental replicates (Table 1), the longevity of adults reared on 5 × 10 6 CFU/g of DELFIN A in low-protein/high-sugar medium was similar to that of non-exposed controls (Fig. 3). Males and females which developed on the two highest doses showed a moderate longevity benefit, higher in females for 10 8 CFU/g (Fig. 3a,b,d,e; Table 1). Males generally survived better than females (Table 1) but their longevity benefit of developing on 10 8 CFU/g was only observed in one experiment (Fig. 3b,e).

The number of offspring produced by the 15 females of each fly group during the longevity experiment varied depending on both the experiment and the DELFIN A dose (Table 1). In the 1st experiment, adults from larvae reared on 10 8 CFU/g had fewer offspring compared to the controls and to adults developed on the other doses whereas the total offspring number varied regardless of the DELFIN A dose in the 2nd experiment (Fig. 3c,f, Table 1).

Table 1. Results of statistical analyses to assess the effect of the dose of formulation/spore production and its interaction with the treatment, the larval instar, the experiment, the sex, the fly strain and the fly species when appropriate. See figures for post hoc comparisons of the doses with the control dose. Significant statistical differences are indicated in bold strains (wild-type Nasrallah and Sefra, and double mutant YW1118) was not impacted at doses up to 10 7 CFU/g of DELFIN A in a high-protein/sugar-free medium. In contrast, the ER of each strain was greatly reduced and DT was increased at higher doses (Fig. 4a,b, Table 1), with no individual reaching the pupal stage at 10 9 CFU/g (LD50 between 10 8 and 10 9 CFU/g). At 10 8 CFU/g, the magnitude of effects on Canton S flies was lower than that observed on the low-protein/high-sugar medium (see Fig. 1a,b). At this dose, the ER varied between strains, the largest reduction being observed for Sefra (Table 1). We observed no dose-dependent bias in SR (Supplementary information S3).

Developmental alterations dependent on

Btk formulation affects differently other Drosophila species. For seven other Drosophila species from different phylogenetic clades that co-occur in the field [START_REF] Benado | An eight-year phenological study of a local drosophilid community in Central Chile[END_REF][START_REF] Nunney | The colonization of oranges by the cosmopolitan Drosophila[END_REF][START_REF] Mitsui | Coexistence of drosophilid flies: Aggregation, patch size diversity and parasitism[END_REF][START_REF] Withers | Les drosophiles de la région Rhône-Alpes (Diptera, Drosophilidae)[END_REF] , doses up to 10 6 CFU/g of DELFIN A in a highprotein/sugar-free medium had no effect on ER and DT, whereas all individuals failed to reach the pupal stage at 10 9 CFU/g (Figs. 5,6). The amplitude of development alterations at 10 7 and 10 8 CFU/g varied among species (Figs. 5,6; Table 1). All species were affected at 10 8 CFU/g as was D. melanogaster (see Fig. 4a for comparison). D. simulans and D. busckii had unchanged ER, but DT was slightly increased for D. simulans (although slightly reduced at 10 7 CFU/g; similar results with a Japanese strain, data not shown) and strongly increased for D. busckii (by 20%, i.e. ~ 4 days) (Figs. 5,6, Table 1). D. yakuba ER and DT were similar to those of D. melanogaster, with an LD50 around 10 8 CFU/g and a moderate DT increase of ~ 1 day (Figs. 5,6, Table 1; similar results with a strain from Sweden, data not shown). The ER of D. hydei and D. subobscura were very low at 10 8 CFU/g (LD50 below this dose), with a high DT (Figs. 5,6; Table 1), while D. immigrans did not survive. No D. suzukii individual emerged at 10 8 CFU/g and development was already moderately impacted at 10 7 CFU/g (Figs. 5,6). No dose-dependent bias in SR was detected for either species (Supplementary information S5).

Development alterations may result from a synergy between formulation components.

Because some additives of commercial formulations might contribute to the observed effects, a DELFIN A suspension was dialyzed to remove low molecular weight additives, resuspended, and mixed with low-protein/high-sugar fly medium. At 10 7 CFU/g, the suspension did not affect ER and DT, while no individual pupated at 10 9 CFU/g (Fig. 7a; Table 1). At 10 8 CFU/g, ER was not modified but DT increased in one experimental set by ~ 1 day, partially reproducing the changes observed without dialysis (Fig. 7a,b; see also Fig. 1a,b, Table 1; 3 independent experiments for ER, 2 independent experiments for DT). The Cry1A profiles of DELFIN A suspensions (dialyzed or not), included a band for the 130-kDa pro-toxins and a band at 60-70 kDa likely representing the activated toxins, but also smaller fragments resulting from the degradation of Cry1A (Fig. 7c). We further explored the respective roles of Btk toxin fragments and spores in the alterations of D. melanogaster development through dialysis experiments followed by successive centrifugations to remove most of the spores and toxin crystals. Despite variation between experiments, ER was strongly affected only in one of the three experiments while DT was always significantly increased in the presence of centrifuged supernatants (Supplementary information S6). Noteworthy, the D. melanogaster development was not impacted in the presence of a homemade production of the Btk strain 4D1 containing spores, toxins, but no additives, even at the highest dose (Supplementary information S7).

Discussion

Our study tested the side-effects of ingestion of Bt bioinsecticide commercial formulations (mainly made of Bt kurstaki strains (Btk) but also of Bt israelensis (Bti)) during the development of eight non-target species of Drosophila naturally present in treated areas. Although the recommended doses for one formulation field spray did not affect the Drosophila development, those 10 and 50 times higher markedly induced mortality and/or developmental delay in at least two of the species tested. We can extrapolate from our data that these doses may affect six of the eight tested species and the four strains of D. melanogaster. The development alterations were already strong at these doses, suggesting an occurrence from lower ingested doses but not visible in our experimental set-up. In addition, in our experimental conditions, a single Drosophila larva could probably not process 1 g of medium during its development. Further analyses, maybe at molecular level, would be required to determine the minimal dose affecting the fly larva. Furthermore, all the tested species except D. simulans were strongly affected at a 100 times the field spray dose, and no or very limited fly development occurred at the highest tested dose, equivalent to 1000 times the maximum field dose but far below the acute intoxication doses classically used in numerous studies [START_REF]WHO specifications and evaluations for public health pesticides: Bacillus thuringiensis subspecies israelensis strain AM65-52[END_REF] . The recommended doses for each spraying of stabilized formulation are given for a homogeneous and dry area, without overlapping. In the field, recommended repeated sprays and post-spray rainfall washouts may increase the concentration of Bt spores and toxins in both space and time. While a dose 1000 times the recommendations would be hardly reached in the field, the minimum doses at which the fly development was impacted and the lower doses from which developmental changes appeared could be reached. Our data also identified a first developmental window of susceptibility to Btk formulation during the 1st larval instar mainly explaining the adverse effects, while a second event of mortality seemed to occur at the pupation period. In testing for generic side effects of Bt formulations, we observed similar patterns of developmental alterations on D. melanogaster but only at higher doses with two other Btk formulations and one Bti formulation (1000-2000 times the recommended spray dose). The three Btk formulations, based on two different bacterial strains, have similar profiles of Cry1A protoxins and activated toxins but differ in their efficient spore content. Thus, the type of formulation and probably the additives, may explain the observed variation in the dose effect.

The impacts of Btk formulations on the development of D. melanogaster are consistent with growing evidence suggesting partial specific targeting of Bt [START_REF] Palma | Bacillus thuringiensis toxins: An overview of their biocidal activity[END_REF][START_REF] Venter | Interactions between Bt crops and aquatic ecosystems: A review[END_REF][START_REF] Van Frankenhuyzen | Specificity and cross-order activity of Bacillus thuringiensis pesticidal proteins[END_REF] .The consensus on the mode of action of Bt after ingestion by insects relied until recently on the key steps of the specific binding of proteolyzed Bt toxins to midgut epithelial cell receptors, defining targets for each Bt subspecies [START_REF] Palma | Bacillus thuringiensis toxins: An overview of their biocidal activity[END_REF][START_REF] Bravo | Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control[END_REF][START_REF] Bravo | Bacillus thuringiensis: A story of a successful bioinsecticide[END_REF] . Several primary and secondary types of toxin receptors have been identified in the Lepidoptera and Diptera mosquitoes such as cadherin-like proteins, aminopeptidases, GPI-anchored alkaline phosphatases [START_REF] Adang | Diversity of Bacillus thuringiensis Crystal Toxins and Mechanism of Action[END_REF] , and more recently the ATP dependent binding cassette reporter C2 [START_REF] Stevens | Expressing a moth abcc2 gene in transgenic Drosophila causes susceptibility to Bt Cry1Ac without requiring a cadherin-like protein receptor[END_REF] . No orthologues of the Lepidoptera cadherin-like Cry receptors were found in Drosophila [START_REF] Stevens | Expressing a moth abcc2 gene in transgenic Drosophila causes susceptibility to Bt Cry1Ac without requiring a cadherin-like protein receptor[END_REF] , supporting the idea of the lack of effect of Btk toxins on these flies. Yet, Drosophila flies may have other types of Cry receptors, therefore explaining the developmental impacts observed, but this remains to be investigated. In addition, the possible lack of solubilization of the protoxin crystals and of proteolytic activation of toxins by proteases in the fly gut, both required for Cry activity in insects' larvae [START_REF] Bravo | Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control[END_REF] , would be possibly compensated by the substantial amounts of active Cry1A toxin fragments in Btk formulations. Other Btk-synthesized toxins present in the formulations could also be players in the observed cross-order activity since some, like Cry2A, have an insecticidal effect on both Lepidoptera and Diptera [START_REF] George | Bacillus thuringiensis applications in agriculture[END_REF] .

Since ingestion of Bacillus subtilis or Btk Cry-free does not affect the development of D. melanogaster, the observed development alterations cannot result solely from a severe disturbance of digestion and nutrient absorption/competition due to the presence of high loads of spores/bacteria in the larval gut throughout development. This suggests a synergistic action of Btk spores and Cry toxins, consistent with the Bt action models on insect larvae, i.e. the breach of the intestinal epithelium allowing colonization of the hemocoele by the gut bacteria, including Bt spores [START_REF] Bravo | Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control[END_REF][START_REF] Bravo | Bacillus thuringiensis: A story of a successful bioinsecticide[END_REF][START_REF] Caccia | Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism[END_REF] . The partial mimicry of mortality rates and developmental delays in preliminary dialysis assays would also support a contribution of diffusible low molecular weight compounds in Btk formulations (e.g. residues of culture media, salts, additives) to these development alterations. Furthermore, there is no impact on the development of D. melanogaster of the ingestion of homemade spores and Cry toxins of the Btk strain 4D1 used without additives even at the highest dose (or HD1, a reference strain used also as a control). Unlike commercial Btk formulations, Btk 4D1 culture contains few activated Cry toxins and smaller toxin fragments, advocating the possible contribution of such fragments to the cross-order activity of Btk formulations on Drosophila. Completion of these preliminary tests is required to further investigate the mechanisms of the harmful effects of Btk formulations on the development of Drosophila and unravel the respective roles of the synergy spores/toxins/crystals and of formulation additives.

As reported for D. suzukii exposed to Btk cultures [START_REF] Cossentine | Biological activity of Bacillus thuringiensis in Drosophila suzukii (Diptera: Drosophilidae)[END_REF] , D. melanogaster mortality on the Btk formulation occurred mainly during early development. Only ∼40% of the 1st and 2nd instar larvae died at the highest dose tested (Fig. 2) while no individual reached the pupal stage, the remaining mortality likely occurring during, or at the end of, the 3rd larval instar, possibly due to the delayed action of the gut accumulated Btk spores and toxins at the onset of pupation. Interestingly, developmental alterations (mortality, delayed emergence) mimicked those typically caused by nutritional stress in insect larvae [START_REF] Nepoux | Effects of inbreeding on aversive learning in Drosophila[END_REF][START_REF] Vantaux | Effects of larvicidal and larval nutritional stresses on Anopheles gambiae development, survival and competence for Plasmodium falciparum[END_REF] . Accordingly, the developmental alterations were partially rescued on a protein rich fly medium, probably by a compensatory protein intake, as in other arthropod species [START_REF] Vantaux | Effects of larvicidal and larval nutritional stresses on Anopheles gambiae development, survival and competence for Plasmodium falciparum[END_REF][START_REF] Moret | Survival for immunity: The price of immune system activation for bumblebee workers[END_REF][START_REF] Kutzer | The effect of diet and time after bacterial infection on fecundity, resistance, and tolerance in Drosophila melanogaster[END_REF] . Also, the sex ratio of flies was strongly biased towards males after development on the Btk formulation dose affecting fly emergence and under protein restriction. This highlights the importance of nutritional conditions such as protein restriction, added to sex-specific differences in larval susceptibility to environmental stressors, here the accumulation of Btk formulation, as already reported previously in D. melanogaster [START_REF] Andersen | Protein and carbohydrate composition of larval food affects tolerance to thermal stress and desiccation in adult Drosophila melanogaster[END_REF] .

The development on sublethal doses of Btk formulation did not dramatically affect the longevity of D. melanogaster adults, nor their lifetime offspring number. Exposure during development to doses of Btk formulation that slightly and strongly reduced the likelihood of reaching the adult stage even provided a dose-dependent longevity benefit to the surviving flies and tended to increase their offspring number. Exposure to the Btk formulation throughout development probably selected resistant and/or tolerant individuals, reminding the increased longevity of adult insects having survived a nutritional stress during development [START_REF] Rion | Evolutionary biology of starvation resistance: What we have learned from Drosophila[END_REF][START_REF] Burger | Dietary restriction affects lifespan but not cognitive aging in Drosophila melanogaster[END_REF] , or withstood environmental stressors [START_REF] Khazaeli | Genetic analysis of extended lifespan in Drosophila melanogaster III. On the relationship between artificially selected and wild stocks[END_REF] . The origin of Drosophila (species, population/strain) influenced the amplitude of the development alterations induced by the Btk formulation. For D. melanogaster, all the tested strains were equally susceptible, but with variation in the dose effect amplitudes. These differences in susceptibility suggest a possible spatial and temporal heterogeneity of the potential impacts of Btk spraying among natural D. melanogaster populations. Among the other seven species tested, differences occurred in the susceptibility to the Btk formulation, in terms of nature of development alterations and effect amplitudes, regardless of their phylogenetic distances. For the subgenus Drosophila, D. simulans was less sensitive than its sister species D. melanogaster, while the African D. yakuba experienced similar development impacts as D. melanogaster. Although phylogenetically close, D. melanogaster and D. simulans would respond very differently to Btk formulations, with a possible advantage for D. simulans in case of competition. D. immigrans, D. subobscura and D. hydei were similarly more sensitive than D. melanogaster. The phylogenetically distant D. busckii (subgenus Dorsilopha) was the least affected of all the species in terms of developmental mortality, but its development was strongly delayed. The species D. melanogaster, D. simulans, D. hydei, D. immigrans, and D. busckii belong to the guild of cosmopolitan domestic Drosophila species, D. subobscura is a sub-cosmopolitan species, and D. busckii is an opportunistic frugivorous species [START_REF] Atkinson | Breeding site specificity in the domestic species of Drosophila[END_REF][START_REF] Walsh | Drosophila suzukii (Diptera: Drosophilidae): Invasive pest of ripening soft fruit expanding its geographic range and damage potential[END_REF][START_REF] Delbac | Drosophila suzukii est-elle une menace pour la vigne?[END_REF] . They all coexist frequently and compete on the same discrete and ephemeral rotting fruit patches, with seasonal variations in the composition of the fly community [START_REF] Haller | Effects of purified or plant-produced Cry proteins on Drosophila melanogaster (Diptera: Drosophilidae) larvae[END_REF][START_REF] Benado | An eight-year phenological study of a local drosophilid community in Central Chile[END_REF][START_REF] Nunney | The colonization of oranges by the cosmopolitan Drosophila[END_REF][START_REF] Atkinson | Breeding site specificity in the domestic species of Drosophila[END_REF] . Differences in species susceptibility to Btk formulations could modify the conditions of larval competition, therefore adding local and temporal variations in the composition of Drosophila communities. The potential side-effects of Bt sprays on non-target Drosophila communities would be hardly predictable as they would depend on spatial patterns of Bt accumulation. A formal mesocosm study of Drosophila community dynamics under exposure to Btk formulation, at least under semi-field conditions, would help to identify the consequences of Bt accumulation on species competition and community composition. The exposure to Btk formulation also impacted the development of the invasive D. suzukii, as recently reported [START_REF] Cossentine | Biological activity of Bacillus thuringiensis in Drosophila suzukii (Diptera: Drosophilidae)[END_REF] , this species being the most susceptible with effects already clearly detectable at only 10 times the recommended spray dose. Compared to the other tested species living on rotten fruits, D. suzukii threatens fruit production since it feeds and lay eggs inside healthy ripening soft fruits [START_REF] Walsh | Drosophila suzukii (Diptera: Drosophilidae): Invasive pest of ripening soft fruit expanding its geographic range and damage potential[END_REF][START_REF] Delbac | Drosophila suzukii est-elle une menace pour la vigne?[END_REF][START_REF] Poyet | Invasive host for invasive pest: When the Asiatic cherry fly (Drosophila suzukii) meets the American black cherry (Prunus serotine) in Europe[END_REF] , colonizing orchards and vineyards earlier during the fruit season. The higher susceptibility of D. suzukii to the accumulation of Btk formulation in the environment might reduce the possible ecological burden of its invasion for local communities of fruit-eating Drosophila in orchards. Alternatively, since D. suzukii attacks on fruits can accelerate their decomposition, its increased susceptibility may reduce the number of fruits available for the rotting fruit-eating Drosophila species.

Overall, our data show that the ingestion of Btk bioinsecticides above the recommended spray doses can potentially impact non-target Drosophila flies, with an effect amplitude depending on both the formulation and the fly species. Although our study was carried out under controlled laboratory conditions, which may considerably differ from those of the field (e.g. temperature, pH, humidity, food availability, presence of predators/ parasites/pathogens, etc.…), standard laboratory strains and flies derived from recently collected populations exhibited similar patterns of developmental alterations, suggesting our results are likely generalizable. Recent studies have reported similar adverse side-effects due to repeated spraying of the Bti formulation on non-target organisms [START_REF] Duguma | Microbial communities and nutrient dynamics in experimental microcosms are altered after the application of a high dose of Bti[END_REF] , and indirectly on predators via food webs [START_REF] Poulin | Red flag for green spray: Adverse trophic effects of Bti on breeding birds[END_REF] . From these studies and our data here, care should clearly be taken when using Bt bioinsecticides to avoid, or at least minimize, potential side-effects on non-target organisms and therefore on biodiversity. At last, D. melanogaster could serve as a model species to identify the mechanisms underlying these side effects and/or the potential emergence of resistance to these bioinsecticides.

Methods

Commercial formulations, Bacillus productions and Colony Forming Unit. Btk brands (serotype 3a, b, c 67 ) were DELFIN (two formulations named A and B; strain SA-11; wettable granules, Valent BioSciences, AMM 9200482, 32,000 UI/mg) and Scutello DF (a Dipel sub-brand; strain ABTS-351; wettable granules, Biobest, AMM 2010513, 540 g/kg). Bti brand (strain HD-14; serotype 14 [START_REF] Zeigler | Bacillus genetic stock center catalog of strains, 7th edition. Part 2: Bacillus thuringiensis and Bacillus cereus[END_REF] ) was VectoBac WG (wettable granules, Bayer, AMM 2020029, 3000 UTI/mg). For each formulation, the number of viable spores was estimated by counting Colony Forming Units (CFUs) developing on LB agar after overnight incubation at 30 °C from serial dilutions of a suspension (Colony Forming Units (CFU) per mg of product). Estimations were 5 × 10 7 CFU/mg for DELFIN A; 2.5 × 10 7 CFU/mg for DELFIN B; 2.2 × 10 7 CFU/mg for Scutello DF; 6 × 10 7 CFU/mg for Vecto- Bac WG, and were stable during the experiments time frame. Our CFU estimations agree with those indicated for the formulations, between 1-5 × 10 13 CFU/kg. Manufacturer-recommended DELFIN doses for one spraying range from 0.15 to 1.5 kg/ha depending on the crop. Based on our CFU estimations, this corresponds to 7.5 × 10 4 to 7.5 × 10 5 CFU/cm 2 of DELFIN A, and 3.75 × 10 4 to 3.75 × 10 5 CFU/cm 2 of DELFIN B. Scutello DF is used at 0.1 to 1 kg/ha, equivalent to 2.2 × 10 4 to 2.2 × 10 5 CFU/cm 2 . VectoBac WG is used at 0.125 to 1 kg/ha, equivalent to 7.5 × 10 4 to 6 × 10 5 CFU/cm 2 .

The acrystalliferous Btk 4D22 strain (depleted for the Cry toxin-encoding plasmids [START_REF] Gonzales | Transfer of Bacillus thuringiensis plasmids coding for δ-endotoxin among strains of B. thuringiensis and B. cereus[END_REF] ; Bacillus Genetic Stock Center, https ://bgsc.org, Columbus, USA), and a Drosophila non-pathogenic Bacillus subtilis (from Dr. E. Bremer, University of Marburg, Germany) were grown at 30 °C in the sporulation-specific medium (Bactopeptone 7. 2) until sporulation (about 14 days). Vegetative cells were eliminated (1 h at 70 °C) and after centrifugation (4500 rpm, 20 min, 4 °C), a spore pellet was collected, washed with sterile water, and lyophilized. Production CFUs was estimated as described above.

Fly stocks. Drosophila melanogaster strains (phylogenetic subgroup: melanogaster) were wild-type Canton S (Bloomington Drosophila Centre) used as a reference strain, "Nasrallah" from Tunisia (strain 1333, Gif-sur-Yvette), a French field-collected strain "Sefra" (Southern France, 2013), and the double mutant standard strain YW1118 (white and yellow mutations; gift from Dr. B. Charroux, IBD, Marseille-Luminy). Other Drosophila species were D. simulans (Gif strain 1132; phylogenetic subgroup: melanogaster), D. yakuba (Gif strain 1880; phylogenetic subgroup: melanogaster), D. hydei (phylogenetic subgroup: hydei) and the invasive D. suzukii (phylogenetic subgroup: immigrans) (both kindly provided by Dr. R. Allemand, LBBE, University Lyon 1), D. immigrans (phylogenetic subgroup: immigrans), D. subobscura (phylogenetic subgroup: obscura), and D. busckii (Dorsilopha subgenus). The populations of the last three species were initiated from individuals collected in South-East of France in Spring 2015.

All the flies were maintained at controlled densities (150-200 eggs/40 ml of fly medium) under standard laboratory conditions (25 °C, or 20 °C for recently collected species, 60% relative humidity, 12:12 light/dark cycle), on a high-protein/sugar-free fly medium (10% cornmeal, 10% yeast, 0% sugar). In our laboratory, the D. melanogaster Canton S was also reared on a standard low-protein/high-sugar medium (8% cornmeal, 2% yeast, 2.5% sugar). For each experiment, eggs, larvae and flies were maintained under standard conditions.

General method of intoxication and dose-response assay.

For the dose-response assays, formulations and spore productions were serially diluted in buffer, and 100 µl of each dilution was homogenized thoroughly with 1 g of fly medium (100 µl/g doses). Eggs and defined larval instar were collected from stock vials and transferred to the intoxication vials and dishes as described below. They were then reared until the fly emergence, and, for larval susceptibility tests, until the desired development stage was reached, or for 24 h for early larvae of the 1st and 2nd instars. Equivalent control groups were transferred on fly medium homogenized with the same volume of buffer alone.

Development-related traits and larval survival. Developmental traits upon intoxication throughout

the entire development of the D. melanogaster strains and the other Drosophila species were evaluated on a precise number of viable eggs collected from mass oviposition and transferred to intoxication vials containing fly medium (high sugar/low protein or high protein/sugar free as indicated) mixed with formulations or spore productions at doses ranging from 1 × 10 5 or 5 × 10 5 CFU/g (mean equivalent to the manufacturer recommendations; Supplementary information S1) to 10 9 CFU/g. Eggs were let to develop until fly emergence. Egg density was set at 8-10 eggs/g of medium (10 eggs/g on 2 g medium in small vials Ø 3.3 cm, surface ~ 8.5 cm 2 , 0.24 g/cm 2 for tests on D. melanogaster Canton S; 8 eggs/g on 6 g medium in large vials Ø 4.6 cm, surface ~ 16 cm 2 , 0.37 g/ cm 2 for strains and species comparisons), except for D. hydei, D. suzukii and D. immigrans for which the egg density was reduced by half because of their reproductive biology (5 eggs/g on 6 g). Numbers and sex of emerging flies were recorded once a day until the day on which the first pupae of the next generation forms. The emergence rate (proportion of flies emerged from the initial eggs), the developmental time (mean number of days for development completion) and the sex-ratio (proportion of male flies) were calculated for each replicate vial.

For the larval susceptibility tests, survival was measured on 20 eggs/larvae of D. melanogaster Canton S at a suitable instar collected from a 4-h mass oviposition and transferred to small dishes (Ø 3 cm, surface ~ 7 cm 2 ) containing 1 g of high-protein/sugar-free fly medium (less limiting for early larval development) homogenized with DELFIN A doses ranging from 10 5 CFU/g to 10 9 CFU/g. First and 2nd instar larvae were used since growth is exponential during these two instars [START_REF] Tennessen | Coordinating growth and maturation-Insights from Drosophila[END_REF][START_REF] Santos | Density-dependent natural selection in Drosophila: Evolution of growth rate and body size[END_REF] and larvae are more likely to be heavily exposed to the bioinsecticide. Proportion of surviving larvae was measured at the indicated developmental stage for the cumulative survival test, or 24 h later. For cumulative survival, unhatched eggs were discarded from the counting. The pH of the fly medium was measured for the Btk dose-responses; neither the presence of Btk formulation nor the dose altered it (Supplementary Information S4).

Adult fitness-related traits. For longevity and total offspring number measurements, D. melanogaster Canton S eggs from mass oviposition were transferred to several vials with low-protein/high-sugar medium mixed with 5 × 10 6 , 5 × 10 7 or 10 8 CFU/g of DELFIN A. For each dose, flies from the vials were pooled 2 days after emergence and groups of 15 males and 15 females were transferred on the same medium without DELFIN A. Flies were transferred to a new vial every 3-4 days and previous vials were incubated for the offspring to develop. Mortality and sex of dead flies were recorded daily until the last fly died. Offspring numbers were counted from the first emergence until pupae of the next generation appeared. Two experimental blocks were set. Due to dif-Dialysis and cry toxin analysis. For some products, additives can be more harmful than the active ingredient [START_REF] Bradberry | Glyphosate poisoning[END_REF] . To eliminate low molecular weight additives present in the formulation, a DELFIN A suspension at 2 × 10 10 CFU was dialyzed against PBS (KH 2 PO 4 1 mM, Na 2 HPO 4 (2H 2 O) 3 mM, NaCl 154 mM, pH 7.2), overnight at 4 °C, using an 8-10 kDa cut-off membrane (ZelluTrans, Roth). CFUs of the dialyzed suspension were estimated as above. The effects on the emergence rate (ER) and developmental time (DT) of D. melanogaster Canton S were analysed on 20 eggs, at 10 7 , 10 8 and 10 9 CFU/g of dialyzed and also centrifuged suspension mixed with 2 g of low-protein/high-sugar fly medium. The dialyzed suspension was subject to a 12.5% SDS-PAGE and compared to the non-dialyzed suspension after silver staining. The presence of Cry1A pro-toxins, activated toxins and toxin fragments was probed by Western-blot using an in-house anti-Cry1A rabbit polyclonal antibody. Data analysis. Data were analysed with mixed-effects models with replicates as random effects. Dose of Btk formulation/spore production, D. melanogaster strain, Drosophila species or developmental stage, experimental block where necessary, and appropriate 2-way interactions between these factors, were included as fixed effects. Main fixed effects and their interactions were tested with log-likelihood ratio tests. Post hoc pairwise comparisons were made for D. melanogaster strains, formulation/spore treatments, and between the control and the other doses.

Data of emergence rate, sex-ratio, and larval survival were analysed with generalized linear models with binomial distribution and logit link; for emergence rate data, the model was also bias-corrected to correct for multiple 0 values, and with replicate as random effect. To run the post-hoc analysis, the same model including replicates as fixed effect was applied to the data of emergence rate and provided similar results. Developmental time (1/x transformed) and offspring number were analysed with linear models. Adult longevity data were analysed with proportional hazard Cox regression models, including fly sex and formulation dose as fixed effects, and replicate as a random effect. Analyses were performed in R 71 using the packages lme4 72 , brglm [START_REF] Kosmidis | brglm: Bias Reduction in Binary-Response Generalized Linear Models[END_REF] , multcomp [START_REF] Horton | Simultaneous inference in general parametric models[END_REF] , survival [START_REF] Therneau | Modeling Survival Data: Extending The Cox Model[END_REF] , and coxme [START_REF] Therneau | Mixed Effects Cox Models. R package version 2[END_REF] .

Figure 1 .

 1 Figure 1. Development of D. melanogaster Canton S flies on Btk and Bti commercial formulations. (a) Emergence rate and (b) developmental time (mean ± s.e.m.) of 20 initial eggs on increasing doses of Btk DELFIN A (red triangles), the Cry-free Btk 4D22 (open lozenges), the mosquito-targeting Bti VectoBac WG (green squares) and the non-pathogenic Bacillus subtilis (light grey circles). For VectoBac WG and B. subtilis, N = 4-7 per dose; for DELFIN A and Btk 4D22, N = 9-12 for the control, N = 3 for 5.10 5 and 10 9 , N = 4-9 for 10 6 , N = 7-14 from 5.10 6 to 10 8 . (c) Emergence rate (mean ± s.e.m.) and (d) developmental time (mean ± s.e.m.) on increasing doses of the two Btk formulations DELFIN B (dark red circles) and Scutello DF (orange squares). N = 4 replicates of 20 eggs per dose and formulation, except for controls and 10 8 CFU/g of DELFIN B (9-10 replicates of 20 eggs). Results of post hoc comparisons of each dose to the control: • 0.05 < P < 0.1; *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001. (e) Immunoblotting with an anti-Cry1A polyclonal antibody on proteins from a suspension of laboratory-produced spores of Cry-free Btk 4D22, the three Btk formulations DELFIN A, B, Scutello DF and a suspension of laboratory-produced Cry1A toxins. Red asterisks indicate the Cry protoxins (~ 130 kDa) and the activated fragments (~ 60 kDa and ~ 70 kDa).

  Btk formulation dose are not specific to D. melanogaster Canton S. As with D. melanogaster Canton S, the development of three other D. melanogaster

Figure 2 .

 2 Figure 2. Survival of D. melanogaster Canton S larval stages on increasing doses of Btk DELFIN A. (a) Proportion of surviving larvae (mean ± s.e.m.) upon Btk formulation exposure from the egg to late 1st instar (open lozenges) and late 2nd instar (black triangles). (b) Proportion of surviving larvae (mean ± s.e.m.) upon 24-h Btk formulation exposure of early 1st instar larvae (open lozenges) and 2nd instar larvae (black triangles). N = 5-7 replicates of 20 individuals per dose. Results of post hoc comparisons of each dose with the control: *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001.

Figure 3 .

 3 Figure 3. Fitness-related traits of adults (longevity and total offspring number) after development on Btk DELFIN A. (a,d) Female longevity (mean survival fraction over time ± s.e.m.), (b,e) Male longevity (mean ± s.e.m.), and (c,f) total offspring number (mean ± s.e.m.), measured on individuals that developed without Btk formulation (blue items) and on 5 × 10 6 CFU/g of Btk DELFIN A (green items), 5 × 10 7 CFU/g (red items), and 10 8 CFU/g (dark red items). Data from 2 experiments (a-c, experiment 1; d-f, experiment 2). For each trait, N = 3-5 replicates of 15 males and 15 females per dose in experiment 1, N = 3 replicates of 15 males and 15 females in experiment 2. Results of post hoc comparisons of each dose with the control: *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001.

Figure 4 .

 4 Figure 4. Development of four D. melanogaster strains on increasing doses of Btk DELFIN A. (a) Emergence rate and (b) developmental time (mean ± s.e.m.) of the strains Canton S (blue lozenges), Nasrallah (yellow triangles), Sefra (green squares), and YW1118 (red circles). N = 4 groups of 50 eggs per dose and fly strain for each trait. Results of post hoc comparisons of each dose to the control: **0.001 < P < 0.01; ***P < 0.001.

Figure 5 .

 5 Figure 5. Emergence rate of seven Drosophila species on increasing doses of Btk DELFIN A. Mean emergence rate (± s.e.m.). N = 4 replicates of 50 eggs per dose for D. simulans, D. yakuba, D. subobscura and D. busckii, N = 4 replicates of 30 eggs per dose for D. hydei, D. suzukii, and D. immigrans. Results of post hoc comparisons of each dose with the control: • 0.05 < P < 0.1; ***P < 0.001.

Figure 6 .

 6 Figure 6. Developmental time of seven Drosophila species on increasing doses of Btk DELFIN A. Mean developmental time (± s.e.m.). N = 4 replicates of 50 eggs per dose for D. simulans, D. yakuba, D. subobscura and D. busckii, N = 4 replicates of 30 eggs per dose for D. hydei, D. suzukii and D. immigrans. Results of post hoc comparisons of each dose with the control: *0.01 < P < 0.05; **0.001 < P < 0.01; ***P < 0.001.

Figure 7 .

 7 Figure 7. Evaluation of the role of small molecular weight components of Btk DELFIN A (dialysis; membrane cut-off: 8-10 kDa) in the altered development of D. melanogaster Canton S. (a) Emergence rate and (b) developmental time (mean ± s.e.m.) on increasing doses of dialyzed DELFIN A. N = 3 experiments of 4 replicates with 20 eggs per dose for the emergence rate, N = 2 experiments of 4 replicates per dose for the developmental time. Results of post hoc comparisons of each dose with the control: ***P < 0.001. (c) Anti-Cry1A probed immunoblot of non-dialyzed (ND) and dialyzed (D) suspensions showing the decrease in the amount of ~ 130/140 kDa migrating protoxins and the increase in that of the potential ~ 60/70 kDa activated toxins after dialysis.

  5 g, KH 2 PO 4 3.4 g, K 2 HPO 4 4.35 g, glucose 7.5 g, PGSM salts (MgSO 4 •7H 2 O, MnSO 4 •H 2 O, ZnSO 4 •7H 2 O, FeSO 4 •7H 2 O) 5 mL, CaCl 2 0.25 M, distilled water qsp 1L, pH 7.

Source of variation/data χ 2 /deviance d.f P value Development on Btk DELFIN A, Btk 4D22, Bti VectoBac WG, Bacillus subtilis

	www.nature.com/scientificreports/
	Emergence rate
	Dose × treatment	285.7	< 0.0001
	Dose for each treatment:
	-DELFIN A	237.5	< 0.0001
	-4D22	7.0	0.40
	-VectoBac WG	165.8	< 0.0001
	-B. subtilis	1.9	0.93
	Developmental time
	Dose × treatment	220.8	< 0.0001
	Dose for each treatment:
	-DELFIN A	68.8	< 0.0001
	-4D22	16.08	0.024
	-VectoBac WG	37.5	< 0.0001
	-B. subtilis	13.5	0.060
	org/10.1038/s41598-020-73145-6

Development on Btk DELFIN B and Scutello DF (dose effect)

	Emergence rate
	-DELFIN B	151.2	< 0.0001
	-Scutello DF	105.1	< 0.0001
	Developmental time
	-DELFIN B	2.5	0.12
	-Scutello DF	30.9	< 0.

0001 Role of formulation components in the development alterations (dialysis)

	Dose effect:
	Emergence rate	459.8	< 0.0001
	Developmental time	13.7	0

.0011 Survival of larval stages on DELFIN A

	Cumulative survival
	Dose × larval instar	16.2	0.0063
	Dose for each instar:
	-Late 1st instar	87.4	< 0.0001
	-Late 2nd instar	25.7	0.0001
	24-h survival
	Dose × larval instar	15.9	0.007
	Dose for each instar:
	-Late 1st instar	55.9	< 0.0001
	-Late 2nd instar	3.76	0.58

Adult fitness-related traits after development on DELFIN A

	Longevity
	Experiment	20.1	< 0.0001
	-1st experiment
	Dose	12.3	0.0065
	Sex (e β coefficient males vs. females ± se: 0.55 ± 0.16)	35.0	< 0.0001
	Dose × sex	20.4	0.00014
	Sexes analyzed separately
	Females (e β coefficients vs. control ± se: 5 × 10 6 : 1.05 ± 0.17, 5 × 10 7 : 0.71 ± 0.16, 10 8 : 0.60 ± 0.21)	12.0	0.0073
	Males (e β coefficients vs. control ± se: 5 × 10 6 : 0.80 ± 0.16, 5 × 10 7 : 0.66 ± 0.16, 10 8 : 1.53 ± 0.18)	20.4	0.00014
	Adult fitness-related traits after development on DELFIN A
	-2nd experiment
	Dose	16.5	0.00090
	Sex (e β coefficient males vs. females ± se: 0.45 ± 0.22)(e β coefficient males vs. females ± se: 0.45 ± 0.22) 31.5	< 0.0001
	Dose × sex	0.69	0.88
	Sexes analyzed separately
	Females (e

β coefficients doses vs. control ± se: 5 × 10 6 : 0.92 ± 0.22, 5 × 10 7 : 0.63 ± 0.21, 10 8 : 0.51 ± 0.21) 13.2 0.0043 Males (e β coefficients doses vs. control ± se: 5 × 10 6 : 1.02 ± 0.22, 5 × 10 7 : 0.70 ± 0.22, 10 8 : 0.64 ± 0.22) 7.01 0.072

Development of other strains of D. melanogaster on DELFIN A (including Canton S)

	Source of variation/data	χ 2 /deviance d.f	P value
	Dose × experiment	28.1	3	< 0.0001
	Dose for each experiment:
	-1st experiment	26.3	3	< 0.0001
	-2nd experiment	4.1	3	0.25
	Emergence rate
	Dose × fly strain	105.5	15	< 0.0001
	Dose for each fly strain:
	-Canton S	588.6	5	< 0.0001
	-Nasrallah	745.3	5	< 0.0001
	-Sefra	900.7	5	< 0.0001
	-YW1118	636.9	5	< 0.0001
	Developmental time
	Dose × fly strain	9.3	12	0.68
	Dose for each fly strain:
	-Canton S	40.3	4	< 0.0001
	-Nasrallah	18.0	4	0.0012
	-Sefra	27.2	4	< 0.0001
	-YW1118	28.9	4	< 0.

0001 Development of other Drosophila species on DELFIN A

	Emergence rate
	Dose × fly species	538.2	30	< 0.0001
	Dose for each species:
	-D. simulans	461.0	5	< 0.0001
	-D. yakuba	750.7	5	< 0.0001
	-D. hydei	596.8	5	< 0.0001
	-D. immigrans	726.3	5	< 0.0001
	-D. subobscura	729.6	5	< 0.0001
	-D. suzukii	725.0	5	< 0.0001
	-D. busckii	586.0	5	< 0.0001
	Developmental time
	Dose × fly species	59.9	22	< 0.0001
	Dose for each species:
	-D. simulans	25.9	4	< 0.0001
	-D. yakuba	34.7	4	< 0.0001
	-D. hydei	11.5	4	0.022
	-D. immigrans	6.01	3	0.11
	-D. subobscura	68.8	4	< 0.0001
	-D. suzukii	11.7	3	0.0085
	-D. busckii	58.8	4	< 0.0001

Scientific RepoRtS | (2020) 10:16241 | https://doi.org/10.1038/s41598-020-73145-6

ferences in the duration of the experiment, the offspring numbers of all vials of each dose of DELFIN A were summed for each experimental block.

Acknowledgements

We thank Xiao Han, Jingru Li and Abir Oueslati for help with preliminary experiments, L. Kremmer, C. Rebuf and O. Magliano for providing and rearing flies and help in preparing fly medium, A. Brun-Barale for the production of Bacillus subtilis spores, D. Pauron for preparation of Cry1A toxin, Hugo Mathé-Hubert for advice on statistical analyses and M. Amichot for helpful discussions. The Cry1A antibody was produced in collaboration with the INRA-PFIE platform (Nouzilly, France).

Funding

This work was supported by the French National Agency for Research (ANR-13-CESA-0003-001 ImBio), the European Union's Seventh Framework Program for research, technological development and demonstration under grant agreement No. 613678 (DROPSA), the "Investments for the Future" LABEX SIGNALIFE (ANR-11-LABX-0028), the INRA Plant Health Department (to MPNE and JLG), the CNRS (to AG), and the University Nice Côte d' Azur (to MP).

Author contributions

A.B., M.P.N.E., A.G., J.L.G. and M.P. designed the experiments. A.B. performed the experiments with contributions of M.P.N.E. A.B performed the statistical analyses and prepared the figures. A.B., J.L.G., and M.P. wrote the manuscript with contributions from all the authors.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-73145 -6.

Correspondence and requests for materials should be addressed to J.-L.G.

Reprints and permissions information is available at www.nature.com/reprints. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Lars-Henrick Lau Heckman

Belén Blanco

An Cools

Edgar Barbara Früh

Garcia Manzanilla

Ilias Kyriazakis

Núria Llanes Baró

Bertrand Méda

Daniel Murta

Katie Owens

EIP-AGRI Focus Group "New feed for pigs and poultry"

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Table of contents

Executive summary

The Focus Group on New Feed for Pigs and Poultry of the European Innovation Partnership for Agricultural Productivity and Sustainability (EIP-AGRI) addressed the question `Which are the promising new sources and strategies to reduce pressure on natural resources while feeding or producing feed for pigs and poultry?` This group of 20 experts from across Europe assessed the challenge and identified possible solutions. The expert group identified 'knowledge gaps' and needs, generated recommendations on how to ensure future implementation of new feed for pigs and poultry, and proposed innovation actions and ideas for Operational Groups.

One of the key issues to be addressed by the Focus Group was to identify and characterise the potential new feed options for pigs and poultry. Based on a rationale focusing on economy, nutritional value and sustainability, outlined in the Starting paper, the experts assessed the most promising new feed options during two workshops held in Finland and Belgium. The Focus Group experts identified the following top five new feed options:

• Bakery products • Green biomass (grass/clover)

• Insects (black soldier fly -Hermetia illucens)

• Micro-algae • Single cell protein (bacteria)

The selected top five new feed options have different pros and cons regarding nutritional value, economy and sustainability (Chapter 4). Overall, bakery products are a potential feed ingredient which would be relatively easy to introduce/use more widely in the EU. Yet, the nutritional value is not as high as the other new feed options in the Focus Group`s top five. Although green biomass (grass/clover) is slightly more mature than insects, micro-algae and single cell protein from bacteria, the remaining four new feeds are relative immature; and require a lot of research and development before they can be fully implemented at both farm and industry level. To promote implementation, several ideas for Operational Groups were suggested for these top five new feed options (Chapter 5).

Recommendations regarding future needs were likewise discussed by the experts both from a practical as well as academic perspective. The consensus was that there is a great need for fast(er) analysis of new feed materials. NIRS (Near InfraRed Spectrometry) was identified as one of the promising techniques to do this. The experts also discussed the need for more insight on the processing of novel (and current) feedstuffs, particularly insects. They also pointed out that more knowledge is needed on the optimal or maximum percentage of these new feed ingredients in feed mixes.

The work of the Focus Group provides a broad overview of how stakeholders from the feed sector can contribute to addressing practical and research related issues.

Introduction

According to the International Feed Industry Federation (IFIF) the global production of compound feed for livestock (including dairy and fish) was estimated at 1 billion tons in 2016 with the European region accounting for approx. 159 million tons 1 . Within the meat production industry, pig and poultry are the fastest growing livestock subsectors; and the demand for pigs and poultry is expected to increase by 38% and 104%, respectively, compared to current production levels 1,2 . Hence, there is a growing need to find feed sources that can supply the expected increase in pig and poultry production, taking into account the increased focus on sustainable farming.

The Focus Group on New Feed for Pigs and Poultry of the European Innovation Partnership for Agricultural productivity and Sustainability (EIP-AGRI) addressed the challenge of identifying new feed sources to improve economy, supply and sustainability for European stakeholders. A group of 20 experts from across Europe assessed this challenge and identified possible solutions.

The overall objective of the Focus Group was to identify the 'knowledge gaps' and needs, and to generate recommendations on how to ensure future implementation of new feed for pigs and poultry, proposing innovation actions and ideas for Operational Groups. More specifically, these objectives and tasks were to:

Make an inventory of alternative strategies and sources for pig and poultry feed, including both new and underused sources.

Analyse the most important pros and cons of these alternative feed sources, strategies and supply chains.

Analyse their general economic and environmental sustainability, analyse their technical viability and safety, and identify potential tools/barriers affecting a broad uptake.

Propose potential innovative actions and ideas for Operational Groups (funded under the Rural Development Programmes) to stimulate the use and improvement of alternative resources. Identify needs from practice and possible gaps in knowledge which may be solved by further research.

Identify how new and emerging alternatives may be transferred to other conditions (location, type of production) and how they may be checked and standardised in a cost-effective way to obtain safe and steady products at farm level. Identify innovative business models for farms and/or third parties.

This report presents the result of the work of the Focus Group experts and is intended to inform stakeholders that have an interest in developing the supply chain of new feed for pigs and poultry. The report summarises the views of a Europe-wide group of experts covering 14 Member States and representing academia, advisers, primary producers and industry stakeholders; highlighting the need for innovative collaboration across sectors, locally, regionally and across Europe.

The members of the Focus Group are listed in Annex B -they contributed as individuals rather than as representatives of an organisation. To open the meeting in Espoo, the Focus Group Starting paper was presented, this document had been prepared by the coordinating expert with support from the EIP-AGRI Service Point. This was followed by breakout sessions where the inventory of suggested new feed for pigs and poultry was further developed and discussed by the expert group in smaller groups and in the plenary. The second part of the meeting focused on outlining the mini-papers (see Annex C), which constitute a significant part of the group's work.

The Brussels meeting started with presentations of the four mini-papers that had been initiated in Espoo covering: i) overview of existing novel feedstuffs; ii) characterisation of novel feed; iii) sustainability; and iv) production and processing of novel feed. Subsequently, success factors and barriers were discussed for a wide range of specific new feeds and from these a list of top five options was identified. The second part of the Brussels meeting focussed on identifying innovative solutions and ideas for EIP Operational Groups and innovative projects as well as identifying gaps related to research and knowledge needs.

The discussions, views and findings of these meetings are the basis of this report.

State of play a. Framing key issues

One of the initial key issues to be addressed by the Focus Group was to identify and characterise the potential new feed options for pigs and poultry. Overall, the new feed options should meet several of the requirements listed below:

Moderate to high protein content (this is a major advantage as protein is in high demand) Good protein digestibility and nutritionally relevant amino acid profile Moderate to high content of relevant micronutrients (minerals and vitamins) No (or limited content of) anti-nutritional factors Health-benefitting properties (e.g. pre-biotics, feed fibres etc.) Sustainable production (this may also cover economics and ethics) Competitive price (preferably comparable to soybean meal but depending on the feed it may also be relevant at fishmeal price levels).

Theoretically, there was a very large number of options to choose from. Based on a rationale focusing on economy, nutritional value and sustainability, the frame work for the Focus Group experts had been outlined in the Starting paper and a decision was made that the Focus Group`s work would focus primarily on new feeds from: i) aquatic biomass, ii) industrial residuals and former foodstuffs, iii) insects and iv) single cell protein.

The experts initially discussed a number of new feed options within these four categories. They also added a fifth category -green biomass -to the overall list. Based on the requirements highlighted above, table 1 below lists the potential new feed options according to the five major categories.

Yeast protein concentrate

Bacterial protein concentrate

Bakery by-product (e.g. batch production intermediates)

Food waste/agrofood industry

In the subsequent progress of the Focus Group, during the second meeting in Brussels, a top five of new feed options (highlighted in bold in Table 1) was selected based on a number of different criteria as outlined in section c of Chapter 4 -these included:

Bakery products Green biomass (grass/clover) Insects (black soldier fly -Hermetia illucens)

Micro-algae Single cell Protein (bacteria)

b. Good practices

As outlined in the introduction there is an increasing demand for sustainable, nutritionally relevant and economically viable feed for pigs and poultry. Yet, many new feed options will, at present, have the disadvantage of being immature in one or several areas (e.g. market-relevant supply); hence, their inclusion in the pig or poultry value chains may initially be slow. This issue is currently relevant for e.g. insects, micro-algae and single cell protein (SCP), while former foodstuffs such as bakery products may not have supply issues. Nevertheless, once developed, insects, micro-algae and SCP may be worthwhile to include, both for nutritional and sustainability reasons. However, currently there are only few examples of good practice with the 'immature' new feed options at full industrial scale; although both the insect and SCP sectors are expanding fast (see example of companies in Annex A).

At farm-level it may be possible to implement some new feed options through local cooperatives or in coproduction with other on-farm activities; an approach that many farmers already use in their daily operation. Several of the new feed options offer great potential to enhance farm circularity. For instance, micro-algae may be produced on slurry on-farm in co-production with pigs or poultry and subsequently be used as feed supplement. Supplemented with locally available biomass (ensuring a nutritionally-balanced diet), insects may also be produced on on-farm vegetable residues from crop or horticulture production, and subsequently used on-farm for pig or poultry production; enabling regional circular bioeconomy (Fig. 1).

Figure 1. On-farm feeding of poultry with insects (© Shutterstock)

Many of the new feed options likewise have great potential to implement circular economy in the pig and poultry value chains. This may, initially, be most obvious regarding application of industrial residues and former foodstuffs such as bakery products. Here several EU-based companies provide good cases of how using former foodstuffs, e.g. from the bakery industry, in feed supplements for poultry and pigs, can provide a sustainable business and supply. Some of the major challenges for this sector are logistics, including securing relevant products based on transportation costs and volumes available at customers, as well as downstream processing (e.g. removal of residual packaging). Nevertheless, bakery products are used widely in the EU, particularly for pigs. Good examples also exist for poultry. For instance, the Dutch farm Kipster produces sustainable eggs using a feed based primarily (97%) on leftovers like bakery products.

c. Success and fail factors

A major outcome of the second meeting in Brussels (Fig. 2) was the identification of a top five of new feed options that the Focus Group experts assessed as the most promising -as outlined above (see Table 1). The top five new feed options were: i) Bakery products, ii) Green biomass (grass/clover), iii) Insects (black soldier fly -Hermetia illucens), iv) Micro-algae and v) Single cell Protein (bacteria). Before getting to this outcome, the experts evaluated several different criteria. Initially, the experts discussed the new feed options from Table 1 to identify how they could be transferred across application and/or geographical levels. They also discussed how the feed options could be standardised and applied in a costeffective way to obtain safe and steady products at farm or industry level. For instance, as illustrated in Figure 3, how can insects be transferred from farm to industry level, or how may Kelp (macro-algae) be utilised to a higher extent at regional level?

Figure 3. Transferring new feed alternatives across application and geographical levels

Algae, bakery products, grass/clover and okara were selected and discussed independently in four sub-workinggroups focussing on possible obstacles related to 'transferring' the new feed option. Table 2 below outlines the baseline of the new feed options discussed followed by different ideas and concerns related to transfer between application and geographical levels. Transferring -Local to regional level

As okara is produced cost-effectively at industry level it may have potential for use by regional producers of compound feed producers Risk Salmonella as well as preservation/oxidation

After discussing the transfer potential (as an overall evaluation of robustness and flexibility) of the new feed alternatives, the experts assessed the success and fail factures of the top five feed options. The overall input from the experts is highlighted in Table 3-7 and summarised below. Additionally, the mini-papers (see Annex C) cover a wide range of relevant parameters that provide an overview of the top five new feeds regarding their: i) maturity as feed (both from an economic, logistical and development/R&D perspective), ii) their nutritional value, iii) sustainability profile as well as iv) how they are produced and processed.

Bakery products (Table 3) is overall an 'easy' applicable new feed that is available in large quantities throughout Europe year round; although it may be scattered in some European regions (e.g. South-East) which would require regional consolidation to ensure that the resources are collected, processed and distributed to local farmers. From a developmental perspective, bakery products are at a relatively mature level. Moreover, using bakery products as feed has very clear sustainability benefits. However, one of the challenges is the lower protein content (mainly a source of carbohydrates) as compared to some of the other new feed options (e.g. SCP and insects); and there may be a risk of residual packaging material (mainly plastics) in the processed bakery products. Green biomass (Table 4), with emphasis on grass/clover, is a relatively mature feed that can be applied with a moderate effort as new feed. Particularly silage from green biomass is a technique that has been used widely and for many years; and it is readily applicable at farm level. On the contrary, protein extracted from green biomass still needs development to be competitive. Green biomass provides good nutrition as feed, although the protein content is low. Moreover, monogastrics, especially pigs, may have increased enteric greenhouse gas (GHG) production when fed on green biomass, which is a challenge from a sustainability perspective. The current lack of production facilities hampers the use of green biomass as a feed ingredient at farm and industry level, despite the potentially large supply. 5) -with special focus on black soldier fly larvae (BSF) -have a high nutritional value for both pigs and poultry; and may provide a sustainable solution if produced on the right substrate(s). Moreover, there may be potential benefits related to improved health and bio-sanitation (i.e. BSF larvae appear to be able to metabolise certain mycotoxins and other contaminants) 3 . Yet, although the insect sector is expected to expand rapidly in the coming years 4 , the volumes and price levels that the BSF producers can deliver are still not fully competitive with established alternatives such as soybean meal and fishmeal. Furthermore, there is lack of knowledge regarding how to ensure optimal use of BSF as feed for both poultry and pigs; e.g. both regarding nutritional needs of BSF, inclusion level of BSF in feed and standardisation to mention a few knowledge needs.

3 See recent articles from AllAboutFeed for more details -health: https://www.allaboutfeed.net/New-Proteins/Articles/2018/3/Insects-3healthy-compounds-for-animal-feed-264878E/ and bio-sanitation: https://www.allaboutfeed.net/Mycotoxins/Articles/2019/10/Can-insectshold-the-key-to-safe-feed-479880E/?dossier=26709&widgetid=1 4 For an overview of the current status of the European insect sector please refer to: http://ipiff.org/ipiff-vision-paper/ Micro-algae production (Table 6) is still a relatively immature sector; although, like insects, there is an increased focus on their potential as a feed of high nutritional value. They contain both macro-nutrients like protein and lipids, but also micro-nutrients such as vitamins and minerals and they provide health benefits related to gut health5 . It could be very interesting to add micro-algae to the drinking water or as liquid feed. On the other hand, one of the main challenges with micro-algae is the very high water content, which requires a lot of energy for either transportation or processing (drying) of the biomass. Micro-algae could potentially be produced and used on-farm, however, from a technical perspective, this is still a few years into the future. Yet, like for insects and SCP (Single Cell Protein, see below), there is in principle no need to use arable land in the production of micro-algae. Single cell protein (SCP) from bacteria (Table 7) has many of the same pros as both insects and micro-algae, e.g. regarding nutritional value and the potential for production in a circular, economic way -looping 'waste' and by-products back into the feed/food chain. From a sustainability perspective, the emission of greenhouse gases associated with the production with SCP from bacteria appears to be low. Yet, as the SCP production platform is still relative immature there is not a lot of data available related to assessing sustainability more widely. Moreover, like with green biomass, insects and micro-algae, there is also lack of knowledge regarding how to ensure optimal utilisation of SCP from bacteria as new feed for both poultry and pigs. In summary, the top five new feed options have different pros and cons regarding nutritional value, economics and sustainability. Overall, bakery products are a potential feed ingredient which would be relatively easy to introduce/use more widely in the EU. Yet, the nutritional value is not as high as the other top five new feed options. Although green biomass (grass/clover) is slightly more mature than insects, micro-algae and SCP, the remaining four new feed options are relative immature and still require a lot of research and development before they can be fully introduced at both farm and industry level. This is further elaborated in Chapter 5 below, and particularly in section b regarding research needs.

What can we do? Recommendations a. Ideas for Operational Groups and other innovative actions

Operational Groups (OGs) are groups of people who come together to work on concrete, practical solutions to a problem or innovative opportunity and whose project is funded by the EU Rural Development policy 6 . OGs are intended to bring together multiple actors such as farmers, researchers, advisers, businesses, environmental groups, consumer interest groups or other NGOs to find innovative solutions for the agricultural and forestry sectors. The OG projects have to share their results through the EIP-AGRI network and other appropriate channels.

After identifying and discussing success and fail factors (section c of Chapter 4), the next task of the Focus Group experts, during the Brussels meeting, was to define and outline a number of ideas for OGs and other innovative actions for each of the top five need feed options. Table 8 below summarises the 21 ideas for OGs and other innovative actions that were generated during this process. It highlights the problem to be addressed, the general idea, where in Europe the innovative action would likely be most relevant, which stakeholders would be the most obvious to include and a contact(s) from the Focus Group for stakeholders requiring more information to develop an OG or other innovative action7 . In summary, the OGs and innovation actions suggested for the respective top five new feed options were very different, highlighting the unique issues with regards to ensuring their future uptake at farm and industry level.

Apart from feed specific OGs, a technical OG was also suggested by a sub-group of experts. As outlined under the section on 'success and fail factors' (section c in Chapter 4), the limited market maturity of most of the new feed options reflects their need for further research and development. This is described in more detail below.

b. Research needs from practice

The final part of the Brussels meeting was dedicated to discussing the needs from practice and the needs for research regarding the top five new feed options. The Focus Group experts were divided into two large subgroups comprising 'practitioners' (farmers, advisers and industry members) and 'academia' (researchers); the former group worked on the needs from practice, while the latter group worked on needs for research. The outcome of the sub-groups is highlighted in Table 9 and Table 10, respectively. 9) and mid-to long-term issues (Table 10). However, there are overlaps in the needs highlighted by the 'practitioners' and 'academia' sub-groups. First, both groups identified a great need for fast(er) analysis of new feed materials; one of the promising techniques identified was NIRS (Near InfraRed Spectrometry). Secondly, more insight into processing methods for novel (and current) feedstuff, particularly insects, was also a common need identified by the two sub-groups. The OG ideas presented in the previous section (see e.g.

Table 8) provide one approach to move forward with (some of) the needs from practice. Moreover, the research needs outlined above (Table 10) will have to be addressed in future European research and innovation programs. Yet, research is already underway that will provide more knowledge on several of the research topics listed above (for more details please refer to Annex D).

c. Other recommendations, including improving take up

Safety and quality standardisation must be ensured in order for the novel feedstuff to be successful as alternative to current feed ingredients. Moreover, for several of the feed options, the number of producers and the level of production must be increased dramatically in order to develop critical mass. The existence of several companies and sources is important for the sectors.

Sustainable feed is particularly important for organic farming. However, additional challenges arise for organic farming in order to meet the requirements for the development and use of the new feedstuffs presented in this report. The separation of the original components or, for example, the substrates for insects are an additional requirement. When developing new feedstuff for the organic farming market, rules on organic production should be taken into account.

Furthermore, some of the new feed options, e.g. BSF, will require coordinated communication with the general public to ensure acceptance and recognition of the novel feed strategies and their impact on the sustainability of animal production.

Feed-a-Gene

Feed-a-Gene is an EU project that aims to better adapt different components of monogastric livestock production systems (i.e., pigs, poultry and rabbits) to improve the overall efficiency and to reduce the environmental impact. This involves the development of new and alternative feed resources and feed technologies, the identification and selection of robust animals that are better adapted to fluctuating conditions, and the development of feeding techniques that allow optimizing the potential of the feed and the animal.

1.

  Executive summary .......................................................................................................................... 2. Introduction .................................................................................................................................... 3. Brief description of the process ......................................................................................................... 4. State of play .................................................................................................................................... a. Framing key issues ....................................................................................................................... b. Good practices ............................................................................................................................. c. Success and fail factors ................................................................................................................. 5. What can we do? Recommendations ................................................................................................ a. Ideas for Operational Groups........................................................................................................... b. Research needs from practice.......................................................................................................... c. Other recommendations, including improving take up ........................................................................ Annex A. Good practices and case studies ............................................................................................... Annex B. Members of the Focus Group .................................................................................................... Annex C. List of mini-papers ................................................................................................................... Annex D. Relevant research projects .......................................................................................................

Figure 2 .

 2 Figure 2. Workshop in Brussels, 30-31 January 2019

  Partnership 'Agricultural Productivity and Sustainability' (EIP-AGRI) is one of five EIPs launched by the European Commission in a bid to promote rapid modernisation by stepping up innovation efforts. The EIP-AGRI aims to catalyse the innovation process in the agricultural and forestry sectors by bringing research and practice closer together -in research and innovation projects as well as through the EIP-AGRI network. EIPs aim to streamline, simplify and better coordinate existing instruments and initiatives and complement them with actions where necessary. Two specific funding sources are particularly important for the EIP-AGRI:  the EU Research and Innovation framework, Horizon 2020,  the EU Rural Development Policy. An EIP AGRI Focus Group* is one of several different building blocks of the EIP-AGRI network, which is funded under the EU Rural Development policy. Working on a narrowly defined issue, Focus Groups temporarily bring together around 20 experts (such as farmers, advisers, researchers, up-and downstream businesses and NGOs) to map and develop solutions within their field. The concrete objectives of a Focus Group are:  to take stock of the state of art of practice and research in its field, listing problems and opportunities;  to identify needs from practice and propose directions for further research;  to propose priorities for innovative actions by suggesting potential projects for Operational Groups working under Rural Development or other project formats to test solutions and opportunities, including ways to disseminate the practical knowledge gathered. Results are normally published in a report within 12-18 months of the launch of a given Focus Group. Experts are selected based on an open call for interest. Each expert is appointed based on his or her personal knowledge and experience in the particular field and therefore does not represent an organisation or a Member State. *More details on EIP-AGRI Focus Group aims and process are given in its charter on: http://ec.europa.eu/eip/agriculture/sites/agri-eip/files/charter_en.pdf

. Brief description of the process The

  Focus Group on New Feed for Pigs and Poultry met on two occasions during 2018 and 2019. The first meeting was held in

	3
	1 IFIF (2017). International Feed Industry Federation Annual report 2016-2017. http://annualreport.ifif.org/wp-content/uploads/IFIF-Annual-
	Report-2016-2017-download.pdf

Espoo, Finland on 7-8 June 2018, while the second meeting was held in Brussels, Belgium on 30-31 January 2019. The

	EIP-AGRI Service Point facilitated the meetings, supported by the
	coordinating expert (see Annex B).

Table 1 : List of potential new feed options for pigs and poultry

 1

	Aquatic Biomass	Industry	Green Biomass	Insects	Single cell
		residuals and			protein (SCP)
		former foodstuffs
	Macro-algae (e.g.	Dairy residuals	Grass/clover (and	Black Soldier Fly
	Kelp)	(e.g. yoghurt, milk)	protein extract	(Hermetia illucens)
			made from
	Micro-algae (e.g.	Hydrolysed animal	grass/clover)	Mealworm
	Chlorella, Spirulina) Duck weed	protein Okara (by-product	Legumes (e.g. Beans)	(Tenebrio molitor and Alphitobius diaperinus)
		of soy
	Mussel meal	fermentation)	Locally-grown	Crickets
			whole plant silage	(Acheta
		Crop by-products	for production and	domesticus)
		(e.g. oil-seed rape,	processing
		cereals, EU soy)
			Tree protein
		Catering waste
			Microbial protein
		Retail waste	from tree
			production
		Bakery products
		(e.g. bread,
		cookies)

Table 2 : Examples of transferring new feed options across application and geographical level Algae Bakery products

 2

	Baseline	Baseline
	A high protein feed that is easy to grow and with	High volume energy-rich feed from mature industry
	potential bioactive properties
	Transferring -Local to regional level	Transferring -Industry to farm level Challenges may involve legislation (e.g.
	R&D example from Denmark: On farm application of	distinguishing between waste and food residues),
	manure in anaerobic digestion (AD) facilities utilised	processing (e.g. residual packaging material in the
	as energy for production of micro-algae production	feed) and availability
	to be used as feed for pigs. Sufficient availability of
	manure and infrastructure to grow micro-algae	Transferring -Local to regional level
		Availability, collection/logistics and demand
	Transferring -Industry to farm level Example from Ireland: Industrial drying of seaweed,	Risk
	for use on farms.	Development of competing industry (e.g. biogas)
	Example from Portugal: Application of industry	Lack of standardisation (regional)
	excess heat from cement works to produce micro-
	algae as feed for pigs

Risk

Excessive use may influence the colour of the meat and feed palatability. An acceptable level of microalgae in feed needs to determined.

Table 3 : Success and fail factors of bakery products

 3

	Success	Barrier
		Technical
	Standard	Additives -risk of dangerous ingredients
		(chocolate/theobromine)
		Packaging
		Standardisation (reactive usage)
		Transportation/collection
		Biological quality (energy)
		Regulatory issues (e.g. organic certification)
		Social
	Re-food	No issues
	Environmental 'credits'
		Economic

Table 4 : Success and fail factors of green biomass (grass/clover)

 4

	Success	Barrier
		Technical
	Silage (low-tech processing)	Technical demand for primary production
		Processing to obtain final concentrate
		High concentration of pigments (chlorophyll)
		For non-ruminants, fresh green biomass may be harder to
		digest
		Requires technology for better use of new fractions
		Seasonally dependent (except if dried or as silage)
		Dependent on local (regional) production, as it will not be
		economically viable to transport green biomass over long
		distances
		Dependent on production system and facilities
		Social
	Natural product	Competition with other species (ruminants)
	Gut health	Antimicrobial use
	High quality co-products	Increase enteric GHG for monogastrics (pigs)
	Reduced GHG ruminants
	Accepted by organic rules
		Economic
	Emerging plant-derived markets demand for	Competition, availability, cost and demand
	new products	Soybean meal dependency
	Use fibre fraction for biofuel
	Insects (Table

Table 5 : Success and fail factors of black soldier fly (BSF)

 5

	Success	Barrier
		Technical
	Re-use pig farms as BSF farms	Managing heat production efficiently
	Easy to produce	Accumulation of heavy metals, etc.
	Opportunities for organic farming	How to separate larvae from substrate
	Upgrading nutritional value of 'waste' (leftovers)	Biosecurity/traceability
	Low environmental impact (especially on 'waste'	Produce constant product on variable substrate
	substrate)	Grow on liquid substrates
	Bio-sanitation function (e.g. regarding	Chitin (can be problematic if content is too high)
	mycotoxins and anti-nutritional factors)
		Social
	Accepted in poultry	Not accepted in swine production
	Suitable as human feed	Insect housing and management in relation to
	Creates new jobs (collection business of 'waste'	communication
	substrate)	Registration as animal protein or separate (i.e. insect
	Use of 'waste' as substrate-circularity	protein) regarding labels/concepts (e.g. vegetable-based)
	Potential reduction in use of antibiotics	Insect welfare unknown
		The idea of eating insects
		Economic
	Use of 'waste' products, biogas products (fat-	Cost price of raw material
	/protein-rich)	Competing for substrate (retrieval systems: logistics of
	Digestibility and protein composition of insect	raw material in relation to cost price)
	meal
	Efficient protein conversion of BSF

Table 6 : Success and fail factors of micro-algae

 6

	Success	Barrier
		Technical
	Energy source	Low dry matter content
	Protein source with good amino acid profile	Risk of contamination with toxic spp. (other micro-algae)
	Carotenoids and other beneficial nutrients (e.g.	Storage tanks or drying capacity needed
	omega-3, vitamins, etc.)	Digestibility
	Add to drinking water or liquid feed	Traceability of heavy metals
	Can be dried
	Boosts immunity, reducing the need for
	antibiotics
		Social
	No competition for land	Need energy for heating

Table 7 : Success and fail factors of single cell protein (SCP) -bacteria

 7

	Success	Barrier
		Technical
	Amino acid profile	Risk of contamination with toxic spp. (other bacteria)
	Easy to handle	Immunology issues
	Production of SCP industry on waste	Content of nucleic acids
	Probiotic effect
		Social
	Production of SCP industry on waste	Consumer perception
	Less antibiotics?	Future legislation
	Palatability	Antibiotic resistance?
		Economic
		Competitiveness

Table 8 : Ideas for Operational Groups and other innovative actions -generated by the Focus Group

 8

	Problem	Idea	Where	Who	Contact
	Bakery products
	Inconsistent quality of	Find methods to	Central Europe	processors who prepare	Kees van
	bread from small(er) scale	guarantee more		feed ingredients for	Gorp
	producers	uniformity of final product Investment in separate		compound feed mills from food waste	Nuria Llanes
		lines		Feed mills
		NIR analyses
	How to analyse and get	Develop fast, robust and	Central Europe	By-product dealers	Kees van
	'on-line' (fast) nutrient value for (by-)products (liquid/solid pasty)	feasible regression methods to analyse and upload results to update the feed formulation Know composition -proximal analysis for	e.g. implement-ted with other interacting by-products (e.g. Okara)	Feed mills Farmers (liquid feeding)	Gorp David Sola-Oriol Nuria Llanes
		starch (carbohydrate)
		fractions and gelatinized
		carbohydrates
	Standardisation of the	Mapping of bakery	Regional/national Bakery by-product	Kees van
	end product	producers and improve		processors	Gorp
		logistics		Bakery industry

6

For more information on the formation of OGs and opportunities for funding please refer to: https://ec.europa.eu/eip/agriculture/en/eipagri-operational-groups-%E2%80%93-basic-principles

Table 9 : Needs from Practice

 9 As expected, the issues displayed in Table9and Table10are very different and mostly represent short-term issues (Table

	Problem	Need for research/knowledge gap	Comments
	Gap between research	Establishment of network	No need for theory
	and practice		(papers) but solutions
	Mapping of available feed	Establishment of database of producers
	(seasonal availability)
	Lack of	Calibration curves for new products
	fast/reliable/cheap	NIRS (Near Infra-Red Spectrometry)
		Mycotoxins

Alexandratos N, Bruinsma J (2012). World agriculture towards 2030/2050: the 2012 revision. ESA Working paper No. 12-03. Rome, FAO. http://www.fao.org/docrep/016/ap106e/ap106e.pdf

See recent article from FeedNavigator: https://www.feednavigator.com/Article/2018/09/11/Microalgae-coproducts-may-boost-healthsupport-growth-in-nursery-pigs

Contact details are available on the Focus Group website: https://ec.europa.eu/eip/agriculture/en/focus-groups/new-feed-pigs-and-poultry

Micro-algae

Annex A. Good practices and case studies

This Swedish research project at SLU, aims to evaluate the usefulness of grass/clover silage as a locally grown feed resource in diets to pigs. The project will evaluate how silage can be utilized at farm level. A special focus will be put on feeding strategies and form of the silage, effect on production, nitrogen utilization, gut health and pig behaviour. Further, the metabolism of phytoestrogens in red clover and its effect on sow fertility and reproduction will be studied.

Coordinator