The black soldier fly, Hermetia illucens, is increasingly studied for its ability to convert organic waste into protein, offering solutions for waste valorisation and livestock feeding. Adult performance is critical for egg production, yet the behavioural and molecular bases of sugar feeding in adult black soldier flies remain poorly understood. To fill this gap, we combined behavioural assays, morphological analyses, electrophysiological recordings and gustatory receptor (GR) repertoires and expression. All the experimental tests conducted in this study converge to show that adult flies can detect and consume sucrose, with females responding more strongly than males. Genome analysis identified 28 GRs, a surprisingly small number for a generalist fly, including only three putative sugar-specific GRs homologous to those of the eight known sugar GRs in Drosophila. Moreover, one of these GRs showed a general high level of expression including in the head and in the antennae whereas the other two displayed tissue-specific patterns of expression. We also identified a high number of GR pseudogenes, including four putative sugar receptor pseudogenes, indicating multiple gene loss events of GRs compared with other dipterans sharing a similar ecological niche. Despite this reduced GR repertoire, adult black soldier flies retained strong behavioural and physiological sensitivity to sugars in their environment. The set of behavioural, morphological and electrophysiological tools developed here provides a foundation for deeper investigations into feeding behaviour in this species of growing agroecological importance.

Here, we present a high-throughput assay for continuously measuring the consumption and feeding choices of individual Drosophila adults over periods of 30 min to several hours. We describe steps for starving flies, introducing flies, filling capillaries, and monitoring consumption from capillary tubes. We then detail procedures for the automated collection of observations and the analysis of the results. This simple, low-cost protocol enables high-precision recording of the consumption and feeding choices of adult flies. For complete details on the use and execution of this protocol, please refer to Colombi1 and Richard et al.2.

Understanding how selection for flowering time affects maize susceptibility to the European corn borer (ECB, Ostrinia nubilalis) is essential in the context of climate change and increasing pest pressures. We investigated the consequences of divergent selection for flowering time on maize defense and susceptibility traits using two genetic backgrounds (MBS847 and F252). Three complementary approaches were combined: leaf palatability tests, ECB field incidence dynamics surveys, and biochemical analyses of maize stem walls. In the MBS847 background, early-flowering families exhibited lower content of defense compounds, higher leaf palatability, and higher field infestation compared to late-flowering families. In contrast, no significant differences were observed between early and late populations in the F252 background. Differences in susceptibility between families were associated with variation in stem biochemical composition, including fiber components which influence stem rigidity and resistance to ECB. Our results indicate that selection for flowering time can modify plant defense traits and susceptibility in a genetic-background dependent manner. These findings highlight the interaction between plant phenology, biochemical defenses, and insect herbivory, with implications for breeding and adaptation under changing environmental conditions. ### Competing Interest Statement The authors have declared no competing interest.

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), poses a significant threat to food security in sub-Saharan Africa. Identification of resistance in tropical maize germplasm is critical for development of FAW-resistant hybrids, which can be a component of Integrated Pest Management (IPM) strategies. This study assessed the general (GCA) and specific (SCA) combining ability, and reciprocal effects for FAW resistance parameters. Seven inbred lines were evaluated for their per se performance, and four lines were selected and crossed in a full diallel to generate 12 hybrids. Both inbred lines and hybrids were tested under artificial FAW infestation under net house conditions. Five FAW neonates were placed in each plant at the V5 growth stage. Leaf feeding damage and larval survival were assessed 14 days after infestation. Significant differences for leaf feeding damage and larval survival were detected among the lines and hybrids. Inbred lines Mp716, CML125, CML71, and CKSBL10008 showed partial resistance to FAW in terms of leaf feeding damage. Significant GCA, SCA, and reciprocal effects for both traits were detected. Additive genetic effects were more important for leaf feeding damage, while both additive and nonadditive genetic effects were more important than reciprocal effects for larval survival. Heterosis associated with increased resistance, ranged from -0.7

Patient’s social environment might influence cancer outcome. This potentially happens in Drosophila , as we previously reported that the social context influences the growth of intestinal tumors. To uncover the underlying social-induced physiological mechanisms, we performed RNA-seq of isogenized beheaded tumorous Drosophila . Importantly, expression of several immune peptides varied according to the social-induced tumor growth effect. Furthermore, ectopic expression in tumors of the apoptotic-inhibitor p35 suppressed the social-induced effect. Next, we challenged the immune peptide Defensin, previously reported to suppress imaginal disc tumor growth through a cell-death/JNK-dependent network. Nonetheless, the social-induced tumor suppression was maintained upon Defensin overexpression or JNKK-knockdown in tumors, and in defensin mutants. Surprisingly, tumor growth was reduced in the latter, indicating that Defensin sustains the growth of these intestinal tumors. In summary, our study indicates that the social context affects the immune response and that a given immune peptide may have opposite effects depending on tumor type.

ABSTRACTPlant secondary metabolites pose a challenge for generalist herbivorous insects because they are not only potentially toxic, they also may trigger aversion. On the contrary, some highly specialized herbivorous insects evolved to use these same compounds as ‘token stimuli’ for unambiguous determination of their host plants. Two questions that emerge from these observations are how recently derived herbivores evolve to overcome this aversion to plant secondary metabolites and the extent to which they evolve increased attraction to these same compounds. In this study, we addressed these questions by focusing on the evolution of bitter taste preferences in the herbivorous drosophilidScaptomyza flava, which is phylogenetically nested deep in the paraphyleticDrosophila. We measured behavioral and neural responses ofS. flavaand a set of non-herbivorous species representing a phylogenetic gradient (S. pallida, S. hsui, andD. melanogaster) towards host- and non-host derived bitter plant compounds. We observed thatS. flavaevolved a shift in bitter detection, rather than a narrow shift towards glucosinolates, the precursors of mustard-specific defense compounds. In a dye-based consumption assay,S. flavaexhibited shifts in aversion toward the non-mustard bitter, plant-produced alkaloids caffeine and lobeline, and reduced aversion towards glucosinolates, whereas the non-herbivorous species each showed strong aversion to all bitter compounds tested. We then examined whether these changes in bitter preferences ofS. flavacould be explained by changes in sensitivity in the peripheral nervous system and compared electrophysiological responses from the labellar sensilla ofS. flava,S. pallida, andD. melanogaster. Using scanning electron microscopy, we also created a map of labellar sensilla inS. flavaandS. pallida. We assigned each sensillum to a functional sensilla class based on their morphology and initial response profiles to bitter and sweet compounds. Despite a high degree of conservation in the morphology and spatial placement of sensilla betweenS. flavaandS. pallida, electrophysiological studies revealed thatS. flavahad reduced sensitivity to glucosinolates to varying degrees. We found this reduction only in I type sensilla. Finally, we speculate on the potential role that evolutionary genetic changes in gustatory receptors betweenS. pallidaandS. flavamay play in driving these patterns. Specifically, we hypothesize that the evolution of bitter receptors expressed in I type sensilla may have driven the reduced sensitivity observed inS. flava, and ultimately, its reduced bitter aversion. TheS. flavasystem showcases the importance of reduced aversion to bitter defense compounds in relatively young herbivorous lineages, and how this may be achieved at the molecular and physiological level.

Physiological status can influence social behavior, which in turn can affect physiology and health. Previously, we reported that tumor growth in Drosophila virgin females depends on the social context, but did not investigate the underlying physiological mechanisms. Here, we sought to characterize the signal perceived between tumorous flies, ultimately discovering that the tumor suppressive effect varies depending on reproductive status. Firstly, we show that the tumor suppressive effect is neither dependent on remnant pheromone-like products nor on the microbiota. Transcriptome analysis of the heads of these tumorous flies reveals social-dependent gene-expression changes related to nervous-system activity, suggesting that a cognitive-like relay might mediate the tumor suppressive effect. The transcriptome also reveals changes in the expression of genes related to mating behavior. Surprisingly, we observed that this social-dependent tumor-suppressive effect is lost in fertilized females. After mating, Drosophila females change their behavior—favoring offspring survival—in response to peptides transferred via the male ejaculate, a phenomenon called “male manipulation”. Remarkably, the social-dependent tumor suppressive effect is restored in females mated by sex-peptide deficient males. Since male manipulation has likely been selected to favor male gene transmission, our findings indicate that this evolutionary trait impedes social-dependent tumor growth slowdown.

Social insects' nests harbor intruders known as inquilines,1 which are usually related to their hosts.2,3 However, distant non-social inquilines may also show convergences with their hosts,4,5 although the underlying genomic changes remain unclear. We analyzed the genome of the wingless and blind bee louse fly Braula coeca, an inquiline kleptoparasite of the western honey bee, Apis mellifera.6,7 Using large phylogenomic data, we confirmed recent accounts that the bee louse fly is a drosophilid8,9 and showed that it had likely evolved from a sap-breeder ancestor associated with honeydew and scale insects' wax. Unlike many parasites, the bee louse fly genome did not show significant erosion or strict reliance on an endosymbiont, likely due to a relatively recent age of inquilinism. However, we observed a horizontal transfer of a transposon and a striking parallel evolution in a set of gene families between the honey bee and the bee louse fly. Convergences included genes potentially involved in metabolism and immunity and the loss of nearly all bitter-tasting gustatory receptors, in agreement with life in a protective nest and a diet of honey, pollen, and beeswax. Vision and odorant receptor genes also exhibited rapid losses. Only genes whose orthologs in the closely related Drosophila melanogaster respond to honey bee pheromone components or floral aroma were retained, whereas the losses included orthologous receptors responsive to the anti-ovarian honey bee queen pheromones. Hence, deep genomic convergences can underlie major phenotypic transitions during the evolution of inquilinism between non-social parasites and their social hosts.

Black soldier fly larvae (BSFL; Hermetia illucens) hold promise for converting biowaste into proteins and lipids for feed. Dietary starch is efficiently digested by the larvae and influences larval performance, but the mechanisms of starch digestion remain poorly understood. This study investigated changes in individual weight and amylase activity in BSFL after 4, 7 and 11 days of feeding for five substrates varying in starch content and type: chicken feed (CF), corn gluten feed (CGF), wheat bran (WB), wheat distillers grain (WDG) and discarded potatoes (DP). Substrate amylase activities were also measured with and without larvae (feeding and fermenting trays, respectively) over time in order to explore external digestion. Feed conversion ratio (FCR) and estimated digestibility (ED) of DM and starch were assessed at the end of the experiment. The ranking for best FCR was CF, WB, CGF, WDG and DP. In feeding trays, ED of DM was 69.8 +/- 1.8, 59.5 +/- 2.9, 58.6 +/- 0.7, 45.4 +/- 0.6 and 19.5 +/- 0.8% in CF, DP, WB, CGF and WDG, respectively. Estimated digestibility of starch reached 100% with WB and CGF, followed by CF (88.2 +/- 2.3%), DP (85. 2 +/- 1.2%) and WDG (43.1 +/- 1.0%). Larval amylase activity increased with growth for all substrates and dropped when approaching pupation. No relationship was found between larval amylase activity and substrate starch or other nutrient content, but a negative correlation was reported with the reducing sugar content of the larvae, suggesting glucose repression of amylase production. Amylase activity decreased with time in all feeding and fermenting substrates except WDG and DP. In vitro degradation assays indicated that BSFL amylase was nine times more efficient on raw corn or wheat starch than on raw potato starch, highlighting that starch structure is a major driver of digestibility. Western blot analysis revealed the presence of BSFL amylase in the feeding substrate, hinting at external digestion. Larval amylase was purified to identify its optimal pH (5.0-6.5) and temperature (70 degrees C). These results highlight that starch content is not a major driver of amylase activity in BSFL and suggest that other noninvestigated factors could have had a crucial impact on the activity of larval digestive enzymes, such as microbial community of the substrate and presence of amylase inhibitors. This study also provides insights into the evolution of BSFL digestive activity during their development and the occurrence of external digestion. (c) 2024 The Author(s). Published by Elsevier B.V. on behalf of The Animal Consortium. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Honeybees (genus: Apis) use a plethora of pheromones for intraspecific communication. The primary compound produced by the queen’s mandibular glands, 9-ODA, is involved in mating in all Apis species. It is the ligand of the most highly expressed olfactory receptor in males of Apis mellifera: AmelOR11. Putative orthologs are found in the genomes of other Apis species: Apis dorsata, Apis florea, and Apis cerana. Modeling of OR11 proteins shows high structure conservation except for AflorOR11. Using heterologous expression in Drosophila and calcium imaging, a broad odorant screening revealed that all OR11 respond predominantly to 9-ODA, but also to secondary ligands, except AflorOR11, which remains specific to 9-ODA. Secondary ligands were confirmed by optical imaging of male antennal lobes in A. mellifera. This work supports a conserved queen sex pheromone detection channel in honeybees, albeit with an extended response spectrum possibly playing a role in reproductive isolation among species.

The fall armyworm (FAW), Spodoptera frugiperda, a pest of maize native to the Americas first reported in West and Central Africa in 2016, severely threatens maize production and food security in Sub-Saharan Africa. Native genetic resistance is one of the best methods of control of insect pests as it is contained in the seed making it more amenable for use by farmers compared to other interventions and it is also compatible with other integrated pest management (IPM) options. An intensive screening against FAW was carried out by artificial infestation in greenhouse conditions in Kenya between 2017 and 2018 on about 3000 inbred lines available in the germplasm collection of the International Maize and Wheat Improvement Center (CIMMYT). Among these lines, only four showed to be resistant to FAW, but the mechanisms of resistance are not yet known. The objective of this study was to determine the resistance mechanisms specifically non-preference and antibiosis to S. frugiperda in these four selected resistant inbred lines. The studies were conducted under laboratory and net house conditions in Kenya from April 2020 to November 2021. Non-preference was assessed estimating the feeding preference by counting the number of FAW neonates found on each leaf portion, silk portion and grain using binary and multiple choice methods under laboratory conditions, while antibiosis was assessed through the relative growth rate (RGR) and developmental time of FAW larvae on leaves, silks and grains under both laboratory and net house conditions. Among the four resistant maize inbred lines tested, two, namely CML71 and CKSBL10008, exhibited the highest level of antibiosis resistance on leaves. Under laboratory conditions, the larval RGR reduced from 13 mg/d on the most susceptible line to 8 mg/d on CML71. CML71 also showed a good non-preference on leaves compared to other tested lines. Only 6% of neonates choose to feed on CML71 whereas more than 10% choose to feed on the other lines (and 15% on the most susceptible) in multiple choice tests. The non-preference for feeding and lower RGR of larvae on CML71 suggest a biochemical involvement resistance to FAW. Through this study, CML71 is revealed as a highly promising line for use in breeding for native genetic resistance to FAW in tropical maize.

Maize is the most-produced cereal in the world, but its production faces constraints such as parasitic attacks from stemborers. We evaluated the resistance of a core-collection of 18 maize lines by measuring their palatability to European Corn Borer (ECB) larvae fed on maize leaf discs. Using an original consumption test device that takes into account the variability of larvae behaviour, we were able to phenotype the resistance of the 18 maize lines. We matched consumption data to existing enzymatic and metabolomic data that characterized the maize core-collection and identified some metabolites such as caffeoyl-lquinate, trocopherol, digalactosylglycerol and tyrosine that are positively or negatively correlated with the palatability to ECB larvae. Altogether, our results confirm the metabolic complexity involved in the establishment of plant defenses. Metabolic changes associated to leaf palatability mostly concern membrane and cell wall composition. Some of them, pointing-out to the phenylpropanoids pathway, were observed independently of plant developmental pace and plant earliness.

Black soldier fly larvae (BSFL) are highly efficient at converting organic substrates into insect biomass suitable for livestock nutrition. BSFL conversion efficiency depends on the nature of their feed, and formulation approaches use macronutrient contents to reach a targeted performance. However, digestibility varies between ingredients and BSFL digestibility data is lacking due to methods unsuitable for insects living in their feed. This study proposes to work under a range of larval densities to reach total ingestion of the feed, and defines Estimated Digestibility (ED) as the difference between distributed feed and frass macronutrient weight, divided by macronutrient weight in distributed feed. Using the same amount of chicken feed, same feeding time and 0 to 29 larvae/cm 2 , ED of dry mass (DM), starch, nitrogen, ether extract (EE), neutral detergent fibre, acid detergent fibre, acid detergent lignin, ash and energy were evaluated, along with survival rate, feed conversion ratio (FCR) and larval individual mass gain on a fresh and dry basis. ED of DM, starch, nitrogen, EE and energy increased with larval density following an asymptotic trend. ED of ash increased between 0 and 11.4 larvae/cm2 but decreased for higher densities. Using a logistic model, asymptotic ED of DM (80.3±1.3%), starch (99.0±2.3%), nitrogen (78.6±1.1%), EE (95.3±1.5%), ash (58.4±1.0%) and energy (80.6±1.2%) were determined. No effect of areal density on ED of fibre fractions was detected. Survival rate and individual mass gain peaked at densities below 5 larvae/cm 2 , FCR (dry to fresh) was improved with increasing density, while FCR (dry to dry) was optimised at 11.4 larvae/cm 2 . Asymptotic ED is a new indicator corresponding to the maximal fraction of macronutrients potentially digestible by BSFL. It should be used to develop a diet formulation approach based on digestible rather than crude macronutrient contents. Further studies should focus on evaluating asymptotic ED in different ingredients.

The primary actors in the detection of olfactory information in insects are odorant receptors (ORs), transmembrane proteins expressed at the dendrites of olfactory sensory neurons (OSNs). In order to decode the insect olfactome, many studies focus on the deorphanization of ORs (i.e., identification of their ligand), using various approaches involving heterologous expression coupled to neurophysiological recordings. The "empty neuron system" of the fruit fly Drosophila melanogaster is an appreciable host for insect ORs, because it conserves the cellular environment of an OSN. Neural activity is usually recorded using labor-intensive electrophysiological approaches (single sensillum recordings, SSR). In this study, we establish a simple method for OR deorphanization using transcuticular calcium imaging (TCI) at the level of the fly antenna. As a proof of concept, we used two previously deorphanized ORs from the cotton leafworm Spodoptera littoralis, a specialist pheromone receptor and a generalist plant odor receptor. We demonstrate that by co-expressing the GCaMP6s/m calcium probes with the OR of interest, it is possible to measure robust odorant-induced responses under conventional microscopy conditions. The tuning breadth and sensitivity of ORs as revealed using TCI were similar to those measured using single sensillum recordings (SSR). We test and discuss the practical advantages of this method in terms of recording duration and the simultaneous testing of several insects.

In adult Lepidoptera the labial palps are best known for their role in CO 2 detection, but they can also bear sensilla chaetica which function is unknown. The number and distribution of sensilla chaetica in labial palps was studied using a bright field microscope. To determine if these sensilla have a gustatory function, we performed single sensillum electrophysiology recordings from palp and antennal sensilla of adult moths of Cydia pomonella (L.), Grapholita molesta (Busck) and Lobesia botrana (Denis and Shieffermüller). Each sensillum was stimulated with 3 doses of one of four test stimulus (sucrose, fructose, KCl and NaCl). Overall, responses (spikes/s −1 ) increased with dose, and were higher in the palps than in the antennae, and higher to sugars than to salts. With sugars the response increased with concentration in the palp but not in the antenna. With salts there was a drop in response at the intermediate concentration. The number and position of sensilla chaetica on labial palps was variable among individuals. Sensilla were located in the most exposed areas of the palp. Differences in sensilla distribution were detected between species. Such differences among species and between palps and antenna suggest that taste sensilla on the palps have an unforeseen role in adaptation.

The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), native to the Americas, was confirmed in West and Central Africa in 2016 and reported in almost all countries of sub-Saharan Africa in 2017, becoming thereafter one of the major constraints on the production of maize, the main staple food crop in the region. Cereals depend on silicon (Si)-based defences to fight off herbivores. Both FAW strains, namely rice and corn strains, have been found to be present in Kenya. The present study tested the influence of Si on larval survival and relative growth rate according to the FAW strain, using potted maize plants treated with 10 and 20 g of Si. The results showed that plants treated with Si disturbed the larval growth of FAW larvae only from the corn strain but not from the rice strain. Overall, the corn strain performed better on maize as compared to the rice strain regardless of Si treatment, explaining why it has become the most abundant strain in Africa.

Understanding how organisms adapt to environmental changes is a major question in evolution and ecology. In particular, the role of ancestral variation in rapid adaptation remains unclear because its trace on genetic variation, known as soft selective sweep, is often hardly recognizable from genome-wide selection scans. Here, we investigate the evolution of chemosensory genes in Drosophila yakuba mayottensis, a specialist subspecies on toxic noni (Morinda citrifolia) fruits on the island of Mayotte. We combine population genomics analyses and behavioral assays to evaluate the level of divergence in chemosensory genes and perception of noni chemicals between specialist and generalist subspecies of D. yakuba. We identify a signal of soft selective sweep on a handful of genes, with the most diverging ones involving a cluster of gustatory receptors expressed in bitter-sensing neurons. Our results highlight the potential role of ancestral genetic variation in promoting host plant specialization in herbivorous insects and identify a number of candidate genes underlying behavioral adaptation.

Insect taste receptors are cuticular structures housing 1–4 gustatory neurons and one mechanoreceptor. The anatomy and localization of these structures is described as well at their molecular physiology. We review how taste neurons encode chemicals presented alone or in mixtures. These issues are illustrated by taking examples from Drosophila and other insects.