Grinding as a slaughter method for farmed black soldier fly (Hermetia illucens) larvae: Empirical recommendations to achieve instantaneous killing.

At least 200 billion black soldier fly (Hermetia illucens) larvae (BSFL) are reared each year as food and feed, and the insect farming industry is projected to grow rapidly. Despite interest by consumers, producers, and legislators, no empirical evidence exists to guide producers in practicing humane - or instantaneous - slaughter for these novel mini-livestock. BSFL may be slaughtered via freezing, boiling, grinding, or other methods; however standard operating procedures (SOPs) and equipment design may affect the likelihood of instantaneous death using these methods. We tested how larval body size and particle size plate hole diameter affect the likelihood of instantaneous death for black soldier fly larvae that are slaughtered using a standard meat grinder. Larval body size did not affect the likelihood of instantaneous death for larvae that are 106-175 mg in mass. However, particle size plate hole diameter had a significant effect on the likelihood of instantaneous death, with only 54% of larvae experiencing an instant death when using the largest particle size plate (12-mm hole diameter) compared to 84% using the smallest particle size plate (2.55 mm). However, a higher percentage of instantaneous death (up to 99%) could be achieved by reducing the proportion of larvae that become stuck in the machine. We conclude by outlining specific recommendations to support producers in achieving a 99% instantaneous death rate through specific SOPs to be used with similarly designed machines. We also develop a protocol for producers that wish to test their own grinding SOPs.

A long-read draft assembly of the Chinese mantis (Mantodea: Mantidae: Tenodera sinensis) genome reveals patterns of ion channel gain and loss across Arthropoda.

Praying mantids (Mantodea: Mantidae) are iconic insects that have captivated biologists for decades, especially the species with cannibalistic copulatory behavior. This behavior has been cited as evidence that insects lack nociceptive capacities and cannot feel pain; however, this behaviorally driven hypothesis has never been rigorously tested at the genetic or functional level. To enable future studies of nociceptive capabilities in mantids, we sequenced and assembled a draft genome of the Chinese praying mantis (Tenodera sinensis) and identified multiple classes of nociceptive ion channels by comparison to orthologous gene families in Arthropoda. Our assembly-produced using PacBio HiFi reads-is fragmented (total size = 3.03 Gb; N50 = 1.8 Mb; 4,966 contigs), but is highly complete with respect to gene content (BUSCO complete = 98.7% [odb10_insecta]). The size of our assembly is substantially larger than that of most other insects, but is consistent with the size of other mantid genomes. We found that most families of nociceptive ion channels are present in the T. sinensis genome; that they are most closely related to those found in the damp-wood termite (Zootermopsis nevadensis); and that some families have expanded in T. sinensis while others have contracted relative to nearby lineages. Our findings suggest that mantids are likely to possess nociceptive capabilities and provide a foundation for future experimentation regarding ion channel functions and their consequences for insect behavior.

Is it time for insect researchers to consider their subjects' welfare?

Recent evidence suggests that at least some insect species might plausibly feel pain. These findings should prompt researchers to think about the welfare implications of insect experiments.

Individual reflectance of solar radiation confers a thermoregulatory benefit to dimorphic males bees (Centris pallida) using distinct microclimates.

Incoming solar radiation (wavelengths 290-2500 nm) significantly affects an organism's thermal balance via radiative heat gain. Species adapted to different environments can differ in solar reflectance profiles. We hypothesized that conspecific individuals using thermally distinct microhabitats to engage in fitness-relevant behaviors would show intraspecific differences in reflectance: we predicted individuals that use hot microclimates (where radiative heat gain represents a greater thermoregulatory challenge) would be more reflective across the entire solar spectrum than those using cooler microclimates. Differences in near-infrared (NIR) reflectance (700-2500 nm) are strongly indicative of thermoregulatory adaptation as, unlike differences in visible reflectance (400-700 nm), they are not perceived by ecological or social partners. We tested these predictions in male Centris pallida (Hymenoptera: Apidae) bees from the Sonoran Desert. Male C. pallida use alternative reproductive tactics that are associated with distinct microclimates: Large-morph males, with paler visible coloration, behave in an extremely hot microclimate close to the ground, while small-morph males, with a dark brown dorsal coloration, frequently use cooler microclimates above the ground near vegetation. We found that large-morph males had higher reflectance of solar radiation (UV through NIR) resulting in lower solar absorption coefficients. This thermoregulatory adaptation was specific to the dorsal surface, and produced by differences in hair, not cuticle, characteristics. Our results showed that intraspecific variation in behavior, particular in relation to microclimate use, can generate unique thermal adaptations that changes the reflectance of shortwave radiation among individuals within the same population.

Challenges in farmed insect welfare: Beyond the question of sentience.

The global Insects as Food and Feed (IAFF) industry currently farms over a trillion individual insects a year and is growing rapidly. Intensive animal production systems are known to cause a range of negative affective states in livestock; given the potential scale of the IAFF industry, it is urgent to consider the welfare of the industry's insect livestock. The majority of the literature on farmed insect welfare has focused on: (i) establishing that insect welfare ought to be of concern; or (ii) extending vertebrate welfare frameworks to insects. However, there are many overlooked challenges to studying insect welfare and applying that knowledge in IAFF industry contexts. Here, we briefly review five of these challenges. We end with practical recommendations for the future study of insect welfare.

Impacts of development and adult sex on brain cell numbers in the Black Soldier Fly, Hermetia illucens L. (Diptera: Stratiomyidae).

The Black Soldier Fly (Hermetia illucens, Diptera: Stratiomyidae) has been introduced across the globe, with numerous industry applications predicated on its tremendous growth during the larval stage. However, basic research on H. illucens biology (for example, studies of their central nervous system) are lacking. Despite their small brain volumes, insects are capable of complex behaviors; understanding how these behaviors are completed with such a small amount of neural tissue requires understanding processing power (e.g. number of cells) within the brain. Brain cell counts have been completed in only a few insect species (mostly Hymenoptera), and almost exclusively in adults. This limits the taxonomic breadth of comparative analyses, as well as any conclusions about how development and body size growth may impact brain cell populations. Here, we present the first images and cell counts of the H. illucens brain at four time points across development (early, mid, and late larval stages, and both male and female adults) using immunohistochemistry and isotropic fractionation. To assess sexual dimorphism in adults, we quantified the number of cells in the central brain vs. optic lobes of males and females separately. To assess if increases in body size during development might independently affect different regions of the CNS, we quantified the larval ventral nerve cord and central brain separately at all three stages. Together, these data provide the first description of the nervous system of a popular, farmed invertebrate and the first study of brain cell numbers using IF across developmental stages in any insect.

Soldier neural architecture is temporarily modality specialized but poorly predicted by repertoire size in the stingless bee Tetragonisca angustula.

Individual heterogeneity within societies provides opportunities to test hypotheses about adaptive neural investment in the context of group cooperation. Here, we explore neural investment in defense specialist soldiers of the eusocial stingless bee (Tetragonisca angustula) which are age subspecialized on distinct defense tasks and have an overall higher lifetime task repertoire than other sterile workers within the colony. Consistent with predicted behavioral demands, soldiers had higher relative visual (optic lobe) investment than nonsoldiers but only during the period when they were performing the most visually demanding defense task (hovering guarding). As soldiers aged into the less visually demanding task of standing guarding this difference disappeared. Neural investment was otherwise similar across all colony members. Despite having larger task repertoires, soldiers had similar absolute brain size and the smaller relative brain size compared to other workers, meaning that lifetime task repertoire size was a poor predictor of brain size. Both high behavioral specialization in stable environmental conditions and reassignment across task groups during a crisis occur in T. angustula. The differences in neurobiology we report here are consistent with these specialized but flexible defense strategies. This work broadens our understanding of how neurobiology mediates age and morphological task specialization in highly cooperative societies.

Experience-expectant brain plasticity corresponds to caste-specific abiotic challenges in dampwood termites (Zootermopsis angusticollis and Z. nevadensis).

Hypotheses for adaptive brain investment predict associations between the relative sizes of functionally distinct brain regions and the sensory/cognitive demands animals confront. We measured developmental differences in the relative sizes of visual processing brain regions (optic lobes) among dampwood termite castes to test whether optic lobe investment matches caste differences in exposure to visually complex environments. The winged primary reproductives (Kings/Queens) on mating flights are the only caste to leave the dark nest cavities and as predicted, Kings/Queens showed greater relative investment in optic lobe tissue than nestbound (neotenic) reproductives and soldiers in two dampwood termite species (Zootermopsis angusticollis and Z. nevadensis). Relative optic lobe size spanned more than an order of magnitude among the castes we studied, suggesting the growth of the optic lobes incurs substantial tissue costs. Optic lobe growth was experience-expectant: the optic lobes of Z. angusticollis brachypterous nymphs, which typically develop into Kings/Queens, were relatively larger than the optic lobes of apterous nymphs, which precede neotenics and soldiers, and relative optic lobe size of nestbound brachypterous nymphs was statistically similar to that of Kings/Queens. Experience-expectant brain tissue growth is rarely documented in insects, likely because it entails high potential costs of tissue production and maintenance and relatively low immediate sensory/cognitive benefits. We develop hypotheses for the conditions under which experience-expectant growth in brain regions could be favored by natural selection.

Neuroanatomical differentiation associated with alternative reproductive tactics in male arid land bees, Centris pallida and Amegilla dawsoni.

Alternative reproductive tactics (ARTs) occur when there is categorical variation in the reproductive strategies of a sex within a population. These different behavioral phenotypes can expose animals to distinct cognitive challenges, which may be addressed through neuroanatomical differentiation. The dramatic phenotypic plasticity underlying ARTs provides a powerful opportunity to study how intraspecific nervous system variation can support distinct cognitive abilities. We hypothesized that conspecific animals pursuing ARTs would exhibit dissimilar brain architecture. Dimorphic males of the bee species Centris pallida and Amegilla dawsoni use alternative mate location strategies that rely primarily on either olfaction (large-morph) or vision (small-morph) to find females. This variation in behavior led us to predict increased volumes of the brain regions supporting their primarily chemosensory or visual mate location strategies. Large-morph males relying mainly on olfaction had relatively larger antennal lobes and relatively smaller optic lobes than small-morph males relying primarily on visual cues. In both species, as relative volumes of the optic lobe increased, the relative volume of the antennal lobe decreased. In addition, A. dawsoni large males had relatively larger mushroom body lips, which process olfactory inputs. Our results suggest that the divergent behavioral strategies in ART systems can be associated with neuroanatomical differentiation.

Potential for Use of Erythritol as a Socially Transferrable Ingested Insecticide for Ants (Hymenoptera: Formicidae).

Ants are significant structural and agricultural pests, generating a need for human-safe and effective insecticides for ant control. Erythritol, a sugar alcohol used in many commercial food products, reduces survival in diverse insect taxa including fruit flies, termites, and mosquitos. Erythritol also decreases longevity in red imported fire ants; however, its effects on other ant species and its ability to be transferred to naïve colony members at toxic doses have not been explored. Here, we show that erythritol decreases survival in Tetramorium immigrans Santschi (Hymenoptera: Formicidae) in a concentration-dependent manner. Access to ad-libitum water reduced the toxic effects of erythritol, but worker mortality was still increased over controls with ad-lib water. Foraging T. immigrans workers transferred erythritol at lethal levels to nest mates that had not directly ingested erythritol. Similar patterns of mortality following erythritol ingestion were observed in Formica glacialis Wheeler (Hymenoptera: Formicidae), Camponotus subarbatus Emery (Hymenoptera: Formicidae), and Camponotus chromaiodes Bolton (Hymenoptera: Formicidae). These findings suggest that erythritol may be a highly effective insecticide for several genera of ants. Erythritol's potential effectiveness in social insect control is augmented by its spread at lethal levels through ant colonies via social transfer (trophallaxis) between workers.

Larval mannitol diets increase mortality, prolong development and decrease adult body sizes in fruit flies (Drosophila melanogaster).

The ability of polyols to disrupt holometabolous insect development has not been studied and identifying compounds in food that affect insect development can further our understanding of the pathways that connect growth rate, developmental timing and body size in insects. High-sugar diets prolong development and generate smaller adult body sizes in Drosophila melanogaster We tested for concentration-dependent effects on development when D. melanogaster larvae are fed mannitol, a polyalcohol sweetener. We also tested for amelioration of developmental effects if introduction to mannitol media is delayed past the third instar, as expected if there is a developmental sensitive-period for mannitol effects. Both male and female larvae had prolonged development and smaller adult body sizes when fed increasing concentrations of mannitol. Mannitol-induced increases in mortality were concentration dependent in 0 M to 0.8 M treatments with mortality effects beginning as early as 48 h post-hatching. Larval survival, pupariation and eclosion times were unaffected in 0.4 M mannitol treatments when larvae were first introduced to mannitol 72 h post-hatching (the beginning of the third instar); 72 h delay of 0.8 M mannitol introduction reduced the adverse mannitol effects. The developmental effects of a larval mannitol diet closely resemble those of high-sugar larval diets.This article has an associated First Person interview with the first author of the paper.

Erythritol Ingestion Causes Concentration-Dependent Mortality in Eastern Subterranean Termites (Blattodea: Rhinotermitidae).

Damage from termite infestations is economically significant and control can be costly when requiring the widespread use of conventional insecticides. Erythritol, a polyalcohol sweetener that is safe for human consumption, causes increased mortality when ingested by some insects, indicating potential as a safe alternative insecticide. Here, we investigated the applicability of erythritol as a novel toxicant method of termite control. Eastern subterranean termites, Reticulitermes flavipes Kollar (Blattodea: Rhinotermitidae), were fed paper foods treated with increasing concentrations of erythritol and were assessed for mortality and bait consumption. Termite survival to 8 d (the duration of the experiment) significantly decreased as erythritol treatment concentration increased, indicating that the lethal effects of erythritol were concentration-dependent. Termites consumed erythritol-treated paper at all concentrations and did not display avoidance in choice assays, suggesting that erythritol may be practical for use as an ingestible bait. These results provide a basis for further development of erythritol as a safe alternative method of termite control.

Brain structure differences between solitary and social wasp species are independent of body size allometry.

Evolutionary transitions in social behavior are often associated with changes in species' brain architecture. A recent comparative analysis showed that the structure of brains of wasps in the family Vespidae differed between solitary and social species: the mushroom bodies, a major integrative brain region, were larger relative to brain size in the solitary species. However, the earlier study did not account for body size effects, and species' relative mushroom body size increases with body size in social Vespidae. Here we extend the previous analysis by measuring the effects of body size variation on brain structure differences between social and solitary vespid wasps. We asked whether total brain volume was greater relative to body size in the solitary species, and whether relative mushroom body size was greater in solitary species, after accounting for body size effects. Both total brain volume and relative mushroom body volume were significantly greater in the solitary species after accounting for body size differences. Therefore, body size allometry did not explain the solitary versus social species differences in brain structure. The evolutionary transition from solitary to social behavior in Vespidae was accompanied by decreases in total brain size and in relative mushroom body size.

Mannitol ingestion causes concentration-dependent, sex-biased mortality in adults of the fruit fly (Drosophila melanogaster).

Mannitol, a sugar alcohol used in commercial food products, has been previously shown to induce sex-biased mortality in female Drosophila melanogaster when ingested at a single concentration (1 M). We hypothesized that sex differences in energy needs, related to reproductive costs, contributed to the increased mortality we observed in females compared to males. To test this, we compared the longevity of actively mating and non-mating flies fed increasing concentrations of mannitol. We also asked whether mannitol-induced mortality was concentration-dependent for both males and females, and if mannitol's sex-biased effects were consistent across concentrations. Females and males both showed concentration-dependent increases in mortality, but female mortality was consistently higher at concentrations of 0.75 M and above. Additionally, fly longevity decreased further for both sexes when housed in mixed sex vials as compared to single sex vials. This suggests that the increased energetic demands of mating and reproduction for both sexes increased the ingestion of mannitol. Finally, larvae raised on mannitol produced expected adult sex ratios, suggesting that sex-biased mortality due to the ingestion of mannitol occurs only in adults. We conclude that sex and reproductive status differences in mannitol ingestion drive sex-biased differences in adult fly mortality.