Assessing Non‐Invasive Methods for Determining Larval Instars in Bumble Bees

The majority of insect species have a clearly defined larval stage during development. Larval nutrition is crucial for individuals’ growth and development, and larval foraging success often depends on both resource availability and competition for those resources. To date, however, little is known about how these factors interact to shape larval development and behaviour. Here we manipulated the density of larvae of the polyphagous fruit fly pest Bactrocera tryoni (‘Queensland fruit fly’), and the diet concentration of patches in a foraging arena to address this gap. Using advanced statistical methods of machine learning and linear regression models, we showed that high larval density results in overall high larval aggregation across all diets except in extreme diet dilutions. Larval aggregation was positively associated with larval body mass across all diet concentrations except in extreme diet dilutions where this relationship was reversed. Over time, larvae in low-density arenas also tended to aggregate while those in high-density arenas tended to disperse, an effect that was observed for all diet concentrations. Furthermore, larvae in high-density arenas displayed significant avoidance of low concentration diets – a behaviour that was not observed amongst larvae in low-density arenas. Thus, aggregation can help, rather than hinder, larval growth in high-density environments, and larvae may be better able to explore available nutrition when at high-density than when at low-density.

Joint effects of brood size and resource availability on sibling competition

To determine the effect of xylem hardness and larval body size on the pupal-chamber tunnels (hereafter PCTs) made by Monochamus alternatus, newly hatched larvae were inoculated on Abies sachalinensis, Picea jezoensis, and Pinus densiflora logs and reared in the laboratory. After most larvae had developed well, larval body size and PCT size were measured. Larval body mass was significantly heavier in P. jezoensis and P. densiflora than in A. sachalinensis. ANCOVA with larval body mass as a covariate indicated that the adjusted mean of the median depth of the PCTs was greater in A. sachalinensis and P. jezoensis than in P. densiflora and that the adjusted mean of the PCT length was greater in P. jezoensis than in the two other tree species. P. densiflora xylem is known to be harder than the two other conifers. Consequently, it can be concluded that larvae of equal body mass make deeper and longer PCTs in soft xylem than in hard xylem.

Deoxynivalenol (DON) is a prevalent mycotoxin in feed, raising concerns about its impact on animal health and feed safety. Insects such as yellow mealworm (Tenebrio molitor) may play a role in the biodegradation of DON-contaminated feed. This study presents the results of a two-week rearing experiment, where 19-week-old yellow mealworm larvae were fed diets with varying concentrations of DON. The larvae were divided into three groups (C, A, and B) that differed in the amount of mycotoxin added to the feed. Larval survival, body mass, and DON accumulation in larvae and their frass were evaluated. A statistical analysis revealed no significant differences in larval survival or body mass gain between the groups. The results point to the low accumulation of DON in larvae, reaching 13.13 ± 2.06 µg/kg (A) and 32.18 ± 4.20 µg/kg (B) after two weeks of feeding. Moreover, at the end of the experiment, DON was detected in larval frass at high concentrations of 507.65 ± 15.31 µg/kg (A) and 862.61 ± 18.53 µg/kg (B), suggesting that larvae are capable of effectively excreting this mycotoxin. The analyzed mycotoxin had no significant effect on larval survival or growth. Deoxynivalenol did not accumulate in the larval biomass and was excreted with frass. These findings enhance our understanding of the interactions between DON and yellow mealworm larvae and have potential implications for using insects in feed production and mycotoxin neutralization within ecosystems. Tenebrio molitor larvae tolerate DON-contaminated feed and effectively excrete the toxin, making them potential candidates for feed detoxification systems.

Larval amphibians are particularly likely to encounter variation in rearing temperature and resource availability due to variation in aquatic breeding habitats. In this study, plasticity in growth rates, larval mass, larval period, and size at metamorphosis were examined in Rana kukunoris Nikolskii, 1918 under different combinations of temperature and food level. Larval period and larval body mass was sensitive to food level, and varied with temperature. Tadpoles metamorphosed at an older age at low temperature than those reared at warm temperature. Food level was a significant affect on larval period at low temperature, but not at warm temperature. Mass was heavier for tadpoles reared at low temperatures than those reared at warm temperatures. The effect of food level depended on temperature, because larvae reared at low temperature that were offered a high food level achieved a larger size than larvae offered a low food level, but this did not occur at warm temperature. Therefore, we suggest that high food availability at low temperature prolonged developmental periods, thus larvae are larger as metamorphs than those reared at warm temperatures.

SummarySystemic signals provided by nutrients and hormones are known to coordinate the growth and proliferation of different organs during development. However, within the brain, it is unclear how these signals influence neural progenitor divisions and neuronal diversity. Here, in the Drosophila visual system, we identify two developmental phases with different sensitivities to dietary nutrients. During early larval stages, nutrients regulate the size of the neural progenitor pool via insulin/PI3K/TOR-dependent symmetric neuroepithelial divisions. During late larval stages, neural proliferation becomes insensitive to dietary nutrients, and the steroid hormone ecdysone acts on Delta/Notch signaling to promote the switch from symmetric mitoses to asymmetric neurogenic divisions. This mechanism accounts for why sustained undernourishment during visual system development restricts neuronal numbers while protecting neuronal diversity. These studies reveal an adaptive mechanism that helps to retain a functional visual system over a range of different brain sizes in the face of suboptimal nutrition.

Nitrogen (N) losses via ammonia (NH3) emissions from manure is one of the main environmental burdens resulting from livestock production. Feeding manure to black soldier fly larvae (BSFL) is envisioned as a new circular strategy to recover manure-N, reduce its environmental impact, and upgrade it into insect proteins to be used in animal feed. However, so far, it remained unknown if BSFL could incorporate N from NH3 in manure into the larval body mass. Here, using the stable isotope 15N in NH3, we demonstrate that at least 13% of pig manure NH3-N can be incorporated into BSFL body mass. Within the larval body, the tracer was found in both insoluble and soluble nitrogen fractions, including proteins. We discuss interventions that could increase the incorporation of NH3-N into larval proteins and with that reduce NH3 emissions from manure. Our results provide the first reliable quantification of NH3-N assimilation in manure-fed larvae and contributes to quantifying the potential of BSFL for manure management, and as a circular protein source.

ABSTRACTAimThis study sets out to understand the variability in larval traits of dispersive life stages of a famous invader, the European shore crab Carcinus maenas, in its native distribution range.LocationNorth East Atlantic coast from the Norwegian Arctic to the southern European distribution limit of C. maenas in Southern Spain.TaxonEuropean shore crab Carcinus maenas (Crustacea, Decapoda).MethodsWe quantified latitudinal patterns in larval body mass, elemental composition (C and N content), and thermal tolerance of the first larval stage. We collected crabs from four populations spanning 25° of latitude (Vigo in Northern Spain; Bergen, Trondheim, and Bodø in Norway) and reanalysed published and unpublished data of body mass and elemental composition of additional populations from Germany, Wales, France, and Southern Spain. Furthermore, we used two laboratory experiments to test the thermal tolerance limits of the first larval stage from Vigo and the Norwegian populations. In the first experiment, we reared larvae from hatching to Zoea II at seven temperatures (9°C–27°C) and from hatching to LT50 at 6°C. In the second experiment, we exposed freshly hatched larvae acutely to increasing or decreasing temperatures (up to 40°C and down to 3°C).ResultsAcross the entire European range, we found a substantial increase in dry mass and carbon and nitrogen content of freshly hatched larvae with latitude. Norwegian populations exhibited higher survival at 9°C than the Vigo population. Furthermore, LT50 at 6°C increased from South to North. All populations showed high survival in the range 12°C–24°C but low survival at 27°C.Main ConclusionsLarval tolerance quantified by using survival to Zoea II is not clearly related to the tolerance quantified with the acute experiments, indicating that each method assesses different aspects of thermal tolerance. Tolerance to low temperature correlated positively to tolerance to high temperature, suggesting that variation among females in larval responses reflects a general physiological quality rather than trade‐offs. We provide evidence for potentially adaptive variations in larval body mass and thermal tolerance across a latitudinal gradient for C. maenas.

Parents can increase the fitness of their offspring by allocating nutrients to eggs and/or providing care for eggs and offspring. Although we have a good understanding of the adaptive significance of both egg size and parental care, remarkably little is known about the co-evolution of these two mechanisms for increasing offspring fitness. Here, we report a parental removal experiment on the burying beetle Nicrophorus vespilloides in which we test whether post-hatching parental care masks the effect of egg size on offspring fitness. As predicted, we found that the parent's presence or absence had a strong main effect on larval body mass, whereas there was no detectable effect of egg size. Furthermore, egg size had a strong and positive effect on offspring body mass in the parent's absence, whereas it had no effect on offspring body mass in the parent's presence. These results support the suggestion that the stronger effect of post-hatching parental care on offspring growth masks the weaker effect of egg size. We found no correlation between the number and size of eggs. However, there was a negative correlation between larval body mass and brood size in the parent's presence, but not in its absence. These findings suggest that the trade-off between number and size of offspring is shifted from the egg stage towards the end of the parental care period and that post-hatching parental care somehow moderates this trade-off.

Simple SummaryTomatoes are a popular crop, and bumble bees and honey bees are its main pollinators. Floral scent usually plays an important role in mediating the foraging behavior of bees, and tomato flowers release special scents. Although it has been found that foraging experience in the tomato greenhouses helped bumble bees develop a strong preference for the scent, honey bees with foraging experience continued to show aversion to tomato floral scent. However, it is currently unknown as to how a single tomato volatile compound regulates the foraging behavior of bees. In the current study, we investigated the foraging behaviors of the widely used pollinator honey bee Apis mellifera and bumble bee Bombus terrestris on tomato flower volatile compounds in order to evaluate whether honey bees and bumble bees show different EAG responses to volatile compounds and how they might influence bee choice behavior. We found that honey bees had a weaker EAG response to the tested compounds compared with bumble bees and that they showed avoidance behavior to these compounds. We conclude that some compounds in tomato floral scents caused the low bias of honey bees to tomato flowers, which may be one driver as to why honey bees dislike tomato, which could be adjusted in order to improve the pollination service efficiency of bees for commercial crops.Bumble bees and honey bees are of vital importance for tomato pollination, although honey bees are less attracted to tomato flowers than bumble bees. Little is known about how tomato flower volatile compounds influence the foraging behaviors of honey bees and bumble bees. In this study, compounds of tomato flower volatiles were detected by gas chromatography–mass spectrometry. Electroantennography (EAG) and a dynamic two-choice olfactometer were used, respectively, to compare the differences of antennal and behavioral responses between Apis mellifera and Bombus terrestris towards selected volatile compounds. A total of 46 compounds were detected from the tomato flower volatiles. Of the 16 compounds tested, A. mellifera showed strong antennal responses to 3 compounds (1-nonanal, (+)-dihydrocarvone, and toluene) when compared with a mineral oil control, and B. terrestris showed 7 pronounced EAG responses (1,3-xylene, (+)-dihydrocarvone, toluene, piperitone, eucarvone, 1-nonanal, and β-ocimene). Additionally, 1-nonanal and (+)-dihydrocarvone elicited significant avoidance behavior of A. mellifera, but not of B. terrestris. In conclusion, bumble bees are more sensitive to the compounds of tomato flower volatiles compared to honey bees, and honey bees showed aversion to some compounds of tomato flower volatiles. The findings indicated that compounds of flower volatiles significantly influenced bee foraging preference for tomato.

Summary Stag beetles are saproxylic insects that live in deadwood colonised by fungi and other microorganisms. Due to the cryptic habits of these species, little is known on their biological aspects, especially for the immature stage. In this study, we investigated morphometric traits of lesser stag beetle, Dorcus parallelipipedus (Linnaeus, 1758) (Coleoptera: Lucanidae) larvae and adults, with comparisons between populations. Instar identification by measuring larval head capsule was performed, and the relationship between larval body mass and head capsule width was investigated for each surveyed population. To assess the differences among populations, the scaling relationships for adults between their body and mandibles length was also considered, especially for adult males. Morphometric relationships were described by exponential equations. Differences between populations were sometimes limited to one or the other feature, and particularly ascribed to the larval body mass. These differences could be imputed to several factors, among with those of environmental or genetic order. The scaling relationship of lesser stag beetle adult males showed a positive allometry but did not differ among populations, indicating that morphological traits within a species and sex may be biologically retained. Findings are discussed from the perspective of D. parallelipipedus life history.

Host symbionts are often considered an essential part of the host phenotype, influencing host growth and development. Bumble bee is an ideal model for investigating the relationship between microbiota and phenotypes. Variations in life history across bumble bees may influence the community composition of gut microbiota, which in turn influences phenotypes. In this study, we explored gut microbiota from four development stages (early-instar larvae, 1st instar; mid-instar larvae, 5th instar; late-instar larvae, 9th instar; and adults) of workers and queens in the bumble bee Bombus terrestris using the full-length 16S rRNA sequencing technology. The results showed that morphological indices (weight and head capsule) were significantly different between workers and queens from 5th instar larvae (p < 0.01). The alpha and beta diversities of gut microbiota were similar between workers and queens in two groups: early instar and mid instar larvae. However, the alpha diversity was significantly different in late instar larvae or adults. The relative abundance of three main phyla of bacteria (Cyanobacteria, Proteobacteria, and Firmicutes) and two genera (Snodgrassella and Lactobacillus) were significantly different (p < 0.01) between workers and queens in late instar larvae or adults. Also, we found that age significantly affected the microbial alpha diversity as the Shannon and ASVs indices differed significantly among the four development stages. Our study suggests that the 5th instar larval stage can be used to judge the morphology of workers or queens in bumble bees. The key microbes differing in phenotypes may be involved in regulating phenotypic variations.

Organisms with complex life cycles, like amphibians, are greatly affected by environmental factors and maternal effects, which in turn influence their morphological variation. The Asiatic toad Bufo gargarizans is found throughout East Asia and is not only valued for its economic medicinal properties in China but also considered a potential indicator species for assessing environmental pollution in inland rivers. However, the specific responses of this species to environmental toxicants, including trace metals, are not well comprehended at present. In this study, we experimentally examined the plasticity in larval period, body mass and size, and survival at metamorphosis in tadpoles of B. gargarizans under different combinations of food quality and zinc (Zn). We found that larval period, survival, SVL and mass at metamorphosis were sensitive to food quality, larval period increased significantly with increasing Zn concentration. The interaction between food quality and zinc primarily influenced the larval period. Tadpoles that were fed a high-energy diet, rich in protein and lipids, tended to have a shorter larval period as zinc concentration increased. Conversely, tadpoles fed a low-energy diet had a longer larval period with increasing zinc concentration. Thus, zinc plays a role in determining the extent to which food quality affects development of B. gargarizans. Additionally, larvae that consume a low-energy diet tend to have higher survival rates as zinc concentration increases, suggesting that moderate levels of zinc concentration (below 150 μg/L) are beneficial for normal physiological and biochemical processes in these organisms. The findings from this study provide valuable insights into the toxicological responses of this species to trace metals, contributing to a better understanding of their impact. Keywords: Bufo gargarizans, acute toxic effect, zinc, interaction effects, age and size at metamorphosis, phenotypic plasticity

The effective use of larvae of the greenbottle fly, Lucilia sericata, in wound debridement requires a working knowledge of how feeding changes over time. Using a laboratory assay and bagged larval dressings, the effect of incubation time on larval feeding rates and body mass was investigated for up to 120 hours at 32°C. The mass of tissue digested increased significantly in incremental 24-hour periods up to 72 hours, with no significant consumption occurring afterwards. Larval mass increased only up to 48 hours. A further test comparing the efficacy of a single 96-hour application of larvae against two consecutive 48-hour applications found that the mass of tissue digested in the latter was 14.3% higher than the former, a difference that was statistically significant. Current clinical guidance suggests a 4-day application period for bagged larvae. Based on these results, an incubation time of 72 hours (3 days) for bagged larvae would be the most effective at the study temperature. However, it is acknowledged that wound temperature can vary, whereby feeding rates would likely differ. In view of this, we conclude that a period of 3 to 4 days is optimum for the application of larvae, and current guidelines should be adhered to.

Effects of Fastac 50 EC on bumble bee Bombus terrestris L. respiration: DGE disappearance does not lead to increasing water loss