Under laboratory conditions, the effects of thiamethoxam were investigated in larvae, pupae and emerging honey bees after exposure at larval stages with different concentrations in the food (0.00001 ng/µL, 0.001 ng/µL and 1.44 ng/µL). Thiamethoxam reduced the survival of larvae and pupae and consequently decreased the percentage of emerging honey bees. Thiamethoxam induced important physiological disturbances. It increased acetylcholinesterase (AChE) activity at all developmental stages and increased glutathione-S-transferase (GST) and carboxylesterase para (CaEp) activities at the pupal stages. For midgut alkaline phosphatase (ALP), no activity was detected in pupae stages, and no effect was observed in larvae and emerging bees. We assume that the effects of thiamethoxam on the survival, emergence and physiology of honey bees may affect the development of the colony. These results showed that attention should be paid to the exposure to pesticides during the developmental stages ofthe honey bee. This study represents the first investigation of the effects of thiamethoxam on the development of A. mellifera following larval exposure.

Due to technical limitations, there have been minimal studies performed on thermal preferences and thermotactic behaviors of aquatic ectotherm species commonly used in ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built and miniaturized electrothermal array for rapid thermal preference behavioral tests. We applied the novel platform to define thermal preferences in multiple invertebrate and vertebrate species. Specifically, Dugesia notogaea (freshwater planarians), Chironomus tepperi (nonbiting midge larvae), Ostracoda (seed shrimp), Artemia franciscana (brine shrimp), Daphnia carinata (water flea), Austrochiltonia subtenuis (freshwater amphipod), Physa acuta (freshwater snail), Potamopyrgus antipodarum (New Zealand mud snail) and larval stage of Danio rerio (zebrafish) were tested. The Australian freshwater water fleas, amphipods, snail Physa acuta as well as zebrafish exhibited the most consistent preference to cool zones and clear avoidance of zones >27 °C out of nine species tested. Our results indicate the larval stage of zebrafish as the most responsive species highly suitable for prospective development of multidimensional behavioral test batteries. We also showcase preliminary data that environmentally relevant concentrations of pharmaceutical pollutants such as non-steroidal anti-inflammatory drug (NSAID) ibuprofen (9800 ng/L) and insecticide imidacloprid (4600 ng/L) but not anti-depressant venlafaxine (2200 ng/L) and (iv) anticonvulsant medications gabapentin (400 ng/L) can perturb thermal preference behavior of larval zebrafish. Collectively our results demonstrate the utility of simple and inexpensive thermoelectric technology in rapid exploration of thermal preference in diverse species of aquatic animals. We postulate that more broadly such technologies can also have added value in ecotoxicity testing of emerging contaminants.

Across the planet, winter de-icing practices have caused secondary salinization of freshwater habitats. Many amphibians are vulnerable because of permeable skin and reliance on small ponds, where salinity can be high. Early developmental stages of amphibians are especially sensitive to salt, and larvae developing in salt-polluted environments must osmoregulate through ion exchange in gills. Though ionoregulation in amphibian gills is generally understood, the role of gill morphology remains poorly described. Yet gill structure should affect ionoregulatory capacity, for instance in terms of available surface area. As larval amphibian gills also play critical roles in gas exchange and foraging, changes in gill morphology from salt pollution potentially affect not only osmoregulation, but also respiration and feeding. Here, we used an exposure experiment to quantify salinity effects on larval gill morphology in wood frogs (Rana sylvatica). We measured a suite of morphological traits on gill tufts—where ionoregulation and gas exchange occur—and on gill filters used in feeding. Larvae raised in elevated salinity developed larger gill tufts but with lower surface area to volume ratio. Epithelial cells on these tufts were less circular but occurred at higher densities. Gill filters showed increased spacing, likely reducing feeding efficiency. Many morphological gill traits responded quadratically, suggesting that salinity might induce plasticity in gills at intermediate concentrations until energetic demands exceed plasticity. Together, these changes likely diminish ionoregulatory and respiratory functionality of gill tufts, and compromise feeding functionality of gill filters. Thus, a singular change in aquatic environment from a widespread pollutant appears to generate a suite of consequences via changes in gill morphology. Critically, these changes in traits likely compound the severity of fitness impacts in populations dwelling in salinized environments, whereby ionoregulatory energetic demands should increase respiratory and foraging demands, but in individuals who possess structures poorly adapted for these functions.

Glyphosate (GLY) is a broad-spectrum herbicide that is the most commonly applied pesticide in terrestrial ecosystems in the U.S. and, potentially, worldwide. However, the combined effects of warming associated with climate change and exposure to GLY and GLY-based formulations (GBFs) on terrestrial animals are poorly understood. Animals progress through several life stages (e.g., embryonic, larval, and juvenile stages) that may exhibit different sensitivities to stressors. Therefore, we factorially manipulated temperature and GLY/GBF exposure in the variable field cricket (Gryllus lineaticeps) during two life stages—nymphal development and adulthood—and examined key animal traits, such as developmental rate, body size, food consumption, reproductive investment, and lifespan. A thermal environment simulating future climate warming obligated several costs to fitness-related traits. For example, warming experienced during nymphal development reduced survival, adult body mass and size, and investment into flight capacity and reproduction. Warming experienced by adults reduced lifespan and growth rate. Alternatively, the effects of GBF exposure were more subtle, often context-dependent (e.g., effects were only detected in one sex or temperature regime), and were stronger during adult exposure relative to exposure during development. There was evidence of additive costs of warming and GBF exposure to rates of feeding and growth in adults. Yet, the negative effect of GBF exposure to adult lifespan did not occur in warming conditions, suggesting that ongoing climate change may obscure some of the costs of GBFs to non-target organisms. The effects of GLY alone (i.e., in the absence of proprietary surfactants found in commercial formulations) were non-existent. Animals will be increasingly exposed to warming and GBFs, and our results indicate that GBF exposure and warming can entail additive costs for an animal taxon (insects) that plays critical roles in terrestrial ecosystems.

The black soldier fly (BSF) Hermetia illucens has a strong tolerance to cadmium stress. This helps to use BSF in entomoremediation of heavy metal pollution. Rich metallothionein (MT) proteins were thought to be important for some insects to endure the toxicity of heavy metal. We identified and characterized three MTs genes in BSF (BSFMTs), including BSFMT1, BSFMT2A, and BSFMT2B. Molecular modeling was used to predict metal binding sites. Phylogenetic analysis was used to identify gene families. Overexpression of the recombinant black soldier fly metallothioneins was found to confer Cd tolerance in Escherichia coli. Finally, functions of BSFMTs in BSF were explored through RNA interference (RNAi). RNAi results of BSFMT2B showed that the larval fresh weight decreased significantly, and the larvae mortality increased significantly. This study suggests that BSFMTs have important properties in Cd detoxification and tolerance in BSF. Further characterization analyses of physiological function about metallothioneins are necessary in BSF and other insects.

Amphibian larvae are commonly used as indicators of aquatic ecosystem health because they are susceptible to contaminants. However, there is limited information on how species characteristics and trophic position influence contaminant loads in larval amphibians. Importantly, there remains a need to understand whether grazers (frogs and toads [anurans]) and predators (salamanders) provide comparable information on contaminant accumulation or if they are each indicative of unique environmental processes and risks. To better understand the role of trophic position in contaminant accumulation, we analyzed composite tissues for 10 metals from larvae of multiple co-occurring anuran and salamander species from 20 wetlands across the United States. We examined how metal concentrations varied with body size (anurans and salamanders) and developmental stage (anurans) and how the digestive tract (gut) influenced observed metal concentrations. Across all wetlands, metal concentrations were greater in anurans than salamanders for all metals tested except mercury (Hg), selenium (Se), and zinc (Zn). Concentrations of individual metals in anurans decreased with increasing weight and developmental stage. In salamanders, metal concentrations were less correlated with weight, indicating diet played a role in contaminant accumulation. Based on batches of similarly sized whole-body larvae compared to larvae with their digestive tracts removed, our results indicated that tissue type strongly affected perceived concentrations, especially for anurans (gut represented an estimated 46–97% of all metals except Se and Zn). This suggests the reliability of results based on whole-body sampling could be biased by metal, larval size, and development. Overall, our data shows that metal concentrations differs between anurans and salamanders, which suggests that metal accumulation is unique to feeding behavior and potentially trophic position. To truly characterize exposure risk in wetlands, species of different life histories, sizes and developmental stages should be included in biomonitoring efforts.

The industrial production of insects for waste management or as a protein source is becoming vital to our society. Large volumes of manure are produced by concentrated animal facilities around the globe that must be managed, utilized, and disposed of properly. Flies offer a partial solution with their abilities to reduce these wastes and heavy metal pollutants. Meat and crop proteins are being supplemented by insect proteins for many feeds across the globe, yet science-based studies behind the mass-rearing of insects are still in their infancy. In the current study, the percent change in the composition of nutrients, heavy metals, and fiber, in dairy, poultry, and swine manure degraded by either black soldier fly (BSF) or house fly (HF) larvae was explored. Pre-digested and post-digested manure samples were collected from four independent studies that differed in production scale (number of larvae and feeding regimen): 1) BSF small-scale (100 larvae fed incrementally), 2) HF small-scale (100 larvae fed incrementally), 3) BSF large-scale (10,000 larvae fed a single time), and 4) HF large-scale (4,000 larvae fed a single time). Results indicate that nitrogen is a key nutrient impacted by larval digestion of manure by both species, regardless of scale. However, scale significantly impacted reductions of other nutrients, as did the type of manure in which the insects were reared. Ultimately, this study demonstrated that manure type and rearing scale impact the ability of BSF and HF larvae to reduce nutrients and heavy metals in manure, and thus insect management procedures need to be congruent with production emphases of the insects for waste management or protein products. Failure to take scale into consideration could lead to inaccurate assumptions related to industrialized efforts on this topic.

Mites are considered the worst enemy of honey bees, resulting in economic losses in agricultural production. In apiculture, flumethrin is frequently used to control mites. It causes residues of flumethrin in colonies which may threaten honey bees, especially for larvae. Still, the impact of flumethrin-induced dysbiosis on honey bees larval health has not been fully elucidated, and any impact of microbiota for decomposing flumethrin in honey bees is also poorly understood. In this study, 2-day-old larvae were fed with different flumethrin-sucrose solutions (0, 0.5, 5, 50 mg/kg) and the dose increased daily (1.5, 2, 2.5 and 3 μL) until capped, thereafter the expression level of two immune genes (hymenoptaecin, defensin1) and two detoxication-related genes (GST, catalase) were measured. Meanwhile, the effect of flumethrin on honey bee larvae (Apis mellifera) gut microbes was also explored via 16S rRNA Illumina deep sequencing. We found that flumethrin at 5 mg/kg triggered the over expression of immune-related genes in larvae, while the larval detoxification-related genes were up-regulated when the concentrations reached 50 mg/kg. Moreover, the abundance and diversity of microbes in flumethrin-treated groups (over 0.5 mg/kg) were significantly lower than control group, but it increased with flumethrin concentrations among the flumethrin-treated groups. Our results revealed that microbes served as a barrier in the honey bee gut and were able to protect honey bee larvae to a certain extent, and reduce the stress of flumethrin on honey bee larvae. In addition, as the concentration of flumethrin increases, honey bee larvae activate their immune system then detoxification system to defend against the potential threat of flumethrin. This is the first report on the impact of flumethrin on gut microbiota in honey bees larvae. The findings revealed new fundamental insights regarding immune and detoxification of host-associated microbiota.

Pesticide exposure is regarded as a contributing factor to the high gross loss rates of managed colonies of Apis mellifera. Pesticides enter the hive through contaminated nectar and pollen carried by returning forager honey bees or placed in the hive by beekeepers when managing hive pests. We used an in vitro rearing method to characterize the effects of seven pesticides on developing brood subjected dietary exposure at worse-case environmental concentrations detected in wax and pollen. The pesticides tested included acaricides (amitraz, coumaphos, fluvalinate), insecticides (chlorpyrifos, imidacloprid), one fungicide (chlorothalonil), and one herbicide (glyphosate). The larvae were exposed chronically for six days of mimicking exposure during the entire larval feeding period, which is the worst possible scenario of larval exposure. Survival, duration of immature development, the weight of newly emerged adult, morphologies of the antenna and the hypopharyngeal gland, and gene expression were recorded. Survival of bees exposed to amitraz, coumaphos, fluvalinate, chlorpyrifos, and chlorothalonil was the most sensitive endpoint despite observed changes in many developmental and physiological parameters across the seven pesticides. Our findings suggest that pesticide exposure during larvae development may affect the survival and health of immature honey bees, thus contributing to overall colony stress or loss. Additionally, pesticide exposure altered gene expression of detoxification enzymes. However, the tested exposure scenario is unlikely to be representative of real-world conditions but emphasizes the importance of proper hive management to minimize pesticide contamination of the hive environment or simulates a future scenario of increased contamination.