Acute Toxicity of Fungicide-Insecticide-Adjuvant Combinations Applied to Almonds During Bloom on Adult Honey Bees.

Beekeepers report significant honey bee deaths during and after almond bloom. These losses pose a major problem for the California almond industry because of its dependence on honey bees as pollinators. The present study aimed to determine if combinations of pesticides applied during almond bloom during daylight hours were a possible explanation for these losses. In this study we aimed to mimic the spray application route of exposure to pesticides using a Potter Spray Tower to treat adult honey bees with commonly encountered pesticides and pesticide combinations at multiples of the maximum recommended field application rates. Tested insecticides included Altacor® and Intrepid®, and tested fungicides included Tilt®, Pristine®, Luna Sensation®, and Vangard®. Synergistic toxicity was observed when the fungicide Tilt (active ingredient propiconazole) was applied with the insecticide Altacor (chlorantraniliprole), though neither caused significant mortality when applied independently. The study also looked at the effect of adding a spray adjuvant, Dyne-Amic®, to pesticide mixtures. Dyne-Amic was toxic to honey bees at concentrations above the maximum recommended field application rate, and toxicity was increased when combined with the fungicide Pristine (pyraclostrobin and boscalid). Addition of Dyne-Amic also increased toxicity of the Tilt and Altacor combination. These results suggest that application of Altacor and Tilt in combination with an adjuvant at the recommended field application rates could cause mortality in adult honey bees. These findings highlight a potential explanation for honey bee losses around almond bloom, emphasize that the safety of spray adjuvants to bees should not be assumed, and provide support for recommendations to protect bees from pesticides through application at night when bees are not foraging. Environ Toxicol Chem 2022;41:1042-1053. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Live calcium imaging of Aedes aegypti neuronal tissues reveals differential importance of chemosensory systems for life-history-specific foraging strategies.

BACKGROUND: The mosquito Aedes aegypti has a wide variety of sensory pathways that have supported its success as a species as well as a highly competent vector of numerous debilitating infectious pathogens. Investigations into mosquito sensory systems and their effects on behavior are valuable resources for the advancement of mosquito control strategies. Numerous studies have elucidated key aspects of mosquito sensory systems, however there remains critical gaps within the field. In particular, compared to that of the adult form, there has been a lack of studies directed towards the immature life stages. Additionally, although numerous studies have pinpointed specific sensory receptors as well as responding motor outputs, there has been a lack of studies able to monitor both concurrently. RESULTS: To begin filling aforementioned gaps, here we engineered Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s. Using this strain, combined with advanced microscopy, we simultaneously measured live stimulus-evoked calcium responses in both neuronal and muscle cells with a wide spatial range and resolution. CONCLUSIONS: By coupling in vivo live calcium imaging with behavioral assays we were able to gain functional insights into how stimulus-evoked neural and muscle activities are represented, modulated, and transformed in mosquito larvae enabling us to elucidate mosquito sensorimotor properties important for life-history-specific foraging strategies.

Parasitoid Jewel Wasp Mounts Multipronged Neurochemical Attack to Hijack a Host Brain.

The parasitoid emerald jewel wasp Ampulex compressa induces a compliant state of hypokinesia in its host, the American cockroach Periplaneta americana through direct envenomation of the central nervous system (CNS). To elucidate the biochemical strategy underlying venom-induced hypokinesia, we subjected the venom apparatus and milked venom to RNAseq and proteomics analyses to construct a comprehensive "venome," consisting of 264 proteins. Abundant in the venome are enzymes endogenous to the host brain, including M13 family metalloproteases, phospholipases, adenosine deaminase, hyaluronidase, and neuropeptide precursors. The amphipathic, alpha-helical ampulexins are among the most abundant venom components. Also prominent are members of the Toll/NF-κB signaling pathway, including proteases Persephone, Snake, Easter, and the Toll receptor ligand Spätzle. We find evidence that venom components are processed following envenomation. The acidic (pH∼4) venom contains unprocessed neuropeptide tachykinin and corazonin precursors and is conspicuously devoid of the corresponding processed, biologically active peptides. Neutralization of venom leads to appearance of mature tachykinin and corazonin, suggesting that the wasp employs precursors as a prolonged time-release strategy within the host brain post-envenomation. Injection of fully processed tachykinin into host cephalic ganglia elicits short-term hypokinesia. Ion channel modifiers and cytolytic toxins are absent in A. compressa venom, which appears to hijack control of the host brain by introducing a "storm" of its own neurochemicals. Our findings deepen understanding of the chemical warfare underlying host-parasitoid interactions and in particular neuromodulatory mechanisms that enable manipulation of host behavior to suit the nutritional needs of opportunistic parasitoid progeny.

Molecular cross-talk in a unique parasitoid manipulation strategy.

Envenomation of cockroach cerebral ganglia by the parasitoid Jewel wasp, Ampulex compressa, induces specific, long-lasting behavioural changes. We hypothesized that this prolonged action results from venom-induced changes in brain neurochemistry. Here, we address this issue by first identifying molecular targets of the venom, i.e., proteins to which venom components bind and interact with to mediate altered behaviour. Our results show that venom components bind to synaptic proteins and likely interfere with both pre- and postsynaptic processes. Since behavioural changes induced by the sting are long-lasting and reversible, we hypothesized further that long-term effects of the venom must be mediated by up or down regulation of cerebral ganglia proteins. We therefore characterize changes in cerebral ganglia protein abundance of stung cockroaches at different time points after the sting by quantitative mass spectrometry. Our findings indicate that numerous proteins are differentially expressed in cerebral ganglia of stung cockroaches, many of which are involved in signal transduction, such as the Rho GTPase pathway, which is implicated in synaptic plasticity. Altogether, our data suggest that the Jewel wasp commandeers cockroach behaviour through molecular cross-talk between venom components and molecular targets in the cockroach central nervous system, leading to broad-based alteration of synaptic efficacy and behavioural changes that promote successful development of wasp progeny.

Ampulexins: A New Family of Peptides in Venom of the Emerald Jewel Wasp, Ampulex compressa.

The parasitoid wasp Ampulex compressa injects venom directly into the brain and subesophageal ganglion of the cockroach Periplaneta americana, inducing a 7 to 10 day lethargy termed hypokinesia. Hypokinesia presents as a significant reduction in both escape response and spontaneous walking. We examined aminergic and peptidergic components of milked venom with HPLC and MALDI-TOF mass spectrometry. HPLC coupled with electrochemical detection confirmed the presence of dopamine in milked venom, while mass spectrometry revealed that the venom gland and venom sac have distinct peptide profiles, with milked venom predominantly composed of venom sac peptides. We isolated and characterized novel α-helical, amphipathic venom sac peptides that constitute a new family of venom toxins termed ampulexins. Injection of the most abundant venom peptide, ampulexin 1, into the subesophageal ganglion of cockroaches resulted in a short-term increase in escape threshold. Neither milked venom nor venom peptides interfered with growth of Escherichia coli or Bacillus thuringiensis on agar plates, and exposure to ampulexins or milked venom did not induce cell death in Chinese hamster ovary cells (CHO-K1) or Hi5 cells ( Trichoplusia ni).

Arizona bark scorpion venom resistance in the pallid bat, Antrozous pallidus.

The pallid bat (Antrozous pallidus), a gleaning bat found in the western United States and Mexico, hunts a wide variety of ground-dwelling prey, including scorpions. Anecdotal evidence suggests that the pallid bat is resistant to scorpion venom, but no systematic study has been performed. Here we show with behavioral measures and direct injection of venom that the pallid bat is resistant to venom of the Arizona bark scorpion, Centruroides sculpturatus. Our results show that the pallid bat is stung multiple times during a hunt without any noticeable effect on behavior. In addition, direct injection of venom at mouse LD50 concentrations (1.5 mg/kg) has no effect on bat behavior. At the highest concentration tested (10 mg/kg), three out of four bats showed no effects. One of the four bats showed a transient effect suggesting that additional studies are required to identify potential regional variation in venom tolerance. Scorpion venom is a cocktail of toxins, some of which activate voltage-gated sodium ion channels, causing intense pain. Dorsal root ganglia (DRG) contain nociceptive neurons and are principal targets of scorpion venom toxins. To understand if mutations in specific ion channels contribute to venom resistance, a pallid bat DRG transcriptome was generated. As sodium channels are a major target of scorpion venom, we identified amino acid substitutions present in the pallid bat that may lead to venom resistance. Some of these substitutions are similar to corresponding amino acids in sodium channel isoforms responsible for reduced venom binding activity. The substitution found previously in the grasshopper mouse providing venom resistance to the bark scorpion is not present in the pallid bat, indicating a potentially novel mechanism for venom resistance in the bat that remains to be identified. Taken together, these results indicate that the pallid bat is resistant to venom of the bark scorpion and altered sodium ion channel function may partly underlie such resistance.