Larval Lethality Research Articles

Overexpression of Lifeact in the C. elegans body wall muscle causes sarcomere disorganization and embryonic or larval lethality

Lifeact is a short peptide that is widely utilized as a probe for actin filaments in live imaging. However, high concentrations of Lifeact can alter actin filament dynamics and cause artificial modifications to the actin cytoskeleton. Here, I evaluated Caenorhabditis elegans strains expressing Lifeact fused to fluorescent proteins in the body wall muscle. I found that, while low-level expression of Lifeact from a single-copy transgene was appropriate for labeling sarcomeric actin filaments, overexpression of Lifeact from an extrachromosomal array causes severe disorganization of muscle sarcomeres and lethality at an embryonic or larval stage. Therefore, for imaging studies in C. elegans, Lifeact needs to be kept at a low level by proper management of the expression system.

Exploring the impact of Agaricus bisporus on mitigating lead reproductive toxicity using the Caenorhabditis elegans model

Given that Agaricus bisporus, an edible mushroom, has demonstrated antioxidant properties, our investigation aimed to assess whether Agaricus bisporus could mitigate the toxic effects of lead (Pb) on Caenorhabditis elegans (C. elegans) model. A dose-response study was conducted involving Pb and Agaricus bisporus to determine appropriate doses. Subsequently, a co-exposure study utilizing C. elegans strains N2 and CL2166 was implemented, with groups designated as Control, Pb, Agaricus bisporus, and Pb + Agaricus bisporus. Our findings revealed that co-exposure to Pb + 100 mg/mL Agaricus bisporus resulted in reduced embryonic and larval lethality, increased brood size, and enhanced motility compared to nematodes exposed solely to Pb. Notably, our observations indicated a transfer of reproductive toxicity from nematode parents to their offspring. Thus, Agaricus bisporus may play a significant role in Pb detoxification, suggesting its potential as a natural antioxidant for neutralizing the detrimental effects of Pb on reproductive health.

Twinstar is a chitin synthase interacting protein with an essential role in insect cuticle biosynthesis

Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton. The membrane-integral β-glycosyltransferase, chitin synthase, has been identified as the central component in chitin biosynthesis. However, the precise roles of other proteins in facilitating chitin synthase in chitin biosynthesis remain unclear. In this study, we employed split-ubiquitin membrane yeast two-hybrid (MYTH) and pull-down assays to demonstrate the physical interaction between Twinstar (Tsr), a small molecular protein in the actin-depolymerizing factor ADF/Cofilin protein family, and chitin synthase Krotzkopf verkehrt (Kkv) in Drosophila melanogaster in vitro. The RNA interference (RNAi)-mediated global knockdown of Tsr in D. melanogaster resulted in larval lethality. Furthermore, targeted suppression of Tsr in the tracheal and epidermal tissues also led to larval mortality, while knocking down Tsr in the wing tissues led to wrinkled wings. Additionally, silencing Tsr not only reduced the chitin content in the first longitudinal vein of the wings but also led to the absence of the chitin lamellar structure. To validate the functional conservation of Tsr in other insect orders, the two agricultural pests Ostrinia furnacalis and Tribolium castaneum, representing lepidoptera and coleoptera insects, respectively, were investigated. Knockdown experiments targeting the Drosophila Tsr orthologues OfTsr in O. furnacalis and TcTsr in T. castaneum produced abnormal larvae during molting or pupation in O. furnacalis and lethality in T. castaneum. Our findings not only improve our knowledge of the chitin biosynthesis machinery in insect cuticles but also provide new potential targets for the control of major agricultural pests.

Biocontrol efficacy of native protease-producing bacterial isolates against the olive leaf moth, Palpita unionalis (Hübner) (Lepidoptera: Pyralidae)

BackgroundThe olive leaf moth (OLM), Palpita unionalis (Hübner) (Lepidoptera: Pyralidae), poses a significant threat to olive cultivation. This study focused on isolating protease-producing bacteria from moribund field strains of P. unionalis larvae and assessing their larvicidal efficacy against laboratory strains through estimated LC30, LC50, and LC90 values. Additionally, the long-lasting impacts of LC50 values on pupal and adult stages were evaluated.ResultsThree entomopathogenic bacteria, Bacillus amyloliquefaciens, Pseudomonas putida, and Bacillus subtilis, were isolated. The larvicidal efficacies of these isolates varied, as B. subtilis exhibiting greater efficacy at LC30 and LC50 values, while P. putida showed the most potent effect at the LC90 value. Conversely, B. amyloliquefaciens demonstrated the lowest larvicidal potentialities at LC50 and LC90 values, with P. putida being the least effective at the LC30 value. The toxicity regression lines indicated increased larval lethality with each unit increase in concentration, as evidenced by the positive slope values. Chi-square (χ2) values suggested a better fit and supported the reliability of the estimated lethal concentrations. Exposure to LC50 values s resulted in significant elongation of larval duration and reduction in weight, whereas pupal lifespan, adult emergence, and sex ratio showed non-significant differences compared to untreated controls.ConclusionsThe study highlights the promising role of these biological candidates in pest control programs as eco-friendly alternatives, potentially enhancing safe plant production. The varied larvicidal efficacies of the bacterial isolates, along with their proteolytic capabilities, underscore their potential as biocontrol pathogens against OLM, contributing to sustainable agricultural practices.

Hyphae of Rhizopus arrhizus and Lichtheimia corymbifera Are More Virulent and Resistant to Antifungal Agents Than Sporangiospores In Vitro and in Galleria mellonella.

Mucorales species cause debilitating, life-threatening sinopulmonary diseases in immunocompromised patients and penetrating wounds in trauma victims. Common antifungal agents against mucormycosis have significant toxicity and are often ineffective. To evaluate treatments against mucormycosis, sporangiospores are typically used for in vitro assays and in pre-clinical animal models of pulmonary infections. However, in clinical cases of wound mucormycosis caused by traumatic inoculation, hyphal elements found in soil are likely the form of the inoculated organism. In this study, Galleria mellonella larvae were infected with either sporangiospores or hyphae of Rhizopus arrhizus and Lichtheimia corymbifera. Hyphal infections resulted in greater and more rapid larval lethality than sporangiospores, with an approximate 10-16-fold decrease in LD50 of hyphae for R. arrhizus (p = 0.03) and L. corymbifera (p = 0.001). Liposomal amphotericin B, 10 mg/kg, was ineffective against hyphal infection, while the same dosage was effective against infections produced by sporangiospores. Furthermore, in vitro, antifungal susceptibility studies show that minimum inhibitory concentrations of several antifungal agents against hyphae were higher when compared to those of sporangiospores. These findings support using hyphal elements of Mucorales species for virulence testing and antifungal drug screening in vitro and in G. mellonella for studies of wound mucormycosis.

FDP-Na-induced enhancement of glycolysis impacts larval growth and development and chitin biosynthesis in fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae)

Fructose 1, 6-diphosphate (FDP) is an endogenous intermediate in the glycolytic pathway, as well as an allosteric activator of phosphofructokinase (PFK). Based on the role in promoting glycolysis, FDP has been widely used as a therapeutic agent for mitigating the damage of endotoxemia and ischemia/reperfusion in clinical practice. However, the effect of exogenous FDP-induced glycolysis activation on insect carbohydrate metabolism and chitin synthesis remains largely unclear. Here, we investigated for the first time the effects of FDP-Na, an allosteric activator of PFK, on the growth and development of Hyphantria cunea larvae, a serious defoliator in agriculture and forestry, especially on glycolysis and chitin synthesis. The results showed that FDP-Na significantly restrained the growth and development of H. cunea larvae and resulted in larval lethality. After treatment with FDP-Na, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) were significantly activated, and HcHK2, HcPFK, HcPK were dramatically upregulated, which suggested that FDP-Na enhanced glycolysis in H. cunea larvae. Meanwhile, FDP-Na also distinctly impacted chitin biosynthesis by disturbing transcriptions of genes in the chitin synthesis pathway, resulting in changes of chitin contents in the midgut and epidermis of H. cunea larvae. Therefore, we considered that FDP-Na caused the growth and development arrest, and impacted chitin biosynthesis, probably by disturbing in vivo glycolysis and carbohydrate metabolism in H. cunea larvae. The findings provide a new perspective on the mechanism by which glycolysis regulates insect growth and development, and lay the foundation for exploring the potential application of glycolysis activators in pest control as well.

Atm Mutation and Oxidative Stress Enhance the Pre-Cancerous Effects of UHRF1 Overexpression in Zebrafish Livers.

The ataxia-telangiectasia mutated (atm) gene is activated in response to genotoxic stress and leads to activation of the tp53 tumor suppressor gene which induces either senescence or apoptosis as tumor suppressive mechanisms. Atm also serves non-canonical functions in the response to oxidative stress and chromatin reorganization. We previously reported that overexpression of the epigenetic regulator and oncogene Ubiquitin Like with PHD and Ring Finger Domains 1 (UHRF1) in zebrafish hepatocytes resulted in tp53-dependent hepatocyte senescence, a small liver and larval lethality. We investigated the role of atm on UHRF1-mediated phenotypes by generating zebrafish atm mutants. atm-/- adults were viable but had reduction in fertility. Embryos developed normally but were protected from lethality caused by etoposide or H2O2 exposure and failed to fully upregulate Tp53 targets or oxidative stress response genes in response to these treatments. In contrast to the finding that Tp53 prevents the small liver phenotype caused by UHRF1 overexpression, atm mutation and exposure to H2O2 further reduced the liver size in UHRF1 overexpressing larvae whereas treatment with the antioxidant N-acetyl cysteine suppressed this phenotype. We conclude that UHRF1 overexpression in hepatocytes causes oxidative stress, and that loss of atm further enhances this, triggering elimination of these precancerous cells, leading to a small liver.

Genetic hyperactivation of Nrf2 causes larval lethality in Keap1a and Keap1b-double-knockout zebrafish

The Keap1–Nrf2 pathway is an evolutionarily conserved mechanism that protects cells from oxidative stress and electrophiles. Keap1 is a repressor of Nrf2 in normal cellular conditions but also a stress sensor for Nrf2 activation. Interestingly, fish and amphibians have two Keap1s (Keap1a and Keap1b), of which Keap1b is the ortholog of mammalian Keap1. Keap1a, on the other hand, is a gene found only in fish and amphibians, having been lost during the evolution to amniotes. We have previously shown that keap1b-knockout zebrafish have increased Nrf2 activity and reduced response to certain Nrf2-activating compounds but that they grow normally to adulthood. This may be because the remaining keap1a suppresses the hyperactivation of Nrf2, which is responsible for the post-natal lethality of Keap1-knockout mice. In this study, we analyzed keap1a;keap1b-double-knockout zebrafish to test this hypothesis. We found that keap1a;keap1b-double-knockout zebrafish, like Keap1-knockout mice, showed eating defects and were lethal within a week of hatching. Genetic introduction of the Nrf2 mutation rescued both the eating defects and the larval lethality, indicating that Nrf2 hyperactivation is the cause. However, unlike Keap1-knockout mice, keap1a;keap1b-double-knockout zebrafish showed no physical blockage of the food pathway; moreover, the cause of death was not directly related to eating defects. RNA-sequencing analysis revealed that keap1a;keap1b-double-knockout larvae showed extraordinarily high expression of known Nrf2-target genes as well as decreased expression of visual cycle genes. Finally, trigonelline or brusatol partially rescued the lethality of keap1a;keap1b-double-knockout larvae, suggesting that they can serve as an in vivo evaluation system for Nrf2-inhibiting compounds.

DOT-1.1 (DOT1L) deficiency in C. elegans leads to small RNA-dependent gene activation

Methylation of histone H3 at lysine 79 (H3K79) is conserved from yeast to humans and is accomplished by Dot1 (disruptor of telomeric silencing-1) methyltransferases. The C. elegans enzyme DOT-1.1 and its interacting partners are similar to the mammalian DOT1L (Dot1-like) complex. The C. elegans DOT-1.1 complex has been functionally connected to RNA interference. Specifically, we have previously shown that embryonic and larval lethality of dot-1.1 mutant worms deficient in H3K79 methylation was suppressed by mutations in the RNAi pathway genes responsible for generation (rde-4) and function (rde-1) of primary small interfering RNAs (siRNAs). This suggests that dot-1.1 mutant lethality is dependent on the enhanced production of some siRNAs. We have also found that this lethality is suppressed by a loss-of-function of CED-3, a conserved apoptotic protease. Here, we describe a comparison of gene expression and primary siRNA production changes between control and dot-1.1 deletion mutant embryos. We found that elevated antisense siRNA production occurred more often at upregulated than downregulated genes. Importantly, gene expression changes were dependent on RDE-4 in both instances. Moreover, the upregulated group, which is potentially activated by ectopic siRNAs, was enriched in protease-coding genes. Our findings are consistent with a model where in the absence of H3K79 methylation there is a small RNA-dependent activation of protease genes, which leads to embryonic and larval lethality. DOT1 enzymes’ conservation suggests that the interplay between H3K79 methylation and small RNA pathways may exist in higher organisms.

The morphogen Hedgehog is essential for proper adult morphogenesis in Bombyx mori

The well-known morphogen Hedgehog (Hh) is indispensable for embryo patterning and organ development from invertebrates to vertebrates. The role of Hh signaling pathway has been extensively investigated in the model organism Drosophila melanogaster, whereas its biological functions are still poorly understood in non-drosophilid insects. In the current study, we describe comprehensive investigation of Hh biological roles in the model lepidopteran insect Bombyx mori by using both CRISPR/Cas9-mediated gene ablation and Gal4/UAS-mediated ectopic expression. Direct injection of Cas9 protein and Hh-specific sgRNAs into preblastoderm embryos induced complete lethality. In contrast, Hh mutants obtained by the binary transgenic CRISPR/Cas9 system showed no deleterious phenotypes during embryonic and larval stages. However, mutants showed abnormalities from the pupal stage and most of adult body appendages exhibited severe developmental defects. Molecular analysis focused on wing development reveal that Hh signaling, Imd signaling and Wnt signaling pathways were distorted in Hh mutant wings. Ectopic expression by using the binary Gal4/UAS system induce early larval lethality. On contrary, moderate overexpression of Hh by using a unitary transgenic system resulted in severe defects in adult leg and antenna development. Our data directly provide genetic evidence that Hh plays vital roles in imaginal discs development and proper adult morphogenesis in B. mori.

Roles for the RNA-Binding Protein Caper in Reproductive Output in Drosophila melanogaster.

RNA binding proteins (RBPs) play a fundamental role in the post-transcriptional regulation of gene expression within the germline and nervous system. This is underscored by the prevalence of mutations within RBP-encoding genes being implicated in infertility and neurological disease. We previously described roles for the highly conserved RBP Caper in neurite morphogenesis in the Drosophila larval peripheral system and in locomotor behavior. However, caper function has not been investigated outside the nervous system, although it is widely expressed in many different tissue types during embryogenesis. Here, we describe novel roles for Caper in fertility and mating behavior. We find that Caper is expressed in ovarian follicles throughout oogenesis but is dispensable for proper patterning of the egg chamber. Additionally, reduced caper function, through either a genetic lesion or RNA interference-mediated knockdown of caper in the female germline, results in females laying significantly fewer eggs than their control counterparts. Moreover, this phenotype is exacerbated with age. caper dysfunction also results in partial embryonic and larval lethality. Given that caper is highly conserved across metazoa, these findings may also be relevant to vertebrates.

Allele-specific alternative splicing of Drosophila Ribosomal protein S21 suppresses a lethal mutation in the Phosphorylated adaptor for RNA export (Phax) gene.

Genetic disruptions to the biogenesis of spliceosomal small-nuclear ribonucleoproteins in Drosophila cause wide-spread alternative splicing changes, including changes to the splicing of pre-mRNA for Ribosomal protein S21 (RpS21). Using a transposon mutant for the Phosphorylated adaptor for RNA export (Phax) gene, we demonstrate that changes in the splicing of RpS21 transcripts have a strong influence on the developmental progression of PhaxSH/SH mutants. Different alleles of the Drosophila RpS21 gene are circulating in common laboratory strains and cell lines. These alleles exhibit differences in RpS21 intron retention and splicing efficiency. Differences in the splicing of RpS21 transcripts account for prior conflicting observations of the phenotypic severity of PhaxSH/SH mutant stocks. The alleles uncover a strong splicing enhancer in RpS21 transcripts that can fully suppress the larval lethality and partially suppress the pupal lethality exhibited by PhaxSH/SH mutant lines. In the absence of the splicing enhancer, the splicing of RpS21 transcripts can be modulated in trans by the SR-rich B52 splicing factor. As PhaxSH/SH mutants exhibit wide-spread splicing changes in transcripts for other genes, findings here establish the importance of a single alternative splicing event, RpS21 splicing or intron retention, to the developmental progression of Drosophila.

Uncovering the role of endocrine factors in decapod physiology

Crustaceans are a valuable food source and commercially important. In order to ensure population stability, environmental diversity and food security, understanding the underlying endocrinology of these arthropods is key. Dr Kenji Toyota, Marine Biological Station, Sado Island Center for Ecological Sustainability, Niigata University, is working on research to shed light on the mechanisms that control key stages in crustacean life cycles and better understand how they have adapted to the environment of Sado Island. The research involves field work, breeding-based experiments with high-performance liquid chromatography (HPLC) and next generation sequencing (NGS) to analyse endocrine factors and related genes, with a focus on the eyestalk-derived hormones found in decapod crustaceans. One of Toyota's key projects is focused on the environmental sex determination (ESD) and toxicogenomics of the water flea Daphnia. He and his collaborators have reported on how insect growth regulators (IGRs) have disrupted non-target arthropod species, which includes larval lethality, defects arising in the metamorphosis process and disruption in sexual differentiation. Toyota's work is highlighting the negative environmental impacts of waste products and chemicals and also uncovering knowledge that can assist in the management of populations in both wild and farmed settings. Toyota has revealed the endocrine mechanisms that control metamorphosis during larval development and uncovered the ecotoxicological impacts of IGRs. The researchers are also investigating the roles of hormones innate JH (methyl farnesoate) and ecdysteroids (20-hydroxyecdysone, 20E) in the progression of Kuruma prawns throughout the larval stages.

Silencing of multiple target genes via ingestion of dsRNA and PMRi affects development and survival in Helicoverpa armigera

RNA interference (RNAi) triggered by exogenous double-stranded RNA (dsRNA) is a powerful tool to knockdown genetic targets crucial for the growth and development of agriculturally important insect pests. Helicoverpa armigera is a pest feeding on more than 30 economically important crops worldwide and a major threat. Resistance to insecticides and Bt toxins has been gradually increasing in the field. RNAi-mediated knockdown of H. armigera genes by producing dsRNAs homologous to genetic targets in bacteria and plants has a high potential for insect management to decrease agricultural loss. The acetylcholinesterase (AChE), ecdysone receptor (EcR) and v-ATPase-A (vAA) genes were selected as genetic targets. Fragments comprising a coding sequence of < 500 bp were cloned into the L4440 vector for dsRNA production in bacteria and in a TRV-VIGS vector in antisense orientation for transient expression of dsRNA in Solanum tuberosum leaves. After ingesting bacterial-expressed dsRNA, the mRNA levels of the target genes were significantly reduced, leading to mortality and abnormal development in larva of H. armigera. Furthermore, the S. tuberosum plants transformed with TRV-VIGS expressing AChE exhibited higher mortality > 68% than the control plants 17%, recorded ten days post-feeding and significant resistance in transgenic (transient) plants was observed. Moreover, larval lethality and molting defects were observed in larva fed on potato plants expressing dsRNA specific to EcR. Analysis of transcript levels by quantitative RT–PCR revealed that larval mortality was attributable to the knockdown of genetic targets by RNAi. The results demonstrated that down-regulation of H. armigera genes involved in ATP hydrolysis, transcriptional stimulation of development genes and neural conduction has aptitude as a bioinsecticide to control H. armigera population sizes and therefore decreases crop loss.

Ppan is essential for preimplantation development in mice†.

PETER PAN (PPAN), located to nucleoli and mitochondria, is a member of the Brix domain protein family, involved in rRNA processing through its rRNA binding motif and mitochondrial apoptosis by protecting mitochondria structure and suppressing basal autophagic flux. Ppan is important for cell proliferation and viability, and mutation of Ppan in Drosophila caused larval lethality and oogenesis failure. Yet, its role in mammalian reproduction remains unclear. In this study, we explored the function of Ppan in oocyte maturation and early embryogenesis using conditional knockout mouse model. Deficiency of maternal Ppan significantly downregulated the expression level of 5.8S rRNA, 18S rRNA, and 28S rRNA, though it had no effect on oocyte maturation or preimplantation embryo development. However, depletion of both maternal and zygotic Ppan blocked embryonic development at morula stage. Similar phenotype was obtained when only zygotic Ppan was depleted. We further identified no DNA binding activity of PPAN in mouse embryonic stem cells, and depletion of Ppan had minimum impact on transcriptome but decreased expression of 5.8S rRNA, 18S rRNA, and 28S rRNA nevertheless. Our findings demonstrate that Ppan is indispensable for early embryogenesis in mice.

SERCA interacts with chitin synthase and participates in cuticular chitin biogenesis in Drosophila

The biogenesis of chitin, a major structural polysaccharide found in the cuticle and peritrophic matrix, is crucial for insect growth and development. Chitin synthase, a membrane-integral β-glycosyltransferase, has been identified as the core of the chitin biogenesis machinery. However, a yet unknown number of auxiliary proteins appear to assist in chitin biosynthesis, whose precise function remains elusive. Here, we identified a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), in the fruit fly Drosophila melanogaster, as a chitin biogenesis-associated protein. The physical interaction between DmSERCA and epidermal chitin synthase (Krotzkopf verkehrt, Kkv) was demonstrated and analyzed using split-ubiquitin membrane yeast two-hybrid, bimolecular fluorescent complementation, pull-down, and immunoprecipitation assays. The interaction involves N-terminal regions (aa 48–81 and aa 247–33) and C-terminal regions (aa 743–783 and aa 824–859) of DmSERCA and two N-terminal regions (aa 121–179 and aa 369–539) of Kkv, all of which are predicted be transmembrane helices. While tissue-specific knock-down of DmSERCA in the epidermis caused larval and pupal lethality, the knock-down of DmSERCA in wings resulted in smaller and crinkled wings, a significant decrease in chitin deposition, and the loss of chitin lamellar structure. Although DmSERCA is well-known for its role in muscular contraction, this study reveals a novel role in chitin synthesis, contributing to our knowledge on the machinery of chitin biogenesis.

Expression of the Human Serotonin 5-HT7 Receptor Rescues Phenotype Profile and Restores Dysregulated Biomarkers in a Drosophila melanogaster Glioma Model.

Gliomas are the most common primary brain tumors in adults. Significant progress has been made in recent years in identifying the molecular alterations involved in gliomas. Among them, an amplification/overexpression of the EGFR (Epidermal Growth Factor Receptor) proto-oncogene and its associated signaling pathways have been widely described. However, current treatments remain ineffective for glioblastomas, the most severe forms. Thus, the identification of other pharmacological targets could open new therapeutic avenues. We used a glioma model in Drosophila melanogaster that results from the overexpression of constitutively active forms of EGFR and PI3K specifically in glial cells. We observed hyperproliferation of glial cells that leads to an increase in brain size and lethality at the third instar larval stage. After expression of the human serotonin 5-HT7 receptor in this glioma model, we observed a decrease in larval lethality associated with the presence of surviving adults and a return to a normal morphology of brain for some Drosophila. Those phenotypic changes are accompanied by the normalization of certain metabolic biomarkers measured by High-Resolution Magic Angle Spinning NMR (HR-MAS NMR). The 5-HT7R expression in glioma also restores some epigenetic modifications and characteristic markers of the signaling pathways associated with tumor growth. This study demonstrates the role of the serotonin 5-HT7 receptor as a tumor suppressor gene which is in agreement with transcriptomic analysis obtained on human glioblastomas.

RNA interference targeting Ras GTPase gene Ran causes larval and adult lethality in Leptinotarsa decemlineata.

RNA interference (RNAi) is a breakthrough technology in pest control. It is highly efficient to Coleopteran pests such as the Colorado potato beetle Leptinotarsa decemlineata, a serious pest defoliator mainly attacking potatoes worldwide. The first step for effective pest control by RNAi is the development of effective and reliable target genes. Our results revealed that continuous ingestion of dsLdRan for 3 days successfully silenced the target gene, inhibited larval growth and killed 100% L. decemlineata larvae. When the bioassay began at the second-, third/fourth-instar larval stages, the larval lethality mainly occurred at the fourth larval instar and prepupal stages, respectively. Importantly, consumption of dsLdRan for 3 days by the newly-emerged males and females effectively knocked down the target transcript, reduced fresh weights and caused 100% of lethality within a week. The LdRan females possessed underdeveloped ovaries. Considering that the larvae, adults and eggs are simultaneously sited on the potato plants, bacterially-expressed dsLdRan is a potential RNAi-based strategy for managing L. decemlineata in the potato field. © 2022 Society of Chemical Industry.

Juvenile hormone synthesis and signaling disruption triggering male offspring induction and population decline in cladocerans (water flea): Review and adverse outcome pathway development.

Juvenile hormone (JH) are a family of multifunctional hormones regulating larval development, molting, metamorphosis, reproduction, and phenotypic plasticity in arthropods. Based on its importance in arthropod life histories, many insect growth regulators (IGRs) mimicking JH have been designed to control harmful insects in agriculture and aquaculture. These JH analogs (JHAs) may also pose hazards to nontarget species by causing unexpected endocrine-disrupting (ED) effects such as molting and metamorphosis defects, larval lethality, and disruption of the sexual identity. This critical review summarizes the current knowledge of the JH-mediated effects in the freshwater cladoceran crustaceans such as Daphnia species on JHA-triggered endocrine disruptive outputs to establish a systematic understanding of JHA effects. Based on the current knowledge, adverse outcome pathways (AOPs) addressing the JHA-mediated ED effects in cladoceran leading to male offspring production and subsequent population decline were developed. The weight of evidence (WoE) of AOPs was assessed according to established guidelines. The review and AOP development aim to present the current scientific understanding of the JH pathway and provide a robust reference for the development of tiered testing strategies and new risk assessment approaches for JHAs in future ecotoxicological research and regulatory processes.

Developmental Toxicology of Metal Mixtures in Drosophila: Unique Properties of Potency and Interactions of Mercury Isoforms.

Mercury ranks third on the U.S. Agency of Toxic Substances and Disease Registry priority list of hazardous substances, behind only arsenic and lead. We have undertaken uncovering the mechanisms underlying the developmental toxicity of methylmercury (MeHg), inorganic mercury (HgCl2), lead acetate (Pb), and sodium arsenite (As). To probe these differences, we used the Drosophila model, taking advantage of three developmental transitions—pupariation, metamorphosis, and eclosion—to differentiate potentially unique windows of toxicity. We elaborated dose response profiles for each individual metal administered in food and accounted for internal body burden, also extending analyses to evaluate combinatorial metal mixture effects. We observed all four metals producing larval lethality and delayed pupariation, with MeHg being most potent. Compared to other metals, MeHg’s potency is caused by a higher body burden with respect to dose. MeHg uniquely caused dose-dependent failure in eclosion that was unexpectedly rescued by titrating in HgCl2. Our results highlight a unique developmental window and toxicokinetic properties where MeHg acts with specificity relative to HgCl2, Pb, and As. These findings will serve to refine future studies aimed at revealing tissue morphogenesis events and cell signaling pathways, potentially conserved in higher organisms, that selectively mediate MeHg toxicity and its antagonism by HgCl2.