Comparing the effects of three neonicotinoids on embryogenesis of the South African clawed frog Xenopus laevis.

Neonicotinoids (NEOs) are widely used insecticides that are ubiquitous in agricultural use. Since NEOs are found in natural waters as well as in tap water and human urine in regions where NEOs are widely used, NEOs pose a potential hazard to non-target organisms such as animals and humans. Some of the commonly detected NEOs are imidacloprid (IMD), thiamethoxam (TMX), and its metabolite clothianidin (CLO). Although previously published scientific information, including an assessment of the environmental risks, particularly for bees, had resulted in a ban on the outdoor use of these three NEOs in the EU - their use is now only permitted in closed greenhouses - these NEOs continue to be used in agriculture in many other parts of the world. Therefore, a detailed study and comparison of the effects of NEOs on the embryonic development of non-target organisms is needed to further define the risk profiles. Embryos of the South African clawed frog Xenopus laevis, a well-established aquatic model, were exposed to different concentrations of IMD, TMX, or CLO (0.1-100 mg/L) to study and compare the possible effects of a single contaminant in natural water bodies on early embryogenesis. The results included a reduced body length, a smaller orbital space, impaired cranial cartilage and nerves, and an altered heart structure and function. At the molecular level, NEO exposure partially resulted in an altered expression of tissue-specific factors, which are involved in eye, cranial placode, and heart development. Our results suggest that the NEOs studied negatively affect the embryonic development of the non-target organism X. laevis. Since pesticides, especially NEOs, pollute the environment worldwide, it is suggested that they are strictly controlled and monitored in the areas where they are used. In addition, the question arises as to whether pesticide metabolites also pose a risk to the environment and need to be investigated further so that they can be taken into account when registering ingredients.

The impact of the insecticide acetamiprid on the embryogenesis of the aquatic model organism Xenopus laevis.

Acetamiprid (ACT) is used extensively in agriculture worldwide, although data on ACT concentrations in natural water bodies and its impact on aquatic organisms are limited. To study whether ACT influences the embryogenesis of the South African clawed frog Xenopus laevis, embryos were incubated in ACT solutions from 0.01 to 100 mg/L. The low concentrations were chosen on the basis of concentrations already found in nature. ACT treatment leads to shorter embryo lengths, intestine malformation and reduced eye areas. It also affects the cranial cartilage and cardiac development as well as the embryo's mobility. The expression of tissue-specific marker genes is affected as well. Thus, our study suggests that pesticides may lead to an increased mortality of non-target organisms and emphasizes the importance of regular testing for ACT concentrations in nature. Our study provides an overview of ACT effects and can therefore be used as a basis for an ACT risk assessment.

Glyphosate without Co-formulants affects embryonic development of the south african clawed frog Xenopus laevis.

BACKGROUND: Glyphosate (GLY) is the most widely used herbicide in the world. Due to its mode of action as an inhibitor of the 5-enolpyruvylshikimate-3-phosphate synthase, an important step in the shikimate pathway, specifically in plants, GLY is considered to be of low toxicity to non-target organisms. However, various studies have shown the negative effects of GLY on the mortality and development of different non-target organisms, including insects, rodents, fish and amphibians. To better understand the various effects of GLY in more detail, we studied the effects of GLY without co-formulants during the embryogenesis of the aquatic model organism Xenopus laevis. RESULTS: A treatment with GLY affected various morphological endpoints in X. laevis tadpoles (body length, head width and area, eye area). Additionally, GLY interfered with the mobility as well as the neural and cardiac development of the embryos at stage 44/45. We were able to detect detailed structural changes in the cranial nerves and the heart and gained insights into the negative effects of GLY on cardiomyocyte differentiation. CONCLUSION: The application of GLY without co-formulants resulted in negative effects on several endpoints in the early embryonic development of X. laevis at concentrations that are environmentally relevant and concentrations that reflect the worst-case scenarios. This indicates that GLY could have a strong negative impact on the survival and lives of amphibians in natural waters. As a result, future GLY approvals should consider its impact on the environment.

The neonicotinoid thiacloprid leads to multiple defects during early embryogenesis of the South African clawed frog (Xenopuslaevis).

There is increasing concern about the health effects of pesticides that pollute natural waters. In particular, the use of neonicotinoids, such as thiacloprid (THD), is causing unease. THD is considered non-toxic to non-target vertebrates. Studies classify THD as carcinogenic, toxic to reproduction, and therefore harmful to the environment. A detailed study of possible THD effects during the amphibian embryogenesis is needed because leaching can introduce THD into aquatic environments. We incubated stage 2 embryos of the South African clawed frog in various THD concentrations (0.1-100 mg/L) at 14 °C to study the potential effects of a one-time THD contamination of waters on the early embryogenesis. We showed that THD has, indeed, negative effects on the embryonic development of the X. laevis. A treatment with THD led to a reduced embryonic body length and mobility. Furthermore, a treatment with THD resulted in smaller cranial cartilages, eyes and brains, and the embryos had shorter cranial nerves and an impaired cardiogenesis. On a molecular basis, THD led to a reduced expression of the brain marker emx1 and the heart marker mhcα. Our results underly the importance of a strict and efficient monitoring of the regulatory levels and application areas of THD.

Impact of glyphosate-based herbicide on early embryonic development of the amphibian Xenopus laevis.

BACKGROUND: Worldwide, amphibian populations are declining drastically. One reason might be the use of pesticides including herbicides. The herbicide glyphosate is an inhibitor of the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase of the plant shikimate pathway, preventing the formation of aromatic amino acids and thus inducing plant death. Due to this specific action, GBH are considered nontoxic to non-target organisms. However, GBH impairs embryonic development of chickens, amphibians and fishes. So far, no detailed tissue- and organ-specific analysis of the effects of GBH during development in amphibians has been performed. RESULTS: We demonstrated that GBH Roundup® LB plus has a negative effect on embryonic development of the South African clawed frog Xenopus laevis. GBH treatment with sublethal concentrations resulted in a reduced body length and mobility of embryos. Furthermore, incubation with GBH led to smaller eyes, brains and cranial cartilages in comparison to untreated embryos. GBH incubation also resulted in shorter cranial nerves and had an effect on cardiac development including reduced heart rate and atrium size. On a molecular basis, GBH treatment led to reduced expression of marker genes in different tissues and developmental stages. CONCLUSION: GBH leads to disturbed embryonic development of Xenopus laevis.

Retinol binding protein 1 affects Xenopus anterior neural development via all-trans retinoic acid signaling.

BACKGROUND: Retinol binding protein 1 (Rbp1) acts as an intracellular regulator of vitamin A metabolism and retinoid transport. In mice, Rbp1 deficiency decreases the capacity of hepatic stellate cells to take up all-trans retinol and sustain retinyl ester stores. Furthermore, Rbp1 is crucial for visual capacity. Although the function of Rbp1 has been studied in the mature eye, its role during early anterior neural development has not yet been investigated in detail. RESULTS: We showed that rbp1 is expressed in the eye, anterior neural crest cells (NCCs) and prosencephalon of the South African clawed frog Xenopus laevis. Rbp1 knockdown led to defects in eye formation, including microphthalmia and disorganized retinal lamination, and to disturbed induction and differentiation of the eye field, as shown by decreased rax and pax6 expression. Furthermore, it resulted in reduced rax expression in the prosencephalon and affected cranial cartilage. Rbp1 inhibition also interfered with neural crest induction and migration, as shown by twist and slug. Moreover, it led to a significant reduction of the all-trans retinoic acid target gene pitx2 in NCC-derived periocular mesenchyme. The Rbp1 knockdown phenotypes were rescued by pitx2 RNA co-injection. CONCLUSION: Rbp1 is crucial for the development of the anterior neural tissue.

Nosip functions during vertebrate eye and cranial cartilage development.

BACKGROUND: The nitric oxide synthase interacting protein (Nosip) has been associated with diverse human diseases including psychological disorders. In line, early neurogenesis of mouse and Xenopus is impaired upon Nosip deficiency. Nosip knockout mice show craniofacial defects and the down-regulation of Nosip in the mouse and Xenopus leads to microcephaly. Until now, the exact underlying molecular mechanisms of these malformations were still unknown. RESULTS: Here, we show that nosip is expressed in the developing ocular system as well as the anterior neural crest cells of Xenopus laevis. Furthermore, Nosip inhibition causes severe defects in eye formation in the mouse and Xenopus. Retinal lamination as well as dorso-ventral patterning of the retina were affected in Nosip-depleted Xenopus embryos. Marker gene analysis using rax, pax6 and otx2 reveals an interference with the eye field induction and differentiation. A closer look on Nosip-deficient Xenopus embryos furthermore reveals disrupted cranial cartilage structures and an inhibition of anterior neural crest cell induction and migration shown by twist, snai2, and egr2. Moreover, foxc1 as downstream factor of retinoic acid signalling is affected upon Nosip deficiency. CONCLUSIONS: Nosip is a crucial factor for the development of anterior neural tissue such the eyes and neural crest cells. Developmental Dynamics 247:1070-1082, 2018. © 2018 Wiley Periodicals, Inc.