Developmental toxicity of pyriproxyfen induces changes in the ultrastructure of neural cells and in the process of skull ossification.

Some studies relate the use of pyriproxyfen (PPF) in drinking water with damage to embryonic neurodevelopment, including a supposed association with cases of microcephaly. However, the effects on neural cells and skull ossification in embryos remain unclear. This study aims to investigate the effects of PPF on the structure and ultrastructure of brain cells and its influence on the skull ossification process during embryonic development. Chicken embryos, used as an experimental model, were exposed to concentrations of 0.01 and 10 mg/l PPF at E1. The findings demonstrated that PPF led to notable ultrastructural alterations such as reduced cilia and microvilli of ependymal cells and damage to mitochondria, endoplasmic reticulum, Golgi bodies, and cell membranes in neural cells. The frequency of changes and the degree of these cell damage between the forebrain and midbrain were similar. PPF induced a reduction in fox3 transcript levels, specific for differentiation of neurons, and a reduction in the NeuN protein content related to mature neurons and dendritic branches. PPF impacted the ossification process of the skull, as evidenced by the increase in the ossified area and the decrease in inter-bone spacing. In conclusion, this study highlights the ability of PPF to affect neurodevelopmental processes by inducing ultrastructural damage to neural cells, concomitant with a reduction in NeuN and fox3 expression. This detrimental impact coupled with deficiencies in skull ossification can prevent the proper growth and development of the brain.

Pyriproxyfen exposure induces DNA damage, cell proliferation impairments and apoptosis in the brain vesicles layers of chicken embryos.

Larvicide pyriproxyfen (PPF), used in drinking water reservoirs to control Aedes mosquitoes, has already been shown as a possible cause of congenital anomalies in the central nervous system. However, the neurotoxic effects of PPF on the development of vertebrate embryos are still underexplored. Thus, the aim of this study was to investigate the effects of PPF on the morphometric parameters of the head and brain, as well as on the cell layers of the forebrain and midbrain, using embryos of Gallus domesticus as a model. Two sublethal PPF concentrations (0.01 mg/L and 10 mg/L), as defined by a survival curve, were tested. Analysis of the biometry of embryos showed significant reduction in body and brain mass and also in measurements of the head and brain. A reduction in cell layer thickness of the forebrain and midbrain was observed, accompanied by a reduction in the numerical density of cells per area. Changes in brain and head sizes and in the thickness of the cell layers of the forebrain and midbrain were significant at 10 mg/L PPF. Notably, PPF caused DNA doublestrand breaks and induced apoptosis in embryos exposed to 10 mg/L, which were accompanied by a reduction in cell proliferation. Regarding neuronal and glial differentiation, no changes were observed in the number of neurons and glial cells on the analyzed layers. Furthermore, PPF did not impact the head ossification process. These findings reveal that PPF is a strong stressor for neurodevelopment, causing damage to the cell architecture of brain vesicles.

Screening Aedes aegypti (Diptera: Culicidae) Populations From Pernambuco, Brazil for Resistance to Temephos, Diflubenzuron, and Cypermethrin and Characterization of Potential Resistance Mechanisms.

Resistance to chemical insecticides detected in Aedes aegypti (L.) mosquitoes has been a problem for the National Dengue Control Program (PNCD) over the last years. In order to provide deeper knowledge of resistance to xenobiotics, our study evaluated the susceptibility profile of temephos, diflubenzuron, and cypermethrin insecticides in natural mosquito populations from the Pernambuco State, associating these results with the local historical use of such compounds. Furthermore, mechanisms that may be associated with this particular type of resistance were characterized. Bioassays with multiple temephos and diflubenzuron concentrations were performed to detect and quantify resistance. For cypermethrin, diagnostic dose assays were performed. Biochemical tests were carried out to quantify the activity of detoxification enzymes. In addition, a screening of mutations present in the voltage-gated sodium channel gene (NaV) was performed in samples previously submitted to bioassays with cypermethrin. The populations under study were resistant to temephos and showed a positive correlation between insecticide consumption and the resistance ratio (RR) to the compound. For diflubenzuron, the biological activity ratio (BAR) ranged from 1.3 to 4.7 times, when compared to the susceptible strain. All populations showed resistance to cypermethrin. Altered enzymatic profiles of alpha, p-nitrophenyl acetate (PNPA) esterases and glutathione-S-transferases were recorded in most of these samples. Molecular analysis demonstrated that Arcoverde was the only population that presented the mutated form 1016Ile/Ile. These findings show that the situation is critical vis-à-vis the effectiveness of mosquito control using chemical insecticides, since resistance to temephos and cypermethrin is widespread in Ae. aegypti from Pernambuco.

Acetylcholinesterase cDNA sequencing and identification of mutations associated with organophosphate resistance in Cochliomyia hominivorax (Diptera: Calliphoridae).

Altered acetylcholinesterase (AChE) has been identified in numerous arthropod species resistant to organophosphate (OP) and carbamate insecticides. The New World screwworm (NWS) Cochliomyia hominivorax (Coquerel), one of the most important myiasis-causing flies in the Neotropics, has been controlled mainly by the application of OP insecticides in its current geographical distribution. However, few studies have investigated insecticide resistance in this species. Based on previous studies about mutations conferring OP resistance in related dipteran species, AChE cDNA was sequenced allowing a survey for mutations (I298V, G401A, F466Y) in NWS populations. In addition, the G137D mutation in the carboxylesterase E3 gene, also associated with OP resistance, was analyzed in the same NWS populations. Only 2/135 individuals presented an altered AChE gene (F466Y). In contrast, a high frequency of the G137D mutation in the E3 gene was found in some localities of Brazil and Uruguay, while the mutant allele was not found in Cuba, Venezuela or Colombia. These findings suggest that the alteration in the carboxylesterase E3 gene may be one of the main resistance mechanisms selected in this ectoparasite. The knowledge of the frequency of these resistance-associated mutations in the NWS natural populations may contribute to the selection of appropriate chemicals for control as part of pest management strategies.

Characterization of mitochondrial control region, two intergenic spacers and tRNAs of Zaprionus indianus (Diptera: Drosophilidae).

The control region in insects is the major noncoding region in animal mitochondrial DNA (mtDNA), and is responsible for a large part of the variation in the DNA sequence and size of the genome of this organelle. In this study, the mtDNA control region, two intergenic spacers and tRNA genes of a Zaprionus indianus strain were cloned, sequenced and compared with other Drosophila species. The overall A+T content in the Z. indianus control region is 94.3%, and a comparison with other Drosophila species demonstrated that the most conserved region appears to be the 420 base pairs nearest to the tRNA(ile), similar to the findings of other authors. We also describe conserved sequence blocks, including a poly-T involved in the replication process of Drosophila mtDNA; a putative secondary structure also involved in the replication process and repeated sequences. tRNA(ile) sequence demonstrated the greatest variability when the tRNA sequences of species were compared.