Malathion is a broad spectrum organophosphate insecticide and many diverse uses in agricultural, home, and garden applications¹. However, concerns remain regarding its widespread use given that it belongs to a family of organophosphorus compounds and can irreversibly block acetycholinesterase that is critical to nerve function both in insects and humans¹. In addition, information on chronic-toxicity from malathion on neural system and its effect on behavior during developmental stages is surprisingly sparse for a compound that has been so extensively used. This study aimed to test the hypothesis that prenatal exposure to malathion leads to a disruption of axonal integrity in the guinea pig offspring using in vivo diffusional kurtosis imaging (DKI).Pregnant guinea pigs were subcutaneously injected with ten consecutive injections during the last ten days of gestation with malathion (25 mg/kg) or Peanut oil (0.5 ml/kg) which served as a vehicle control. DKI was performed on both male (n=15 malathion, n=14 vehicle) and female (n=16 malathion, n=14 vehicle) offspring at postnatal-day 70. In vivo MRI experiments were performed on a Bruker Biospec 7.0 T scanner. Fast spin-echo T₂-weighted MR images were obtained with a TR/TE_eff of 6197/60 ms, 8 echo-trains, a 35 x 35 mm² field of view, and 20 slices with 1 mm thickness in the coronal view. Diffusion kurtosis images that matched the prescription of the T₂-weighted images were acquired using a single-shot spin-echo EPI sequence with a TR/TE_eff of 8500/45 ms, 96 x 96 matrix, 30 gradient-directions, and two b-values (1000/2000 s/mm²). DKI results were processed according to a previously published method². Maps of the various diffusion parameters including fractional anisotropy (FA), mean diffusivity (MD), radial (RD) and axial (AD) diffusivity, axial (AK) and radial (RK) kurtosis and mean kurtosis (MK) were generated. ROIs (Fig 1) queried from diffusion images included corpus callosum (CC), cerebral cortex (Cor), striatum (Str), hippocampus (Hip), amygdala (Amy),and thalamus (Tha). Statistical comparisons were made between male and female groups and malathion and vehicle treatments using two-way ANOVA with multiple comparisons.Kurtosis metrics and fractional anisotropy measurements indicated a sex difference among control animals on RK in the CC (p=0.040) and FA in the Cor (p=0.034) (Fig 2).There were main malathion treatment effects in FA (F=4.299, P=0.043), MK (F=5.425, P=0.024), and RK (F=6.094, P=0.017) in the CC. Post hoc test revealed significant reduction of MK (p=0.023), RK (p=0.007), and FA (p=0.016) in the CC in male offspring only in the malathion treated animals (Fig 3). No malathion effect was detected in female offspring using any diffusion measures in any of the six selected regions.The study demonstrated for the first time that the corpus callosum microstructure in male offspring of mothers that were exposed prenatally to malathion is altered significantly. Early prenatal exposure appears to have a profound lasting effect among the offspring. The CC is important for sustained and divided attention. Changes in CC have been implicated in various disorders such as attention deficit hyperactivity disorder (ADHD) and autism spectrum disorders (ASD)^3,4. Furthermore, studies have shown that children with high levels of organophosphate insecticide exposure are likely to have neurodevelopment and neuropsychological disorders leading to ADHD and ASD^5-10.Our current results demonstrating structural damage associated with prenatal exposure further corroborates the notion that the organophosphorus agent malathion can have long lasting effects in the central nervous system. In our recent study⁸ on prenatal exposure of guinea pigs to chlorpyrifos, another organophosphorus insecticide, significant reductions in FA and kurtosis measures (MK, AK, and RK) in the CC in female offspring were found. Although male guinea pigs were not used in that study, the results from these studies suggest that the lasting effects from these pesticides may follow different mechanisms in a gender dependent way. In conclusion, the findings reveal the lasting effect of prenatal exposure to malathion and the danger of mother to child transmission of malathion in the environment which is more pronounced in the males than in females at the micro-structural level in the corpus callosum. Future studies will probe into the underlying mechanism of these gender differences.