Neonicotinoids, some of the most widely used pesticides in the world, act as agonists to the nicotinic acetylcholine receptors (nAChRs) of insects, resulting in death from abnormal excitability. Neonicotinoids unexpectedly became a major topic as a compelling cause of honeybee colony collapse disorder, which is damaging crop production that requires pollination worldwide. Mammal nAChRs appear to have a certain affinity for neonicotinoids with lower levels than those of insects; there is thus rising concern about unpredictable adverse effects of neonicotinoids on vertebrates. We hypothesized that the effects of neonicotinoids would be enhanced under a chronic stressed condition, which is known to alter the expression of targets of neonicotinoids, i.e., neuronal nAChRs. We performed immunohistochemical and behavioral analyses in male mice actively administered a neonicotinoid, clothianidin (CTD; 0, 10, 50 and 250 mg/kg/day), for 4 weeks under an unpredictable chronic stress procedure. Vacuolated seminiferous epithelia and a decrease in the immunoreactivity of the antioxidant enzyme glutathione peroxidase 4 were observed in the testes of the CTD+stress mice. In an open field test, although the locomotor activities were not affected, the anxiety-like behaviors of the mice were elevated by both CTD and stress. The present study demonstrates that the behavioral and reproductive effects of CTD become more serious in combination with environmental stress, which may reflect our actual situation of multiple exposure.

ABSTRACT. Clothianidin (CTD) is a neonicotinoid developed in the 1990s as an insecticide having selective toxicity, but it was later found to cause reproductive abnormalities in rats through oxidative stress. There is an attempt to preserve endangered animals, including the Japanese crested ibis, in Japan. However, there is a concern that neonicotinoid affects the reproduction of this bird, since it is used in its habitat. CTD toxicity in the birds is poorly understood, so we investigated whether or not the daily oral administration of CTD has any deleterious effects on the reproductive functions of mature male quails as experimental animals. The animals were randomly divided into four groups of 6 or 7 quails each, treated orally with 0, 0.02, 1 or 50 mg CTD/kg body weight (Control, CTD0.02, CTD1 and CTD50). After that the males bred with untreated females to estimate the egg weights, and rates of fertilization and normal development, the testes, liver and spleen were examined histologically. Vacuolization and the number of germ cells having fragmented DNA in seminiferous tubules, and the number and size of vacuoles in hepatocytes increased dose-dependently. There were no significant differences in egg weights and fertilization rates between the groups, but some eggs of the CTD1 and CTD50 groups failed to develop, and embryonic length decreased dose-dependently. Thus, it was found that CTD affected the reproduction of the male quail through the fragmentation of germ cells and the inhibition or delay of embryonic development.

Neonicotinoids are novel systemic pesticides acting as agonists on the nicotinic acetylcholine receptors (nAChRs) of insects. Experimental studies have revealed that neonicotinoids pose potential risks for the nervous systems of non-target species, but the brain regions responsible for their behavioral effects remain incompletely understood. This study aimed to assess the neurobehavioral effects of clothianidin (CTD), a later neonicotinoid developed in 2001 and widely used worldwide, and to explore the target regions of neonicotinoids in the mammalian brain. A single-administration of 5 or 50mg/kg CTD to male C57BL/6N mice at or below the no-observed-adverse-effect level (NOAEL) induced an acute increase in anxiety during the elevated plus-maze test. In addition, mice in the CTD-administered group spontaneously emitted human-audible vocalizations (4-16kHz), which are behavioral signs of aversive emotions, and showed increased numbers of c-fos immunoreactive cells in the paraventricular thalamic nucleus and dentate gyrus of the hippocampus. In conclusion, mice exposed to NOAEL-dose CTD would be rendered vulnerable to a novel environment via the activation of thalamic and hippocampal regions related to stress responses. These findings should provide critical insight into the neurobehavioral effects of neonicotinoids on mammals.

Neonicotinoids, which were developed in the 1990 s as an insecticide having selective toxicity, were later found to cause reproductive abnormalities in experimental animals. In Japan there is an attempt to preserve endangered animals, including the Japanese crested ibis, and there is a question of whether neonicotinoids affect the reproduction of this bird, since they are used in its habitat. Hence, we investigated whether the daily oral administration of the neonicotinoid clothianidin (CTD) has any deleterious effects on the reproductive function of mature male only or both young male and female quails as experimental animals. Vacuolization and the number of germ cells having fragmented DNA in seminiferous tubules, as well as the number and size of vacuoles in hepatocytes, increased dose-dependently. The ovaries showed abnormal histology in the granulosa cells, which produce progesterone. There were significant differences in egg-laying rates and embryo weights between the groups. Glutathione Peroxidase 4 (GPx4) and Manganese Superoxide Dismutase (Mn-SOD), which protect the organism from oxidative damage, showed a dose-dependent decrease. Thus, it is possible neonicotinoids affect the bird's reproductive system through oxidative stress, reflecting an imbalance between the production of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. Responding to our study, Sado Island has since succeeded in breeding Japanese crested ibis in the wild without the use of neonicotinoids.