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.

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.

Mammalian sexual fate is determined by the presence or absence of sex determining region of the Y chromosome (Sry) in the “bipotential” gonads. Recent studies have demonstrated that both male and female sexual development are induced by distinct and active genetic pathways. Breeding the Y chromosome from Mus m. domesticus poschiavinus (POS) strains into C57BL/6J (B6J) mice (B6J-XYPOS) has been shown to induce sex reversal (75%: bilateral ovary, 25%: true hermaphrodites). However, our B6N-XYPOS mice, which were generated by backcrossing of B6J-XYPOS on an inbred B6N-XX, develop as males (36%: bilateral testis with fertility as well as bilateral ovary (34%), and the remainder develop as true hermaphrodites. Here, we investigated in detail the expressions of essential sex-related genes and histological features in B6N-XYPOS mice from the fetal period to adulthood. The onsets of both Sry and SRY-box 9 (Sox9) expressions as determined spatiotemporally by whole-mount immunohistochemistry in the B6N-XYPOS gonads occurred 2–3 tail somites later than those in B6N-XYB6 gonads, but earlier than those in B6J-XYPOS, respectively. It is possible that such a small difference in timing of the Sry expression underlies testicular development in our B6N-XYPOS. Our study is the first to histologically show the expression and ectopic localization of a female-related gene in the XYPOS testes and a male-related gene in the XYPOS ovaries. The results from these and previous experiments indicate that the interplay between genome variants, epigenetics and developmental gene regulation is crucial for testis development.

Although neonicotinoid pesticides are expected to have harmful influence on mammals, there is little animal experimental data to support the effect and mechanisms. Since acetylcholine causes the release of dopamine, neonicotinoids may confer a risk of developmental disorders via a disturbance in the monoamine systems. Male mice were peripubertally administered dinotefuran (DIN) referring to no observed effect level (NOEL) and performed behavioral and immunohistological analyses. In an open field test, the total locomotor activity was increased in a dose-dependent manner. The immunoreactivity of tyrosine hydroxylase in the substantia nigra was increased in DIN-exposed mice. These results suggest that exposure to DIN in peripubertal male mice causes hyperactivity and a disturbance of dopaminergic signaling.

Neonicotinoids are pesticides used worldwide. They bind to insect nicotinic acetylcholine receptors (nAChRs) with high affinity. We previously reported that clothianidin (CTD), one of the latest neonicotinoids, reduced antioxidant expression and induced germ cell death in the adult testis of vertebrates. Here, we investigated the male reproductive toxicity of prenatal and early postnatal exposure to CTD, because it is likely that developmental exposure more severely affects the testis compared to adults due to the absence of the blood-testis barrier. Pregnant C57BL/6 mice were given water gel blended with CTD (0, 10 or 50 mg/kg/day; no-observed-adverse-effect-level [NOAEL for mice]: 47.2 mg/kg/day) between gestational day 1 and 14 days post-partum. We then examined the testes of male offspring at postnatal day 14. The testis weights and the numbers of germ cells per seminiferous tubule were decreased in the CTD-50 group, and abnormal tubules containing no germ cells appeared. Nevertheless, the apoptotic cell number and proliferative activity were not significantly different between the control and CTD-exposed groups. There were no significant differences in the androgen-related parameters, such as the Leydig cell volume per testis, the Sertoli cell number and the tubule diameter. The present study is the first demonstration that in utero and lactational exposures to CTD at around the NOAEL for mice reduce the germ cell number, but our findings suggest that these exposures do not affect steroidogenesis in Leydig cells during prenatal or early postnatal life.