The findings of the current study suggest that exposure of carp fingerlings to three different sublethal concentrations of imidacloprid for 28 days showed low to moderate histological alterations and significant upregulation in (HSP70 and CYP1A) gene expression of liver and gills.
Histological analysis
The general histological examination revealed low to moderate damage in gill and liver tissues of common carp after exposure to three different sublethal concentration of IMI (T1=26.04 mg/L, T2= 20.38 mg/L, T3=17.36 mg/L) or 28 days. Tissues obtained from the control groups showed normal features (Fig 1A and 2A). Hydropic degeneration (Fig 1B) and cellular infiltration (Fig 1C) in liver as well as telangiectasis in secondary lamellae (Fig 2B), epithelial lifting and oedema (Fig 2C) in gills were observed in the evaluation of samples exposed to IMI.
Histological examinations have been proven to be very sensitive tool to evaluate the effects of toxicants on the exposed fish tissues
(Dawood et al., 2020). Fish liver is regarded as the main organ of several metabolic pathways and changes in the liver histology is being widely used as biomarkers of toxicant and carcinogen exposure damage
(Khafaga et al., 2020). Hepatic degeneration withing hepatocytes and hepatopancreatic cells was a common phenomenon to toxicant exposure.
Ozdemir et al., (2018) noticed hydropic degeneration in hepatocytes and necrosis in liver tissues, mononuclear cell infiltration that induced inflammation of liver and passive hyperaemia and disruption of hepatic tissue organisation when exposed to various concentration of IMI for 96 hr.
Owing to susceptible nature, fish gill tissues are often considered as water quality monitors and bioindicators of toxicant exposure
(Erkmen et al., 2017). The hyperplasic alterations are nonspecific defence responses delineated to protect the organism due to increasing uptake of many xenobiotics by augmenting distance between the chemical and the blood vessels
(Gunal et al., 2020). Similar kind of observations like epithelial lifting, proliferation and fusion of the secondary lamella on
Oreochromis mossambicus was observed by
Ozdemir et al., (2018).
Gene expression studies
The results were analysed and the fold changes in the expression of the gene (HSP70 and CYP1A) in gill and liver samples are shown in Fig (3, 4, 5 and 6). The mRNA level of HSP70 in gill was significantly upregulated in treatment T1 and T2 on day 7, 14, 21 and 28 (Fig 3A, 4A, 5A and 6A), whereas HSP70 in the liver was significantly upregulated in all the treatments T1, T2 and T3 on day 7, 14, 21 and 28 except T3 on day 7 (3B, 4B, 5B and 6B) (p<0.0001). Similarly, CYP1A gene in gill and liver was significantly upregulated in treatment T1 and T2 on days 7, 14, 21 and 28 (Fig 3C, 4C, 5C and 6C) and T1 on day 7, T1 and T2 on day 14 and 21 and T1, T2 and T3 on day 28 (Fig 3D, 4D, 5D and 6D) respectively (p<0.05).
The physiological response of a fish to external stimuli typically includes HSP70 expression. Numerous studies have revealed that environmental stressors such as metal pollution and toxic chemicals have significant influence on fish HSP70
(Dang et al., 2010). In the present study, significant increase in the expression of the HSP70 gene was observed between three different sub-lethalconcentrations of IMI in dose and time dependent manner. Similar kind of findings like increase in mRNA levels of HSP70 in brain, kidney and gill of common carp was reported by
Xing et al., (2012) when exposed to different concentration of atrazine and chlorpyrifos. Pesticides may impair cells’ ability to cope with oxidative stress, resulting in the production of free radicals or enzyme systems that scavenge free oxygen radicals. Oxidative stress can also cause early-stage HSP70 mRNA expression, implying that oxygen radicals, particularly superoxide anions, have a role in HSP70 expression
(Kukreja et al., 1994).
CYP1A, a member of the cytochrome P450 (CYP) enzyme superfamily, is a key enzyme in drug and chemical metabolism in fish and other animals
(Tsuchiya et al., 2005). Thus, it may provide useful information about fish behaviour and health resulting from environmental stress
(Uno et al., 2012). Higher levels of CYP1A mRNA in the liver in the treatment groups could be due to
de novo synthesis rather than CYP1A mRNA stability or degradation rates. CYP1A gene was significantly upregulated in both gills and liver during the experimental period which might be due to increase in ROS production cause by IMI toxicity (
Narr, 2014). Similar, kind of findings like increases in CYP1A mRNA level upon sublethal exposure to IMI was also reported by
Ozdemir et al., (2018) in
C. carpio.
Li et al., (2015) also reported the upsurge expression of CYP1A mRNA in liver, gills and muscle treated with biocide tributylin (TBT) in common carp.