The present data exhibited that the behaviour of the organism is extensively associated with the concentration of insecticide and the demise rate is exposure period-dependent (Fardisi et al. 2017). In the present experiment, we focus on the 96h exposure period LD50% and LD95% confidence levels calculated in both the glass jar and bait test, and both extracted results are below 5% variation. The symptoms of both insecticide intoxication are unpredictable cockroach activities, shadowed by shocks and immobility. In both experiments after application (glass jar and bait test), the LD50% values of IMI are found to be 7.7 mg/L and the CLO 2.5 mg/L respectively at the end of the 96h period. These insecticide statistics confirm that the lethal effects upsurge with the quantity and the period of exposure (Fardisi et al. 2017; Fardisi et al. 2019).
The AChE was discovered in both the cholinergic and non-cholinergic nervous systems, with its activity advancing in motor neurons and combatively in sensory neurons (Massoulie et al. 1993). In the communication process, the neurotransmitter ACh is released from the synaptic split and binds to the ACh receptor at the postsynaptic membrane, transmitting the signal after the neuron, whereas AChE hydrolyses or decomposes the ACh and dismisses the transmission signal. The insect central nervous system exhibits various nicotinic acetylcholine receptors (nAChR) at internal and external synaptic levels and an astonishing neurotransmitter at the synapses (Casida 2011; Calas-List et al. 2013; Crossthwaite et al. 2017). To prevent acetylcholine accretion in the synapse, acetylcholinesterase (AChE) interferes with the hydrolysis of the acetylcholine into choline and acetic acid. Inhibition of the enzyme causes constant and extreme acetylcholine accumulation in nerve synapses. The AChE rapidly participates in the catalytic mechanism to hydrolyze acetylcholine and clears the path of communication of neuronal impulses, thereby protecting the organism’s functions (Zimmerman and Soreq 2006).
The IMI and CLO are nicotinoid compounds that act as agonists on insect acetylcholine receptors (nAChR) involving the central nervous system (Makoto et al. 2006; Tan et al. 2007; Honda et al. 2006; Crossthwaite et al. 2017). Cockroaches have two categories of receptors impervious, i.e., antagonist nAChR1, which is delicate to IMI and the other receptor is nAChR2 which is delicate to CLO (Bordereau-Dubois et al. 2012; Calas-List et al. 2013). CLO insecticidal efficiency is greater than IMI because both exhibited similar empathy toward American cockroach nAChRs, but CLO hydrophobicity and antagonist activities remained inferior to IMI entities (Makoto et al. 2006; Crossthwaite et al. 2017). Because of the effects of CLO, cockroach efferent dorsal neurons fire calcium pathway mechanisms (CaMKII), which control nAChR function (Taillebois et al. 2013; List et al. 2014). The IMI and metabolites have more potency for neurotransmitter-blocking activity, and the insecticidal strength is well connected to the nerve activity after the hydrophobic aspect of the compounds (Shinzo et al. 2004). To circumvent the phenomenon of ROS effects, insects’ primary regulators are corpora cardiaca/allata, and prothoracic glands (Gilbert et al. 2005) and they also have extremely developed antioxidant defence mechanisms (Felton and Summers 1995). Present results indicating that continuous exposure to IMI and CLO alter the AChE activity in the cockroach nervous system.
The insect has different kinds of resistant mechanisms such as esterases, enzymes, antioxidants and P450s. These detoxifying enzymes play vital roles such as catabolization, hydrolysis and biosynthesis of the toxicants. The monooxygenase P450 enzymes are complex in texture in insects, play a vital role in the xenobiotic compound breakdown in the endogenic metabolism and are important in adaptation to toxic chemicals in the host medium (Pang et al. 2020). The P450 enzymes conjugate with oxygen molecules and obtain energy from NADPH to penetrate oxygen into the substrate/insecticide. P450 monooxygenase enzyme activity has been linked to insecticide resistance in various studies (Siegfried and Scot 1992; Pridgeon et al. 2003; Wolfe and Scharf 2022) and regular prolonged intoxication results in elevated enzyme activity (Hu et al. 2020; El-Samad et al. 2022). In the current study, continuous long-term exposure to imidacloprid and clothianidin disrupted the P450 system in cockroaches and the insecticide-induced changes in the activities of enzymes involved in metabolism.
The ROS stress generates free radicals in the electron transport system of the mitochondria such as O2•‒ (superoxide), OH• (hydroxyl), RO2 (peroxyl) HO2• (hydroperoxyl), oxygen and H2O2 (hydrogen peroxide), OCl‒ (hypochlorite ion) and O3 (ozone) (Farooqui and Farooqui, 2009). The antioxidant enzymatic resistance versus ROS, established by the activities of superoxide dismutase (SOD) and catalase (CAT) is on the frontlines, and cockroaches have established protuberant resistance against insecticides (Barbehenn 2002). SOD activity was increased by scavenging free radicals (O2•‒ and OH+) to protect the organism from insecticide-induced toxicity, as well as by increased superoxide radical absorption to combat oxidative stress by converting them to low-toxic hydrogen peroxide (H2O2) (Gilbert et al. 2005). The CAT converts the less harmful H2O2 into harmless H2O (water) and O2 (Oxygen) in the cellular catalysis reaction (Escobar et al. 1996). The organism's endo and exogenous systems and the CAT-SOD pathway antioxidant mechanism provide the primary resistance against oxygen-free radical toxicity (Singh et al. 2017; Večeřa et al. 2012). The current results explain that IMI and CLO cause significant damage/alteration, and the SOD and CAT enzymes are scavenging the front-line antioxidant enzyme mechanisms of B. germanica, indicating that the excess production of H2O2 is converted into water and oxygen molecules.
Glutathione S-transferases (GSTs) are classified into cytosolic, microsomal, and mitochondrial/ peroxisomal in aerobic organisms and the enzymes protect against oxidative stress and endogenous and exogenous detoxification (Enayati et al. 2005; Casida 2011). The elevated activities of GST contributed to escalating the detoxification aptitude and providing resistance to the insects from several pollutants and they actively participated in intra-cellular transport and several biosynthetic pathways. The insects, especially cockroaches, developed protuberant resistance in contradiction of pesticides, which has been accredited to increased GST activities (Ma and Chang 2007). Previous biochemical statistics revealed that the significant increase in GST activities in Blattella germanica was caused by exposure to various concentrations of several insecticides (Habes et al. 2006) and collaboration by the P450 system in the detoxifying process (Gondhalekar and Sharf 2012). The exposure of imidacloprid and clothianidin insecticides triggered a high-level elevation of GST activity in the Blatella germanica, possibly due to an effective mechanism for detoxification.
As such, GSTs conjugate to non-enzymatic reduced GSH in phase II bio-transformation reactions, which takes place through substrate conjugation, the stimulation of the thiol-group of GSH and a later nucleophilic hit by the an-ionic GSH on the hydrophobic complex (Habes et al. 2006; Casida 2011; Rodrigues et al. 2013). GSH regulates intracellular redox homeostasis and is responsible for the supply and abundance of thiols in widely distributed cells (Li 2009). The GSH is required to decrease the intracellular stress environment in order to protect cells from pro-oxidative damage. Lipo-peroxidation caused by nicotinoid insecticides such as IMI and CLO has been linked to low GSH levels (Casida 2011). Huang et al. (2012) concluded that the elevation of GST activity and inhibition of GSH levels by insecticides disclose an initiation of oxidative stress and an elicitation of the detoxification system. The elevated GST activity and decreased GSH levels indicate that the GST catalyses the unification of the tri-peptide glutathione to the centres of lipophilic complexes via a nucleophilic replacement response, consequently creating more dissolved conjugates that are easily defecated from cells. The unstable lipid peroxides are derivates of polyunsaturated fatty acids, they are split into numerous extremely reactive compounds, which are combined with several active functional molecules such as proteins, lipoproteins and DNA (Janero 1990). Malondialdehyde, which is measurable as thiobarbituric acid and widely used as an oxidative stress biomarker, is the most common degradation product of LPO. The current results indicate that the exposure to IMI and CLO triggered a high-level elevation of LPO activity in Blatella germanica, possibly due to an effective mechanism for the detoxification process. The previous research reports also indicated that increased LPO levels were observed in different classes of chemical compound exposure in cockroaches (Rodrigues et al. 2013; Adedara et al. 2015). At the same time, the substantial elevation in H2O2 and TBRS evidences an elevated LPO index and is also considered an insecticide that induces stress since GSH is vital and responsible for defending against oxidative stress (Meister 1991). In the present study, there is evidence that the elevating LPO caused by both insecticides may be unswervingly associated with reduced GSH content.
Phosphatases (alkaline and acidic) are hydrolytic; they split the ester-bond between the phosphate and the pollutant's organic deposit, which involves the biological activities of the lysosomal hydrolytic enzyme (Csikós and Sass 1997). The ALP activity is highly intense in the plasma membrane, whereas the ACP is allied with lysosomes and is involved in different important metabolic activities. Phosphatase activities in the insects were low and increase slowly after moulting, maximum activity is observed in the starvation stage in the fifth instar nymph and the nethermost appears in the developed larval stages (Miao 2002). The ACP enzyme hydrolyses foreign phosphomonoesters through the lysosomal endocytosis activity and ALP plays a vital role in phosphomonoester surface hydrolysis and transphosphorylase extant in the plasma membrane (Sakharov et al. 1989). The elevation trends indicate the insecticides seriously damaged the phosphorylation pathways and the organism's effort to recuperate from the stress mechanism. The LDH is a tetrameric catalytic protein (glycolytic) and a variable conversion mechanism of pyruvate to lactate and glucose to source energy in the organism (Diamantino et al. 2001). It poses a key role in carbohydrate metabolism in the anaerobic mechanism, elevation activities of the enzyme are extensively recognised as increasing in the anaerobic mechanism owing to the circumstances of environmental tension (Ribeiro et al. 1999). Therefore, the significant elevation of LDH activity in the organism indicates an increase in the glycolytic mechanism to counter the insecticide effects (Senthil-Nathan 2013). The present results verify the literature and increase information about neonicotinoids’ ability to cause oxidative trauma in cockroaches by displaying the harmful activities of imidacloprid and clothianidin to the oxidative and antioxidant protective mechanisms in B. germanica.
Insecticide resistance escalates the cost of regulatory programs; for example, DDT partially failed to control malarial vector control programs and the cost increased by nearly 8–10 folds (WHO 1992). Widespread applications of neurotoxic insecticides have led to the development of a retention mechanism for insecticides in the German cockroach (Chai and Lee 2010; Fardisi et al. 2017; Fardisi et al. 2019). Three types of resistance mechanisms, including i) Decreased circulation and diffusion, which confer less than three folds of insecticide resistance. ii) Increased metabolic detoxication iii) Target site insensitivity (mixed oxidase function systems play a vital role in detoxication). Previous studies suggest that Blattella germanica developed insecticide resistance mechanisms through adaptation of enzymatic activities such as target site insensitivity, detoxification, reduced cuticular penetration, and behavioural escaping, as well as being habituated in comparatively closed populations, which aids rapid selection to acquire high-level resistance (Chai and Lee 2010; Sparks and Nauen 2015; Fardisi et al. 2017; Fardisi et al. 2019). A small amount of insecticide disrupts the behavioural response of insects to sex-pheromones, resulting in unexpected reproduction abilities in target insects (Guedes et al. 2010). The insect gut is connected with malpighian tubules, which is a single-cell layered, long, closed-ending tubules floating in haemolymph, that maintain the homeostasis of haemolymph and play a vital role in the ROS detoxification system (Dow et al. 2018). Symbiotic microbes in the insect gut play an important role in interacting with pest control compounds via catabolic activities, producing pheromones, vitamin synthesis, and activation of antioxidant enzymes, all of which contribute significantly to physiology (Ozdal et al. 2016).