In-vitro Toxicity of Synthetic Insecticides against Subterranean Termites, Coptotermes heimi (Isoptera: Rhinotermitidae)

Subterranean termites cause significant damage to agricultural crops and wooden infrastructures worldwide. Coptotermes and Odontotermes were found as the most abundant and damaging genera of subterranean termites in Pakistan. Many conventional synthetic insecticides are being used to combat termite infestations with often unsatisfactory control results. This study assessed the comparative toxicity of some prevailing synthetic insecticides with different modes of action against subterranean termites Coptotermes heimi Wasmann (Isoptera: Rhinotermitidae) which was found as a dominant termite species in district Sargodha. Filter paper disc-based bioassays revealed that all insecticides showed a significant impact ( P < 0.001) on the mortality of C. heimi workers and this mortality response was directly proportional to insecticidal concentrations and exposure times. Significantly higher mortality was recorded by chlorpyrifos (100.0%) and fipronil (95.0%) at 72 h post-exposure with minimum LC 50 values of 1.29 and 2.04%, respectively. Similar trend of effectiveness was exhibited by their LT 50 values. Minimum mortality of C. heimi workers was recorded by the formulations of chlorantraniliprole and abamectin. Based on overall study results, it is concluded that chlorpyrifos and fipronil are effective synthetic termiticides and are recommended to the indigenous farmers for combatting subterranean termite infestations.


INTRODUCTION
T ermites belong to order Isoptera with 12 families and 3500 species described so far (Davies et al., 2021). These invertebrates constitute a major insect fauna of humid and temperate regions and contribute to the ecosystem in terms of both beneficial and harmful aspects (Brauman et al., 2015). These invertebrates play a key role in ecological processes such as biodegradation of plant based organic matter and nutrients cycles (Majeed, 2012). However, many termite species are economic pests of agricultural crops, forest trees and other wooden structures (Rouland-Lefèvre, 2010). In Pakistan, about 53 species of termites have been identified in various ecological zones that are damaging many agricultural crops and wooden infrastructures (Manzoor and Mir, 2010).
Coptotermes, Microtermes and Odontotermes are the most prevalent genera of pest termites in Indo-Pak region (Rajagopal, 2002). Among 80 subterranean termite species, 38 belong to genus Coptotermes (Rust and Su, 2012;Krishna et al., 2013). These termites are highly destructive pests of a wide array of agricultural crops and wooden household structures (Ahmed and Qasim, 2011;Manzoor et al., 2011). Their infestation has been challenging in both agricultural crops and urban areas (Katsumata et al., 2007;Gazal et al., 2014).
Synthetic insecticides have been the prime option to combat subterranean termite infestation worldwide. Various synthetic insecticides are used against subterranean termites including carbosulfan, deltamethrin, DDT,

O n l i n e F i r s t A r t i c l e
chlorpyrifos and triazophos etc. Manzoor et al., 2012;Paul et al., 2018). These traditional synthetic insecticides remain highly persistent with longterm residual effects causing the eradication of beneficial organisms and other soil microbes, and secondary pests' outbreak is another concern (Desneux et al., 2007;Paul et al., 2018). With extensive use of synthetic chemicals, resistance against these insecticides has developed in various insects including termites (Zhu et al., 2016). Novel insecticides with different chemistry and modes of action have always been a viable source of protection against different pests including termites in various insecticide resistance management programs (Iqbal and Saeed, 2013;Paul et al., 2018). Thus, there is a need to explore some differential chemistry insecticides that should be environment friendly and effective to control termite population. Instead of traditional methods, novel chemistry insecticides have become promising tools for mitigating the problems of pest resistance and environmental contamination. These insecticides are not only target-specific but also safer for non-target fauna, i.e., predators and parasitoids. Previous studies have shown that novel chemical insecticides are effective against different species of termites (Mao et al., 2011;Rashid et al., 2012;Iqbal and Saeed, 2013;Akbar et al., 2019). The pesticide resistance and environmental pollution caused by traditional insecticides with limited modes of action necessitate screening out currently available synthetic insecticides with different modes of action against indigenous subterranean termite species. We therefore evaluated some selected synthetic insecticides having different modes of action viz; abamectin, chlorantraniliprole, chlorpyrifos, deltamethrin, emamectin benzoate, fipronil, lambda-cyhalothrin, lufenuron, and profenofos against subterranean termites Coptotermes heimi (Isoptera: Rhinotermitidae) which is a dominant wood infesting subterranean termite species in Pakistan. Some of these selected insecticides are registered against termites such as chlorpyrifos, fipronil.

Collection and maintenance of termites
First of all, an extensive survey was conducted in different localities of district Sargodha (Punjab, Pakistan) in order to determine the prevailing status of termite infestation in study area. For this purpose, small land-hold farmers, and rural and urban dwellings were surveyed randomly and samples of termite infested materials (crop stubbles and wooden infrastructures) were collected and brought to the laboratory of Entomology, University of Sargodha for identification. Termite soldier individuals were observed under an inverted trinocular microscope (XDS-3, Optika SRL, Italy). Coptotermes and Odontotermes were found as the most abundant and damaging genera of subterranean termites.
For in-vitro evaluation of different synthetic insecticides as detailed in Table I, C. heimi (Wasmann) (Isoptera: Rhinotermitidae) was used as model species because it was the most abundant species among the collected termite samples. Intact portions of C. heimi colony along with the termite individuals were collected from a fallen infested log of sheshum (Dalbergia sissoo DC.) and were maintained for few days in a rearing glass box (30 × 30 × 30 cm) under controlled conditions in dark (25±2 ℃ and 65±5% RH).

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Screening bioassay against C. heimi In first bioassay, nine different synthetic insecticides having different modes of action were tested against C. heimi as per their label-recommended doses. Although most of these insecticides do not have recommended dose rates against termites, we tested them in our preliminary screening as per their label-recommended dose rates against other target insect pests. In the control treatment, tap water was used. Filter paper disc bioassay method as described in Akbar et al. (2019) was used to assess the toxicity of these insecticides against C. heimi. Experimental design was completely randomized (CRD) with eight replications per treatment. Filter paper discs were dipped into each treatment solution for 5-10 sec and were air-dried at ambient temperature (26 ℃) for 10-15 min before placing them into Petri-plates (9 × 2.5 cm) lined with 1.0 mm layer of 1.5% agar. Ten active and healthy termite individuals (9 workers and 1 soldier) were released on each treated filter paper disc and Petri-plates were incubated at 25±2 ℃ and 65±5% RH in an environment chamber. Mortality data was recorded after 3, 6, 12, 24, 48 and 72 h of exposure. Moribund individuals showing no movement were considered as dead.
Four most effective insecticides resulted out from the screening experiment were further bioassayed using five concentrations (i.e., 5, 10, 20, 40 and 80% of the labelrecommended dose) of each insecticide. Experimental protocol was similar as described above for the first bioassay except number of replications for each treatment was six in this bioassay. Mortality data were recorded at regular time intervals as mentioned above.

Statistical analyses
Mortality data of termites in response to different insecticidal treatments were analyzed by factorial analysis of variance (ANOVA) keeping insecticides and exposure time as main factors. The percent mortality was corrected with Abbott's formula (Abbott, 1925). Means were further compared by Tukey HSD test at 95% significance level. The analyses were performed by using Minitab 17.0 software. Median lethal concentration (LC 50 ) and time (LT 50 ) values were calculated by probit analysis using PoloPlus ® software.

RESULTS
In screening bioassay, all insecticides exhibited significant (F = 530.5, p < 0.001) mortality of C. heimi individuals at different exposure time (F = 242.4, p < 0.001). The highest mean mortality of C. heimi individuals was recorded by chlorpyrifos (89.0%) and fipronil (85.0%) recorded at 48 h of application and this mortality was increased to 100.0 and 95.0%, respectively at 72 h post-exposure. Deltamethrin, lambda-cyhalothrin, lufenuron and emamectin benzoate exhibited intermediate mortality (65.0-76.0%) of C. heimi at 72 h of application. The least effective chemicals were chlorantraniliprole and profenofos causing 26.0 and 37.0% mortality, respectively at 72 h post-exposure (Fig. 1).
The toxicity of four most effective insecticides with different concentrations was tested against C. heimi individuals in second toxicity bioassay. Moreover, this mortality trend increased along with the insecticidal concentrations and exposure time. At minimum concentration, chlorpyrifos and fipronil exhibited 34.9 and 31.2% mean mortality of termites, respectively which increased to 69.6 and 60.5% at their higher concentrations followed by 53.5% by lambda-cyhalothrin and 37.8% by lufenuron. Lower concentrations of lambda-cyhalothrin and lufenuron caused less than 20.0% mean mortality of termites (Table II). According to the dose response probit regression analysis of the mortality data, lowest LC 50 value was recorded in the case of chlorpyrifos at 72 h (1.29%) and 48h (5.36%) than other insecticides. In case of fipronil, LC 50 values were 2.04% at 72 h and 8.06% at 48 h postexposure. LC 50 values for lambda-cyhalothrin were 3.68% at 72 h and 10.23% at 48 h, while the minimum effectiveness was observed by lufenuron having maximum values of LC 50 (Table III). Similar trend of effectiveness was observed in case of LT 50 values. Minimum LT 50 values were recorded for chlorpyrifos (6.58 h) and fipronil (10.11 h), while lambda-cyhalothrin and lufenuron showed LT 50 values of 14.14 and 30.14 h, respectively (Table IV).

DISCUSSION
Subterranean termites such as C. heimi cause considerable economic loss to agricultural crops and wooden infrastructures worldwide, and are considered as major threat to agro-forestry and urban sectors, particularly in the tropical and subtropical countries (Evans, 2021). Although many control tactics including chemical, physical and biological techniques can be employed to manage termite infestations, however chemical termiticides have been commonly used combating subterranean termites Su, 2011;Kuswanto et al., 2015).
We conducted a preliminary survey in district Sargodha to assess the local farmers and civil community's perception about subterranean termites, their infestation, identification and control measures. Unfortunately, most of the community did not know how to identify and how to combat termite infestations. Conventional insecticides were being used by them as sole control option with no or unsatisfactory control of termites and other insect pests as documented previously (Manzoor et al., 2012;Majeed et al., 2022). Furthermore, Coptotermes and Odontotermes and C. heimi were found as the most important subterranean termites' genera and the most abundant termite species, respectively in the study area.

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In-vitro Toxicity of Synthetic Insecticides against Termites These findings are in line with previous studies (Manzoor et al., 2011(Manzoor et al., , 2013Rasib and Ashraf, 2014;Dugal and Latif, 2015;Sarmad et al., 2020). We evaluated some currently available synthetic insecticides including some reduced-risk insecticidal formulations against C. heimi in the laboratory. Termites showed a differential response to all tested insecticides. This difference in toxicity of insecticides would definitely be due to their differential chemistry and modes of action (Li et al., 2012;Rashid et al., 2012;Iqbal and Saeed, 2013). Out of nine tested chemicals, chlorpyrifos and fipronil exhibited highest toxicity against C. heimi termites. Our findings are in accordance with Iqbal and Saeed (2013) and Zhang et al. (2022) who also reported chlorpyrifos and fipronil as the best insecticidal options to manage subterranean termites Microtermes mycophagus and Coptotermes formosanus, respectively. Some insecticides including chlorantraniliprole, avermectins and profenofos were least effective and caused lowest termite mortality. These differences in susceptibility of various insecticides could be the result of differential rates of penetration, insensitivity of target sites and metabolic resistance due to some enhanced level of detoxifying enzymes (Valles et al., 2000;Osbrink et al., 2001;Valles and Woodson, 2002;Zhou et al., 2021). However further biochemical studies are needed to better understand this phenomenon.
In the second bioassay, different concentrations of the four most effective insecticides were tested against C. heimi. The results showed that the mortality of termite individuals was greater with the application of a higher concentration (80%) of chlorpyrifos and fipronil. Lower LC 50 and LT 50 values also confirmed the higher toxicity of these chemicals. In previous studies, the toxicity of chlorpyrifos and fipronil has been reported against termites (Ahmed et al., 2005;Khan et al., 2021). Upadhyay et al. (2010) reported that fipronil is an effective chemical to manage the infestation of O. obesus. Similarly, chlorpyrifos is a very effective chemical to control termite infestation, and it has been confirmed by previous studies as well (Ahmed et al., 2017). It is a repellent termiticide and controls termites' movement in the soil and limits the access of termites to food leading to termites' mortality O n l i n e F i r s t A r t i c l e (Ahmed et al., 2015). Similarly, Ahmed et al. (2017) also reported that fipronil is an effective compound to control subterranean termites as a pre-construction treatment; it does not allow the termites to penetrate through the treated soil.

CONCLUSION
Our findings showed that chlorpyrifos and fipronil at recommended doses were the most effective synthetic insecticides against C. heimi termites. Overall, this laboratory study suggests that subterranean termites' infestation such as of C. heimi can be eliminated by using these two synthetic insecticides in agricultural sector and in urban dwellings.

ACKNOWLEDGEMENT
Authors are thankful to Dr. Muhammad Asam Riaz for identification of the termite species.

Funding
The study was financially supported by the internal grant of the Department of Entomology, University of Sargodha, Pakistan.

Ethical statement
Authors declare that this study did not require ethical committee's approval or any other ethical considerations.

Statement of conflicts of interest
The authors have declared no conflict of interest.

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In-vitro Toxicity of Synthetic Insecticides against Termites O n l i n e