Virulence of four Steinernema species as a biological control agent in controlling the termite, Coptotermes heimi (Wasmann) (Isoptera: Rhinotermitidae)

Subterranean termites are an ancient group of social insects, broadly spread, known primarily as economically important pests for the destruction of wooden structures and also as agricultural pests. Many of the banned chlorinated hydrocarbon insecticides used to be recommended for the control of termites. Hence, it has become necessary to find alternative measures for termite control in the natural diverse habitats as well as in the cultivated soil to diminish use of these chemicals. Therefore, in the present study, 4 strains of entomopathogenic nematodes (EPN) belong to the genus Steinernema were assessed against Coptotermes heimi (Wasmann). These EPN included Steinernema pakistanense NNRC-AS.04, S. siamkayai NNRC-As.12, S. bifurcatum NNRC-As.65, and S. maqbooli NNRC-As.88. Virulence of all strains was determined at 3 different EPN inocula in plastic containers layered with sand. A significant nematode inoculum effect was detected for all the tested EPN species. NNRC-AS.04 and NNRC-As.65 showed the highest virulence effects of 95 and 100%, respectively at 150 IJs/ml.


Background
Worldwide, termites are a massive dilemma in both urban and agricultural areas, as they are the source for considerable devastation to plants, agricultural crops, wood structures, and account for financial loss. High-quality wood products are often preferred by customers, but physical or biological damages reduce their worth (Uzunovic et al. 2008). The incidence of termites is habitually not eagerly observed as of concealed behavior. They act as decomposers as well as herbivores feeding on a spacious variety of dead, rotten, or fresh plant material (Traniello andLeuthold 2000 andBignell andEggleton 2000). Coptotermes heimi (Wasmann) (Isoptera: Rhinotermitidae) has been reported from urban and agricultural fields of Pakistan as a serious pest Mir 2010 andManzoor et al. 2011). Successful management for termite colonies needs many particular skills depending on the species origin invasion. Knowledge of termite ecology and its identification can help to spot damage and ways of control (Khan et al. 2016). Chemical troubles made a big impact on the agricultural society and drew attention to the use of biocontrol agents as a safe and effective biopesticide alternative. It can be used in many diverse agricultural systems rather than an immediate solution. Therefore, biocontrol should be considered as a long-term research aim. The two important nematode families falling in the group of entomopathogenic nematodes (EPNs) are Steinernematidae Travassos, 1927, andHeterorhabditidae Poinar, 1975, which are considered as one of the most successful examples of biological tools used to control soil dwelling insect pests. They possess virtually all the attributes of an ideal biological control agent. They enter the host through the natural openings such as mouth, anus or spiracles, or sometimes by abrasing the intersegmental membranes of the insect cuticle (e.g., Heterorhabditis spp.) (Grewal et al. 2005), reach the hemocoel, release there the cells of symbiotic bacteria from their intestine, which ultimately results in killing the host within 48 h (Askary 2010;Askary 2012). Because the nematode symbiotic bacterium kills insects, so quickly, there is no intimate host parasite relationship as characteristic for other insect parasitic nematodes (Shapiro-Ilan et al. 2012).
The aim of this research was to assess the virulence of EPNs, Steinernema species against C. heimi, under laboratory conditions.

Target pest
Alive colonies of C. heimi were collected from infested fallen wooden logs of Mangifera indica (Sapindales: Anacardiaceae) from main campus University of Karachi, Karachi, Pakistan (24.9418°N, 67.1207°E) and identified with a help of the key of (Akhtar 1983). The termite colonies were bought to the culturing room of National Nematological Research Center (NNRC), University of Karachi (Uok), Karachi, Pakistan and maintained in 1000 ml plastic containers with wooden logs at 28°C and 75-80% RH till the experiment was executed.

Virulence assay
Active C. heimi termite individuals were collected from a rearing container for virulence assay of 4 EPN species in a plastic container (280 × 160 × 80 mm) separately for each nematode species and concentration. Containers were layered with 45-g sterilized moist soil. Twenty termites were added in each container exposed to 3 different numbers of nematodes viz., 50, 100, and 150 IJs/ml in 2.5 ml distilled water suspension covered with a plastic lid. Concentrations were dropped evenly in containers by a 1000 μl pipette, to evade mingle sterile pipette tips that were changed after each conduct. Simple distilled water 2.5 ml was dispensed in a control treatment. Mortality rate was calculated after 48 h of exposure and the containers were kept at 28 ± 2°C. Dead termites were transferred in a plastic cavity block (4.5 × 4.5 cm) layered with moist filter paper disk (Whattmann No. 1) to record a nematode emergence. Experiment was carried out twice with 3 replicates at each concentration and EPN species.

Statistical analysis
Data were subjected to analysis of variance in SAS (ver. 9.1, SAS Institute, Cary, NC). If the interaction in EPN species and numbers was significant, it was used to explain results. If the interaction was non-significant (P < 0.05), means were separated with DMRT Duncan's multiple range test (Duncan 1955). Lethal concentration 50 and 90% (LC 50 and LC 90 ) values, intercept, and chisquare values were analyzed by PROC PROBIT routine of SAS, 2000. Abbott (1925) formula was used to correct mortality percentages as follows.

Results and discussion
The comparative virulence assay of the 4 EPN species against C. heimi termite was investigated plastic container layered with 45-g sterilized moist soil at 28 ± 2°C in laboratory of NNRC conditions. The analysis of variance showed significant differences among nematode species efficacy on termites (ANOVA F = 201.5; df = 3; P = 0.05). Nematode inocula also differed significantly (ANOVA F = 6.6; df = 3; P = 0.05) and interaction of the 3 inocula with 4 nematode species, also had marked effect on the pest (ANOVA F = 0.8; df = 3; P = 0.05). Results demonstrated that the nematode could suppress the populations of C. heimi termite. S. pakistanense NNRC-AS.04 and S. bifurcatum NNRC-As.65 showed higher effects at all application concentrations than S. siamkayai NNRC-As.12 and S. maqbooli NNRC-As.88. The highest mortality rate was achieved when EPNs were applied at the concentrations of 150 IJs/ ml after 48 h (Fig. 1). At the lowest concentration of 50 IJs/ml after 48 h of exposure time, at least 50% of termite were killed by S. pakistanense NNRC-AS.04, showing significant differences with S. siamkayai NNRC-As.12 30% and S. maqbooli NNRC-As.88 25%. Concentrations had a great impact on the efficacy of species of nematode (Trdan et al. 2009). The increased mortality of termite caused was concentration dependent. At 150 IJs/ml, the highest mortality rate was induced by NNRC-AS.04 (95 and 100%) by NNRC-As.65, while the lowest one was (62%) by NNRC-As.88. Nematode progenies can reproduce in termites, they were clearly seen when dead termites were transferred to vacant cavity block. The LC 50 , LC 90 values with P value are shown in Table 1. The overall result showed that S. pakistanense and S. bifurcatum were highly virulent against the target subterranean termite, C. heimi 48 h after application. If nematode reproduction can occur in the target insect, long-term management might be achievable. Similar results of maximum mortality response against termite species Macrotermes in a sand and filter paper assay caused by S. pakistanense were also stated by Shahina and Tabassum (2010). Razia and Sivaramakrishnan (2016) evaluated 3 species of EPN, S. siamkayai, S. pakistanense, and H. indica against subterranean termites, Reticulitermes flavipes and Odontotermis hornei under laboratory conditions. In sand assay method, S. pakistanense showed significant results, causing 100% mortality of both pests within 24 h, followed by S. siamkayai and Heterorhabditis indica, which were applied at 250 IJs/ml at 48 h. Wilson-Rich et al. (2007) reported that Zootermopsis angusticollis termite of wet timber showed a susceptibility to concentration-dependent by S. carpocapsae (Mexican strain). Under field conditions, few research studies have been accomplished, using EPNs as a biocontrol agent for termites (Dolinski and Lacey 2007). For the pathogenicity against the termite, Macrotermes bellicosus one strain of H. indica and 29 strains of H. sonorensis (Beninese) were tested by Zadji et al. (2014) and reported that 73% of the isolates parasitized more than 80% of the termite and was influenced by a grouping of biotic and abiotic factors, nematode strain and species. Divya and Sankar (2009) reported 50% mortality of the termite species, Odontotermes obesus after 36 h of post H. indica application. Termites, strained by chemical and pathogen sub-lethal doses, were more vulnerable by EPNs (San-blass and Gowen 2008). Termites exist and feed in environment that are cool, damp, and with no direct sunlight such as wood materials or soil. These ecological surroundings are perfect for the endurance and association of EPNs, providing the source for the concerned pest management. Investigations suggested that the nematodes are effective to control termite colonies as environmentally secure approach (Askari et al. 2012). During the last 30 years of research, EPNs gain common acceptance, and their  commercial applications are being developed as environmentally alternatives to chemical pesticides further noticed in decision of nematodes as better application methods against subterranean termites (Khan et al. 2016). In the fields, species of EPNs that are noted as heat tolerant or environmental stress are requisite for management of termites.

Conclusion
Obtained results are evidence that EPNs can be efficient tactics to control the termites but further studies under field conditions are required.