Environmental Entomology, 49(2), 2020, 412–421 doi: 10.1093/ee/nvaa014 Advance Access Publication Date: 10 March 2020 -Microbial Interaction Research

Entomopathogenic Nematodes as Potential Biological Control Agents of Subterranean , Microcerotermes diversus (: Termitidae) in Iraq Jawad B. Al-Zaidawi,1 Javad Karimi,1,3, and Esmat Mahdikhani Moghadam2 Downloaded from https://academic.oup.com/ee/article-abstract/49/2/412/5802319 by guest on 15 April 2020 1Biocontrol and Insect Pathology Lab., Department of Plant Protection, Ferdowsi University of Mashhad, Mashhad, Iran, 2Department of Plant Protection, Ferdowsi University of Mashhad, Mashhad, Iran and 3Corresponding author, email: [email protected]

Subject editor: Angel Acebes-Doria applyparastyle "fig//caption/p[1]" parastyle "FigCapt" Received 5 September 2019; Editorial decision 5 February 2020 applyparastyle "fig" parastyle "Figure" Abstract The infectivity of three species of entomopathogenic nematodes (EPNs) such as Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae), Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), and H. bacteriophora Poinar (IRQ.1 strain) were examined against subterranean termite Microcerotermes diversus (Silvestri) (Blattodea: Termitidae) that is the most economically destructive termite in Iraq. Laboratory and field NList_dot_numeric2=HeadC=NList_dot_numeric=HeadC efficacy of these strains were evaluated to test the feasibility of indigenous EPNs to be used in a biological control NList_dot_numeric3=HeadC=NList_dot_numeric1=HeadC program. The biological traits examined included pathogenicity, penetration, and reproduction of EPN species. NList_dot_numeric2=HeadD=NList_dot_numeric=HeadD Filter paper and wood bioassays were conducted using six concentrations: 25, 50, 100, 200, 400, and 600 IJs/termite. NList_dot_numeric3=HeadD=NList_dot_numeric1=HeadD In both tests, all strains were virulent against M. diversus workers. The LC of S. carpocapsae in both petri dishes NList_dot_numeric2=HeadE=NList_dot_numeric=HeadE 50 and in containers with sawdust was (57.9 and 15.7 IJs/termite) less than both indigenous (274.2 and 60.8 IJs/termite) NList_dot_numeric3=HeadE=NList_dot_numeric1=HeadE and commercial (139.6 and 52.6 IJs/termite) Heterorhabditis bacteriophora, respectively. In the field, the percent mortality of the tested workers ranged from 22.5–80 ± 8.3%, 37.5–96.2 ± 8.9%, and 28.7–67.5 ± 6.8% for commercial H. bacteriophora and S. carpocapsae and native H. bacteriophora, respectively. All EPN strains successfully penetrated the M. diversus workers under field conditions, while the results showed that there was a significant difference between the three EPN strains. The percent mortality caused by native H. bacteriophora against was higher (43.6 ± 2.7%) than both commercial strains of S. carpocapsae (36.9 ± 1.6%) and H. bacteriophora (29.9 ± 1.4 %). These results highlight the efficiency of EPNs for the control ofM. diversus workers.

Key words: Entomopathogenic nematode, Heterorhabditis bacteriophora, Microcerotermes diversus, Biological control

One of the most common social documented in many coun- to manage subterranean termites in Iraq mainly includes application tries around the world are termites, which are reported as economic of chemical pesticide (Al-Jassany 1996). The main insecticide fre- and destructive pests, especially in Iraq. Some species of termite at- quently used in the country to control this subterranean termite is tack wood sources above the soil surface, while others cause damage Chlorpyrifos 48% TC. Estimates of the cost to import Chlorpyrifos to trees or crops underneath the soil (Josens 1983). The most eco- in 1998 and Fipronil in 2001 into Iraq were 1.1 and 2 million USD, nomically important termite species in agricultural areas of Iraq is respectively (Al-Zubaidy and Al-Jassany 2013). The ingredients of Microcerotermes diversus (Silvestri) (Blattodea: Termitidae). This these compounds have adverse environmental effects. For example, species can attack fruit trees and agricultural crops in various re- the use of high concentrations of chemical pesticides for a long time gions, as well as buildings causing massive property damage. could contaminate the crops and may cause a serious problem to Furthermore, the termite tunnel on stem trees causes weakness to the human and their agro-ecosystem (Rola and Pingali 1993, Potter and stems by consuming the carbohydrate ultimately causing collapse of Hillery 2001). Another method conducted to control the termites trees (Al-Jassany 1996). in Iraq was commercial formulation of fungi Metarhizium aniso- Currently, several control approaches are applied against this pliae which was used effectively to protect olive trees against termite costly pest, including cultural, botanical, and chemical controls. Of species such as M. diversus (Hussain et al. 2011, Al-Zubaidy and these methods, chemical control involves the use of synthetic insecti- Al-Jassany 2013). Because of concerns about the side effects of pro- cides such as bifenthrin, fipronil, cypermethrin, and deltamethrin longed pesticide application and positive results of using biocontrol (Wagner et al. 2003). Currently, the most common method applied agents, it is crucial to reduce these applications by finding alternative

© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: [email protected]. 412 Environmental Entomology, 2020, Vol. 49, No. 2 413 approaches to control subterranean termites, including the use of they were allowed to warm up to room temperature (25 ± 1°C) for entomopathogenic nematodes (EPN) (Gouge 2005). The cryptic 2 h. Also, their viability for motion was checked using dissected habitat of subterranean termite nesting and foraging primarily in microscope. the soil provides suitable conditions for application of EPNs (Epsky and Capinera 1993). Collection and Maintenance of Termites The families Steinernematidae and Heterorhabditidae (Poinar The termite species (M. diversus) were collected from infested trees 1990) are widely available commercially and have been used com- in citrus and palm orchards in central Baghdad. The termite colony monly to control a number of different soil insect pests (Kaya and used in this study was transferred to laboratory and maintained in Gaugler 1993, Kaya et al. 2006). These are obligate lethal parasitic plastic containers (80 × 70 × 70 cm) with pieces of wood in a dark pathogens of insect pests. The ‘infective juveniles (IJs) of the nema- chamber at 25 ± 1°C and 65–70% relative humidity. For the experi- todes’ live symbiotically with bacteria of the genera Xenorhabdus ment, a modified aspirator was used to collect the termite workers and Photorhabdus, respectively (Poinar 1990, Ciche et al. 2003). from the colony. The nematodes can enter the target insect pest through natural openings such as mouth, anus, and sometimes via the cuticle causing Downloaded from https://academic.oup.com/ee/article-abstract/49/2/412/5802319 by guest on 15 April 2020 Laboratory Bioassay mortality of pests (Grewal et al. 2005). The EPNs are widely distrib- EPNs’ Efficacy, Reproduction, and Penetration in G. mellonella uted in soils throughout the world (Kaya 1990). Moist, cool, and Three isolates of EPN were used in the experiments. The applied dark conditions of termite’s habitat are ideal conditions for the sur- isolates were commercial strains of S. carpocapsae (Capsanem) and vival and movement of Steinernematid and Heterorhabditid nema- H. bacteriophora (Larvanem) supplied by Koppert B.V. (Berkel en todes (Lewis et al. 1992; Kaya and Gaugler 1993, 1997; Glazer et al. Rodenrijs, the Netherlands) and H. bacteriophora IRQ.1 strain 2001). Preliminary studies have indicated that some termites are sus- (Al-Zaidawi et al. 2019). All strains were reared under laboratory ceptible to Steinernema species (Chouvenc et al. 2011). Indigenous conditions with IJs being collected. The bioassay was conducted to natural enemies are highly recommended and important to save the determine the lethal concentration of EPNs against last instar larvae time of adoption and importation of new species as well as to avoid of G. mellonella under laboratory conditions. Five concentrations possible side effects on nontarget species. For example, native EPNs of EPNs (100, 200, 400, 600, and 800 IJs/larva) were inoculated in isolated from Iran have been used effectively against fruit fly Dacus Petri dishes (9 cm in diameter) with wetted filter papers. Four rep- ciliates Loew (Diptera: Tephritidae) which was the most serious pest licates (10 larvae) per treatment (concentration) and untreated con- of cucumber, melon, musk melon, and other related fruits (Kamali trol were used in this bioassay. Each replicate received 1 ml of EPNs et al. 2013). Although applying nematodes against various insect suspension. Larvae mortality was recorded 48 h postinfection. Half pests all over the world is considered as a safe and successful control of the infected larvae were dissected under a stereomicroscope after method, the use of biological control agents against termites is still rinsing with distilled water. Next, the number of IJs found per larva very limited and more research is required to establish which biocon- was counted to evaluate the penetration of each EPN species. The trol agent is effective under which conditions (Yu 2009). other half of the infected larvae were transferred to White traps indi- The main objective of the current study was to evaluate the ef- vidually and maintained for 2 wk to provide an appropriate time for ficacy of local EPN isolated from citrus and palm orchard soils of EPN reproduction. The Petri dishes were kept and moistened with Iraq against the workers of termites in both laboratory and field distilled water periodically for nematode development. The number conditions. of emerged IJs was recorded every day until the last IJs emerged from the cadaver. Materials and Methods Soil Sampling and Isolation of EPNs Filter Bioassay of M. diversus Soil samples were collected from citrus and date palms orchards in The worker casts were collected from the termite colony and trans- central Iraq (33°17′43.5″N 44°25′30.9″E) during May–September ferred to Petri dishes with filter papers (Whatman No. 1). The nema- 2017–2018. The samples were taken from a depth of up to 20 cm, todes suspended in 1 ml of deionized water were added to each Petri using a hand spade, from underneath the canopy of the trees. The dish in six concentrations of 25, 50, 100, 200, 400, and 600 IJs/ter- bait method using G. mellonella larvae (Bedding and Akhurst 1975) mite. Only deionized water was added to Petri dishes in the untreated was used to isolate the nematodes from the soil. The colony of control. After 60 min, 20 workers of M. diversus were placed in each G. mellonella was maintained via an artificial medium (Han and plate. Then, the Petri dishes were kept under laboratory conditions Ehlers 2000) and used as suitable food source for the wax moth (25 ± 2°C) inside large plastic containers and covered with aluminum larvae. The last larval stage of this insect was used to maintain and foil for darkness. The EPN species were considered as treatment and propagate the nematodes throughout the entire period of time in each treatment had six concentrations, each concentration included this study. five replicates and the whole experiment was performed twice. The A 250 g plastic container was used for splitting the samples control treatment received 1 ml of deionized water without nema- for each site. Then, the last instar G. mellonella larvae (10 larvae/ todes. The mortality of the termites was checked 72 h after treat- container) were placed at mid-surface of each soil sample. During ment. The immobile termites which had changed color because of a 5- to 7-day period, the samples were kept under laboratory con- infection were considered dead. Dead termites were dissected under ditions with suitable moisture of soil. The containers were checked a stereomicroscope to confirm the presence of nematodes. daily, and the larvae, appearing to be infected by nematodes in terms of color and shape, were transferred to a White trap (White 1927). Wood (Sawdust) Bioassay The emerged nematodes (offspring production) were collected and Small plastic containers (4 cm diameter, 4 cm high) (Sahar Plast, used to confirm Koch’s postulates for pathogenicity. The harvested Tehran) with a hole in the lid (approximately 0.5 cm in diameter) IJs were kept at 10–12°C for experiments for less than a week be- covered with tarpaulin (poplin) to allow air exchange were used in fore they were applied in tests (Kaya and Stock 1997). Before use, this experiment. Sawdust (15 g) was added into each container as a 414 Environmental Entomology, 2020, Vol. 49, No. 2 food source (Zadji et al. 2014, Razia and Sivaramakrishnan 2016). analyzed through analysis of variance (two-way ANOVA), factorial EPNs were applied in six concentrations (25, 50, 100, 200, 400, and design (EPN species × concentrations) analyzed considering that ter- 600 IJs/termite). Then, the moisture was adjusted to 10% (w/w). mite mortality was the independent variable, while nematode strain Deionized water was added only to the containers in the untreated and concentration were the independent variables (Minitab Inc., PA, control. Four replicates with 20 termites each were prepared. After USA, 2014). The mean (± SEM) number of penetrating nematodes 10 min, the insects were placed in the containers and covered with from the two nematode species and their reproduction potential a holed lid. The containers were incubated inside closed plastic con- were compared by two-way ANOVA (concentrations × EPN spe- tainers at room temperature and covered with aluminum foil. After cies). Furthermore, the difference in penetration and reproduction 72 h, the experiment was checked and the termite mortality was re- of EPN species was tested using the Fisher LSD method (P ≤ 0.05). corded. Infected insects were dissected under a stereomicroscope to In the field test, mortality of workers was analyzed by two-way confirm the presence of EPNs. The entire test was conducted twice analyses of covariance (ANCOVA) in which nematode species and under the same conditions. concentrations were considered as the main factors, while the total number of termite workers was introduced as a covariate. Wherever Field Experiment Against Termites the covariate was significant (P ≤ 0.05), differences between the Downloaded from https://academic.oup.com/ee/article-abstract/49/2/412/5802319 by guest on 15 April 2020 The field experiment was conducted in an orchard located in the means were determined by LSD test comparisons. SAS software, ver- center of Baghdad (Al- Jadryia), Iraq (33°17′43.5″N 44°25′30.9″E) sion 9.1 was used for statistical analyses of the field test (SAS 2001). in September 2018. The field was a citrus and date palm orchard naturally infested with M. diversus. The soil type was loam and trial Results conditions were R.H:16.1, temperature: 28 ± 3°C and pH 6.2. The field had not been treated with pesticides throughout the previous Laboratory Bioassay year. Bait stations were placed around infested trees 3 mo before conducting the field experiment. Twenty-four bait stations were pre- Filter Paper: Lethal Concentration and Efficacy of EPNs on pared with each bait station being a plastic container, with four holes G. mellonella oppositely located 8 × 4 cm in diameter, and eight holes in the base All three strains of EPNs (commercial and native) caused mortality of container to allow entrance of termites. on the last instar larvae of G. mellonella. After 48 h at a rate of 100 Six natural infested citrus trees were selected and four holes were IJs/larva, G. mellonella exposed to the three EPN strains exhibited made around each tree with 30 cm in depth. Each replicate had four increased mortality as the concentrations rose. S. carpocapsae was stations for each infested tree including a control treatment. The bait more virulent (40–93%) than both commercial (30–90%) and indi- stations were buried and covered with soil up to their lids. Palm genous (24–84%) H. bacteriophora strains. There were significant frond woods were used as a desired trap for termites (Al-Jassany differences between the treatments (EPNs strains) (F2, 46 = 11.86, 1996). Before inserting the pieces of wood inside each container, they df = 2, P < 0.002). The penetration of nematodes into G. mellonella were cut down to small pieces (10 cm) and grouped together as a was significantly affected by the nematode strains. The penetration pack. Then, they were sterilized using electric oven at 120°C for 48 h of IJs increased by raising the concentrations of nematodes ran- and placed inside the bait station. The bait stations were checked ging within 1.7–56, 1.8–64.5, and 1.4–39 IJs/larva for commercial regularly to confirm their invasion by termites. The stations took 3 H. bacteriophora, S. carpocapsae, and native H. bacteriophora, re- mo to be infected with termites. Three strains of nematodes includ- spectively. There was a significant difference between EPN strains ing two commercial strains of S. carpocapsae and H. bacteriophora (F2, 138 = 6.00, df = 2, P < 0.01) and concentrations (F6, 138 = 96.79, and indigenous H. bacteriophora were used in the test. A prelim- df = 6, P < 0.001). inary test was performed to evaluate the application rate. EPNs were applied on the wood surface within the bait stations at two rates Virulence of EPNs on M. diversus Workers in Filter Paper and 2 (400 and 600 IJs/ cm ) using a manual sprayer. After IJs suspension Wood Bioassay was added, the final moisture of station was 10% of each replicate The wood bioassay facilitates infection of termite by nematodes as (station). The control treatment was received only 5 ml of sterilized indicated by lower LC for all EPNs compared to the filter paper water. After treatment, the lids covered the stations without soil; the 50 assay (Table 1). In the filter paper bioassay, the LC50 value of com- conditions were 28 ± 5°C and relative humidity 65 ± 10%. After mercial S. carpocapsae (57.9 IJs/larva) revealed greater virulence 1 wk, mortality was determined. than both commercial H. bacteriophora (139.6 IJs/larva) and native H. bacteriophora (274.2 IJs/larva) and diminished upon prolonga- Statistical Analysis of Data tion of the exposure time. In the wood bioassay, LC50 value for Insect mortality was adjusted for mortality in the control treatments commercial S. carpocapsae (15.7 IJs/larva) was lower than that of using Abbott’s correction (Abbott 1925). Normality and homo- both commercial and native commercial H. bacteriophora strains geneity of variance were tested by Kolmogorov–Smirnov test and (52.6 and 60.8 IJs/larva, respectively). Rayan–Joiner test (Minitab 2014), respectively. The corrected mor- All EPN strains were effective against the termite workers and tality was subjected to multifactorial analysis of variance (ANOVA), their virulence was statistically different between the three EPN where the mean was separated by the LSD test (Minitab 2014). To strains in both filter paper and wood experiments. measure susceptibility of M. diversus workers to EPN species in the In the filter paper assay, the corrected mortality of workers in-

Petri dish assay, mortality resulting from nematode species and con- creases with IJ concentrations (F5, 54 = 186.5, df = 5, P < 0.003). The centration were subjected to Probit analysis to assess the LC50 for results of bioassay showed significant differences between the tested each nematode at P ≤ 0.05. It was done by comparing the propor- isolates; it was higher for S. carpocapsae (80 ± 8.3%) than for both tions of dead insects between treatments and control using POLO commercial H. bacteriophora (96.2 ± 8.9%) and native H. bacteri- plus program (LeOra Software 2006). Quantitative data obtained ophora (67.5 ± 6.8%) (F2, 54 = 132.5, df = 2, P < 0.002), which was from laboratory tests with both filter paper and wood trial were clear at high concentrations (400 and 600 IJs /termite). In addition, Environmental Entomology, 2020, Vol. 49, No. 2 415 there was a significant difference in the interaction between the significantly enhanced upon elevation of the concentrations of IJs in nematode species and concentrations (F10, 54 = 2.6, df = 10, P < 0.01). all tested EPNs species when using both filter paper and wood. The maximum mortality (96.2 ± 8.9) was achieved when they were In the filter paper bioassay, the number of IJs of S. carpocapsae treated with S. carpocapsae 600 IJs/termite (Fig. 1). was greater than of both H. bacteriophora species (commercial and

On the other hand, the results of wood bioassay showed that native) at all concentrations used in this bioassay (F2, 72 = 46, df = 2, the total percentage of termite mortality induced by three EPN P < 0.01) (Fig. 3). The IJs number ranged within 15.2–72 IJs/ter- strains was greater than that caused by the same EPN strains in the mite for S. carpocapsae compared with 5.6–66.6 and 4.8–64.4 for filter paper experiment. The mortality rate caused by S. carpocap- both commercial and native H. bacteriophora respectively. In add- sae (57.5–97 ± 6.1%) was significantly greater than that obtained ition, the concentrations of EPN species significantly affected the IJs by both commercial (25–90 ± 11.9%) and native H. bacteriophora penetration (F5, 72 = 249.8, df = 5, P < 0.001). Also, the interaction

(27.5–92 ± 11.1%), especially at low concentrations (F2, 54 = 36.6, between EPN species and concentrations significantly affected the df = 2, P ≤ 0.002). Also, the concentrations of three nematodes penetration (F10, 72 = 2.2, df = 10, P < 0.02). significantly affected the workers’ mortality (F5, 54 = 127.3, df = 5, Similarly, in the wood bioassay, the total number of IJs pene- P ≤ 0.001). Furthermore, the interactive effect of nematode strains trated into termite workers was significantly affected by EPN spe- Downloaded from https://academic.oup.com/ee/article-abstract/49/2/412/5802319 by guest on 15 April 2020 and IJs concentrations were statistically different (F10, 54 = 5.4, cies. A greater number was recorded for S. carpocapsae species df = 10, P < 0.00) among all EPN species (Fig. 2). The percentage at concentration 600 (74.6 IJs/termite) which had no significant mortality caused with all tested EPNs was significantly influenced effect for both commercial (70.2) and native (70.6) H. bacterio- by substrates. The percentage mortality was greater for all exam- phora at the same concentration. However, low concentrations (25 ined EPN species under wood conditions than through filter papers and 50 IJs/termite) showed a considerable difference between the

(F1, 18 = 71.6, df = 1, P < 0.00) (Fig.2). EPN species (F2, 72 = 19.6, df = 2, P < 0.05). Also, the IJs con-

centration significantly affected the IJs penetration (F5, 72 = 145.4,

Penetration Value of EPNs Against Termite on Filter Paper and df = 5, P < 0.05) where the two factors showed an interaction (F10,

Wood Bioassays 72 = 2.29, df = 10, P < 0.05). High concentrations of EPNs caused In this experiment, there was a correlation between penetration and greater IJs penetration into termite workers (Fig. 4). There was a IJs concentrations; the invasion of IJs into the termite workers was positive correlation between penetration and mortality across EPN

Table 1. The calculated LC50 value (IJs/termite) for commercial H. bacteriophora, S. carpocapsae and native H. bacteriophora on termite workers in filter paper and in wood bioassays.

EPN species LC50 (95% confidence limits P-valuea ×2b Slope ± SEc Experiment

H. bacteriophorad 139.6 (104.4–187.1) 0.4 17.5 0.95 ± 0.12 Filter paper S. carpocapsae 57.9 (38.3–78.7) 0.6 24.7 1.07 ± 0.13 H. bacteriophoraf 274.2 (191.6–452.2) 0.4 10.6 0.71 ± 0.12 H. bacteriophorad 52.6 (37.7–68.2) 0.5 20.5 1.74 ± 0.22 Wood S. carpocapsaee 15.7 (5.17–27.6) 0.7 10.3 1.21 ± 0.23 H. bacteriophoraf 60.8 (43.4–79.5) 0.5 7.5 1.61 ± 0.21

aP values represent the probability of the slope. bPearson × 2 of the slope. cSE indicates standard error. dCommercial H. bacteriophora. eCommercial S. carpocapsae. fNative H. bacteriophora.

Fig. 1. Mean percent mortality (±SE) of M. diversus workers exposed to commercial S. carpocapsae and H. bacteriophora and indigenous H. bacteriophora after 72 h from treatment (filter bioassay). Means that do not share a letter are significantly differentP ( < 0.05). 416 Environmental Entomology, 2020, Vol. 49, No. 2 Downloaded from https://academic.oup.com/ee/article-abstract/49/2/412/5802319 by guest on 15 April 2020

Fig. 2. Mean percent mortality (±SE) of M. diversus workers exposed to commercial S. carpocapsae and H. bacteriophora and indigenous H. bacteriophora after 72 h from treatment (wood bioassay). Means that do not share a letter are significantly different P( < 0.05).

Fig. 3. Means (±SE) IJs of EPN in infected workers of M. diversus at 48 h exposure in filter paper bioassay. Bars with same letter are not significantly difference (P < 0.05). species and concentrations as shown in Supp File 1 (online only) reproduction rate was recorded for commercial H. bacteriophora at (R2 = 0.54, P < 0.01). 600 IJs (27,700 IJs/ termite) compared with the IJs which emerging from both commercial S. carpocapsae (24,800 IJs/termite) and indi- EPNs’ Reproduction From M. diversus Workers genous H. bacteriophora (17,300 IJs/ termite) at the same concen- All tested EPN species (commercial and native) successfully repro- tration (Fig. 6). duced and emerged from workers of M. diversus within 9–15 d. In the filter paper bioassay, the reproduction rates ranged within Field Experiment Against Termites 6,100–27,600, 6,300–2,300, and 5,500–16,600 IJs/termite for com- Under the field conditions, after 1 wk from the application, the mor- mercial H. bacteriophora, commercial S. carpocapsae and native tality results showed that there was a significant difference between H. bacteriophora, respectively (Fig. 5). The results were statistically the three EPN species (F2, 11 = 16.2, df = 2, P = 0.005) (Fig. 7). The significantly different among the EPN species (F2, 72 = 114.7, df = 2, mortality slightly increased with elevation of the IJs concentra- P < 0.05). In addition, the reproduction rates were affected by dif- tions for all tested EPN species, but the effect was not statistically ferent concentrations of experimental EPN species (F = 536.2, 5, 72 significant (F1,11 = 1.29, df = 1, P = 0.28). The native H. bacterio- df = 5, P < 0.05). Also, the interaction between EPN species and IJs phora caused higher mortality (43.6 ± 2.7%) than both commercial concentrations (F10, 72 = 34.7, df = 10, P < 0.05) had a significant ef- S. carpocapsae (36.9 ± 1.6%) and H. bacteriophora (29.9 ± 1.4 %) fect on the total number of IJs emerging from cadavers. did. The mortality rate of termites ranged within (35.8–43.6 %), Similar results were obtained in the wood bioassay. The difference (27.8–29.9 %), and (33.8–36.9 %) for native, commercial H. bac- between the treatments (EPN species) was significant (F2,72 = 76.2, teriophora, and S. carpocapsae, respectively. Also, there were no sig- df = 2, P < 0.05), especially at high concentrations of EPN species nificant interactions between nematode species and concentrations (400 and 600 IJs/ termite). Furthermore, the concentration signifi- for all EPN species (F 2,11 = 0.33, df = 2, P = 0.72). Nonetheless, the cantly affected (F5,72 = 441.7, df = 5, P < 0.05) the reproduction rate total number of termites (209–426 termite/station) significantly af- among all EPN species. There was a significant interaction among fected the mortality value (F1,11 = 146.01, df = 1, P < 0.001). There EPNs and IJs concentrations on reproduction values. The highest was a positive correlation between the total number of termites into Environmental Entomology, 2020, Vol. 49, No. 2 417 Downloaded from https://academic.oup.com/ee/article-abstract/49/2/412/5802319 by guest on 15 April 2020

Fig. 4. Means (±SE) IJs of EPN in infected workers of M. diversus at 48 h exposure in wood bioassay. Bars with same letter are not significantly difference (P < 0.05).

Fig. 5. Mean reproduction (±SE) of three EPN species of commercial H. bacteriophora and S. carpocapsae and indigenous H. bacteriophora within M. diversus workers in filter paper bioassay. Means that do not share a letter are significantly differentP ( < 0.05). the stations with mortality values (R2 = 0.79, P < 0.01) as shown in and absence of any ecological or behavioral barriers (Gaugler Supp File 2 (online only). et al. 1997). The results of the current study clearly showed that the mortality rate of termite increased within elevation of the IJs concentrations Discussion and vice versa for all tested EPN species. This has obviously been The nematode virulence test under laboratory or field conditions illustrated by many researchers who have reported that IJs concen- revealed that entomopathogenic nematode species showed diverse trations and host mortality have a positive relationship with each levels of pathogenicity against termite workers. This study pro- other (Glazer and Navon 1990, Peters and Ehlers 1994, Razia and vides the first data on susceptibility of M. diversus workers to in- Sivaramakrishnan 2016). The pathogenicity bioassays under la- digenous and commercial EPNs. Each EPN strain was evaluated boratory conditions indicated that M. diversus (Silvestri) workers for its pathogenicity based on their ability to kill the termite and were highly susceptible to the commercial and indigenous EPN spe- to penetrate as well as reproduce IJs within the body of cadavers. cies used in this study. These results agreed with Wang et al. (2002) The ability of indigenous EPN species to influence and kill termite who found that both that S. carpocapsae Weiser and H. bacterio- under both conditions (laboratory and field) was an important phora Poinar showed high levels of pathogenicity against termite finding in the present study. Although the indigenous species had workers of R. flavipes under laboratory conditions. These results less virulence (10–75%) compared with two commercial EPN spe- are in congruence with other reports of EPN efficacy against ter- cies under laboratory conditions, this species had high virulence mites such as H. bacteriophora (Mauldin and Beal 1989), S. glaseri against termite under field conditions. Generally, virulence of EPN (Danthanarayana and Vitarana 1987), and S. carpocapsae (Epsky species under laboratory conditions could be higher than in field and Capinera 1988). In addition, H. bacteriophora and S. carpocap- conditions because of the optimal environmental circumstances sae showed high mortality against termites, along with Heterotermes 418 Environmental Entomology, 2020, Vol. 49, No. 2 Downloaded from https://academic.oup.com/ee/article-abstract/49/2/412/5802319 by guest on 15 April 2020

Fig. 6. Mean reproduction (±SE) of three EPN species commercial H. bacteriophora and S. carpocapsae indigenous H. bacteriophora within M. diversus workers in wood bioassay. Means that do not share a letter are significantly different P( < 0.05).

Fig. 7. Means percent mortality (±SE) of M. diversus workers exposed to three commercial H. bacteriophora and S. carpocapsae and indigenous H. bacteriophora in field conditions. Means that do not share a letter are significantly differentP ( < 0.05). aureus under laboratory conditions (Yu et al. 2006). In contrast, It is notable that in the current study, the virulence of all tested Manzoor (2012) reported that neither H. bacteriophora nor S. car- EPN species under wood conditions was greater compared to the pocapsae had a significant effect on termite mortality when used filter paper conditions (Plate). The differences between wood and alone against Reticulitermes flavipes termite. plate bioassays agreed with other studies; they reported that the LC50 In this study, S. carpocapsae was the best of the three strains of H. bacteriophora within a natural medium such as soil (45.89 2 2 tested in terms of infectivity. Its LC50 value was lower than the value IJs/cm ) was lower than by a filter paper (325.68 IJs/cm ) (Kamali for H. bacteriophora strains against M. diversus workers in petri et al. 2013). Similarly, in previous studies, Xenorhabdus nematoph- dishes and containers with sawdust. The current outcomes on the ila, which is associated with S. carpocapsae, indicated higher efficacy virulence of S. carpocapsae are in line with the results reported by in sand than in filter paper. The Xenorhabdus cells were found to be Maketon et al. (2010). They documented that S. carpocapsae caused more effective against G. mellonella under sand conditions as com- 100% mortality to adults of German cockroaches as compared to pared with filter paper (Mahar et al. 2005). other nematodes used in this study after 7 d from treatment. In add- Penetration trait of EPN species is a crucial characteristic which ition, S. carpocapsae showed great virulence on the same insects could be considered as a criterion for successful primary and sec- compared with others ENP species with high mortality on females ondary infestation (Susurluk and Ehlers 2008, Salame et al. 2010). In of B. germanica being found on the first day of exposure (Baker et al. the present study, all tested EPN species (commercial and native) ef- 2012). Amongst all known EPN species, S. carpocapsae hast most fectively penetrated into M. diversus workers under laboratory con- often been reported to infect adult insects besides larvae which might ditions. The penetration value of S. carpocapsae was greater than explain why it is also superior in infecting termite workers. that of both commercial and indigenous H. bacteriophora species, Environmental Entomology, 2020, Vol. 49, No. 2 419 especially at low concentrations (25 and 50 IJs/termite). The value in disagreement with the findings of a previous study conducted increased by elevating the IJs concentrations within both plate and by Epsky and Capinera (1988). They reported that S. carpocapsae wood bioassays. In previous studies, the number of heterorhabditid had no significant effect on Reticulitermes tibialis using 107 IJs/ m2 IJs which penetrated into shoot borer larvae, Chilo infuscatellus under field conditions. Also, the outcomes the current study are (Lepidoptera: Crambidae), was lower than that of steinernematid dissimilar with the results of Mauldin and Beal (1989) who found (Sankaranarayanan et al. 2011). The results of the current study on that Reticulitermes flavipes species was not susceptible to EPNs of penetration were also similar to those reported by recent researchers H. bacteriophora when IJs of these nematodes were applied on in- who observed that the level of penetration for S. carpocapsae was fested wood under field conditions. Termites are more active in their far higher than that of other tested EPN species such as S. glaseri, natural habitat, which could be a reason of this outcome. This result S. feltiae, S. riobravis and two strains of H. bacteriophora (Epsky agreed with Yee and Lacey (2003) who documented that EPNs viru- and Capinera 1993, Caroli et al. 1996, Ricci et al. 1996). lence was lower than that under laboratory conditions. Reproduction is another important characteristic for EPN popu- The moisture percentage in the bait stations was less than that lation for enhancing their opportunity to establish and to persist suc- in both filter paper and sawdust assay. It was 40 ± 10% for stations, cessfully in the host’s environment (Phan et al. 2005). In addition, while it was 65 ± 5% for both sawdust and filter paper assay. This Downloaded from https://academic.oup.com/ee/article-abstract/49/2/412/5802319 by guest on 15 April 2020 multiplication and recycling of EPN species play an essential role in could provide appropriate conditions for examining the behavior of their persistence and effectiveness in the host habitat which is con- nematodes to find their host effortlessly. However, foraging strategy sidered an effective factor for pest control (Harlan and de Wet 1971, of EPNs could influence their infectivity in controlling insect pests Georgis and Hague 1981). The three EPN strains used in this study (Lezama-Gutie´rrez et al. 2006). Although the nictation strategy of reproduced well in M. diversus workers under both filter paper and S. carpocapsae is important factor for it to be more effective in soil wood conditions. However, the reproduction rate varied among the (Yee and Lacey 2003), high levels of energy consumed could be a species; the highest production rate of IJs was obtained with com- reason why movement of S. carpocapsae diminished which could mercial H. bacteriophora recorded at high IJs concentrations (400 influence their efficacy in controlling insect pests. This explanation and 600 IJs/termite), a trait which might be desirable for commer- can be a reason why H. bacteriophora was more virulent than S. car- cial issues. However, the progeny emerging from termite cadavers was pocapsae. The replicates received optimal level of relative humidity similar among the tested EPN strains at low concentrations (25, 50, and temperature under the laboratory conditions. This could set the 100, and 200 IJs/termite) within both plate and wood bioassays. The ground for for IJs’ activity and ultimately enhance their efficacy. One results of this study are also in line with the findings reported by Salari issue is the importance of bait station condition for improving the et al. (2014). They documented that the reproduction rate of H. bac- field efficacy of EPNs. Based on the results, filling the bait station teriophora was greater than that of S. carpocapsae when these were with saw-dust or/and sand provide a better matrix for nematode ac- applied against Leopard moth borer, Zeuzera pyrina (Lepidoptera: tivity. This would also be important for further studies on this topic. Cossidae) larvae under laboratory conditions. These differences could Integrated protection against termite is still an urgent issue. be due to differences in methodology (Barbosa-Negrisoli et al. 2009). Therefore, further experiments are currently ongoing to assess the Note that field studies using EPN species to manage and con- compatibility of indigenous EPN species with some selective pesti- trol termite species are limited not just in Iraq but also in different cides that have been registered. The goal is to provide accurate re- countries all over the world (Wilson-Rich et al. 2007, Baimey et al. commendations on using this agent with a chemical pesticide within 2015). The EPNs virulence and their efficacy were far lower under an integrated method. In addition, temperature plays a critical role field conditions than under laboratory conditions (Yee and Lacey in EPNs activity and impacts the mortality rate (Hazir et al. 2001). 2003). The suspension was sprayed directly on the infested bait sta- Thus, the temperature of bait stations should be evaluated to provide tions, while the nematodes that could be lost with running water a recommendation of optimal temperature for EPNs. were not recorded in this study. After 7 d termite mortality was as- sessed with the captured termites within each station. There results showed that the three tested EPN strains caused lower mortality rate Supplementary Data on workers in the field than in laboratory conditions which ranged Supplementary data are available at Environmental Entomology within (35.8–43.6%), (27.8–29.9%), and (33.8–36.9%) for native, online. commercial H. bacteriophora, and S. carpocapsae, respectively. The termite mortality had a positive correlation with the total number of termite bait stations; increased number of termites within the bait Acknowledgments station could provide a greater opportunity for IJs to penetrate and This manuscript is a part of Ph.D. dissertation of the first author which was sup- infect termites. Indeed, a large number within a station showed high ported by a grant to JK (p.3/45368) provided by research deputy of Ferdowsi mortality at these stations. Similarity, Dolinski and Lacey (2007) re- University of Mashhad ‘FUM’. We thank Ferdowsi University of Mashhad and ported that colonies of Neotermes, which attack palms and other the Ministry of Science and Technology for financial support this own initia- trees in the South Pacific islands, can be successfully controlled by tive project. The authors appreciate staff members of Biocontrol and Insect Heterorhabditis sp. In addition, The colonies of Glyptotermes dil- Pathology Laboratory in School of Agriculture at Ferdowsi University spe- cially Reyhaneh Darsouei and Shokoofeh Kamali for their assistance. Finally, atatus were successfully eliminated when Heterorhabditis sp. Was the authors also thank the editors and three anonymous reviewers for pro- applied in 4,000 and 8,000 ml of nematode at rates of 40 and 30 ml viding insightful comment to improve our manuscript. per tea bush, respectively (Danthanarayana and Vitarana 1987). However, the results of the current study are incongruent with the outcomes reported by Koppenhöfer et al. (2000). They docu- References Cited mented that nematodes have less ability to control subterranean Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. species because of their behavior. Meanwhile, the repellence could J. Econ. Entomol. 18: 265–267. be the significant reason that could affect the virulence of EPN spe- Al-Jassany, R. 1996. 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