Sustainable Grain Protectants: Recruiting Entomopathogenic Nematodes against Stored-Product Coleopterans

Abstract

Stored-product commodities are attacked by numerous insect species. The adulticidal effects of entomopathogenic nematodes (EPNs) on grains remain uninvestigated. Thus, in the current study, seven doses of the EPNs Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae), and Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) were inoculated on wheat kernels against adults of Trogoderma granarium Everts (Coleoptera: Dermestidae), Tenebrio molitor L. (Coleoptera: Tenebrionidae), and Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae). Complete mortality (100.0%) of T. granarium was recorded after exposure for eight days to the highest dose of 50,000 Infective Juveniles/mL (IJs/mL) of all tested EPN species. At the same exposure interval, 62.2%, 85.6%, and 76.7% of T. molitor were killed by 50,000 IJs/mL of H. bacteriophora, S. carpocapsae, and S. feltiae, respectively. The highest mortality of A. diaperinus (11.1%) was documented eight days post-exposure to 50,000 IJs/mL of H. bacteriophora. In general, T. granarium was highly susceptible, followed by T. molitor and A. diaperinus. Concerning EPN species, S. carpocapsae exhibited the highest insecticidal capacity, followed by S. feltiae and H. bacteriophora. Trogoderma granarium and T. molitor can be sufficiently managed by the highest dose of 50,000 IJs/mL of all three EPNs and by S. carpocapsae, respectively. However, A. diaperinus was not affected by any EPNs.

1. Introduction

Entomopathogenic nematodes (EPNs) are obligate or on occasion facultative roundworm insect parasites that can be employed within a sustainable control regime of stored-product pests since they are not harmful for humans, non-target organisms, and environment [1,2,3,4,5,6,7,8,9,10,11,12,13]. They penetrate the host body through natural entrances i.e., spiracles, anus, oral cavity, or sometimes the cuticular intersegmental membrane, when they reach the third, infective stage of juveniles (IJ) [14,15]. These organisms are carriers of intestinal entomopathogenic bacteria that belong to the genera Photorhabdus (Enterobacterales: Morganellaceae) and Xenorhabdus (Enterobacterales: Morganellaceae) [15,16,17,18,19]. These microorganisms exit EPN bodies through the anus, oral cavity or by defecation. Then, they proliferate into the nutrient-rich haemocoel of insects [14,17,18,20,21]. The microorganisms secrete proteins and toxins that cause septicemia and eventually kill the insects [22,23,24,25]. Additionally, Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae) was recently reported to be able to kill insects by releasing venom proteins [17].

Entomopathogenic nematodes are members of two genera: Steinernema (Steinermatidae) and Heterorhabditis (Heterorhabditidae). Their life cycle comprises six stages: egg, the first, second, third, and fourth juvenile stages (indicated as J1-4), and adult. Stage J3 is also referred to as IJ (infective juvenile), since it is the stage at which the EPN enters the host body. Infective juveniles are soil-inhabiting, free-living, and non-feeding, and they are the only stage occurring outside the body of the host insect [9]. When they enter the host body, IJs release mutualistic bacteria that produce toxins and suppress the host’s immune system, causing its septicemic or toxemic death, within 24–48 h. Insects that die on account of the Steinernema–Xenorhabdus duo become pale or greyish depending on the species of the EPN, whereas those killed by the Heterorhabditis–Photorhabdus duo acquire a dark-red color due to the production of luminescent anthraquinone pigments by the bacteria [26,27].

To date, the most effective EPN species are S. carpocapsae, Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), and Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) [5,11]. In the past, these three EPNs have been evaluated against several stored-product insects. Ramos-Rodríguez et al. [4] tested S. carpocapsae, S. feltiae and Steinernema riobrave Cabanillas, Poinar & Raulston (Rhabditida: Steinernematidae) on filter paper against adults, pupae and larvae of Ephestia kuehniella Zeller (Lepidoptera: Pyrallidae), Trogoderma variabile Ballion (Coleoptera: Dermestidae), Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae), Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), Tenebrio molitor L. (Coleoptera: Tenebrionidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), as well as Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) and Sitophilus oryzae (L.) (Coleoptera: Curculionidae) adults. The least susceptible species was S. oryzae, while the most susceptible was E. kuehniella. Similarly, Yuksel et al. [28] studied two strains of S. feltiae and two strains of H. bacteriophora, inoculated on filter paper, against adults of R. dominica and S. oryzae and larvae of E. kuehniella. Eight days post-exposure, E. kuehniella larvae were the most susceptible species followed by S. oryzae and R. dominica adults. Furthermore, three isolates of S. feltiae applied on filter paper caused variable mortality rates to S. oryzae adults at variable temperatures [29].

Trogoderma granarium Everts (Coleoptera: Dermestidae), T. molitor, and Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) are species that cause high qualitative damage to stored commodities, as well as allergies that can potentially harm public health [30,31,32,33,34,35,36,37]. More specifically, T. granarium, T. molitor, and A. diaperinus are documented to infest 94, 46 and 77 commodities, respectively, e.g., grains, cereals, seeds, nuts, spices, tobacco and products of animal origin [33]. Trogoderma granarium is a primary stored-product pest, while the other two species are secondary pests [32,38]. All of them are cosmopolitan and can usually be found in storage facilities, grains, pet shops, and mills [32,33,38]. Research efforts tend to natural insecticides and/or biological control for their efficient management to avoid environmental and human health risks [39,40,41,42,43]. Therefore, even though essential oils (EOs), nanoemulsions (NEs), microemulsions (MEs), diatomaceus earths (DEs), and entomopathogenic fungi have been used as grain protectants [42,43,44,45,46,47,48,49], there is little knowledge about the efficacy of EPNs as grain protectants [5,11].

Previously, Athanassiou et al. [5] inoculated H. bacteriophora, S. carpocapsae and S. feltiae on wheat against E. kuehniella larvae, R. dominica adults, S. oryzae adults, and Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) adults or larvae at 20 °C and 30 °C. Entomopathogenic nematodes provided low-to-moderate mortalities at both temperatures. Similarly, Karanastasi et al. [11] studied the aforementioned EPN applied on wheat kernels to manage the tolerant T. granarium larvae [43]. Results were promising for both sizes of larvae, since mortality reached 98.9% and 87.8% for small and large larvae, respectively, after an 8-day exposure to 50,000 IJs/mL S. feltiae. Yet, there are no data about the adulticidal effects of EPNs H. bacteriophora, S. carpocapsae, and S. feltiae against T. granarium, T. molitor, and A. diaperinus adults. Thus, the current study examines the efficacy of these three EPNs inoculated on stored wheat against adults of the aforementioned coleopterans.

2. Materials and Methods

2.1. Insects and Commodity

All three insect species were kept at the Laboratory of Agricultural Zoology and Entomology (Agricultural University of Athens) at 30 °C, 65% relative humidity (RH) and an absence of light [44,45,50,51]. To culture T. granarium, whole wheat was used as the rearing medium [45]. To culture T. molitor, oat bran plus potato cuts were used as the rearing medium [44]. Finally, to culture A. diaperinus, yeast (1/4) plus wheat bran (3/4) plus apple cuts were used as the rearing medium [51]. Potatoes or apples are used to enhance the moisture content of the culture [44,45,51]. The unsexed selected adult individuals of T. granarium, A. diaperinus and T. molitor were <1, 7 or 14 days old, respectively [44,45,51]. The commodity that was used for the experimentations was Triticum durum Desf. (Poales: Poaceae) (var. Claudio). The grains were clean from pests, pesticides and impurities. Prior to the trials, the wheat moisture content was estimated at 11.8% with a calibrated mini GAC plus moisture meter (Dickey-John Europe S.A.S., Colombes, France).

2.2. Entomopathogenic Nematodes

All tested EPNs that were utilized in the current study were provided by Bio-insecta (Nea Silata, Greece).

2.3. Bioassays

After conducting preliminary trials by using all examined coleopterans, the EPN doses that were used in the current study were 100 IJs/mL, 500 IJs/mL, 1000 IJs/mL, 5000 IJs/mL, 10,000 IJs/mL, and 50,000 IJs/mL. The experiments were conducted in glass vials (12.5 cm height; 7.5 cm diameter). Lids of the vials had one circular opening (1.5 cm diameter) for the ample aeration of their inside space. The upper internal side of these vials was covered with polytetrafluoroethylene (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to prevent insects from escaping. Three 10 g wheat samples were placed inside each vial after being weighed by a Precisa XB3200D electronic compact balance (Alpha Analytical Instruments, Gerakas, Attica, Greece), conducted on a filter paper. Afterwards, the wheat was inoculated with 1 mL of tap water that contained the aforementioned doses of EPNs, and subsequently the grains were hand-shaken for one minute to ensure the even distribution of the EPNs to the whole mass of the commodity [11]. The tested doses were equivalent to 10 IJs/adult, 50 IJs/adult, 100 IJs/adult, 500 IJs/adult, 1000 IJs/adult, and 5000 IJs/adult [5,11]. The moisture of kernels was then recalculated, reaching 13.7%. Vials that were inoculated only with tap water were used as control. Thereupon, ten adults of each species were separately inserted into each vial. The prepared containers were conveyed into incubators at 30 °C and 65% RH until the end of the experimental period. Mortality data were acquired after 4 days and 8 days under an Olympus SZX9 stereomicroscope (Bacacos S.A., Athens, Attica, Greece). An individual was declared dead after no motion was detected when the adults were poked with a brush. The dead insects were withdrawn from the vials and subsequently dissected (Figure 1) to confirm the presence of EPNs in the dead insect bodies. The entire bioassay was repeated two more times using fresh EPNs, insects, vials and grains. In total, 5670 individuals were used in the experiments (3 replications × 3 subreplications (i.e., three dishes with wheat inoculated with a given dose of EPN) × 10 individuals × 7 doses/control × 3 EPN species × 3 insect species).

2.4. Data Analysis

Control mortality was <5%; hence, no correction of data was required. To acquire a normal variance, prior to the analysis, data were converted to log (x + 1) [52,53]. The repeated-measures model (MANOVA) was utilized for each insect separately [54]. The repeated factor, response variable and main effects were exposure, mortality and nematode dose/nematode species, respectively. Means were discriminated with the Tukey–Kramer test HSD (compare all pairs) at the 5% significance level [55]. Analysis was conducted on JMP 16.2 software [56].

3. Results

3.1. Mortality of Trogoderma granarium Adults

All main effects and interactions of T. granarium adults between and within exposure intervals were significant except for the exposure × nematode species × nematode dose (

Table 1. MANOVA parameters for main effects and associated interactions for mortality of Trogoderma granarium, Tenebrio molitor, and Alphitobius diaperinus adults between and within exposure intervals (error DF = 144).

Effect		Trogoderma granarium		Tenebrio molitor		Alphitobius diaperinus
Between exposure intervals
Source	DF	F	p	F	p	F	p
Intercept	1	39,131.4	<0.01	2600.6	<0.01	29.9	<0.01
Nematode species	2	26.3	<0.01	4.3	0.02	4.4	0.02
Nematode dose	5	27.0	<0.01	33.4	<0.01	8.3	<0.01
Nematode species × nematode dose	10	2.0	0.04	1.4	0.19	1.0	0.41
Within exposure intervals
Exposure	1	571.0	<0.01	371.5	<0.01	25.3	<0.01
Exposure × nematode species	2	10.6	<0.01	18.2	<0.01	9.2	<0.01
Exposure × nematode dose	5	5.5	<0.01	1.4	0.21	7.5	<0.01
Exposure × nematode species × nematode dose	10	1.1	0.36	9.1	<0.01	1.9	0.05

). For the first 4 days of the experiment, all doses of H. bacteriophora, S. carpocapsae and S. feltiae provided low-to-moderate mortalities, i.e., 21.1–36.7% at 100 IJs/mL and 46.7–72.2% at 50,000 IJs/mL (

Table 2. Mean mortality of (% ± SE) Trogoderma granarium adults after 4 and 8 days on wheat treated with Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema feltiae applied at six doses. Within each row, means followed by the same uppercase letter are not significantly different in all cases. DF = 5, 53; Tukey–Kramer test at p = 0.05. Within each column, means followed by the same lowercase letter are not significantly different, in all cases DF = 5, 53; Tukey–Kramer test at p = 0.05.

		100 IJs/mL	500 IJs/mL	1000 IJs/mL	5000 IJs/mL	10,000 IJs/mL	50,000 IJs/mL	F	p
4 Days	H. bacteriophora	21.1 ± 3.5 Cc	23.3 ± 3.7 BCc	28.9 ± 3.1 ABCe	51.1 ± 7.2 ABb	53.3 ± 4.4 Acd	55.6 ± 3.4 Ac	7.3	<0.01
	S. carpocapsae	36.7 ± 5.0 Bbc	48.9 ± 5.9 ABb	51.1 ± 5.6 ABcd	52.2 ± 6.2 ABb	68.9 ± 4.2 Abc	72.2 ± 3.6 Ab	7.0	<0.01
	S. feltiae	23.3 ± 4.1 Cc	24.4 ± 1.8 BCc	37.8 ± 2.2 ABde	38.9 ± 4.2 ABb	42.2 ± 7.0 ABd	46.7 ± 5.0 Ac	5.7	<0.01
8 Days	H. bacteriophora	55.6 ± 5.0 Bab	57.8 ± 3.6 Bab	58.9 ± 4.6 Bbc	83.3 ± 4.7 Aa	84.4 ± 2.4 Aab	100.0 ± 0.0 Aa	17.6	<0.01
	S. carpocapsae	76.7 ± 4.4 Ba	78.9 ± 4.6 Ba	86.7 ± 2.4 ABa	96.7 ± 1.7 Aa	97.8 ± 1.5 Aa	100.0 ± 0.0 Aa	11.4	<0.01
	S. feltiae	61.1 ± 5.6 Cab	73.3 ± 6.5 BCa	80.0 ± 3.7 ABCab	84.4 ± 2.9 ABa	85.6 ± 4.4 ABab	100.0 ± 0.0 Aa	7.0	<0.01
	F	11.1	31.1	26.2	16.1	18.9	34.6
	p	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01

). About half of the individuals (51.1%) were dead after exposure for 4 days to 1000 IJs/mL S. carpocapsae. Eight days post-exposure, all EPNs provided complete mortality when the dose was 50,000 IJs/mL. Steinernema carpocapsae killed 96.7% and 97.8% of T. granarium adults at 5000 IJs/mL and 10,000 IJs/mL. The lowest dose of the three EPN species (100 IJs/mL) caused 55.6–76.7% mortality at the end of the experiments. Control mortality (0 IJs/mL) provided 1.1% mortality at the end of the experiments.

3.2. Mortality of Tenebrio molitor Adults

The main effects were significant between the exposure intervals for T. molitor adults. Within the exposure intervals, exposure × nematode species and exposure × nematode species × nematode dose were significant (Table 1). Mortalities caused by the three EPNs were low at all tested doses at 4 days post-exposure, not exceeding 7.8% for the lowest dose of 100 IJs/mL and 27.8% for the highest dose of 50,000 IJs/mL (

Table 3. Mean mortality of (% ± SE) Tenebrio molitor adults after 4 and 8 days on wheat treated with Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema feltiae applied at six doses. Within each row, means followed by the same uppercase letter are not significantly different in all cases. DF = 5, 53; Tukey–Kramer test at p = 0.05. Within each column, means followed by the same lowercase letter are not significantly different in all cases. DF = 5, 53; Tukey–Kramer test at p = 0.05.

		100 IJs/mL	500 IJs/mL	1000 IJs/mL	5000 IJs/mL	10,000 IJs/mL	50,000 IJs/mL	F	p
4 Days	H. bacteriophora	7.8 ± 2.2 Bab	12.2 ± 4.7 ABab	14.4 ± 1.8 ABa	16.7 ± 1.7 ABc	17.8 ± 2.8 ABd	20.0 ± 4.1 Ab	3.6	0.01
	S. carpocapsae	7.8 ± 2.8 Bab	13.3 ± 2.4 ABa	16.7 ± 1.7 Aa	21.1 ± 3.5 Abc	24.4 ± 2.4 Ad	26.7 ± 4.1 Ab	6.4	<0.01
	S. feltiae	2.2 ± 2.2 Bb	3.3 ± 1.7 Bb	3.3 ± 1.7 Bb	23.3 ± 4.1 Abc	26.7 ± 4.1 Acd	27.8 ± 2.8 Ab	24.0	<0.01
8 Days	H. bacteriophora	16.7 ± 4.4 Ca	20.0 ± 4.4 BCa	25.6 ± 3.4 ABCa	43.3 ± 5.5 ABa	46.7 ± 2.4 Aab	62.2 ± 8.5 Aa	6.9	<0.01
	S. carpocapsae	12.2 ± 4.0 Dab	18.9 ± 3.1 CDa	27.8 ± 4.0 BCa	51.1 ± 6.8 ABa	70.0 ± 4.1 ABa	85.6 ± 3.4 Aa	13.4	<0.01
	S. feltiae	15.6 ± 3.4 Ca	20.0 ± 2.9 BCa	24.4 ± 3.8 BCa	40.0 ± 5.8 ABab	42.2 ± 4.9 ABbc	76.7 ± 6.7 Aa	10.6	<0.01
	F	3.3	4.8	20.0	8.1	19.8	24.7
	p	0.01	<0.01	<0.01	<0.01	<0.01	<0.01

). Mortality values 8 days post-exposure were higher but still moderate for the doses 100 IJs/mL, 500 IJs/mL, 1000 IJs/mL, and 5000 IJs/mL for all EPNs. The doses 10,000 IJs/mL and 50,000 IJs/mL of S. carpocapsae killed 70.0% and 85.6%, respectively, while 50,000 IJs/mL of H. bacteriophora and S. feltiae did not exceed 62.2% and 76.7% mortality, respectively, at the same exposure interval. Control mortality was 0% after 8 days of exposure.

3.3. Mortality of Alphitobius diaperinus Adults

Between and within exposures, the main effects and interactions were significant, except those of nematode species × nematode dose interaction for A. diaperinus adults (Table 1). Mortality values were 0.0% after 4 days and 8 days of exposure to 100–10,000 IJs/mL of S. feltiae and 100–1000 IJs/mL of S. carpocapsae, while no mortality (0.0%) was recorded 4 days or 8 days post-exposure to 100–5000 IJs/mL or 100–1000 IJs/mL of H. bacteriophora, respectively (

Table 4. Mean mortality of (% ± SE) Alphitobius diaperinus adults after 4 days and 8 days on wheat treated with Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema feltiae applied at six doses. Within each row, means followed by the same uppercase letter are not significantly different in all cases. DF = 5, 53; Tukey–Kramer test at p = 0.05. Within each column, means followed by the same lowercase letter are not significantly different in all cases. DF = 5, 53; Tukey–Kramer test at p = 0.05.

		100 IJs/mL	500 IJs/mL	1000 IJs/mL	5000 IJs/mL	10,000 IJs/mL	50,000 IJs/mL	F	p
4 Days	H. bacteriophora	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	1.1 ± 1.1 ab	1.1 ± 1.1 b	0.8	0.56
	S. carpocapsae	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	1.1 ± 1.1	2.2 ± 2.2 ab	2.2 ± 2.2 b	0.6	0.70
	S. feltiae	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0 b	1.1 ± 1.1 b	1.0	0.43
8 Days	H. bacteriophora	0.0 ± 0.0 B	0.0 ± 0.0 B	0.0 ± 0.0 B	6.7 ± 3.3 AB	7.8 ± 3.2 ABa	11.1 ± 2.6 Aa	6.7	<0.01
	S. carpocapsae	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	2.2 ± 1.5	2.2 ± 2.2 ab	5.6 ± 2.9 ab	1.9	0.11
	S. feltiae	0.0 ± 0.0 B	0.0 ± 0.0 B	0.0 ± 0.0 B	0.0 ± 0.0 B	0.0 ± 0.0 Bb	3.3 ± 1.7 Aab	4.0	<0.01
	F	-	-	-	2.2	2.4	3.6
	p	-	-	-	0.07	0.05	0.01

). The highest doses of 50,000 IJs/mL killed 1.1–2.2% of A. diaperinus adults after the first four days of experimentation and 3.3–11.1% at the termination of the experimental period. Control mortality was 0% at 8 days post-exposure.

4. Discussion

The current study sheds light for the first time on the adulticidal efficacy of H. bacteriophora, S. carpocapsae and S. feltiae against T. granarium, T. molitor and A. diaperinus as grain protectants. Regarding T. granarium adults, the results revealed the remarkable efficacy of all EPN species at the highest dose. The effective management of this stage is crucial for this species, since its offspring at the egg or larva stages are tolerant to numerous synthetic or natural insecticides [43,46,57,58,59]. Therefore, adults determine the growth of the population of this species and the establishment of larvae in storage facilities, leading to dangerous population outbreaks [45,49]. Recently, Karanastasi et al. [11] reported almost total mortality to small T. granarium larvae and ~88% to large T. granarium larvae. Taking these results into account along with those of the current study, we can conclude that the use of H. bacteriophora, S. carpocapsae and S. feltiae on wheat can efficiently control adults and larvae (large and small) of this noxious insect. Furthermore, we propose the dose of 50,000 IJs/mL for all EPN species to achieve complete or almost complete adult or larval mortalities.

Regarding T. molitor adults, S. carpocapsae provided higher mortality (85.6%) than H. bacteriophora (62.2%) or S. feltiae (76.7%). Previously, 10 IJs/adult of S. carpocapsae, S. feltiae, and S. riobrave have been tested against T. molitor adults on filter paper [4]. Steinernema carpocapsae and S. riobrave could kill 90% and 95% of the exposed individuals, while S. feltiae could kill only 20% after 4 days of exposure. Virulence differences within the same EPN species can be attributed to the different strains of EPN that are used to manage T. molitor adults, a fact that could partially explain the high mortality related to the utilization of S. feltiae in our results vs. the low levels of mortality reported by Ramos-Rodríguez et al. [4]. For instance, four strains of H. bacteriophora caused the death of 30–90% of the exposed T. molitor larvae [7]. Similarly, Mbata and Shapiro-Ilan [3] noticed significant differences in the mortality values exhibited by H. bacteriophora strains against P. interpunctella adults. In a recent study, 2000 IJs/mL S. riobrave or 3000 IJs/mL H. bacteriophora applied on filter paper could kill 10.0% or 20.0% of the exposed T. granarium individuals, three days post-exposure [13]; these are very low percentages compared to our findings (100.0%).

In the case of A. diaperinus, former studies have confirmed that mortality levels depend on the strain of the EPN. Ten strains of Heterorhabditis sp. applied on filter paper killed between 1.0 and 40.0% of the exposed A. diaperinus adults [2]. The results of this study revealed that none of the three EPNs could kill more than 11.1% of the individuals 8 days post-exposure. In general, the existing studies of EPN efficacy against A. diaperinus adults show tolerance of this species to EPN. For instance, 60 or 120 IJs/adult Steinernema glaseri (Steiner) (Rhabditida: Steinernematidae) killed 0.0% or 3.3% of the A. diaperinus individuals, respectively, after 7 days of exposure on filter paper. On the other hand, 60 IJs/adult S. carpocapsae led to 26.7% mortality of the A. diaperinus individuals after 7 days of exposure on filter paper [1]. Furthermore, different strains of insects may have variable susceptibility to EPNs. For example, Koc et al. [60] studied four geographical strains of A. diaperinus. After 5 days of exposure to 200 IJs/mL S. carpocapsae, adult mortalities ranged between 40.0% and 66.6%, while larval mortalities ranged from 73.3% to 90.0%. It is worth noting that mortalities caused by EPNs can vary when they are applied to different types of materials. For example, Del Valle et al. [8] revealed that the strains CUL of Steinernema rarum (Doucet) (Rhabditida: Steinernematidae) and SMC of H. bacteriophora killed 32.7% and 60.0% of A. diaperinus adults when they were applied to filter paper while they killed only 2.7% and 16.7% when they were applied to dry rice hull, 4 days post-exposure, respectively. In the same study, when the rice hull was wet, mortalities were higher, reaching 9.3% and 33.3% for the S. rarum CUL and H. bacteriophora SMC, respectively.

Another factor affecting the virulence of the EPN is temperature. For instance, the B49 and B30 strains of S. feltiae caused the death of more S. oryzae adults at 20 °C than at 15 °C, 25 °C, or 30 °C, vs. the 3162 strain, which caused higher mortality at 25 °C than at all other temperature levels [61]. Similarly, S. carpocapsae and S. feltiae killed more E. kuehniella larvae at 20 °C than at 30 °C, but the same temperature increase yielded opposite results regarding H. bacteriophora [5]. Interestingly, the same temperature change (from 20 °C to 30 °C) increased the efficacy of S. feltiae against R. dominica adults and decreased the virulence of H. bacteriophora against T. confusum larvae and adults. However, mortalities caused by S. carpocapsae, H. bacteriophora and S. feltiae to S. oryzae adults were approximately the same when temperature increased [5]. At 30 °C, Karanastasi et al. [11] provided >91% and >98% mortality of T. granarium small larvae after 8 days of exposure to 50,000 IJs/mL of S. carpocapsae and S. feltiae, respectively, an issue that was confirmed in the case of adults in the current study. Whether the complete mortality (100.0%) of T. granarium adults found in the current study remains the same under lower or higher temperatures merits further investigation. Moreover, Kung et al. [62] revealed that as RH levels decreased from 100% to 25%, the numbers of S. carpocapsae and S. glaseri IJ gradually decreased. Interestingly, the same RH decrease led to gradually lower pathogenicity of the aforementioned EPNs. Therefore, abiotic factors such as temperature and RH play a significant role in the survival and the efficacy of the EPN. Due to variable findings, apparently there is no general trend between temperature and the virulence of EPN.

5. Conclusions

In conclusion, the highest dose (50,000 IJs/mL) of the EPNs H. bacteriophora, S. carpocapsae and S. feltiae inoculated on wheat are highly efficient for the management of adults of T. granarium, an important quarantine species in several countries of the world. The same dose of S. carpocapsae can provide adequate protection of wheat from T. molitor adults, while all EPNs were ineffective against A. diaperinus adults. The susceptibility rank (from most to least susceptible) of the tested insect species was T. granarium > T. molitor > A. diaperinus. The EPN efficiency rank (from most to least efficient) was S. carpocapsae > S. feltiae > H. bacteriophora. Entomopathogenic nematodes have already been suggested for insect management in soil-less cryptic environments, as they are an excellent substitute for chemical control. The current study provides additional information on their adulticidal ability; nonetheless, to assess their entire potential as grain protectants, additional experimentation is required, including experimentation on more EPN species/strains and the use of a wide variety of grain commodities against stored-product insects and their developmental stages under a series of variable abiotic conditions.