Rhynchophorus ferrugineus Olivier is an invasive pest of palm worldwide. The use of insecticides by farmers for its management has been found insignificant. This study evaluated the potential use of entomopathogenic fungi for R. ferrugineus management with a particular focus on the fungal infection on the activities of different detoxification enzymes.
Area of study:
Grubs and adults of R. ferrugineus were collected from various infested date palm fields in the four provinces of Pakistan.
Material and methods:
Fungi Isaria fumosorosea (If-02) and Metarhizium anisopliae (Ma-M2) were evaluated against R. ferrugineus, and its immune responses were biochemically characterized.
Main results:
The highest mortality rate was recorded at concentration 3×108 spores mL-1 on the 7th day post infection in the populations treated with M. anisopliae from Punjab, Khyber Pakhtunkhwa (KPK), Sindh and Baluchistan (93.75, 90.0, 90.0 and 81.25% respectively). M. anisopliae with lowest LC50 (1.1×106 spores mL-1) from Sindh also proved to be the most lethal fungus against R. ferrugineus. Maximum acetylcholinesterase (AChE) and glutathione S-transferases (GSTs) activities were observed in Baluchistan (26.28 and 24.0 μmol min-1 mg-1 protein, respectively) and maximum esterases (EST) activity (35.4 μmol min-1 mg-1 protein) was observed in the KPK population on the 3rd-day post I. fumosorosea infection.
Research highlights:
Fungal infection by I. fumosorosea caused a significant increase in AChE, GST and EST activities which may hinder R. ferrugineus development. However, M. anisopliae, to some extent, also inhibited enzyme activities and yielded a sudden increase in mortality. Future bio-pesticides could be developed for integrated pest management (IPM) of palm weevil.
Supplementary material (Tables S1, S2, S3) accompanies the paper on SJAR’s website.
Funding:
The authors received no specific funding for this work.
Introduction
The palm weevil, Rhynchophorus ferrugineus Olivier, is a destructive pest of palm trees, affecting approximately 29 different species of palm (Dembilio & Jacas, 2011; Hussain et al., 2013) including Phoenix dactylifera L. (date palm) (Giblin-Davis, 2001; Dembilio et al., 2009; Al-Dosary et al., 2016). R. ferrugineus is present in almost 50% of P. dactylifera growing countries (Faleiro, 2006; Dembilio & Jaques, 2015) and causes severe production losses ranging from 0.7 to 10 tons ha-1 (Singh & Rethinam, 2005). R. ferrugineus is considered endemic to subcontinental countries; its first appearance in Pakistan was reported around the early 1980’s which declined date production by 10–20% (Baloch et al., 1992) and by 30% in the Gulf region, while the Middle-East has lost US$ 103.66 million owing to injury caused by R. ferrugineus (El-Sabea et al., 2009). The geographic origin of R. ferrugineus is thought to be Melanesia and South East Asia (Ferry & Gomez, 2002) and dispersion has occurred due to the transportation of different palms around the globe (El-Mergawy & Al-Ajlan, 2011).
R. ferrugineus presents enormous management challenges due to its cryptic life cycle. Conventional methods for the control of R. ferrugineus consist of sanitary actions, use of pheromone traps, year-round surveillance, and the continued use of synthetic chemical insecticides which are applied by many methods, including axil filing, wound dressing, injection, spraying and fumigation (Cabello et al., 1997; Abraham et al., 1998; Al-Rajhy et al., 2005; Giblin-Davis et al., 2013). Undoubtedly, the use of synthetic insecticides reduces R. ferrugineus infestations (Dembilio et al., 2010b). However, many factors, including environmental hazards, applicator safety, residue persistence and evolution of resistance have led experts to search for more environmentally friendly alternatives to control R. ferrugineus (Hussain et al., 2013). During the last decade, the development of sterile insect techniques and pheromone traps has shown some promise (Al-Ayedh & Rasool, 2010; Abdel-Moety et al., 2012). However, knowledge on the potential for microbiological control of R. ferrugineus remains scarce. Laboratory research on the infection of R. ferrugineus with various microorganisms, including bacteria (Bacillus thuringiensis) (Manachini et al., 2009), nematodes (Steinernema affine) (Llácer et al., 2010) and fungi (Isaria fumosorosea, Metarhizium anisopliae and Beauveria bassiana) (Ghazavi & Avand-Faghih, 2002; Shaju et al., 2003; Gindin et al., 2006; Dembilio et al., 2010a; Güerri‐Agulló et al., 2010; Hussain et al., 2013; Cito et al., 2014) has yielded diverse results in terms of insect mortality. Entomopathogenic fungi are an effective alternate to insecticides because they are cheap, environmentally friendly, and safe for non-target insects. With the ability to readily capture the host defense and degrade cuticle, high adoption rate, entomopathogenic fungi are cost-worthy choice (Hussain et al., 2014; 2015). The main benefit of using entomopathogenic fungi is their distinctive mode of action, which looks very promising as a low-impact alternative to chemical pesticides. Much effort has been invested in developing strains for the control of agricultural pests (Naeem et al., 2020). However, little is known about the potential for application of fungi to control R. ferrugineus in the field, as it passes almost all stages of life in the trunk of the tree, making the insect inaccessible for direct or contact treatment.
In response to infection with microbial agents or insecticides, many immune responses and/or metabolic processes can be triggered in an insect. Detoxifying enzymes are important modulators of the immune response of insects and refer to melanization and phagocytic ability (Gopalakrishnan et al., 2011). Enzymes activity can be stimulated by various xenobiotics and is associated with resistance to toxic chemicals (Müller et al., 2007). Increased enzyme activity may enhance the immune system ability to respond to xenobiotic challenges and may support healing (Cerenius & Söderhäll, 2004). Many mechanisms aid resistance to xenobiotics i.e., changed target site modification, increased excretion or decreased metabolic resistance and penetration mediated by detoxifying enzymes such as acetylcholinesterase (AChE), esterases (EST) and glutathione S-transferases (GST) help to develop resistance against xenobiotics (Kliot & Ghanim, 2012). These enzymes enable insects to escape xenobiotics by degrading the toxic chemicals before reaching target sites (Bogwitz et al., 2005). The increased activity of GST and EST produced via biotransformation of toxic chemicals is associated with increased metabolic detoxification of toxicant target sites, leading to development of resistance (Sokolova & Sundukov, 1999). Acetylcholinesterase plays a very important role in detoxification of xenobiotics by hydrolyzing the neurotransmitter acetylcholine at the cholinergic synapses, which leads to disruption of normal synaptic transmission (Kono & Tomita, 2006; Colovic et al., 2013). In general, the increased activity of these detoxifying enzymes reduces the insect’s sensitivity to toxic chemicals (Serebrov et al., 2006). However, the functions of detoxifying enzymes in R. ferrugineus following fungal infection remain unexplored. Therefore, the current study was conducted to evaluate the effect of I. fumosorosea and M. anisopliae infections on the activities of detoxification enzymes, such as AChE, EST and GST, as related to pathogenicity to R. ferrugineus, in order to develop improved management strategies for this pest.
Material and methods<em>R. ferrugineus</em> collection and rearing
Grubs and adults of R. ferrugineus were collected by hand from various infested date palm fields in four provinces of Pakistan i.e., Khyber Pakhtunkhwa (KPK), Baluchistan, Punjab and Sindh. Palm weevil infestation was recognized by the symptoms of oozing frass and sap along the date palm trunk. The insects were placed in plastic cages (30×60×60 cm) covered with mesh screens and reared at 27 ± 2°C, relative humidity 70 ± 5% and L/D photoperiod 12/12 h. The adults were raised on fresh, clean and uninfected sugarcane stems (refreshed after 24 h), while the grubs were raised in plastic cups with Ahmed & Freed (2021a)´s artificial diet (45 g corn flour, 47 g wheat flour, 45 g yeast, 4 g ascorbic acid, 1.6 g sorbic acid, 2 g benzoic acid, two capsules of Pharmaton (1.55 g capsule-1), 2 capsules Tetracycline (250 mg) and 37 g agar in 875 mL of distilled H2O).
Insect pathogenic fungal isolates
Local isolates of insect pathogenic fungi I. fumosorosea (If-02) and M. anisopliae (Ma-M2) used for the experiments were identified and maintained in the Laboratory of Insect Microbiology, Department of Entomology, Bahauddin Zakariya University, Multan, Pakistan. The isolates If-02 and Ma-M2 were grown on potato dextrose agar (PDA) plates, placed at 27°C in dark at 75-85% relative humidity for 15 days, and after 15 days of fungal growth, the spores were stored at 5°C for further use against the insects. Different concentrations were prepared for the fungal bioassays by scrapping spores into Tween® 80 solution (0.05%); the spore concentrations were counted by haemocytometer and then the required concentrations were prepared by the serial dilution method.
Fungal bioassay
To check the efficacy of different fungal isolates, we followed the method of Ahmed & Freed (2021b): third-stage R. ferrugineus grubs were taken and immersed for 25 s in five different conidial concentrations of M. anisopliae and I. fumosorosea (1 × 106, 1 × 107, 1 × 108, 2 × 108 and 3 × 108 spores mL-1) (n = 80 grubs for each concentration were exposed to four replicates). In the control treatment the grubs were immersed in 0.05% Tween® 80 aqueous solution. A total of 480 grubs were tested in each bioassay including the control. Thereafter, the treated grubs were carefully placed on dry tissue paper to absorb excess moisture; and then were placed in plastic Petri plates containing the artificial diet as described above. For sporulation and mycosis, the treated grubs were placed at >75% relative humidity. Mortality data were recorded on the 3rd, 5th, and 7th day of treatment. Live grubs were collected from all treatments with M. anisopliae and I. fumosorosea on the above days and placed in a freezer (-20°C) prior to biochemical assay. The assays were performed under the previously described laboratory conditions.
Enzyme assay and protein determination
The frozen grubs (n = 4) from fungal treatments and controls were crushed in ~ 80 µL of 0.15 M NaCl, in 2 mL Eppendorf tubes with a small pestle. After crushing, the final volume was adjusted to 700 µL per replicate for centrifugation as described by Caballero et al. (2008) with modifications. The homogenates were centrifuged at 10,000 rpm for 5 min and supernatants were used to determine AChE, GST and EST activities.
The protein contents of R. ferrugineus grub treated with M. anisopliae and I. fumosorosea were determined according to the procedure of Bradford (1976). AChE activity was measured according to Ellman et al. (1961) using the substrate acetylcholine iodide (0.075 M); changes in absorbance were recorded at λ=412 nm after 4 min for 30-s interval. GST activity was assessed following Caballero et al. (2008) using as substrate 1 mM 1-chloro-2, 4-dinitrobenzene (CDNB); changes were recorded at λ=340 nm after 4 min at 30-s intervals. EST activity was determined following the method of Damayanthi & Karunaratne (2005) using 1 mM 4-nitrophenyal acetate as substrate; changes were determined at λ=405 nm after 4 min 30-s intervals. The spectrophotometer used was UV3000 (O.R.I. Germany).
Statistical analyses
The mortality data of fungal-treated R. ferrugineus were analyzed by using the US. Environmental Protection Agency (EPA) Probit software 1.5 (EPA, 1992) to estimate lethal concentrations (LCs), their 95% fiducial limits (FLs), chi square values and slope (Finney, 1971). The probabilities were calculated by chi-square (χ2) and degrees of freedom (df). We considered LC50 values significantly different if 95% FLs were non-overlapping (Litchfield & Wilcoxon, 1949).
Effect of fungal concentration, location and post-infection time on percent mortalities and release activities of enzymes (i.e., AChE, GST and EST) post-infection were assessed using linear mixed-effect models, with replications fitted as random effect, and concentration, location, post-infection time and their interactions as fixed effects. The effects were studied separately between M. anisopliae and I. fumosorosea isolates. The data were tested for normality and homoscedasticity using Kolmogorow-Smirnov and Levene’s tests, respectively. The percent mortality data were arcsine-square root transformed to improve compliance with these assumptions. The assumptions were approximately satisfied for enzyme activities data, so no transformation was required. Significant (p<0.05) treatment means, for each concentration, location, and post-infection time were separated by multiple comparison test of Tukey’s honest significant difference (HSD). As the interaction between concentration and post-infection time was typically significant at most events, therefore, the concentration effects on percent mortality and enzyme activities were further compared between three post-infection times, separately within districts, using repeated measures (RM) analysis of variance (ANOVA), followed by Tukey’s honest significant difference (HSD) multiple comparison treatment for separating significant effects. As we were interested in comparing concentration effects between post-infection times rather than between locations, ANOVAs performed did not compare locations. All analyses were performed in SPSS software, vers. 21 (IBM Corporation, Chicago, IL, USA). All means and standard errors in text and figures were calculated with untransformed data. Graphs were prepared by using GraphPad Prism, vers. 6.02.
ResultsPathogenicity of <em>I. fumosorosea</em> and <em>M. anisopliae</em> against 3rd instar grubs of <em>R. ferrugineus</em>
The pathogenicity of I. fumosorosea and M. anisopliae or LC50 values against R. ferrugineus were not significantly different (95% FLs overlapped) among the tested populations. However, the lowest LC50 value (1.1×106 spores mL-1) was recorded in the Sindh population treated with M. anisopliae which proved to be more potent given the higher LC50 values (3.4 × 107) for R. ferrugineus treated with I. fumosorosea on the 7th-day post-fungal treatment (Table 1).
Pathogenicity against <em>Rhynchophorus ferrugineus</em> of <em>Isaria fumosorosea</em> and <em>Metarhizium anisopliae</em> entomopathogenic fungi isolates obtained from different locations.
Fungi
Provinces
LC50 (spores mL-1)
95% FL
Slope
X2
df
p
N
M. anisopliae
Punjab
1.2×106
1.8×105-3.8×106
0.44±0.07
7.14
4
0.1289
480
Baluchistan
2.8 ×106
1.1×105-1.1×107
0.28±0.07
4.86
4
0.3023
480
Sindh
1.1 ×106
1.6×105-3.85×106
0.43±0.07
7.15
4
0.1281
480
KPK
3.8×106
7.1×105-10.1×107
0.44±0.08
6.60
4
0.1585
480
I. fumosorosea
Punjab
2.3×106
1.2×105 -1.0×107
0.28±0.13
8.99
4
0.0613
480
Baluchistan
3.9 ×106
2.1×105-1.4×107
0.27±0.07
6.12
4
0.1903
480
Sindh
3.4 ×107
3.1×106-2.5×108
0.22±0.07
0.73
4
0.9478
480
KPK
4.8×106
3.7×105-1.6×107
0.29±0.08
0.62
4
0.9608
480
FL = Fiducial limits. p = probability. N = number of R. ferrugineus exposed. df = degree of freedom.
<em>Rhynchophorus ferrugineus</em> post infection mortalities and enzyme activities
Mixed linear model analyses revealed significant effects for concentration, location, and post-infection time on post-infection mortalities and AChE, GST and EST activities in both M. anisopliae- and I. fumosorosea-treated R. ferrugineus (Tables 2, 3). The mortalities and activities of enzymes in M. anisopliae- or I. fumosorosea-treated R. ferrugineus increased in a highly concentration-dependent as well as time-dependent manner, i.e., mortalities and enzyme activities increased with increasing concentration and time of infection. Isaria fumosorosea-treated R. ferrugineus mortality was greatest in populations from Punjab and KPK and lowest in populations from Sindh and Baluchistan. The Punjab and KPK populations showed the greatest EST activities, whereas, Baluchistan populations had the highest AChE and GST activities. The lowest activities of these three enzymes were recorded from Sindh, KPK and Punjab populations, respectively. Metarhizium anisopliae-treated R. ferrugineus showed greater mortalities for Punjab and KPK populations, and lowest for Baluchistan populations. The highest AChE, GST and EST activities were found in Punjab populations and the lowest in Baluchistan populations. Effects for treatment × day and treatment × day × location interactions were typically significant towards mortalities and enzyme activities; therefore we decided to investigate concentration effects according to post-infection time separately between districts.
Analysis (linear mixed effect model) results for percent mortalities and release activities of detoxification enzyme in the <em>Rhynchophorus ferrugineus</em> after infection with <em>I. fumosorosea</em> and <em>M. anisopliae</em> across three post-infection times and four different provinces of Pakistan.
df
F
p
F
p
F
p
% mortality
M. anisopliae
I. fumosorosea
Treatment (T)
5
442.09
<0.001
670.22
<0.001
Location (L)
3
31.48
<0.001
19.24
<0.001
Day (D)
2
704.08
<0.001
710.98
<0.001
T × L
15
2.20
0.007
1.64
0.064NS
T × D
10
24.88
<0.001
13.46
<0.001
L × D
6
2.30
0.036
8.41
<0.001
T × L × D
30
0.438
0.996NS
1.92
0.004
Error (df)
213
442.09
<0.001
670.22
<0.001
Enzyme activity against M. anisopliae
AChE
EST
GST
Treatment (T)
5
601.64
<0.001
352
<0.001
239.7
<0.001
Location (L)
3
37.82
<0.001
32.9
<0.001
38.87
<0.001
Day (D)
2
81.33
<0.001
19.7
<0.001
62.15
<0.001
T × L
15
3.48
<0.001
3.07
<0.001
3.39
<0.001
T × D
10
9.83
<0.001
3.93
<0.001
3.3
0.001
L × D
6
2.89
0.011
9.22
<0.001
7.41
<0.001
T × L × D
30
1.75
0.016
2.64
<0.001
1.42
0.089NS
Error (df)
142
Enzyme activity against I. fumosorosea
AChE
EST
GST
Treatment (T)
5
1999
<0.001
2803
<0.001
1052.1
<0.001
Location (L)
3
70.97
<0.001
310.9
<0.001
225.42
<0.001
Day (D)
2
2862
<0.001
3117
<0.001
1268
<0.001
T × L
15
16.02
<0.001
55.8
<0.001
23.79
<0.001
T × D
10
477.6
<0.001
442.1
<0.001
279.09
<0.001
L × D
6
43.05
<0.001
60.17
<0.001
178.12
<0.001
T × L × D
30
11.88
<0.001
15.69
<0.001
20.06
<0.001
Error (df)
142
NS non-significant results (p>0.05). p = probability. df = degree of freedom. AChE = acetylcholinesterase. EST = esterases. GST = glutathione S-transferases.
Mean (±SE) percent mortalities and enzyme release activities in the <em>Rhynchophorus ferrugineus</em> after infection with <em>Isaria fumosorosea</em> and <em>Metarhizium anisopliae</em> across three post-infection times and four different provinces of Pakistan.
I. fumosorosea (spores mL-1)
M. anisopliae (spores mL-1)
% Mortality
AChE
EST
GST
% Mortality
AChE
EST
GST
Treatment
1×106
34.58±1.61 e
3.53±0.17 e
3.29±0.16 e
3.35±0.24 e
38.23±1.69 e
2.26±0.06 e
2.06±0.07 d
2.25±0.08 e
1×107
42.19±1.72 d
5.25±0.28 d
5.68±0.39 d
4.38±0.26 d
46.04±1.98 d
2.61±0.08 d
2.60±0.05 c
2.65±0.08 d
1×108
48.02±1.76 c
7.05± 0.51 c
9.76± 0.88 c
5.32± 0.46 c
52.71±2.12 c
3.08± 0.71 c
2.72± 0.05 b
3.16± 0.08 c
2×108
53.23±1.91 b
9.48± 0.83 b
14.24±1.26 b
7.19± 0.61 b
59.27±2.55 b
3.51± 0.06 b
3.04± 0.06 b
3.46± 0.09 b
3×108
58.13±2.13 a
13.16±1.38 a
16.93±1.62 a
9.40±0.98 a
66.28±2.97 a
3.69±0.05 a
3.43±0.09 a
3.70±0.11 a
Control
14.38 ±0.59 f
1.45 ±0.03 f
1.52 ±0.05 f
1.71 ±0.03 f
19.06±1.11 f
1.43 ±0.03 f
1.33 ±0.04 e
1.61 ±0.05 f
Location
Punjab
44.24±2.24 a
6.97±0.86 b
9.67±1.23 a
4.23±0.40 c
50.42±2.57 a
2.99±0.12 a
2.69±0.11 a
3.16±0.14 a
KPK
43.06±2.31 a
6.02±0.55 c
9.51±1.24 a
4.48±0.42 c
46.04±2.53 b
2.77±0.12 b
2.33±0.09 b
2.82±0.11 b
Sindh
40.28±1.86 b
6.22±0.81 c
6.10±0.67 c
5.80±0.45 b
49.17±2.67 a
2.73±0.11 b
2.69±0.11 a
2.67±0.09 bc
Baluchistan
39.44±2.29 b
7.41±0.86 a
9.01±1.01 b
6.39±0.78 a
42.50±2.25 c
2.57±0.11 c
2.41±0.09 b
2.56±0.12 c
Day
3 dpi
29.64±1.13 c
10.64±0.91 a
13.84±1.27 a
7.67±0.69 a
32.03±1.13 c
2.52±0.09 c
2.65±0.11 a
2.51±0.08 c
5 dpi
41.88±1.61 b
5.59±0.37 b
6.45±0.47 b
4.32±0.21 b
46.25±1.69 b
2.82±0.11 b
2.41±0.08 c
2.84±0.11 b
7 dpi
53.75±1.09 a
3.73±0.16 c
5.43±0.54 c
3.69±0.15 c
62.81±1.26 a
2.95±0.11 a
2.53±0.08 b
3.07±0.06 a
Means sharing similar letters within columns, for each tested variable, do not differ significantly (Tukey’s HSD test, p > 0.05). dpi = days post-infection.
Effects of post-infection time on <em>R. ferrugineus</em> mortalities
Treatment, sampling time and their interaction significantly affected mortality and AChE, GST, and EST enzyme activities (repeated measures ANOVAs; Tables S1, S2, S3 [suppl]). Table 4 shows that the mortality response of I. fumosorosea for 3×108 spores mL-1 (62.5 ± 0.87% to 78.75 ± 0.68%), 2×108 spores mL-1 (60.0 ± 0.52% to 71.25 ± 0.55%) and 1×108 spores mL-1 (58.5 ± 0.80% to 65.0 ± 0.50%), was similar between 5th and 7th-day, and these two were similarly higher than on the 3rd day, suggesting that this fungus requires at least 5 days to cause maximum mortality. The mortalities by M. anisopliae were comparatively similar between tested concentrations up to the 5th day, but upon approaching the 7th day, mortalities significantly increased in 3×108 spores mL-1 (81.25 ± 0.27% to 95.00 ± 0.44%) and 2×108 spores mL-1 (71.25 ± 0.55% to 86.25 ± 0.27%) concentrations, meaning this fungus requires at least 7 days post inoculation to cause maximum mortality (Table 5).
Mean (±SE) percent mortalities of <em>Rhynchophorus ferrugineus</em> from different locations after exposure of <em>Isaria fumosorosea</em> across three post-infection times.
Day
Concentration (spores mL-1)
Punjab
KPK
Sindh [a]
Baluchistan
3 dpi
1×106
23.75± 0.50 hi
22.50± 0.57 ij
23.75± 0.50 c
20.00± 0.81 hi
1×107
32.50± 0.52 gh
30.00± 0.69 hi
31.25± 0.42 b
25.00± 0.74 gh
1×108
38.75± 0.42 fg
35.00± 0.69 gh
35.00± 0.60 b
28.75± 0.80 f-h
2×108
42.50± 0.43 e-g
37.50± 0.45 f-h
42.50± 0.43 a
32.50± 0.48 fg
3×108
43.75±0.71 d-g
42.50±0.43 e-g
42.50±0.45 a
36.25±0.75 e-g
Control
13.75 ±0.64 i
11.25±0.79 j
11.25±0.79 d
8.75 ±0.79 i
5 dpi
1×106
35.00± 0.69 g
35.00± 0.64 gh
32.50± 0.48 c
32.50±0.52 fg
1×107
41.25± 0.71 e-g
42.50± 0.91 e-g
41.25± 0.39 b
38.75±0.39 d-f
1×108
47.50± 0.43 d-f
50.00± 0.60 de
50.00± 0.60 a
45.00±0.60 c-e
2×108
55.00± 0.55 b-d
55.00± 0.50 cd
52.50± 0.40 a
50.00±0.39 b-d
3×108
68.75±0.59 a
67.50±0.34 ab
55.00± 0.55 a
52.50±0.40 bc
Control
16.25 ±0.64 i
13.75 ±1.51 j
15.00±1.29 d
12.50 ±0.91 i
7 dpi
1×106
52.5± 0.40 c-e
47.50± 0.74 d-f
40.00± 0.60 b
50.00± 0.60 b-d
1×107
62.5± 0.62 a-c
56.25± 0.78 b-d
52.50± 0.96 a
52.50± 0.70 bc
1×108
65.0± 0.50 ab
65.00± 0.50 a-c
58.75± 0.64 a
58.50± 0.80 ab
2×108
68.75± 0.86 a
71.25± 0.55 a
60.00± 0.52 a
71.25± 0.55 a
3×108
72.50±0.80 a
75.00±0.84a
62.50±0.87 a
78.75±0.68 a
Control
16.25 ±1.07 i
17.50 ±0.64 j
18.75 ±0.55 c
17.50±0.64 hi
Table is showing post-ANOVA statistics for concentration effects according to significant treatment by day interaction; see Table S1 [suppl]. [a] When the treatment by day interaction was non-significant, the pair-wise mean comparison between concentrations was performed within each of three post-infection times, separately. Means labelled with similar letters within columns do not differ significantly from one another (Tukey’s HSD test, p > 0.05). SE denotes standard error. dpi = days post-infection.
Mean (±SE) percent mortalities of <em>Rhynchophorus ferrugineus</em> from different locations after exposure of <em>Metarhizium anisopliae</em> across three post-infection times.
Day
Concentration (spores mL-1)
Punjab
KPK
Sindh
Baluchistan
3 dpi
1×106
27.50± 0.58 h-k
25.00± 0.82 h-j
26.25± 0.50 ij
25.00± 0.82 h-k
1×107
35.00± 0.65 g-j
28.75± 0.46 h-j
33.75±0.81 hi
28.75±0.46 h-j
1×108
41.25± 0.37 f-h
32.50± 1.02 g-i
40.00±0.61 gh
32.50± 1.02 g-i
2×108
45.00± 0.61 fg
35.00± 0.69 f-i
45.00± 0.61 f-h
35.00± 0.69 f-h
3×108
48.75±0.37 e-g
38.75±1.35 e-h
46.25±0.68 fg
38.75±1.35 e-h
Control
18.75 ±2.86 k
13.75 ±1.51 j
13.75±1.51 k
13.75 ±1.51 k
5 dpi
1×106
37.50± 0.48 g-i
37.50± 0.45 e-h
35.00± 0.69 g-i
35.0±0.64 f-h
1×107
45.00±0.57 fg
45.00± 0.57 d-g
46.25± 0.67 fg
41.25±0.39 e-g
1×108
52.50±0.96 ef
52.50± 0.38 de
55.00± 0.55 d-f
47.50±0.43 d-f
2×108
61.25±1.01 c-e
57.50± 0.38 cd
60.00.± 0.52 c-e
52.50±0.38 c-e
3×108
73.75±0.28 bc
70.00±0.48 bc
70.00±0.50 bc
56.25±0.32 cd
Control
22.50 ±2.47 jk
21.25 ±4.15 ij
20.00±2.23 jk
15.00 ±2.23 jk
7 dpi
1×106
55.00± 0.52 d-f
50.00± 0.77 d-f
52.50± 0.38 ef
52.50± 0.96 c-e
1×107
67.50± 0.62 cd
60.00± 0.71 cd
65.00± 0.50 b-d
56.25± 1.06 cd
1×108
73.75± 0.28 bc
68.75± 0.31 bc
73.75± 0.28 b
62.50± 0.36 bc
2×108
83.75± 0.72 ab
78.75± 0.55 ab
86.25± 0.27 a
71.25± 0.55 ab
3×108
93.75±0.50 a
90.00±0.43 a
95.00±0.44 a
81.25±0.27 a
Control
25.00 ±0.91 i-k
23.75 ±1.67 h-j
21.25 ±0.55 jk
20.00±1.58 i-k
Table is showing post-ANOVA statistics for concentration effects according to significant treatment by day interaction; see Table S1 [suppl]. Means labelled with similar letters within columns do not differ significantly from one another (Tukey’s HSD test, p > 0.05). SE denotes standard error. dpi = days post-infection.
AChE, EST and GST post infection responses to <em>I. fumosorosea</em>
In I. fumosorosea-treated R. ferrugineus, the post-infection activities of AChE, EST and GST decreased as time post-infection increased (Fig. 1). The release rates were highest at 3 days post-infection (dpi) and typically for the highest exposure concentration (3×108 spores mL-1) that resulted in maximum AChE activities i.e., 26.95 ± 0.15, 16.28 ± 0.52, 26.28 ± 0.52, 24.95 ± 0.15 μmol min-1 mg-1 protein in Baluchistan, KPK, Punjab and Sindh, respectively (Fig. 1). The KPK population of R. ferrugineus treated with I. fumosorosea showed the maximum EST activities at 3 dpi (35.71±0.64 μmol min-1 mg-1 protein) followed by Baluchistan, Punjab and Sindh population with maximum EST activities (25.71 ± 0.56, 34.71 ± 0.46, 20.04 ± 0.99 μmol min-1 mg-1 protein, respectively), at the highest exposure concentration. The Baluchistan population of R. ferrugineus infected by I. fumosorosea showed the maximum activity of GST (24.14 ± 0.37 μmol min-1 mg-1 protein) at 3 dpi with the highest concentration of 3×108 spores mL-1, followed by the 5 dpi and 7 dpi (5.78 ± 0.01 and 5.81 ± 0.07 μmol min-1 mg-1 protein, respectively).
Mean (±SE) activities of enzymes (AChE, EST, GST) in <em>Isaria fumosorosea</em>-treated <em>Rhynchophorus ferrugineus</em> across three post-infections times for populations from different provinces of Pakistan. Figure panels are showing post-ANOVA statistics for concentration effects according to significant treatment by day interaction; see Table S3 [suppl]. SE denotes standard error. Bars within each panel labelled with similar letters do not differ significantly from one another, according to Tukey’s HSD test, <em>p</em> > 0.05.
AChE, EST and GST post infection responses to <em>M. anisopliae</em>
Metarhizium anisopliae-treated R. ferrugineus populations showed lower AChE, EST and GST responses at 3 dpi than I. fumosorosea-infected R. ferrugineus populations. Unlike I. fumosorosea-infected R. ferrugineus, the release rates for three enzymes in M. anisopliae-treated R. ferrugineus populations were generally similar at 3 dpi for higher concentration (Fig. 2). The AChE releases rates were 3.76 ± 0.04, 3.82 ± 0.04, and 3.72 ± 0.16 μmol min-1 mg-1 protein in KPK, Baluchistan and Sindh, respectively, at 5 dpi with M. anisopliae and in Punjab, a non-significant difference of AChE activity was observed at 3 dpi, 5 dpi and 7 dpi. The maximum EST activity induced by M. anisopliae infection was detected in the Punjab population at 3 dpi (4.44 ± 0.24 μmol min-1 mg-1 protein) at highest concentration (3×108 spores mL-1) followed by Baluchistan and Sindh (3.44 ± 0.26 and 4.11 ± 0.28 μmol min-1 mg-1 protein respectively) at 3 dpi, and KPK (3.18 ± 0.04 μmol min-1 mg-1 protein) at 7 dpi with M. anisopliae. The Punjab population infected by M. anisopliae showed the maximum GST activity at 7 dpi (4.84 ± 0.05 μmol min-1 mg-1 protein) at the highest concentration (3×108 spores mL-1) followed by Baluchistan and KPK (4.51 ± 0.30 μmol min-1 mg-1 protein) at 7 dpi and Sindh (3.6714 ± 0.23 μmol min-1 mg-1 protein) at 5 dpi with M. anisopliae.
Mean (±SE) activity of enzymes (AChE, EST, GST) in <em>Metarhizium anisopliae</em>-treated <em>Rhynchophorus ferrugineus</em> across three post-infection times for populations from different provinces of Pakistan. Figure panels are showing post-ANOVA statistics for concentration effects according to significant treatment by day interaction; see Table S2 [suppl]. SE denotes standard error. Bars within each panel labelled with similar letters do not differ significantly from one another, according to Tukey’s HSD test, <em>p</em> > 0.05.
Discussion
Entomopathogenic fungi are potential bio-control agents for many species of insects including R. ferrugineus (Cito et al., 2014). However, these fungi stimulate the immune system and alter the activity of detoxifying enzymes, so their effectiveness and modes of action against particular pest species need to be evaluated (Etebari et al., 2007; Lopes et al., 2011). Detoxification enzymes eliminate xenobiotics and maintain regular insect physiological processes (Nussenbaum & Lecuona, 2012). The role of detoxifying enzymes in induction of insect resistance using pathogens remains poorly explored. Hence, the aims of this study were to investigate the effect of M. anisopliae and I. fumosorosea on percent mortality and determine the role of detoxifying enzyme activities in the formation of insect resistance by the palm weevil, R. ferrugineus. We documented differences in mortality of R. ferrugineus with respect to regional populations, fungal conidial concentration and the role of detoxifying enzymes in survival of R. ferrugineus. The highest mortality (93.75%) was recorded in M. anisopliae-treated populations from Punjab, followed by populations from KPK, Sindh and Baluchistan (90.0%, 90.0% and 81.25% respectively), at the highest concentration (3×108) at 7 dpi. This is probably due to greater penetration of M. anisopliae spores into the host integument in the population from Punjab. Hyphae grow through the insect integument into the haemocoel, invade the tissues or organs, deplete nutrients and eventually cause death (Liu et al., 2011). The direct effect of physical damage caused by penetration of the hyphae into the insect integument is also an important aspect that requires additional research to develop measurements for the physical damage caused by the fungus itself.
Our results are also consistent with those of Fong et al. (2018), who reported M. anisopliae as the most pathogenic fungus, causing 100% grub mortality within 12 days of infection and a significant increase in mortality with an increase in conidial concentration. Similar results were reported by Francardi et al. (2012), who observed the highest efficacy of M. anisopliae against R. ferrugineus grubs and adults with 100% cumulative mortality. Other studies also suggest that M. anisopliae is the most potent fungus against R. ferrugineus (Gindin, 2006; Sewify & Fouad, 2006; Sabry et al., 2011; Kontodimas & Vassiliou, 2015; Abdel-Rahman & Abdel-Raheem, 2018; Yasin, 2019).
Preliminary studies have shown that an insect’s enzymatic system is activated by entomopathogenic fungi infection and this may enable the insect to maintain regular physiological functions (Song et al., 2002; Jun et al., 2003). In the present study, the activities of detoxification enzymes EST, GST and AChE were estimated 3, 5, and 7 days following fungal infection. The population of Baluchistan showed maximum activities of AChE and GSTs (26.28 and 24.0 μmol min-1 mg-1 protein, respectively), whereas, the maximum activity of EST in the KPK population (35.4 μmol min-1 mg-1 protein) was reached after 3 dpi with I. fumosorosea. These detoxifying enzymes contribute to defense reactions at the early stage of severe fungal infection (Dubovskiy et al., 2012). Comparable outcomes were previously documented with Verticillium lecanii, M. anisopliae and B. bassiana isolates (Kawachi et al., 2001). The results of our research are consistent with those of Vidhya et al. (2016) showing maximum GST activity in Spodoptera litura (Fabricius) after fungal infections. Similarly, Naeem et al. (2020) reported elevated activity of GST and EST in Diaphorina citri (Kuwayama) post fungal infection. Elevated activities of detoxifying enzymes in insects against fungal infection may result from activation of the immune system (Xu et al., 2017). Serebrov et al. (2006) reported elevated activities of GST and EST in Galleria mellonella L. post fungal infection. Moreover, AlJabr et al. (2017) reported elevated activities of EST and GST in R. ferrugineus post treatment with natural pesticides such as coumarin, methyl eugenol, diniconazole and rosmarinic acid.
Detoxifying enzymes make it possible for insects to avoid microbial agents by degrading xenobiotics before they reach target sites (Bogwitz et al., 2005). Our results showed that the use of different tested concentrations of I. fumosorosea isolate If-02 on R. ferrugineus increased EST, AChE and GST activities at 3 dpi. Similar results were reported by Dubovskiy et al. (2011) in G. mellonella, in which there was a significant increase in EST and GST activity after fungal infection. In our experiments, AChE activity increased after I. fumosorosea infection. Similarly, Bilal et al. (2017) reported elevated activity of AChE in H. armigera post-I. fumosorosea infections. According to Serebrov et al. (2006), G. mellonella caterpillars showed no significant increases in EST activity after M. anisopliae infection. Similarly, R. ferrugineus in our research showed no significant increase in EST activity after M. anisopliae infection. Similar results were obtained by Cao et al. (2016) showing inhibition of AChE and EST in Locusta migratoria L. after infection with M. anisopliae. Inhibited activities of detoxification enzymes showed that they play no significant role in the breakdown of the xenobiotics and may instead increase the susceptibility of insects to these toxic chemicals (Bakr et al., 2013).
The activity of detoxification enzymes in insects due to entomopathogenic fungi may vary both among and within species. Our study revealed differences in detoxification enzyme activities among R. ferrugineus populations from different provinces of Pakistan reared under the same laboratory conditions and subjected to the same entomopathogenic fungal treatments. Such differences may be related to adaptation to different environmental conditions in different palm growing regions or adaptive responses to prior exposure to fungal pathogens in some populations. The possible impacts of such population-level differences on development of effective pest management strategies should be explored through further research.
In conclusion, the present study demonstrated enhanced detoxification enzyme activities in R. ferrugineus after infection with two fungal species, I. fumosorosea and M. anisopliae. This suggests that M. anisopliae can be used for eco-friendly management due to its lower inductive effects on the inhibitory detoxifying enzyme activities. This research opens up novel options for developing effective biological products based on entomopathogenic fungi in combination with enzyme inhibitors. More specifically, this may provide an effective pest management technique for the date palm industry through development of myco-insecticides targeting R. ferrugineus.
Acknowledgments
We sincerely would like to thank Prof. Dr. Ahmet Bayram, Department of Plant Protection in Diyarbakır, Turkey for providing his help with technical editing of the manuscript.
Author's ContributionsConceptualization: S. Freed, R. Ahmed.Data curation: R. Ahmed, S. Freed.Formal analysis: A. Naeem, S. Freed, R. Ahmed.Funding acquisition: S. Freed.Investigation: S. Freed, R. Ahmed.Methodology: R. Ahmed.Project administration: S. Freed.Resources: S. Freed.Software: A. Naeem.Supervision: S. Freed.Validation: S. Freed.Visualization: S. Freed, M. Akmal, Christopher H. Dietrich.Writing – original draft: R. Ahmed, S. Freed.Writing – review & editing: S. Freed, A. Naeem, Christopher H. Dietrich.ReferencesAbdel-MoetyELotfyHRostomY2012Trace determination of red palm weevil, Rhynchophorus ferrugineus, pheromone at trapping locations under Egyptian climate.Int J Agr Food Sci21320Abdel-RahmanIAbdel-RaheemM2018Using entomopathogenic fungi as bio agents control on the red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae).J Entomol Zool Stud6387390AbrahamVShuaibiMAFaleiroJAbozuhairahRVidyasagarPS1998An integrated management approach for red palm weevil Rhynchophorus ferrugineus Oliv. a key pest of date palm in the Middle East.J Agric Marine Sci37783https://doi.org/10.24200/jams.vol3iss1pp77-83AhmedRFreedS2021aBiochemical resistance mechanisms against chlorpyrifos, imidacloprid and lambda-cyhalothrin in Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae).Crop Prot14310556843https://doi.org/10.1016/j.cropro.2021.105568AhmedRFreedS2021bVirulence of Beauveria bassiana Balsamo to red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae).Egyp J Bio Pest Control3114https://doi.org/10.1186/s41938-021-00422-5Al-AyedhHYRasoolKG2010Determination of the optimum sterilizing radiation dose for control of the red date palm weevil Rhynchophorus ferrugineus Oliv. (Coleoptera: Curculionidae).Crop Prot2913771380https://doi.org/10.1016/j.cropro.2010.06.019Al-DosaryNMAl-DobaiSFaleiroJR2016Review on the management of red palm weevil Rhynchophorus ferrugineus Olivier in date palm Phoenix dactylifera L.Emir J Food Agric:3444https://doi.org/10.9755/ejfa.2015-10-897AlJabrAMHussainARizwan-ul-HaqMAl-AyedhH2017Toxicity of plant secondary metabolites modulating detoxification genes expression for natural red palm weevil pesticide development.Molecules22169https://doi.org/10.3390/molecules22010169Al-RajhyDHusseinHIAl-ShawafAMA2005Insecticidal activity of carbaryl and its mixture with piperonylbutoxide against red palm weevil Rhynchophorus ferrugineus (Olivier) (Curculionidae: Coleoptera) and their effects on acetylcholinesterase activity.Pak J Biol Sci8679682https://doi.org/10.3923/pjbs.2005.679.682 , , , , , , . : -.
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