Metamasius spinolae (Gyllenhal, 1838) (Coleoptera: Curculionidae), is a main pest of the cacti crop (Opuntiasp) in Mexico. It reduces crop yield   and is distributed throughout the country with the highest incidence in Milpa Alta, D. F. with yield losses of 51.73%  . Larvae are inserted into the base in the older plant stalks, feeding on internal tissue, forming galleries and damaging the support structure. They also favor the entry of pathogens to the plant, and adults feed on the young pads.  . Due to the economic and cultural importance of cacti, it is necessary to propose alternatives to control M. spinolae and to replace the use of pesticides   . Cyclocephala lunulata (Burmeister, 1847) (Coleoptera: Melolonthidae) white grub feeds on the roots of sugar cane (Saccharumofficinarum L.), maize (Zea mays L.), alfalfa (Medicagosativa L.), potato (Solanumtuberosum L.), asparagus (Asparagus officinalis L.), coffee plant (Coffeaarabica L.), beet (Betavulgaris L.), lettuce (Lactucasativa L.), onion (Alliumcepa L.) and pastures (Poaceae)   . In order to control populations of C. lunulata, are sprayed systemic insecticides as Furadan®, hazardous to human health and the environment  . Entomopathogenic fungi (EF) represent one of the most efficient biological control alternatives. Beauveria bassiana (Bálsamo) Vuillemin (Ascomycota: Hypocreales), which infects many insect’s orders   , is a non-chemical alternative; it can degrade and penetrate the insect cuticle by synthesizing hydrolytic enzymes such as lipases, proteases and chitinases   . Insecticidal and antifeedant activity of enzymes of B. bassiana, such as subtilisin type protease (Pr1), which alters the peritrophic membrane of insects, blocking the formation or regeneration, has been tested  . Lipases start penetration of the insect integument, degrading lipids, waxes and lipoproteins that prevent water loss and protection against pathogenic organism  . After degradation of the epicuticle, chitinases and proteases are secreted to degrade proteins that surround chitin, core biopolymer which forms integument of insects  . Degradation products serve as nutrients to the fungus. The most important characteristic of this is that its application and management do not pose human health risks and the agricultural products are safe  . In this study we were evaluated the insecticidal activity and reduce the time of death of M. spinolae and C. lunulata for the conidia and enzymatic extract of B. bassiana obtained from solid state fermentation.
2. Materials and Methods
2.1. Fungal Isolates
An isolate (11) of B. bassiana was cultured in potato dextrose agar (PDA) at 25˚C for 15 days. The spores were suspended in a solution of Tween 80 (0.05%) in sterile distilled water and stored at 4˚C.
2.2. Culture Conditions
Solid-state fermentation of B. bassiana was performed in 250 ml Erlenmeyer flasks. Culture medium was prepared as described by Barranco et al.  . For enzymatic induction shrimp shell was used (60 g∙l−1). A concentration of 1 × 107 spores was inoculated per gram of humid matter. The culture conditions were RH 75%, pH 5.0 and temperature of 25˚C.
2.3. Extraction of Conidia
A solution Tween 80 (0.05%) was added to a solid-state fermentation from 15 days in sterile distilled water at a ratio of 1:1 (w/v). The mixture was homogenized, filtered and the suspension was stored at 4˚C in sterile tubes. The conidia were counted using a hemocytometer (Neubauer, United States Patent PLANE Charles A. Neubauer, Baltimore, Md, 1970). The suspension was adjusted to a concentration of 1 × 108 conidia mL−1  .
2.4. Obtaining the Crude Enzymatic Extract
Distilled water was added to a solid-state fermentation for five days at a ratio of 1:1 (w/v). The mixture was homogenized, pressed and centrifuged at 10,000 rpm for ten minutes at 4˚C. The supernatant was filtered through a membrane Millipore of 0.45 µm and the extract was stored at 4˚C in sterile tubes.
2.5. Determination of Lipase Activity
For the enzyme assay, the substrate was an emulsion of olive oil and gum arabic at 10% (1:1), 1 mL of this substrate, 2 mL of phosphate buffer 50 mM, pH 6.8 and 1 mL of crude enzyme extract were mixed. The reaction mixture was incubated one hour at 37˚C under constant stirring. The reaction was stopped by adding 4 mL of a solution of acetone ethanol 1:1 containing 0.09% of phenolphthalein. Enzyme activity was determined by titrating free fatty acids using 5 mL of NaOH  . One international unit was defined as enzyme activity that produced 1 µmoL of fatty acid min−1.
2.6. Determination of Protease Activity
Protease assay: 1) subtilisin (Pr1) succinyl-alanina 2 proline-phenylalanine-p-nitroani- line (Sigma-Aldrich, Inc.
2.7. Determination of Endochitinase Activity
0.5 ml of chitin colloidal 0.5% (w/v) was used as substrate along with 0.5 mL of enzyme extract and 1.0 mL phosphate buffer citrate 0.2 M, pH 5.6. The mixture was incubated at 50˚C for one hour, and the reaction was stopped adding 3 mL of dinitrosalicylic acid (DNS). The mixture was heated in a water bath for five minutes. The solution was centrifuged at 10,000 rpm and read at 575 nm  . One international unit was defined as enzyme activity that produced 1 nmoL of reducing sugars min−1.
2.8. Determination of Exochitinase Activity
The substrate used was p-nitrophenyl-N-acetyl-β-D-glucosamine (Sigma-Aldrich, Inc.
Adults of M. spinolae were collected in San Juan Tepenahuac, Milpa Alta, México, and moved to the insectarium laboratory of the Xochimilco Metropolitan Autonomous University (UAM-X). They were kept individually in plastic cups 1 L. They were fed 20 g of spiny tender cactus, changing every two days. The larvae of C. lunulata were collected on the lawn of the sports area of the UAM-X, by sampling five golds, on area of 50 × 50 cm2. The material was placed individually in plastic cups 1 L with 200 g of soil from the site of collection and were fed with a grass root ball of garden, changing according to the degree of wilting showed that each root ball. 20 larvae were separated until complete their development. With adults, taxonomic determination was made by Roberto Alejandro Terron Sierra from UAM-X. The individuals were acclimatized for seven days with a photoperiod of 12:12 L/D, 25˚C ± 2˚C and 50% RH in the bioclimatic chamber LUMISTELL®.
Inoculation. Adults of M. spinolae were immersed in each treatment for 10 s. In order to remove excess water, they were placed on absorbent paper for 10 s  . Larvae of C. lunulata, on the back of the head capsule, were injected with 10 μL of each treatment  with a gas chromatography syringe (Haminton Microliter TMSerie 700. Sigma-Al- drich).
Experimental Design. Completely randomized, with six treatments: T1: negative control (0.05% Tween 80); T2: crude enzymatic extract; T3: 1 × 108 conidia; T4: conidia more crude enzymatic extract; T5: positive control (Bea Tron®) 5 × 108; T6: enzymatic extract more positive control. M. spinolae had 48 replications with a total of 288 insects per treatment and C. lunulata had 10 replications per treatment with a total 60 insects. The observations were made every 24 hours for seven days. The experiments were established in the bioclimatic chamber LUMISTELL® under the conditions described above.
2.11. Statistical Analysis
Data were analysed with the software JMP® 8. The independent analyses were made for the bioassay in M. spinolae. Chi square tests to mortality rate and Kruskal-Wallis to average time death, because the model did not meet the assumptions of normality and homoscedasticity. In C. lunulata larvae, analysis of variance and post hoc multiple comparison of mean values were carried out by Tukey test. All experiments described in this paper were for triplicate. For all analysis, differences of means were considered significant at p values <0.05.
3.1. Enzymatic Activities
The enzymatic activities obtained of the crude extract of B. bassiana from five days correspond to lipases (820.83 U), subtilisin-type protease Pr1 (0.422 ± 0.077 U), trypsin-type protease Pr2 (0.095 ± 0.029 U), endochitinases (3.239 ± 0.382 U) and exoquitinases (2.41 ± 0.07 U). These enzymes are responsible for breaking the integument of insects. The production of the subtilisin-type protease (Pr1) and trypsin (Pr2) was high: Pr1 activity is 4.4 times greater than the activity of Pr2. This is the first report of the use of enzymatic extracts and conidia of B. bassiana together to reduce the time of death of M. spinolae and Clunulata under controlled conditions.
Mortality. M. spinolae: there are significant differences among treatments, χ2 (5) = 91.271, p < 0.001. The best results were obtained by the positive control treatment Bea Tron® with crude enzymatic extract (T6) with 77% mortality; 75% in the positive control Tron® Bea (T5), followed by the crude enzymatic extract (T2), conidia (T3) and conidia with crude enzymatic extract (T4) with 29, 27 y 31% (Figure 1); there was significant differences between treatments (Table 1).
Mortality. In larvae C. lunulata, the results showed significant difference, χ2 (5) = 46.614, p < 0.001, among treatments: crude enzymatic extract (T2), conidia (T3) and conidia more crude enzymatic extract (T4) had 100% mortality, the positive control with crude enzymatic extract (T5) had 90% and positive control (T6) had 50% (Figure 2). The negative control (T1) was no mortality in organisms. 100% survival in the negative control showed that the conditions under which the bioassay was established were appropriate and the mortality in treatments was due to its insecticidal effect (Table 2).
Time of Death. M. spinolae: The results showed significant differences between treat- ments by applying the Kruskal-Wallis test: χ2 (5) = 78,548, p < 0.0001. In the treatments: crude enzymatic extract (T2), conidia (T3) and conidia more crude enzymatic extract (T4) observed that there were shorter times of death of four days on average. While, in treatments: positive control (Bea Tron®) (T5) and positive control (Bea Tron®) more crude enzymatic extracts (T6) were nine days on average (Table 1).
Time of Death. C. lunulata: In this case, the results showed significant differences between treatments by applying analysis of variance (F (5, 298.84) = 35.93; p < 0.0001). The treatments: crude enzymatic extract (T2) and conidia more crude enzymatic extract (T4) caused the death of the larvae in a time of 1.2 days on average, Respect to
Figure 1. Mortality percentage to adult of M. spinolae under laboratory conditions.
Table 1. Mortality and time of death of adult M. spinolae.
N.D. It was not determined. Means signed by the same letter differ not significantly according to Tukey. N = 48.
positive control (Bea Tron®) more enzymatic extracts (T6) was in 1.7 days, these treatments showed reduced the time of death. The treatments conidia (T3) and positive control (Bea Tron®) caused death in a longer time with 2.8 and 8.8 days, respectively. In both bioassays, the negative controls (T1) not cause the death of any insect (Table 2).
In entomopathogenic fungi, enzymes that degrade insect cuticle play an important role in initiating the infectious process  . Shrimp shell is similar to the insect cuticle  . Its structure is a suitable substrate to induce the synthesis of proteases, chitinases and lipases   . The proteolytic activities Pr1 and Pr2 were determined, being higher
Figure 2. Mortality percentage to larvae of C. lunulata under laboratory conditions.
Table 2. Mortality and time of death of larvae of C. lunulata.
N.D. It was not determined. Means signed by the same letter differ not significantly according to Tukey. N = 10.
activity of Pr1 in relation to Pr2. These results are similar to those obtained by Dias et al.  using cuticle of the coffee berry borer (Hypothenemus hampei). According to St. Leger et al.  , the difference between the activity of Pr1 and Pr2 is because trypsin is a complement of the subtilisin enzyme. The ability to produce subtilisins is a virulence factor of B. bassiana  that allows infecting various orders of insects. By contrast, trypsins work as a suppressor of the immune system of the insect  . In addition, the lipolytic activities determined using the shell of shrimp as substrate, while not a specific inducer, and are attributed to an adjustment mechanism cAMP dependent signal trans- duction  to which the enzymes are regulate. Lipases are fundamental for starting epicuticle penetration, first barrier of protection against insects of these microorganisms. Without these enzymes, infection would not be established   . Lipases of Isaria fumosorosea have been used against diamondback moth Plutella xylostella L. (Lepidoptera: Plutellidae)  . Chitinases are enzymes of interest as agents for control of pests and plant diseases   . Exo and endo chitinase activity was determined in the extract, these enzymes act synergistically with proteases  , even though the production of exoquitinases in this solid culture was lower than that obtained by Barbosa et al.  , who used silkworm pupae. However, induction of chitinases by using shrimp shell has been demonstrated  .
The insecticidal action was mainly due to softening of the integument by the action of enzymes contained in the crude enzymatic extract, an effect that allowed the germ tube to penetrate the structure more easily to finally reach the haemocoel and promote infection in less time. So, the enzymatic extracts of B. bassiana showed insecticide when applied topically to adult of M. spinolae, alone or together with conidia, which re- presents the first report for this pest. Enzymes have their specific action on the cuticle of each component. Lipases hydrolyze the ester bonds of lipoproteins, fats and waxes epicuticle   , the Pr1 and Pr2 proteases degrade the proteinaceous material and finally, chitinases degrade the network of chitin  , the main structural component of the exoskeleton in the walls, so that the cuticle lose rigidity and facilitating penetration. The insecticidal effect of enzymatic extracts was probably due to penetration by the pore channels, the base of silk, or the intersegmental membranes of the integument  , including the mouth, spiracles and other openings outside of the insect. In dead individuals of M. spinolae, treated with enzymatic extract (T2), contraction of the three pairs of legs and weaker binding, abdomen and pronotal flexibility was observed, possibly due to degradation and joint intersegmental membranes, both with lower levels of sclerotization (Figure 3(b) and Figure 3(c)). Insects in the control group showed no symptoms associated with altered activity in the food or displacement or anatomy during the experiment (Figure 3(a)).
The bodies of M. spinolae treated with conidia (T3) presented mycelium of B. bassiana, mainly on the coxa and intersegmental space pronotum and abdomen (Figure 3(e) and Figure 3(f)). In other bodies, it also emerged from the side of the elytra and the joints between the femur and tibia (Figure 3(d)) which remained completely rigid and extended as a result of the invasion of the mycelium in the muscle tissue. In other orders of insects this sign has been reported  . On the other hand, in the bodies of treatment with conidia and enzymatic extract (T4) showed numerous groups of conidiogenous cells and aerial mycelium that emerged from the intersegmental spaces (Figure 3(g)). Most of the body surface of the insects was colonized by B. bassiana (Figure 3(h) and Figure 3(i)), unlike bodies with conidia treatment. Possibly the use of extracts with conidia facilitated the emergence of mycelium allowing their proliferation.
Use of enzymatic extracts with conidia of B. bassiana resulted in symptoms such as feeding activity inhibition: in three or four days the insect stops feeding and alterations in its locomotor system. Besides, the extracts could facilitate the emergence of mycelium, giving the fungus the greatest attributes in terms of persistence and spread. From
Figure 3. M. spinolae: (a) Negative control (b) and (c) Enzymatic extract, showing contraction of the legs; (d) Conidia, dorsal view of the head. (e): dorsal view with mycelium in the intersegmental space between pronotum (1) and abdomen (2), (f): ventral view from the coxa (1), knuckles, side of the elytra (2) and anus (3): Conidia more enzymatic extract. (g): dorsal view from the antennas (1), head (2), joints of femur and tibia (3); pronotum and abdomen (4); (h) ventral view covered by aerial mycelium; (i) ventral view from antennas and head with aerial mycelium (1); the articulation of the femur and tibia (2) and coxae (3).
an agricultural point of view, this raises their viability in the field as it becomes a new source of infection to be dispersed by abiotic factors such as the air and the breeze or contamination between individuals  .
Injection of the enzyme extract in larvae of C. lunulata caused a toxic effect. The enzymes Pr1 and Pr2 contained in the enzyme extract caused pigmentation of cuticles as a response of the immune system to the presence of foreign enzymes inside the bodies (Figure 4(b)). The basic function of Pr1 is to degrade cuticle proteins and facilitate penetration of the integument. In addition, Pr1 is also toxic when introduced into the haemocoel to degrade proteins of hemolymph and melanization of integument  or secondary metabolites such as bassiacridin that cause the formation of melanised spots on tracheae and air sacs and melanised nodules in contact with the fat body  . The responsible compound for pigmentation of the cuticle is melanin, whose biological property is its fungicidal activity, and is synthesized by phenoloxidase in the hemolymph of arthropods, which is an inactive proenzyme called prophenoloxidase (proPO) likely to be active for the recognition of foreign agents  . Once proPO cascade system sets are activated in haemocytes, defense reactions as phagocytosis, encapsulation for removing foreign agent  ; proPO insect system is activated by serum proteases
Figure 4. C. lunulata larvae: (a) Negative control. Side view with normal coloration; (b) Enzymatic extract, showing melanization throughout the body mainly among spaces intersegmental and regions near spiracles and loss of firmness of the tissue (side view); (c) Conidia (side view), the tissue became more rigid (d) Conidia more enzymatic extract, appearance of larvae with melanization tone dark brown throughout the body coupled with the emergence of mycelium from oral apparatus and leg joints and anal opening.
 present in an inactive form in haemocytes, reacting in the presence of β-1, 4 glucans from yeast, bacterial peptidoglycans or lipopolysaccharides of Gram bacteria  . This complex interacts with the surface of haemocytes to activate the prophenoloxidase system.
In larvae of C. lunulata, various pigmentary changes were observed on the tegument. Negative control larvae maintained their characteristic creamy white and soft texture and body (Figure 4(a)). However, the bodies of larvae of the enzyme extract (T2) took brownish pigmentation attributed to melanization, although the highest intensity in areas near spiracles, and body tissue lost firmness (Figure 4(b)).
In the bodies of larvae inoculated with conidia (T3), were observed changes in pigmentation to an orange color in the body. Tissue of the bodies also varied. It was observed with greater rigidity and later emerged mycelium (Figure 4(c)). The larvae inoculated with conidia more extracts (T4) acquired a dark brown pigmentation and mycelium emerged from natural openings and firm body tissue (Figure 4(d)). The synergy between extracts and conidia caused combining melanization and mycelium, in order to persist in the environment  . Lower mortality was observed in the commercial product probably because of B. bassiana strain is less virulent for C. lunulata larvae; however the commercial product plus the extract increased mortality. Melanization in larval cuticle has been reported by Fuguet and Vey  which was observed in larvae of Galleria mellonella (Lep: Pyralidae) by injection with conidia of different isolates of B. bassiana, where different tones and degrees of pigmentation were observed as well as patterns that were categorized, pointing to the processes of melanization in the integument, located at points where mycelium emerged. In this work, in the bodies of C. lunulata inoculated with conidia of B. bassiana, the melanization was not in dark colors, but it was in orange pigmentation Chavez et al.  reported the same results with injecting crude extract of B. bassiana to Phyllophaga ssp. However, in larvae injected with crude enzyme extract, the death was 24 hr post inoculation.
The insecticidal activity of enzymes such as chitinases and proteases of B. bassiana has already been tested in some pests of agricultural importance when topically applied as a powder, it showed insecticidal activity on cotton aphid Aphis gossypii Glover  or with crude proteins of Isaria fumosorosea that affect the growth and metamorphosis of diamond back moth Plutella xylostella  . Similar results were reported by Quesada-Moraga et al.  for the use of crude proteins of Metarhizium anisopliae (01/58- His) in Spodoptera littoralis larvae, causing inhibition of feeding activity. These enzymes in the intestine cause damage significant to the structure of the peritrophic membrane, resulting in the inability of feeding and death   .
The crude enzymes extract of B. bassiana obtained by solid-state fermentation have insecticidal potential to be used in the biological control of pests of agricultural importance, given that they are constituted by a mixture of lipases, proteases and chitinases, which caused a toxic effect on M. spinolae and C. lunulata. Also, to use it as an adjuvant with conidia reduced the time of death of the insects, opening new prospects for the development of bio-insecticides with less impact to the environment, because they are a sustainable option to replace and reduce the use of chemical insecticides. It is necessary to continue studies to assess the stability and effectiveness of the enzymatic extracts in other models and different biological states and perform histological studies of insects treated, to determine the effect of these biomolecules on insect’ physiology.
We are grateful to John A. Goolsby, Ph.D., Research Entomologist Biological Control and Integrated Pest Management of Ticks, Insects and Weeds. United States Department of Agriculture, Agricultural Research Service, for their valuable comments and correcting the manuscript.This research was support by Consejo Nacional de Ciencia y Tecnología (CONACYT), Lluvia de Carolina Sánchez Pérez beneficiary of a grant.
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