Effects of Lectin Preparations from Microgramma vacciniifolia Rhizomes on the Survival, Digestive Enzymes, and Acetylcholinesterase Activity of Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

Abstract

In this study, we evaluated the susceptibility of Alphitobius diaperinus larvae and adults to saline extract (SE), lectin-rich fraction (FR), and isolated lectin (MvRL) from Microgramma vacciniifolia rhizomes. To determine immediate effects, larvae and adults were exposed to SE (10.5 mg/mL), FR (7.5 mg/mL), or MvRL (1.0 mg/mL) for 48 h. Live insects were evaluated for acetylcholinesterase (AChE) activity. The delayed effects of SE (10.5 mg/mL), FR (7.5 mg/mL), and MvRL (0.2 and 0.4 mg/mL) were checked by incubating the adults for 16 days with a diet containing the preparations. In vitro effects on gut digestive enzymes were investigated. All preparations showed immediate larvicidal effect but had no effect on adult survival. Extracts from FR-treated larvae showed higher AChE activity than control insects. In the delayed effect assay, the adults lost biomass after consuming SE and FR. FR was the most effective inhibitory agent of trypsin-like and amylase activities (88% and 65% inhibition, respectively). All preparations inhibited endoglucanase activity in 94–98%, while SE and FR inhibited exoglucanase activity in 93.2 and 94.1%, respectively. In conclusion, M. vacciniifolia rhizomes contain compounds (including MvRL) that affect the survival and physiology of A. diaperinus, acting as potential natural insecticides for controlling this pest.

1. Introduction

Poultry farming is one of the agricultural sectors of greatest socio-economic importance. Brazil is a successful world producer and exporter in the poultry industry because of investments in the technological modernization of the process that led to significant increases in production efficiency. Currently, this country is the world’s second largest producer of chicken meat and its derivatives, and remains the largest exporter [1,2].

The Brazilian chicken farms are, in general, free of important contagious diseases, such as avian influenza, which have been plaguing exporting countries, including the United States and China [3]. However, there are still major obstacles to poultry production. One of them is the presence of Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae), a flying and cold-tolerant insect commonly known as the lesser mealworm.

The A. diaperinus life cycle comprises the phases of eggs, larvae (8 to 11 instars), prepupae, pupae, and adults. The complete cycle, from oviposition to adult hatching, can take from 34 to 165 days, according to temperature [4]. The adult females live for four months to one year and each one lays about 3.5 eggs/day, singly on or within the substrate. Alphitobius diaperinus is mainly found in poultry houses, stables, henhouses, mills, or associated with stored products, including wheat, barley, rye, nuts, herbs, beans, and seeds, as well as products such as flour, bran, and hay [5,6]. In poultry farms, A. diaperinus lives in litter and feces, feeding on manure and feed. They are voracious eaters that assume cannibalistic behavior or can attack small vertebrates when food is scarce [4].

Due to the short life cycle and high viability of offspring, it is difficult to control A. diaperinus in the conditions of temperature, humidity, and food that are common in the poultry environment [7]. A. diaperinus larvae and adults are vectors of pathogens, transmitting bacterial infections (Escherichia, Bacillus, and Streptococcus) and avian viral diseases (Marek disease, Newcastle disease, Gumboro disease, and avian influenza), as well as the parasitic diseases coccidiosis and chicken tapeworm [8,9]. Furthermore, the ingestion of both adults and larvae by chickens may lead to obstruction of the gastrointestinal tract and has been related to hypoglycemia spiking mortality syndrome of broilers and viral poultry enteritis complex “running stunting syndrome” [10]. The receding health of poultry due to A. diaperinus infestation blunts the growth of the animals, therefore raising the cost of production [11]. In addition, A. diaperinus may also pose a threat to public health, not only as a vector of zoonotic diseases, but also because their abdominal glands produce quinones [12], compounds that can be carcinogenic [13].

The most common method of controlling A. diaperinus populations is the use of synthetic insecticides, such as pyrethroids, organophosphates, and cypermethrin. These compounds are applied by spraying the floor and walls of chicken coops before the replacement of the litter for the next breeding cycle, aiming to avoid direct contact with birds [7]. However, these chemicals are unable to kill high percentages of the beetles and have been associated with insect resistance and environmental toxicity [14,15]. Thus, alternative methods of control are needed, and plant products have aroused interest because they are efficient and environmentally friendly, with lower nontarget toxicity and a higher degree of degradability [15,16]. Cinnamon (Cinnamomum zeylanicum) oil and its nanoemulsion killed A. diaperinus larvae and adults [17], while the Illicium verum walnut’s essential oil increased glutathione-S-transferase activity and decreased acetylcholinesterase activity, as well as caused the loss of refuge-seeking capacity and the loss of locomotor ability in the insects [18].

Microgramma vacciniifolia (Langsd. and Fisch) Copel., popularly known as snakefern in English or “cipó-cabeludo” in Portuguese, is an epiphytic plant of the Polypodiaceae family with far-reaching distribution worldwide, though it is found preferentially in the tropics. Its rhizome contains a lectin (carbohydrate-binding protein) called MvRL whose toxicity to lung carcinoma (NCI-H292) cells, Fusarium oxysporum f. sp. lycopersici, and Biomphalaria glabrata was previously reported [19,20]. MvRL also showed toxicity to Nasutitermes corniger workers and soldiers [21] and Sitophilus zeamais adults [22]. According to the authors, the insecticidal mechanisms of MvRL may involve the imbalance of digestive enzymes.

Since MvRL showed insecticidal potential on insect pests, this study evaluated the susceptibility of A. diaperinus larvae and adults to lectin preparations of M. vacciniifolia rhizomes. The effects of rhizome extract, lectin-rich fraction, and MvRL on A. diaperinus survival, digestive enzymes, and acetylcholinesterase activity are described here. This is the first time that lectin preparations were evaluated for insecticidal activity on this insect.

2. Materials and Methods

2.1. Insects

Adults and larvae of A. diaperinus were collected in poultry beds at Paudalho, Pernambuco, Brazil. The insects used in the bioassays have been maintained at the Laboratório de Biofísica Teórico Experimental e Computacional (LABTEC) from the Departamento de Morfologia e Fisiologia Animal of the Universidade Federal Rural de Pernambuco. Insects were reared in glass boxes (38 cm × 17 cm × 10 cm) that contained a diet composed of (1:1, w/w) oat flakes (Quaker Oats Company, Chicago, IL, USA), crushed rabbit food (Presence, Paulínia, São Paulo, Brazil), and some pieces of broiler litter. Adults and larvae were packaged in different glass boxes and maintained in the dark at 25 ± 2 °C and 75–80% humidity.

2.2. Lectin Preparations from M. vacciniifolia Rhizome

Saline extract (SE), lectin-rich fraction (FR), and MvRL from M. vacciniifolia rhizomes were obtained according to Albuquerque et al. [21]. Rhizomes were collected at the campus of the Universidade Federal de Pernambuco (UFPE) with authorization (no. 72024) from the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). The study was recorded (no. A347889) at the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen). The rhizomes were submitted to air drying for 8 days. Then, they were crushed in a multiprocessor to obtain a flour (40 mesh), which was homogenized (10 g) with 0.15 M NaCl (100 mL). After agitation for 16 h at 4 °C, filtration through gauze, and centrifugation (15 min at 8000× g, 4 °C), the supernatant corresponded to saline extract (SE).

The addition of solid ammonium sulfate (60% saturation) [23] to SE was performed followed by gentle agitation for 4 h. After centrifugation (15 min, 8000× g, 4 °C), the precipitated material was solubilized and dialyzed against distilled water and 0.15 M NaCl. The resulting material corresponded to the lectin-rich fraction (FR).

MvRL was isolated from FR by chromatography on a chitin (Sigma-Aldrich, St. Louis, MO, USA) column, eluted using 1.0 M acetic acid, and subsequently dialyzed against distilled water (6 h) to remove the eluent and dried by lyophilization to a final concentration of 1.8 mg/mL. The purity of the MvRL was checked by polyacrylamide gel electrophoresis under denaturing conditions (presence of sodium dodecyl sulfate), according to Laemmli [24]. Protein concentration in all purification steps was determined according to Lowry et al. [25] using bovine serum albumin (31.25–500 μg/mL) as the standard.

Carbohydrate-binding ability was monitored through the hemagglutinating activity (HA) assay, which was carried out according to Carvalho et al. [26]. Briefly, the assay was performed in 96-well V-bottom plates, where SE, FR, or MvRL (50 μL) was two-fold serially diluted in 0.15 M NaCl. Next, a suspension (2.5%, v/v) of rabbit erythrocytes, previously fixed with glutaraldehyde, was added to each well [27]. After incubation for 45 min at 28 °C, the HA was determined as the reciprocal of the highest dilution of the sample promoting full agglutination of erythrocytes. Specific HA was defined as the ratio between HA and protein concentration (mg/mL). The erythrocytes were collected after approval by the Ethics Committee on Animal Use of the Universidade Federal de Pernambuco (process 23076.033782/2015–70). The HA of MvRL was also evaluated after incubation (30 min) with 200 mM D(+)-mannose before adding erythrocytes [21].

2.3. Immediate Effects of SE, FR, and MvRL on Larvae and Adults of A. diaperinus

The bioassays were performed according to Zafeiriadis [28] with adaptations. Larvae (fourth instar) and adults of A. diaperinus were transferred to Petri dishes (90 mm × 15 mm) and exposed to SE (10.5 mg/mL), FR (7.5 mg/mL), or MvRL (1.0 mg/mL) for 48 h. For this, each plate received 250 μL of one of the treatments or distilled water (control) that was mixed all over the plate for 30 s before 10 larvae or 10 adults were put in each plate. After 48 h, the mortality was recorded, and the live insects were collected to evaluate acetylcholinesterase (AChE) activity as described in the next section. Two independent experiments were performed with five replicates.

2.4. Determination of Acetylcholinesterase (AChE) Activity

Live larvae or adults (n = 10) from each treatment (SE, FR, or MvRL) or the control were immobilized at 4 °C for 10 min. After this period, the elytra of adults were removed. Next, larvae or adults were homogenized in 1 mL of 0.1 M potassium phosphate, pH 7.5, using a tissue grinder. The mixture was centrifuged (9000× g, 4 °C, 10 min) and the supernatant corresponded to the larva body extract (LBE) or adult body extract (ABE). The protein content in the insect extracts was determined according to Bradford [29].

AChE activity was determined using 2.0 mL microcentrifuge tubes by incubating 10 μL of LBE from control, SE, FR, or MvRL treatments (2.08, 1.22, 1.27, and 0.47 mg of protein, respectively) or 10 μL of ABE from control, SE, FR, or MvRL treatments (0.52, 0.63, 0.31, and 0.95 mg of protein, respectively) with 10 μL of 0.1 M potassium phosphate, pH 7.5, and 20 μL of 7.5 mM acetylthiocholine iodide (Sigma-Aldrich). Ten minutes later, 200 μL of DTNB (5,5′-dithiobis-2-nitrobenzoic acid, 0.8 mM; Sigma-Aldrich) was added and the assay was incubated for 3 min at 25 °C. The release of thiocholine was monitored by measuring the absorbance at 405 nm at time 0 and after 3 min [30]. One unit of AChE activity was defined as the amount of enzyme required to hydrolyze 1 μmol of acetylthiocholine per minute. Two independent experiments were performed in triplicate.

2.5. Delayed Effect of SE, FR, and MvRL on the Survival of Adults of A. diaperinus

The bioassays were performed according to Rice and Lambkin [31]. Adults at 20–30 days of age were used. The weights of the insects were determined before the start of the test. In petri dishes, 1 mL of SE (10.5 mg/mL), FR (7.5 mg/mL), MvRL (0.2 and 0.4 mg/mL), or distilled water (control) was added to 1 g of oat flakes + 0.1 g of rabbit food. Next, the diet was dried in an oven (56 °C) for 30 min. To each plate, 10 adults were added, and the assays were incubated under the same conditions used for the maintenance of the colony. Mortality and insects’ weight were recorded after 16 days. The adults were considered dead when they did not react when touched with a needle. Two independent experiments were performed with five replicates.

2.6. Digestive Enzyme Activities of A. diaperinus Adults

2.6.1. Gut Extracts from A. diaperinus Adults

To obtain gut extracts, A. diaperinus adults (n = 50) were separated from the colony and immobilized by cooling at 4 °C for 10 min. Next, their guts were dissected with a needle and homogenized in 1 mL of 0.1 M Tris-HCl, pH 8.0 (containing 0.02 M CaCl₂ and 0.15 M NaCl), using a glass tissue grinder (2 mL). Thereafter, the mixture was centrifuged (9000× g, 4 °C, 15 min) and the supernatant (gut extract in Tris buffer, GETb) was collected to determine the protein content [29]. The gut extract in acetate buffer (GEAb) was obtained as described above, but using 0.1 M sodium acetate, pH 5.5 (containing 0.02 M CaCl₂ and 0.15 M NaCl). The enzymatic activities of GETb and GEAb were assayed as follows.

2.6.2. Trypsin-Like Activity

This assay was performed in 96-well microtiter plates and the trypsin-like activity in GETb was evaluated according to Kakade et al. [32]. GETb (10 μL, 150 μg of protein) was incubated (60 min, 37 °C) with the peptidomimetic substrate BApNA (5 μL, 8 mM, N-benzoyl-DL-arginine-ρ-nitroanilide) in Tris buffer (185 μL), and trypsin-like activity was measured by the absorbance at 405 nm. One unit of trypsin-like activity corresponded to the concentration of enzyme in GETb that hydrolyzes 1 μmol of BApNA per minute under the conditions employed here. To ensure that these conditions were suitable for the investigation of trypsin-like activity, the above procedure was performed using bovine trypsin (5 μL, 0.5 mg) instead of GETb. Two independent experiments in triplicate were performed.

2.6.3. A-Amylase Activity

The investigation of α-amylase activity was performed according to Bernfeld [33] with adaptations. The trial was prepared in 2.0 mL microcentrifuge tubes. GEAb (25 μL, 220 μg of protein) was incubated (56 °C, 10 min) with soluble starch in an acetate buffer (1% w/v, 200 μL) as substrate. To stop a possible reaction, the reagent 3,5-dinitrosalicylic acid (DNS, 250 μL) was added and the mixture was heated (100 °C, 6 min) in a water bath. After cooling on ice (15 min), the absorbance (540 nm) was measured. The concentrations of reducing sugars in the mixtures were determined using a standard curve of a glucose (1.0–10.0 mg/mL) reaction with DNS. One unit of α-amylase activity was assumed to be the enzyme concentration in GEAb required to produce 1 μmol of glucose per minute. Reaction blanks were performed by submitting GEAb to the same steps, but in the absence of starch.

2.6.4. Endoglucanase and Exoglucanase Activities

GEAb (25 μL, 275 μg of protein) was incubated (50 °C, 10 min), in 2.0 mL microcentrifuge tubes with 200 μL of 1% (w/v) carboxymethylcellulose for the evaluation of endoglucanase activity or Avicel for exoglucanase activity [34]. Both substrates were prepared in sodium acetate, pH 5.5, containing 0.15 M NaCl. Afterwards, the reaction was ended by adding DNS (250 μL), followed by heating at 100 °C for 6 min. Next, the mixtures were cooled (15 min) and the absorbance at 540 nm was measured. The amount of reducing sugars was determined using the glucose-DNS standard curve mentioned above. One unit of enzyme activity corresponded to the amount of enzyme in GEAb that catalyzed the production of 1 μmol of glucose per minute. Blanks were performed by submitting GEAb to the same reaction steps described above without the addition of substrate. Two independent experiments were performed in triplicate.

2.7. Effects of SE, FR, and MvRL on Digestive Enzyme Activities

Gut extracts (GETb and GEAb) of A. diaperinus adults were incubated (1:1, v/v) with SE (260 μg of protein), FR (190 μg of protein), or MvRL (50 μg) for 15 min at 28 °C. Next, the mixtures were used to determine the residual activities of digestive enzymes as described above. Reaction blanks were performed using SE, FR, and MvRL. Two independent experiments were run in triplicate.

2.8. Statistical Analysis

Significant differences between the treatment groups were analyzed by a one-way analysis of variance (ANOVA), followed by Tukey’s test, with a significance level at p < 0.05, using the GraphPad Prism software. The data are represented as mean ± standard error of the mean (SEM).

3. Results

3.1. Lectin Preparations from Rhizomes

SE, FR, and MvRL from M. vacciniifolia rhizomes were successfully obtained with protein concentrations of 10.46, 7.5, and 1.8 mg/mL and specific HAs of 24.4, 136, and 1137, respectively. The purification factor of MvRL (ratio between the MvRL-specific HA and that of the previous stage) was 46.5 and 8.4 regarding SE and FR, respectively. MvRL HA was inhibited by about 90% in the presence of D(+)-mannose, and SDS-PAGE confirmed it as a single polypeptide of about 17 kDa (Figure 1a).

3.2. Insecticidal Effects on A. diaperinus

Lectin Preparations from M. vacciniifolia Rhizomes Induce Mortality of A. diaperinus Larvae and Alterations in the AChE Activity of Larvae and Adults

The survival rates of A. diaperinus fourth instar larvae and adults after 48 h exposure to SE, FR, and MvRL can be seen in Figure 1. The statistical analysis showed that the mortality of larvae (Figure 1b) was induced by all treatments with the M. vacciniifolia lectin preparations (F_3,9 = 24.95, R²: 0.8618; p < 0.0001), while no effect on adult survival (Figure 1c) was recorded (F_3,16 = 2.667, R²: 0.2286; p = 0.0678).

Figure 2 shows the AChE activities of body extracts from larvae (LBE) and adults (ABE) that remained alive after 48 h of starting treatments. The results revealed significant differences between treatments for both larvae (Figure 2a; F_3,5 = 43.85, R²: 0.8976; p < 0.0001) and adults (Figure 2b; F_3,5 = 43.81, R²: 0.8976; p < 0.0001). LBE from individuals exposed to FR showed higher (p < 0.05) AChE activity in comparison with LBE from control insects. Conversely, in the case of adults, ABE from insects treated with SE and MvRL showed higher (p < 0.05) AChE activity than control insects.

3.3. Lectin Preparations from M. vacciniifolia Rhizomes Have Antinutritional Effects on A. diaperinus Adults

The survival rates of adult insects incubated for 16 days with a diet containing SE, FR, or MvRL (delayed effect) are presented in

Table 1. Survival rate (%) of Alphitobius diaperinus adults incubated for 16 days (delayed effect) with diets containing saline extract (SE), lectin-rich fraction (FR), or lectin (MvRL) from Microgramma vacciniifolia rhizomes.

Treatment	Survival Rate (%)
	Days
	1st	8th	16th
Control	100 ± 0.0	100 ± 0.0	90 ± 3.33
SE (10.5 mg/mL)	100 ± 0.0	90 ± 2.98	70 ± 3.65 *
FR (7.5 mg/mL)	100 ± 0.0	90 ± 2.58	70 ± 4.71 *
MvRL (0.2 mg/mL)	100 ± 0.0	100 ± 0.0	100 ± 0.0
MvRL (0.4 mg/mL)	100 ± 0.0	100 ± 0.0	100 ± 0.0

(*) Significant difference (p < 0.05) in comparison with control. Each value corresponds to the mean ± SEM of two independent experiments performed in quintuplicate.

. It was observed that SE and FR induced significant (p < 0.05) adult mortality at day 16. The insects that did not die presented changes in locomotion, moving antennae and paws when touched, but without leaving the position in which they were. In contrast, the insects in the control group remained alive and actively moving. No mortality was observed with MvRL treatments at 0.2 and 0.4 mg/mL, and no changes in the motility or behavior of insects were detected.

Statistical analysis revealed that there were significant differences between the groups regarding the weight variation during the delayed effect assay (F_3,9 = 101.7, R²: 0.9187; p < 0.0001). Adults incubated with the diets containing SE and FR showed a negative variation (p < 0.05) in their weight after 16 days, indicating a loss of biomass (Figure 3). On the other hand, MvRL treatment did not cause loss of weight by the insects, similarly to the control.

Gut extracts (GETb and GEAb) of A. diaperinus adults showed trypsin-like (0.25 ± 0.017 mU/mg), endoglucanase (1.79 ± 0.15 mU/mg), exoglucanase (0.34 ± 0.04 mU/mg), and amylase (7.57 ± 0.27 mU/mg) activities (Figure 4). Trypsin-like activity was altered when GETb was incubated with all the lectin preparations (F_3,5 = 55.65, R²: 0.9176; p < 0.0001), with FR being the most effective inhibitory agent (88% reduction). SE, FR, and MvRL were also able to inhibit the endoglucanase activity (F_3,5 = 105.6, R²: 0.9548; p < 0.0001) of GEAb, showing similar effects between them (94–98% inhibition). The evaluation of GEAb exoglucanase activity showed that this enzyme activity was inhibited (F_3,5 = 20.13, R²: 0.8010; p < 0.0001) by SE and FR (93.2 and 94.1% inhibition, respectively), but not by MvRL. Finally, GEAb amylase activity was inhibited by all preparations (F_3,5 = 362.7, R²: 0.9864; p < 0.0001), with FR being the most effective (65% inhibition).

4. Discussion

The insecticidal potential of plant lectins has been extensively investigated against agricultural insect pests and disease vectors belonging to various orders, such as Coleoptera [35,36,37,38]. Previous reports have indicated that lectins can induce changes in all stages of insect development, interfering with behavior, survival, nutrition, development, and reproduction. Among the insecticidal mechanisms of lectins, the resistance to proteolysis and interactions with glycoconjugates of the digestive tract and/or digestive enzymes can be highlighted, which consequently cause damage to the morphology of the digestive tract, disruption of the peritrophic membrane, and inhibition or induction of enzymatic digestion [38,39].

A. diaperinus is one of the most common pests in poultry farms and it has been proven to be extremely difficult to control, as spinosad (an allosteric modulator of nicotinic acetylcholine receptors) is one of the few insecticides allowed for use in organic agriculture. This fact can lead to future problems because the constant use of insecticides without a planned scheme of rotation of action mechanisms can accelerate the emergence of resistant insect populations [15,40]. In addition, the use of synthetic insecticides in agriculture can lead to bioaccumulation and, therefore, the residual contamination of products, bringing risks to the health of consumers. In this sense, the demand for foods free of toxic residues requires alternative control strategies that result in minimal residual contamination and greater safety for consumers of chicken meat [40,41].

It is well known that chemical insecticides of reduced polarity tend to accumulate in living organisms and soil [15], and it was based on this knowledge that we investigated here the M. vacciniifolia rhizome preparations, which have high solubility in water and had been previously reported as potential insecticides against other species. The presence of protein content and hemagglutinating activity in SE, FR, and MvRL confirmed that the protocol established by Albuquerque et al. [21] was successfully reproduced. The inhibition of lectin activity by D(+)-mannose ensures that its domain for carbohydrate recognition remained active after elution. In addition, the MvRL electrophoretic profile ensured that the lectin had been isolated from other SE constituents, as well as that it was the same protein previously described [19,20,21].

When the insects were exposed for 48 h solely to the samples (immediate effect), the larvae were more sensitive than the adults. This was expected due to the immature condition of the biochemical and physiological apparatus, as well as the less-elaborated cuticle, of insect larvae [42]. The SE and FR concentrations tested were 10.5 and 7.5 times greater than that of MvRL. These values of the extract and fraction were lower than the purification factor of the lectin, indicating that MvRL was more concentrated in the isolated sample than in SE and FR, which allowed us to evaluate whether the lectin would be the main active principle. Since the mortality rates of larvae treated with SE, FR, and MvRL were not statistically different, the lectin is probably not the single larvicidal agent in FR and SE.

Acetylcholine is the main excitatory neurotransmitter in insects, while AChE catalyzes the degradation of this neurotransmitter, and inhibition of this enzyme leads the insect to paralysis due to nervous hyperexcitation [43]. Since AChE is the site of action of currently used insecticides, including carbamates and organophosphates, we evaluated whether the inhibition of this enzyme could be associated with the immediate larvicidal effect of M. vacciniifolia preparations. The data suggest that the larvicidal mechanisms of SE and MvRL involve other targets of larvicidal action unrelated to AChE. On the other hand, the increased AChE activity in larvae treated with FR may be linked to the insecticidal activity of this preparation. Bittencout and Souza [44] reported that 2-phenylethynyl-butyltellurium (PEBT), an insecticidal organotellurium compound, increased the AChE in the heads of Drosophila melanogaster. These authors called attention to a hypothesis that both decreases or increases in AChE activity can impact the locomotion of insects. In the case of increased AChE activity, there will be a decrease in acetylcholine levels, stopping nervous impulse transmission, which can affect locomotion and cause neuron death [45]. These data also indicate that other extract compounds, in addition to MvRL, may have been concentrated in FR, but eliminated after the lectin purification process. A previous study showed that saline extract from M. vacciniifolia rhizomes contains cinnamic acid derivatives and chlorogenic acid, while these metabolites are not present in FR [46]. Thus, it can be evaluated in the future whether FR contains other proteins than MvRL which were also concentrated after ammonium sulfate precipitation and that can be related to the differences in the results found for this preparation in comparison with SE and MvRL. When analyzing the results obtained for A. diaperinus adults, an increase in AChE activity in individuals treated with SE and MvRL was observed; however, this effect seems to be not enough to cause the death of the insects. This leads us to believe that other events that could have contributed to the death of the adults did not occur here.

Since A. diaperinus adults were not sensitive to 48 h exposure to M. vacciniifolia preparations, we evaluated whether SE, FR, and MvRL would have a delayed effect when ingested by the insects through incorporation in their diet. The reduction in body weight of insects when treated with SE and FR shows that they promoted negative effects on processes associated with the acquisition of biomass, such as ingestion, digestion, and absorption of nutrients. This datum corroborates with the fact that insect mortality caused by the ingestion of SE and FR occurred only at the 16th day, revealing a chronic effect. It is important to emphasize that, similarly to what was observed in the immediate assay with larvae, FR is a more efficient insecticide than SE to A. diaperinus adults, since it was evaluated at a concentration 1.4 times lower. The absence of mortality in adults that received a diet containing MvRL is consistent with the absence of changes in insect body weight and indicates that, unlike SE and FR, the lectin did not cause chronic effects until the 16th day of assessment. This result reinforces that SE and FR contain other insecticidal ingredients that were eliminated by chitin chromatography. The investigation of the delayed effect was not carried out with A. diaperinus larvae due to their recognized cannibalism habit [4,47], making it impossible to maintain the assay for long periods.

Since the results of the delayed effect assay suggested antinutritional effects of SE and FR on A. diaperinus adults, we evaluated the in vitro effects of M. vacciniifolia preparations on trypsin-like and glucosidase activities from insects’ gut extracts. Interestingly, SE, FR, and MvRL were able to inhibit the activity of the digestive enzymes evaluated. However, it is clear that FR was the most active preparation, indicating that other compounds than MvRL are linked to the delayed insecticidal effect.

In conclusion, M. vacciniifolia rhizome extract, lectin-rich fraction, and MvRL are larvicidal agents against A. diaperinus; additionally, the extract and fraction caused chronic effects on adults, reflected in the decrease in body weight. The preparations caused an imbalance of digestive enzymes and interfered with the AChE activity of the insects. In this sense, the damages of M. vacciniifolia preparations to the survival and physiology of A. diaperinus stimulate future studies on their use as biomaterials for controlling this pest, as well as on possible formulations or modes of application.