Differences in Midgut Phosphatases Activity and Hemolymph Composition in Lymantria dispar and Euproctis chrysorrhoea Larvae Exposed to the Polycyclic Aromatic Hydrocarbon Fluoranthene

TL;DR

This study shows how the pollutant fluoranthene affects enzyme activity and energy levels in two pest insect larvae, leading to reduced growth.

Contribution

The study reveals species-specific physiological responses to fluoranthene exposure in two insect species, highlighting differences in defense mechanisms.

Findings

Fluoranthene reduced non-lysosomal acid phosphatase activity in Lymantria dispar larvae.

Euproctis chrysorrhoea larvae showed increased acid phosphatase activity as a possible defense response.

Fluoranthene exposure reduced larval mass in both species, indicating resource allocation for defense.

Abstract

This study investigated the effects of the environmental pollutant fluoranthene on the activity of the digestive enzymes called phosphatases, as well as the level of energy compounds like lipids and trehalose in the larvae of two pest insect species Lymantria dispar and Euproctis chrysorrhoea. In Lymantria dispar larvae fluoranthene reduced the activity of non-lysosomal acid phosphatase, while in Euproctis chrysorrhoea larvae this pollutant caused an increase in the activity of total and lysosomal acid phosphatases. Additionally, the concentration of trehalose, a sugar that provides energy, decreased in the hemolymph of Lymantria dispar larvae exposed to fluoranthene, but lipid levels were unchanged in both species. Importantly, the mass of fifth instar larvae of both species was reduced by fluoranthne exposure, suggesting that this pollutant can negatively impact the growth and development of these common forest pests. These findings can also serve as a foundation for further research on the ecotoxicological impacts of fluoranthene and other similar environmental pollutants on the wide range of organisms including man.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants that can exert significant effects on living organisms. This study investigated the effects of fluoranthene, a representative PAH, on the activities of alkaline and acid phosphatases in the midgut, and on total lipid and trehalose concentrations in the hemolymph, of larvae of the forest insect species Lymantria dispar and Euproctis chrysorrhoea (Lepidoptera: Erebidae). The results revealed species-specific differences in midgut phosphatase responses to dietary fluoranthene exposure. In L. dispar larvae, the activity of non-lysosomal acid phosphatase decreased at the higher fluoranthene concentration. In contrast, the activities of total acid and lysosomal acid phosphatases increased in E. chrysorrhoea larvae exposed to the lower fluoranthene concentration, consistent with a defensive role. Zymogram analyses revealed different patterns of midgut phosphatase isoform expression in the two species. While lipid concentrations in the hemolymph were unaffected, a significant decrease in trehalose concentration was observed in L. dispar larvae exposed to the lower fluoranthene concentration. Furthermore, fluoranthene exposure resulted in reduced larval mass in both species, indicating the allocation of resources toward defense. These responses specify the significance of relationships between physiological changes and mass reduction in L. dispar and E. chrysorrhoea larvae affected by pollutant, and contribute to understanding their defense mechanisms and energy metabolism for coping with this environmental stressor.

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1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a group of complex organic compounds that are widely distributed in the environment. They originate from both natural sources, such as volcanic activity and forest fires, and more commonly from anthropogenic activities. These include industrial processes, incomplete combustion of fossil fuels, automobile exhaust, residential heating, cooking, and tobacco use [1,2,3]. PAHs are of significant public concern because of their environmental persistence, potential for bioaccumulation, and numerous adverse effects on human health and ecosystems. The United States Environmental Protection Agency [4] has classified 16 PAHs, including fluoranthene, as compounds of greatest environmental relevance, considering their recognized toxicity to both animals and humans.

Fluoranthene is often used as an indicator of overall environmental exposure to PAHs. It is a known pollutant with toxic and co-carcinogenic effects [3,5,6,7,8]. Thus, phytotoxicity of fluoranthene and other PAH compounds has been reviewed [9], reporting possible destruction of the photosynthetic pigment molecules in leaves of pea plants due to fluoranthene influence, than its accumulation in lipophilic cell compartments of experimental tobacco cell model, such as biomembranes, and their disturbed permeability due to ROS formation, or the harmful effects on stomatal conductance, on the contents of total chlorophyll etc., in Pinus densiflora. The results of Šepič et al. have shown that fluoranthene and primary metabolic products of its biodegradation by bacterial strain Pasteurella sp., were toxic for crustacean species Daphnia magna, while its metabolite of low molecular weight was toxic for Thamnocephalus platyurus [8]. The exposure of Capitella sp. I to fluoranthene led to DNA damage, related to impact of its metabolite [6]. Results of Saunders et al. have shown behavioral toxicity in 8-week-old female and male F-344 rats treated with fluoranthene [7], while non-cytotoxic doses of fluoranthene have increased carcinogenic potential of benzo[a]pyrene in a non-tumorigenic mouse C 10 cells, suggesting its co-carcinogenic properties [5].

Fluoranthene and other PAHs, detected in the leaves of various forest and other plant species, affect their physiological processes [9,10,11] and consequently influence phytophagous insects. Leaves of deciduous plant species serve as food for the larvae of widespread polyphagous forest pests, including Lymantria dispar and Euproctis chrysorrhoea, which are model systems in our research. The larvae, particularly in later instars, maintain direct contact with the substrate and feed continuously. They consume large quantities of leaves and can potentially bioaccumulate environmental toxicants [12,13].

In our previous studies [14,15], significant changes were detected in the activities of certain detoxification and antioxidant enzymes in the midgut and hemolymph of larvae of these two lepidopteran species exposed to long-term effects of fluoranthene, as well as in the activities of digestive enzymes.

Phosphatases play a significant role in the detoxification of various xenobiotics, processes that are energy-intensive and require resources for the activation of defense mechanisms aimed at mitigating their harmful effects. Alkaline (ALP) and acid (ACP) phosphatases are enzymes that catalyze the hydrolysis of phosphomonoesters in alkaline or acidic media [16]. The activity of both enzymes is highest in the insect midgut. ALP is present on the microvillar membrane of the midgut in Lepidoptera species, whereas ACP is soluble in the cytosol of midgut cells, although it can also be detected in other tissues [17]. These enzymes are involved in transphosphorylation reactions, digestion, carbohydrate and phospholipid metabolism, and other physiological processes with high energy demands [18,19]. While the overall activity of ACP is often equated with that of the lysosomal forms, it has been found that non-lysosomal phosphatases are frequently overlooked. The detoxifying role of insect phosphatases in defense against various xenobiotics has been previously investigated [20,21]. According to the available literature, the effects of PAHs on midgut phosphatases have been studied in L. dispar larvae exposed to benzo[a]pyrene [22]. Other studies have examined the effects of cadmium (Cd) on midgut phosphatase activity in L. dispar larvae [23,24], the influence of different host plants on alkaline phosphatase activities in Bemisia tabaci and Trialeurodes vaporariorum [25], and the impact of insecticides on the alkaline phosphatases of the honeybee Apis mellifera [26].

Hemolymph, the insect equivalent of vertebrate blood, circulates throughout the body in direct contact with tissues. It consists of a liquid phase (plasma) and cellular components known as hemocytes. Hemolymph plays a key role in transporting nutrients, xenobiotics, and their metabolites to other tissues [27]. The primary response to stressors is controlled by insect neurohormones, which regulate, among other processes, the energy supply of insects exposed to stress. Neurohormones can act in the hemolymph to control lipid and carbohydrate metabolism, which is part of the defense mechanism [28]. Lipids and carbohydrates are the main energy compounds in insects. Many insects, including some lepidopterans, utilize carbohydrates to provide energy. Energy substances can be transported from the fat body to the hemolymph, or stored in the hemolymph and transported to tissues and organs that require them [29]. Lipids comprise a broad class of natural compounds, including fats, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triacylglycerols, and phospholipids [30]. They are essential components of insect membranes and energy reserves, and serve as precursors for several secondary metabolites [31]. Trehalose is a non-reducing disaccharide composed of two glucose units joined by an α,α-1,1-glycosidic bond. In animals, trehalose was first described in the hemolymph of insects [32] at the larval and pupal stages [33]. In adult insects, trehalose levels rapidly decrease during energy-demanding activities, such as flying [34], highlighting its role as a readily mobilizable energy source. To date, no studies have investigated the effects of fluoranthene on insect hemolymph composition. However, several studies have reported reductions in protein, glycogen, and lipid content in the hemolymph of various arthropods, including insects, following exposure to chemical pollutants [35,36,37,38]. These depletions may be attributed to the mobilization of energy reserves to cope with pollutant-induced stress [39,40].

The presence of plant defense compounds, as well as inorganic and organic pollutants in food, can significantly impact the life history traits of phytophagous insects. Silva et al. [41] investigated four concentrations of rutin—a flavonoid glycoside from citrus—on Spodoptera frugiperda larvae and observed a linear relationship between increased rutin concentration and prolonged larval development, along with reduced larval mass. Matić et al. [42] reported a dose-dependent decrease in mass and prolonged development in fifth-instar L. dispar larvae exposed to two concentrations of Cd. Several studies have documented the effects of PAHs on the life history traits of phytophagous insects [43,44,45]. In response to xenobiotics, insects often reallocate resources toward defense mechanisms, resulting in reduced growth and reproductive performance [46].

As a continuation of our previous research [14,15,47,48], this study was conducted to evaluate the long-term effects of fluoranthene-supplemented diet on the activities of alkaline and acid phosphatases in the midgut, and total lipid and trehalose concentrations in the hemolymph of fifth-instar larvae of L. dispar and E. chrysorrhoea, as well as the differences in the responses to PAH pollutant between these two lepidopteran species. Since the induction of defense mechanism is energetically expensive, the effect of PAH pollutant on larval mass was also determined in both species. In addition, physiological changes, such as alterations of enzymes activity, may serve as early indicators of stress and environmental changes. Also, there is no scientific information available regarding the effects of fluoranthene on the examined parameters in these two important forest Lepidoptera species. The results of this study are of fundamental importance and may contribute to fluoranthene monitoring in forest ecosystems, which are increasingly threatened by organic pollutants.

2. Materials and Methods

2.1. Insect Rearing

As previously described [14,15], egg masses of L. dispar were collected in November from a mixed oak forest near the town of Majdanpek (eastern Serbia), while winter nests of E. chrysorrhoea were collected in February from a mixed oak forest near the town of Prijepolje (southwestern Serbia). These localities belong to the protected areas (https://www.zzps.rs, accessed on 20 November 2024), and are considered free from industrial pollution. The winter nests were stored at a temperature of 4 °C until late March (E. chrysorrhoea) while egg masses were stored at the same temperature until April (L. dispar), and then they were transferred to constant laboratory conditions suitable for larval development. Larvae of both species were randomly divided into control groups (C) and fluoranthene-treated groups (Fl and Fh). The control groups were fed a high wheat germ diet (HWG) [49], which is optimal for laboratory rearing. Larvae of the Fl and Fh groups were fed HWG diets supplemented with 6.7 ng/g dry food and 67 ng/g dry food of fluoranthene, respectively. The fluoranthene concentrations were chosen based on previously reported levels in leaves of oak and other deciduous tree species [11], and fall within the range found in other plant species [50,51]. Larvae were checked daily for molting, and fresh food was provided every 48 h. They were reared until the third day of the fifth instar, at which point they were weighed and subsequently sacrificed.

2.2. Preparation of Homogenates for Detection of Midgut Enzymes Activity and Hemolymph Concentrations of Lipids and Trehalose

On the third day of the fifth larval instar of both species, the last pair of legs was cut off and the hemolymph of each larva collected into tubes containing phenylthiourea. The larvae were then decapitated and the midgut dissected, with all procedures performed on ice. The midgut and hemolymph samples were stored at −20 °C until further use. The midgut of each larva was weighed and homogenized on ice in 0.15 M NaCl (final tissue concentration 100 mg/mL) using an Ultra Turrax (IKKA-Werke, Staufen, Germany), and then centrifuged at 10,000× g for 10 min at 4 °C (Eppendorf 5417R, Hamburg, Germany). The resulting supernatants were used for the determination of phosphatase activity.

The protein concentration was determined according to Bradford [52], using bovine serum albumin as a standard.

2.3. Midgut Phosphatases Activity

2.3.1. Alkaline Phosphatases

The activity of alkaline phosphatases was assessed using the method of Nemec and Socha [53]. This method is based on the hydrolysis of p-nitrophenyl phosphate (pNPP) under alkaline conditions, resulting in the formation of p-nitrophenol. The sample was added to a reaction mixture containing 100 mM Tris-HCl buffer (pH 8.6), 5 mM MgCl_2_, and 5 mM pNPP. After 30 min of incubation at 30 °C, the reaction was terminated by adding 500 mM NaOH, and absorbance was measured at 405 nm.

A modified procedure based on Allen et al. [54] was used for the detection of alkaline phosphatase activity via native electrophoresis on a 12% polyacrylamide gel. Protein samples (10 µg per well) were subjected to electrophoresis at a constant voltage of 100 V at 4 °C. Following electrophoresis, the gel was rinsed with distilled water and incubated in a buffer containing 100 mM Tris-HCl (pH 8.6), 0.13% α-naphthyl phosphate, and 0.1% Fast Blue B. Colored bands formed at room temperature, indicating enzyme activity.

2.3.2. Total Acid Phosphatases

The activity of total acid phosphatases was determined using the same method [53], adapted for acidic conditions. Sample was added to a reaction mixture consisting of 100 mM citrate buffer (pH 5.6), 5 mM MgCl_2_, and 5 mM pNPP. After 60 min of incubation at 30 °C, the reaction was terminated by adding 500 mM NaOH. Absorbance was recorded at 405 nm to quantify the formation of p-nitrophenol.

Detection of acid phosphatase activity on native gels was performed using a modified version of the method of Allen et al. [54]. Protein samples (10 µg per well) were loaded onto a 12% polyacrylamide gel and separated at a constant voltage (100 V) at 4 °C. The gel was rinsed with distilled water and incubated in 50 mM acetate buffer (pH 5.2) at 30 °C for 10 min. A nitrocellulose membrane was then incubated in a 0.13% solution of α-naphthyl phosphate in acetate buffer. The membrane was placed on the gel surface and incubated in a humid chamber in the dark at 30 °C for one hour. After incubation, it was immersed in a 0.3% solution of Fast Blue B in acetate buffer until bands of acid phosphatase activity appeared.

2.3.3. Lysosomal and Non-Lysosomal Acid Phosphatases

Lysosomal acid phosphatase activity was estimated indirectly using the procedure described by Amlabu et al. [55] and Holtzman [56]. Total acid phosphatase activity was determined as previously described, with 50 mM sodium fluoride (a specific inhibitor of lysosomal phosphatases) added to distinguish non-lysosomal enzyme activity. Absorbance at 405 nm reflected non-lysosomal acid phosphatase activity, and lysosomal activity was calculated by subtracting this value from the total.

Specific enzyme activities are expressed as units per mg of protein.

Differences in band intensities were analyzed using ImageJ 1.42q software (NIH, Bethesda, MD, USA).

Midgut samples (individual midgut homogenates, N = 7–10 for L. dispar larvae and N = 8–10 for E. chrysorrhoea larvae, per experimental group) were measured in duplicate using 2 blank samples and 2 non-catalytic probes.

2.4. Lipid and Trehalose Concentrations in Hemolymph

2.4.1. Lipid Concentration

Total lipid concentration in larval hemolymph was determined according to the method of Zöllner and Kirsch [57]. Concentrated sulfuric acid was added to hemolymph samples, followed by boiling in a water bath for 10 min. After cooling to room temperature, vanillin reagent (0.19% vanillin in 85% phosphoric acid) was added. The samples were shaken vigorously and incubated for 30 min to allow color development. Absorbance was measured at 546 nm, and lipid concentration was calculated based on a standard curve prepared from a triacylglycerol mixture.

2.4.2. Trehalose Concentration

Trehalose concentration was determined according to the method of Wyatt and Kale [32]. Hemolymph samples were mixed with 10% trichloroacetic acid and centrifuged at 5500 rpm for 5 min. Anthrone reagent (0.15% anthrone in 72% sulfuric acid) was added to the supernatant, and the mixture was incubated in a boiling water bath for 15 min. Samples were cooled in an ice bath for 10 min, then mixed vigorously. Absorbance was measured at 630 nm. Trehalose concentration was calculated from a standard curve prepared with known concentrations of trehalose.

Individual hemolymph samples used for the assays (N = 12–15 for L. dispar as well as for E. chrysorrhoea larvae, per experimental group, and measured in duplicate using 2 blank samples and 2 non-catalytic probes.

2.5. Larval Mass

The mass of larvae of both species was measured on the third day of the fifth instar. Sample sizes for experimental groups were N = 77–91 for L. dispar larvae, and N = 67–79 for E. chrysorrhoea larvae.

2.6. Statistical Analyses

Results are presented as means and standard errors of the mean. Following the assessment of normality and homogeneity of variances (Kolmogorov–Smirnov and Shapiro–Wilk tests, and Levene’s test, respectively), significant differences in mean values of analyzed parameters between experimental groups were estimated by one-way ANOVA and Tukey’s test. Analyses were performed on log-transformed values of phosphatase specific activities [58]. The influence of dietary fluoranthene on the mass of L. dispar and E. chrysorrhoea on the third day of the fifth larval instar was analyzed by Kruskal–Wallis ANOVA and multiple comparisons of mean ranks for all experimental groups [59]. Statistical significance was considered at p < 0.05. Canonical discriminant analysis was employed to evaluate species-level differences, and cluster analysis (unweighted pair-group average, using city-block–Manhattan distances) was performed to classify all measured parameters into homogeneous groups. All statistical analyses were conducted using Statistica 12.0 (StatSoft, Inc., Tulsa, OK, USA).

3. Results

3.1. Phosphatases Activity in the Midgut of L. dispar Larvae

No significant changes were observed in the specific activity of alkaline phosphatases in the midgut of L. dispar larvae following fluoranthene exposure compared with the control group [F_(2,27)_ = 1.011; p > 0.05]. Zymogram analysis revealed three isoforms (I1, I2, I3). Isoforms I1 and I2 were present across all three groups, whereas isoform I3 was absent in larvae exposed to the lower fluoranthene concentration. Isoform I2 showed the highest intensity, which decreased in a concentration-dependent manner (Figure 1a).

There were no statistically significant changes in total acid phosphatase activity in the midgut of L. dispar larvae after long-term exposure to fluoranthene [F_(2,27)_ = 1.583; p > 0.05]. Zymogram analysis confirmed the presence of three isoforms in all groups. Isoform I2 exhibited the highest intensity, which declined with increasing fluoranthene concentration (Figure 1b).

No significant differences in lysosomal acid phosphatase activity were detected between the control and treated groups [F_(2,24)_ = 1.582; p > 0.05] (Figure 1c). However, a significant reduction in non-lysosomal acid phosphatase activity was observed in larvae exposed to the higher fluoranthene concentration [F_(2,23)_ = 5.200; p < 0.05] (Figure 1d).

3.2. Phosphatases Activity in the Midgut of E. chrysorrhoea Larvae

The specific activity of alkaline phosphatase was significantly lower in the group exposed to the higher fluoranthene concentration compared with the group exposed to the lower one [F_(2,27)_ = 3.920; p < 0.05]. Zymogram analysis revealed a single isoform present in all groups, with visibly higher intensity in fluoranthene-treated groups compared with the control (Figure 2a).

Total acid phosphatase activity in the midgut of E. chrysorrhoea larvae increased significantly after exposure to fluoranthene at a concentration of 6.7 ng/g dry food [F_(2,27)_ = 14.049; p < 0.05]. Zymographic analysis detected four isoforms. Isoform I1 appeared exclusively in the lower-concentration treatment group and was absent in the other two. Isoforms I2 and I3 showed the strongest intensity in the group exposed to the lower fluoranthene concentration, whereas isoform I4, the dominant form, showed no visible changes across groups (Figure 2b).

Lysosomal acid phosphatase activity was significantly elevated in larvae exposed to the lower fluoranthene concentration compared with the control [F_(2,27)_ = 18.846; p < 0.05] (Figure 2c). However, no statistically significant changes were observed in non-lysosomal acid phosphatase activity between the control and treated groups, [F_(2,25)_ = 1.206; p > 0.05] (Figure 2d).

Original, uncropped images of the native gel electrophoresis (Figure 1a,b and Figure 2a,b) are provided in the Supplementary Materials.

3.3. Phosphatase Activity Patterns

Canonical discriminant and cluster analyses were used to assess the similarities and differences in phosphatase activities between L. dispar and E. chrysorrhoea larvae in response to long-term exposure to a fluoranthene-supplemented diet.

At the lower fluoranthene concentration, canonical discriminant analysis revealed clear separation between groups. The first canonical function accounted for 69.8% of the variability, primarily influenced by the activities of lysosomal and non-lysosomal acid phosphatases. The second function accounted for 30.1%, with total acid phosphatase activity being the major contributing factor (Figure 3a).

At the higher fluoranthene concentration, group separation was less pronounced. The first canonical function explained 50.9% of the variability, and the second function 47.8%. The major contributors to separation along the first function were alkaline, lysosomal, and non-lysosomal acid phosphatases, whereas the specific activity of alkaline phosphatases dominated the second function (Figure 3b).

3.4. Lipid and Trehalose Concentrations in Hemolymph of L. dispar and E. chrysorrhoea Larvae

Long-term exposure to dietary fluoranthene had no significant effect on lipid concentration in the hemolymph of L. dispar larvae [F_(2,38)_ = 1.234; p > 0.05]. However, trehalose concentration significantly decreased in larvae exposed to the lower fluoranthene concentration (6.7 ng/g dry food), compared with the control group, [F_(2,38)_ = 4.005; p < 0.05] (Figure 4a).

No significant differences were observed in lipid [F_(2,41)_ =2.557; p > 0.05] and trehalose [F_(2,37)_ = 0.622; p > 0.05] concentrations in the hemolymph of E. chrysorrhoea larvae across experimental groups (Figure 4b).

3.5. Larval Mass of L. dispar and E. chrysorrhoea

In L. dispar, a significant reduction in the mass of fifth-instar larvae was observed following exposure to the lower concentration of dietary fluoranthene [H (2, N = 251) = 7.025; p < 0.05] (Figure 5a).

In E. chrysorrhoea, larval mass decreased at both fluoranthene concentrations compared with the control [H (2, N = 216) = 46.665; p < 0.05] (Figure 5b).

4. Discussion

In this study, no significant changes were observed in the specific activities of alkaline, total acid, or lysosomal acid phosphatases in the midgut of L. dispar larvae after exposure to fluoranthene-supplemented diet. Only the activity of non-lysosomal acid phosphatase was sensitive to fluoranthene, with significant inhibition observed at the higher concentration compared with the control. Similarly, no significant changes were recorded in alkaline phosphatase activity in fifth-instar larvae of Glyphodes pyloalis exposed to Cd. In contrast, acid phosphatase activity was significantly reduced in treated larvae compared with controls, which may be explained by Cd-induced ROS damaging cells and reducing enzyme activity [21]. Inhibition of acid phosphatases has also been observed in larvae of Cnaphalocrocis medinalis chronically exposed to plant insecticides and Bacillus thuringiensis toxins [60]. The authors concluded that reduced acid phosphatase activity under the influence of plant insecticides and bacterial toxins indicates a decreased release of phosphorus required for energy metabolism, a reduced metabolic rate, and lower metabolite transport, potentially due to direct effects on enzyme regulation. The same species (C. medinalis) exposed to limonoids from Azadirachta indica also showed reduced acid phosphatase activity [61]. Reduced phosphatase activity has been reported in the hepatopancreas and ovary of the crab Scylla tranquebarica from a naphthalene-polluted habitat [62]. Although the activities of other phosphatases did not change, a higher concentration of fluoranthene in the diet was sufficient to reduce only the activity of non-lysosomal phosphatase in the midgut of L. dispar larvae. This could be a consequence of disrupted energy metabolism and/or oxidative stress caused by the pollutant, consistent with previously observed induction of antioxidant enzyme activity in the midgut tissue of this species [15].

In E. chrysorrhoea, alkaline phosphatase activity remained unchanged following fluoranthene exposure, compared with the control group. However, the activities of total acid and lysosomal acid phosphatases increased significantly in larvae exposed to the lower pollutant concentration. Similar findings were reported by Jiang and Yan [63] in L. dispar larvae reared on poplar leaves contaminated with heavy metals, in which increased acid phosphatase levels were detected, and by Matić et al. [64], who documented increased acid phosphatase activity in L. dispar larvae after exposure to Cd. Increased acid phosphatase activity has also been observed in Galleria mellonella larvae fed with plant growth regulators [65]. Significantly increased acid and alkaline phosphatase activities were recorded in the midgut tissue of Agrotis ipsilon larvae fed on Ricinus communis leaves collected from metal-contaminated areas [66]. Braeckman et al. [67], while studying the effect of Cd on acid phosphatases in the C6/36 cell line of the insect Aedes albopictus, also reported increased activity, indicating activation of the lysosomal system for xenobiotic sequestration. High acid phosphatase activity has similarly been detected in the hemolymph of mussels exposed to low-molecular weight PAHs, such as phenanthrene and anthracene [68], likely reflecting lysosomal membrane destabilization in hemocytes. Disruption of lysosomal membrane integrity may lead to acid phosphatase release into the cytosol [69,70], causing cellular damage and increased cell death. The high activity of acid phosphatases in the hemolymph of these shellfish may indicate increased synthesis in viable hemocytes to enhance defense against the toxic effects of xenobiotics. Considering the important roles of acid phosphatases, an increase in their activity was expected in E. chrysorrhoea larvae in response to pollutant exposure. A similar response has been reported in third-instar larvae of the beetle Xanthogaleruca luteola treated with a botanical insecticide, suggesting the role of the enzyme in the detoxification process [20].

A large number of different enzyme isoforms are involved in these functions. The expression of specific isoforms and their differential activities are crucial for the capacity of enzymes to respond to the presence of particular pollutants and mitigate their harmful effects.

The divergent enzyme responses observed between L. dispar and E. chrysorrhoea larvae may result from differences in the expression of enzyme isoforms. Zymogram analysis revealed three alkaline phosphatase isoforms in L. dispar larvae, with the dominant isoform decreasing in intensity as dietary fluoranthene concentration increased. In contrast, only one alkaline phosphatase isoform was detected in E. chrysorrhoea larvae, and its intensity increased following fluoranthene exposure. Acid phosphatase zymograms revealed three isoforms in L. dispar and four in E. chrysorrhoea, with isoform I1 present only in E. chrysorrhoea larvae treated with the lower fluoranthene concentration. This isoform may be primarily responsible for the observed increase in specific activity, suggesting species-specific differences in enzyme response to dietary fluoranthene. Canonical discriminant analysis further confirmed these species-specific differences in phosphatase responses to fluoranthene exposure.

Insect hemolymph is a key source of information regarding the mechanisms controlling nutrient uptake. Nutrients newly absorbed from food in the gut or generated by metabolic activities of the fat body and other tissues enter the hemolymph. Nutrients required for somatic and reproductive growth, respiration, and excretion are also drawn from the hemolymph via the fat body and other tissues. Thus, the current concentrations of constituents in the hemolymph can provide information about the timing, quantity, and nutrient content of the insect’s last meal and its current nutrient requirements [71,72,73]. The hemolymph is important not only for transporting nutrients but also for transporting xenobiotics and their metabolites to tissues [27]. Lipids serve as the primary energy reserves during non-feeding periods [27,74], and a significant portion of dietary carbohydrates is converted to lipids during larval development [75]. Stored lipids are metabolized by lipases to meet energy demands during energetically costly processes, such as metamorphosis [76]. Lipid depletion may result from mobilization in response to stress, as shown in Pimpla turionellae and Galleria mellonella larvae exposed to cypermethrin and cadmium, respectively [36,77]. Altuntaş [78] proposed that the reduced lipid content in G. mellonella exposed to ethephon may reflect lipid and glucose utilization for lipoprotein synthesis, which aids cellular repair under stress. Similar findings were reported in Tenebrio molitor females exposed to malathion, attributed to disruption of adipokinetic hormone regulation [79]. In marine organisms, such as crabs from naphthalene-polluted environments, lipid concentrations in key organs were also found to be reduced [62]. However, in this study, no significant changes in lipid concentration were observed in the hemolymph of L. dispar or E. chrysorrhoea larvae exposed to fluoranthene, suggesting that lipid catabolism was not a primary stress response.

In contrast, a significant decrease in trehalose concentration was detected in L. dispar larvae exposed to the lower fluoranthene concentration. Although the physiological role of trehalose as an energy source during insect development is not fully understood, its mobilization is considered critical for maintaining metabolic homeostasis [80]. Trehalose is a non-reducing glucose disaccharide that serves as the basic form of carbohydrate transfer from glycogen depots in the fat body to tissues requiring energy [81], both under normal and stress conditions. Carbohydrates play an essential role in the structure and function of all insect tissues [82], and their hemolymph content is an important indicator of metabolic rate [83]. In general, trehalose concentration in insect hemolymph is high, as it is hydrolyzed into two glucose molecules to supply energy to tissues and organs [84]. Several studies have reported decreases in carbohydrate content in specific tissues of different insect species following insecticide exposure [85,86,87,88]. Decreased trehalose concentrations have also been observed in the hemolymph of L. dispar larvae infected with Gliptapanteles liparidis parasites and following Cd exposure [35]. Similarly, trehalose and glucose levels in hemolymph decline in insects under starvation stress [89]. The recorded decrease in trehalose concentration in L. dispar larvae may be due to the fact that defense against stressful conditions requires the provision of an energy source. Moreover, because insect neurohormones are involved in regulating energy supply during stress and influence carbohydrate metabolism in the hemolymph, some of these hormones may modulate trehalose concentration under stressful conditions [28]. Interestingly, trehalose concentrations in the hemolymph of E. chrysorrhoea larvae were lower than those measured in L. dispar larvae across all experimental groups. This could indicate that E. chrysorrhoea larvae efficiently utilize trehalose for energy production under both normal and stressful conditions, even at lower concentrations.

In addition to enzyme activity and hemolymph composition, fluoranthene also affected larval performance. Effects on life history traits were observed in the larvae of both species in response to fluoranthene-supplemented food [14,47]. In the present study, we recorded the effects of fluoranthene on larval mass in both species; in E. chrysorrhoea, this effect was apparent at both lower and higher concentrations of dietary fluoranthene. Insects often allocate resources toward defense mechanisms under environmental stress, which can reduce growth and reproduction [46,90]. Food quantity and quality significantly influence life history traits, and the nutritional intake and energy reserves acquired during the larval stage affect adult survival and fecundity [91]. Nutritional deprivation during development can reduce mass and growth rates, prolong development, and impair reproductive capacity [92,93,94]. Previous studies have shown that nicotine in artificial diets reduces mass and growth in Spodoptera eridania [95], while Cd exposure reduces mass in L. dispar larvae [96]. The mass reduction observed in L. dispar and E. chrysorrhoea larvae may therefore be a consequence of the allocation of resources towards defense under stressful conditions. Insect development and other processes are also controlled by hormones, and disruption of hormonal balance can lead to abnormal development [97]. For example, in Chironomus tentans, exposure to fluoranthene and pentachlorobenzene reduced cumulative adult emergence, potentially due to interference with hormone levels or metabolic regulation [98]. Thus, the reduction in larval mass observed in L. dispar and E. chrysorrhoea in the present study may similarly result from altered hormonal regulation triggered by fluoranthene or its metabolites.

5. Conclusions

The results of this study revealed changes in midgut phosphatases activity in L. dispar and E. chrysorrhoea larvae, and reduction in larval mass in both species treated with dietary fluoranthene. This study provides unique insight into the differential responses of two lepidopteran species to fluoranthene exposure. It highlights the complexity of insect physiological responses to environmental pollution, demonstrating that even related species can exhibit markedly different strategies for coping with stress induced by pollutants, and also the potential for adjustment to stressful conditions. Canonical discriminant analysis confirmed differences between species in the responses of phosphatase enzymes to fluoranthene in food. The research further underscores the importance of considering multiple physiological responses, as changes in enzyme activities, energy metabolism, and also growth can all contribute to the insect’s overall response to environmental stress. Since insects encounter a variety of environmental pollutants, these findings have implications for understanding the ecological consequences of PAH pollution and the potential of insects to develop sensitivity or tolerance to such compounds over time. In addition, isozyme analysis can be used in biomonitoring of environmental pollution, as chronic exposure to fluoranthene leads to differences in the expression of phosphatase enzymes isoforms in the midgut of larvae of both species.