Abstract
Pollination by honey bees (Apis mellifera) is crucial for maintaining biodiversity and crop yields. However, the widespread use of pesticides may threaten bees’ survival by contaminating their resources. Lambda-cyhalothrin, a neurotoxic insecticide commonly used in agricultural pest control, poses particular risks. In insects, the midgut and fat body serve as primary barriers against xenobiotics, and exposure to these chemicals during larval development can impact adult bees. This study aimed to assess whether the residual concentration of lambda-cyhalothrin in pollen grains affects the midgut and fat body of larval A. mellifera workers after chronic exposure. The midgut epithelium of larvae exposed to a lambda-cyhalothrin-based insecticide (λ-CBI) exhibited autophagic vacuoles, apical cell protrusions, apocrine secretion, nuclear pyknosis, and high levels of polysaccharides and glycoconjugates in the cytoplasm, with smaller amounts in the brush border. Histochemical analysis revealed areas of vacuolation and damage to cell integrity in the midgut. In fat body cells, the insecticide increased polysaccharide storage and decreased lipid droplet diameter. Despite the histopathological damage, no effects were found in the larval development and adult emergence. These findings suggest the occurrence of apoptosis and autophagy in midgut cells and alterations in nutrient storage in the fat body of A. mellifera larvae exposed to the λ-CBI, potentially impacting the physiology and development of this pollinator with possible effects on adult workers.
Introduction
Bees are insects belonging to Hymenoptera, which also includes wasps and ants, and they exhibit richness and diversity of species with global distribution (Orr et al., 2021). Among the Hymenoptera, bees play a crucial role in the global economy, whether through the extraction of their products, including honey, royal jelly, wax, propolis, and apitoxin, or the pollination of native and cultivated plants (Castilhos et al., 2016; Cornara et al., 2017; Khalifa et al., 2021).
The honey bee Apis mellifera Linnaeus, 1758 (Apoidea: Apidae) accounts for approximately the same economic benefits as all other bee species combined (Kleijn et al., 2015), which are commercially used in the pollination of crops (Khalifa et al., 2021; Klein et al., 2007).
In recent decades, there has been a general decline in the number of pollinators (Potts et al., 2010; Rhodes, 2018; Zattara & Aizen, 2021), as well as colony collapse disorder, which affects colonies of A. mellifera (Evans & Chen, 2021; Roy et al., 2018). The decline in bee populations has been attributed to the occurrence of pathogens and parasites (Gregorc et al., 2016; Smith et al., 2013), climate change (Le Conte & Navajas, 2008), dietary restrictions (Grab et al., 2019), introduction of exotic species (Iwasaki & Hogendoorn, 2022; Page & Williams, 2023a, 2023b), excessive use of pesticides (Abati et al., 2021; Kiljanek et al., 2016; Sanchez-Bayo & Goka, 2014), and interactions among the aforementioned factors (Cornelissen et al., 2019; Dicks et al., 2021; Goulson et al., 2015; Neov et al., 2021; Potts et al., 2010; Siviter et al., 2021). Population growth and high demand for food have stimulated the development of new technologies to improve food production (Popp et al., 2013), including the use of pesticides, which are indispensable for large-scale agriculture. However, since they are extensively employed in pest control, they contaminate soil, water, and air, besides damaging non-target organisms (Abraham et al., 2018; Ceuppens et al., 2015; Johnson, 2015; Verma & Bhardwaj, 2015).
Due to the reliance on pesticides, new compounds for agricultural use have been developed. Lambda-cyhalothrin is a synthetic organic pyrethroid that remains effective for long periods, with a half-life of up to three weeks. It is efficient in controlling various pest insects, including beetles, leafhoppers, caterpillars, sap-sucking bugs, and flies, in several crops such as cotton, potatoes, coffee, beans, corn, soybeans, tomatoes, and wheat (He et al., 2008).
Pyrethroids act on the insect nervous system, modulating the activation of voltage-dependent sodium channels present in neurons that affect calcium channels, whose activity is important in nerve impulse conduction causing paralysis or death (Davies et al., 2007). Lambda-cyhalothrin is classified as a type II pyrethroid, characterized by a cyano group, which, in addition to modulating the activation of voltage-dependent sodium channels present in neurons, affects calcium channels, whose activity is important in nerve impulse conduction (Davies et al., 2007). At lethal concentration applied topically, it crosses the insect cuticle causing a knock-down effect, cessation of food consumption, loss of muscle control, paralysis, and death (Bradberry et al., 2005; Soderlund, 2012).
The focus on lethality and pest control, coupled with the false perception of lambda-cyhalothrin’s environmental safety, results in limited studies on other non-target animals, including vertebrates and invertebrates that may benefit crops, such as natural enemies and pollinators (Desneux et al., 2007). Some studies reveal that the adverse effects of lambda-cyhalothrin include multiple impairments in different aspects of the health of these animals, such as hematological, hepatic, pulmonary, renal, and reproductive alterations in rabbits and rats (Basir et al., 2011; Fetoui et al., 2010; Yousef, 2010); hematological changes, gill tissue damage, cardiac, hepatic, and renal tissue damage, and increased mortality rate in fish (Amin et al., 2020; Çavaş & Ergene-Gözükara 2003; Li et al., 2014; Wang et al., 2007); hematological changes, behavioral alterations, and increased mortality rate in amphibians (Campana et al., 2003; Nkontcheu et al., 2017); cytoplasmic vacuolization and necrosis in the liver in the lizard Eremias argus Peters, 1869 (Chang et al., 2016); embryonic death, growth delay, and developmental abnormalities in embryos in chicken Gallus gallus domesticus (Linnaeus, 1758; Sadaf et al., 2022); and increased mortality rate in coccinellids (Jalali et al., 2009; Rodrigues et al., 2013).
Negative effects of lambda-cyhalothrin are also observed in bees, including reduced food consumption, reproductive rate, and increased mortality in adults of the bumblebee Bombus terrestris (Linnaeus, 1758; Ceuppens et al., 2015), and histopathological damage to the midgut, hypopharyngeal glands, nervous system, alterations in locomotion pattern, social interaction, memory, longevity, and mortality in adults of A. mellifera (Abdel Razik, 2019; Arthidoro de Castro et al., 2020; Bailey et al., 2005; Ingram et al., 2015; Liao et al., 2018; Melisie et al., 2015).
In addition to the effects of direct pesticide application on non-target organisms, some chemicals are found in the field in residual concentrations, associated with substrates that are collected by worker bees and used in the feeding of the whole colony, such as nectar and pollen grains (Mullin et al., 2010; Zioga et al., 2020; 2023). In a realistic field scenario, pesticides are sprayed mixed with adjuvants that increase the spreading and sticking of the active molecules facilitating their penetration into the pest body. Although adjuvants are classified as inert ingredients, some studies reveal that they may be toxic for non-target insects, including bees (Mullin, 2015; Shannon et al. 2023a). Therefore, assessing the effects of pesticide formulation at residual concentrations found in the field on both adult and immature individuals can provide insights into how pesticides affect the biological aspects of bees and how this may affect the colony dynamics.
Apis mellifera undergoes complete metamorphosis, comprising the stages of egg, five larval instars, pupa, and adult. The duration of each larval instar is distinct, and the total process takes up to five days (Michelette & Soares, 1993; Rembold et al., 1980).
Although the targets of pyrethroids are the protein channels in the nervous system, the midgut is the first organ to come into contact with the insecticide molecules when they are ingested with contaminated food (Denecke et al., 2018). Additionally, the insecticide can cross the barrier formed by the intestinal epithelium and circulate through the hemolymph, reaching other organs such as the fat body (Denecke et al., 2018). Thus, despite not being the target organs of lambda-cyhalothrin it is necessary to evaluate its sublethal effects on the cells of the midgut and fat body of A. mellifera.
The midgut is the main organ of the digestive system in insects, where digestion and nutrient absorption take place. It is composed of a simple epithelium with columnar digestive cells, which are the most numerous, along with endocrine and regenerative cells, all onto a thin basal lamina and a double layer of circular and longitudinal muscles (Caccia et al., 2019).
The digestive cells are involved in the production and secretion of digestive enzymes, peritrophic matrix, and nutrient absorption (Hegedus et al., 2009; Serrão & Cruz-Landim, 1996a; 2000). Endocrine cells are few and regulate the midgut functions through hormone secretion (Neves et al., 2003; Serrão & Cruz-Landim, 1996b). Regenerative cells, which are found throughout the midgut, clustered in nests near the base of the digestive cells, work in the replacement of the other epithelial cells that die (Dias et al., 2024; Fernandes et al., 2010; Martins et al., 2006).
Similarly to other holometabolous insects, the digestive tract of bees undergoes extensive remodeling during metamorphosis (Gonçalves et al., 2017). In the pupal stage, midgut cells detach toward the lumen, while regenerative cells give rise to the new epithelium through mitosis and differentiation (Martins et al., 2006; Neves et al., 2002).
The fat body in bee larvae consists of two types of cells: trophocytes, the most abundant cell type of mesodermal origin, and oenocytes, found more sparsely and of ectodermal origin. This organ may be parietal close to the integument and perivisceral surrounding the internal organs (Oliveira & Cruz-Landim, 2003). During the larval stage, the trophocytes store energy reserves for metamorphosis, including lipids, carbohydrates, and proteins. Additionally, the fat body plays a role in innate immunity, besides helping in the detoxification of xenobiotics, intermediary metabolism, hormonal regulation, and synthesis of hemolymph components (Feyereisen, 1999; Li et al., 2007; 2019). Oenocytes are large, spherical-shaped cells with well-defined borders, responsible for the production of hydrocarbons and waxes present in the body cuticle. However, they may also be involved in organismal homeostasis, such as the detoxification of xenobiotics and innate immunity (Martins & Ramalho-Ortigão, 2012).
In addition to their intrinsic functions in insect development, the digestive tract and fat body also participate in detoxifying the organism and developing pesticide resistance. Thus, it is necessary to understand how pesticides affect the cells of these organs during larval development to elucidate gaps concerning how these chemicals affect organs, developmental stages, and non-target organisms. The present study aimed to test the hypothesis that the chronic ingestion of residual concentrations of the insecticide lambda-cyhalothrin found in pollen grains has cytotoxic effects on non-target organs, namely, the midgut and fat body of A. mellifera larvae.
Materials and methods
Insect collection
Frames of Africanized A. mellifera brood were collected from five colonies at the Central Apiary of the Federal University of Viçosa, Viçosa, Minas Gerais, Brazil (20° 45' N 42° 52' W) and transferred to the laboratory to obtain the larvae. The apiary is kept near to a natural area without use of pesticide or other anthropogenic pollutants.
Exposure to insecticide
To evaluate the effect of the insecticide on the larvae, the bioassays were conducted using 384 first instar larvae (head capsule 0.32 mm, weight 0.11–0.30 mg; Michelette & Soares, 1993) of A. mellifera. The control group was formed by 192 of them, while 192 larvae were exposed to the commercial lambda-cyhalothrin-based insecticide Karate Zeon 50CS (λ-CBI; 50 g L−1 active ingredient Syngenta, São Paulo, Brazil). The bioassay followed the guidelines of Organisation for Economic Co-operation and Development (OECD) 239 (OECD, 2016). The first instar larvae were collected and transferred to artificial queen cells (9 mm in diameter and 8 mm in depth) with 20 µL of diet (Table 1). The queen cells were placed in 48-well cell culture plates containing a piece of cotton moistened with a sterilizing solution (15% w/v glycerol and 0.2% w/v methylbenzethonium chloride) and kept in a hermetic chamber, at 34 ± 1 °C, 85 ± 5% relative humidity, in the dark. The artificial larval diet contained 50% (w/w) sugars (D-fructose, D-glucose, and yeast extract) and 50% (w/w) royal jelly. From the third to the sixth day of the larval development, control larvae were fed on an artificial diet, and the treated larvae were fed on a diet containing λ-CBI, at the concentrations summarized in Table 1.
Diet supplied daily to Apis mellifera larvae with the correspondent amount of the insecticide lambda-cyhalothrin active ingredient according to Organisation for Economic Co-operation and Development guidelines 239 (OECD, 2016).
| Day | Diet volume per larvae (µL) | D-Glucose (% w/w) | D-fructose (% w/w) | Yeast extract (% w/w) | Royal jelly (% w/w) | Lambda-cyhalothrin 5 × 10−10 (g mL−1) |
|---|---|---|---|---|---|---|
| 1 | 20 | 12 | 12 | 2 | 50 | – |
| 2 | – | – | – | – | – | – |
| 3 | 20 | 15 | 15 | 3 | 50 | 0.48 |
| 4 | 30 | 18 | 18 | 4 | 50 | 0.86 |
| 5 | 40 | 18 | 18 | 4 | 50 | 1.47 |
| 6 | 50 | 18 | 18 | 4 | 50 | 2.24 |
| Day | Diet volume per larvae (µL) | D-Glucose (% w/w) | D-fructose (% w/w) | Yeast extract (% w/w) | Royal jelly (% w/w) | Lambda-cyhalothrin 5 × 10−10 (g mL−1) |
|---|---|---|---|---|---|---|
| 1 | 20 | 12 | 12 | 2 | 50 | – |
| 2 | – | – | – | – | – | – |
| 3 | 20 | 15 | 15 | 3 | 50 | 0.48 |
| 4 | 30 | 18 | 18 | 4 | 50 | 0.86 |
| 5 | 40 | 18 | 18 | 4 | 50 | 1.47 |
| 6 | 50 | 18 | 18 | 4 | 50 | 2.24 |
Note. “–” = not supplied.
Diet supplied daily to Apis mellifera larvae with the correspondent amount of the insecticide lambda-cyhalothrin active ingredient according to Organisation for Economic Co-operation and Development guidelines 239 (OECD, 2016).
| Day | Diet volume per larvae (µL) | D-Glucose (% w/w) | D-fructose (% w/w) | Yeast extract (% w/w) | Royal jelly (% w/w) | Lambda-cyhalothrin 5 × 10−10 (g mL−1) |
|---|---|---|---|---|---|---|
| 1 | 20 | 12 | 12 | 2 | 50 | – |
| 2 | – | – | – | – | – | – |
| 3 | 20 | 15 | 15 | 3 | 50 | 0.48 |
| 4 | 30 | 18 | 18 | 4 | 50 | 0.86 |
| 5 | 40 | 18 | 18 | 4 | 50 | 1.47 |
| 6 | 50 | 18 | 18 | 4 | 50 | 2.24 |
| Day | Diet volume per larvae (µL) | D-Glucose (% w/w) | D-fructose (% w/w) | Yeast extract (% w/w) | Royal jelly (% w/w) | Lambda-cyhalothrin 5 × 10−10 (g mL−1) |
|---|---|---|---|---|---|---|
| 1 | 20 | 12 | 12 | 2 | 50 | – |
| 2 | – | – | – | – | – | – |
| 3 | 20 | 15 | 15 | 3 | 50 | 0.48 |
| 4 | 30 | 18 | 18 | 4 | 50 | 0.86 |
| 5 | 40 | 18 | 18 | 4 | 50 | 1.47 |
| 6 | 50 | 18 | 18 | 4 | 50 | 2.24 |
Note. “–” = not supplied.
The concentration of lambda-cyhalothrin found as residue in pollen grains (1.7 ng g−1; Mullin et al., 2010) was used, and calculated according to the total amount of pollen consumed during the larval stage (5.4 mg; Rortais et al., 2005). The proportions and volumes of the diet used followed the OECD 239 guidelines (OECD, 2016). The total concentration of λ-CBI expected to reach the larvae through feeding was 0.057 ng mL−1 (maximum ingestion of 0.0080 ng larva−1), calculated according to the equation described by Tadei et al. (2020):
where Cd is the estimated concentration of insecticide in the diet (ng mL−1); Cp represents the concentration of insecticide found in pollen grains (ng g−1); Pc is the total amount of pollen consumed during the larval phase (g); and Vd is the total volume of diet consumed during the larval phase (mL).
Histopathology
On the seventh day, after collecting the larvae, corresponding to the fifth instar of development, 10 individuals from the control group and 10 from the treatment group were collected and cold-immobilized for 3 min at −4°C. The head and the tip of the abdomen were cut off, and the larvae were transferred to Zamboni fixative solution (Stefanini et al., 1967), for 24 hr, at room temperature. Subsequently, the samples were dehydrated in a graded ethanol series (70%, 80%, 90%, 95%, and 99.5%), for 20 min each, and embedded in historesin (Leica); 3-μm-thick sections were obtained using a Leica RM2255 rotary microtome, stained with hematoxylin (15 min) and eosin (30 s), analyzed under an Olympus BX60 light microscope.
Histochemistry
Some unstained sections, obtained as described above, were submitted to histochemical tests for the detection of total proteins and neutral polysaccharides and glycoconjugates. All samples were analyzed and photographed using an Olympus BX 60 light microscope.
Protein detection
The samples were transferred to a mercury-bromophenol solution (100 mL of 2% acetic acid; 0.05 g of bromophenol blue; 1.5 g of mercury(II) chloride) for 135 min. Next, they were washed with 0.5% acetic acid for 10 min and in running water for 15 min, then mounted and analyzed under a light microscope.
Neutral polysaccharides and glycoconjugates detection
The samples were transferred to 0.4% periodic acid for 30 min, quickly washed in distilled water, and then incubated into Schiff’s reagent (1.5 g of basic fuchsin; 4.5 g of potassium metabisulfite; 1 g of activated charcoal; 45 mL of 1% HCl; 300 mL of H2O), for 1 hr, in the dark. After washing in running water for 30 min, the samples were mounted and analyzed under a light microscope.
Semiquantitative analysis of cellular chemical components
To quantify the changes in the amount of proteins, polysaccharides, and lipids in the fat body cells of the larvae, the samples stained with hematoxylin and eosin and submitted to histochemical tests were photographed using a 40×, numerical aperture 0.75 objective lens, and the same illumination parameters, in three random fields of the fat body for each control (n = 10) and the treatment (n = 10) larva. Changes in the amounts of proteins, polysaccharides, and lipids were quantified by the GIMP 2.10 software system (https://www.gimp.org/), which allows converting the color corresponding to proteins (blue), polysaccharides (magenta), and lipids (non-stained droplets) into grayscale followed by transformation into pixel density (Solomon, 2009). To perform the analysis of the size of lipid droplets, 20 droplets from the treatment and 20 from the control, for each of the three selected slice fields, had their diameter or longer axis measured using the Image J software system (https://imagej.nih.gov/; Fujii et al., 2022).
Transmission electron microscopy
On the seventh day, after collecting the larvae corresponding to the fifth instar of development, 10 individuals from the control group and 10 from the treatment group fed with a diet containing 0.057 ng/mL of λ-CBI were dissected in 0.2 M sodium cacodylate buffer pH 7.2. The midguts and fragments of the visceral fat body were transferred to a fixative solution containing 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer and 0.2 M sucrose, for 4 hr, at room temperature. The samples were post-fixed in 1% osmium tetroxide in the same buffer for 2 hr, washed in buffer, dehydrated in a graded ethanol series (70%, 80%, 90%, and 100%), and embedded in LR White resin (London Resin Company). Ultrathin sections (70–90 nm thick) were obtained with a diamond knife on a Sorvall MT2-BMT2-B ultramicrotome (Sorvall Instruments, Wilmington, DE, USA), stained with 1% aqueous uranyl acetate and lead citrate (Reynolds, 1963), and examined under a Zeiss EM 109 transmission electron microscope (Carl Zeiss, Jena, Germany).
Honey bee development
After the histopathological evaluation of the larval midgut and fat body, an additional analysis was performed to evaluate if the sublethal concentration of the λ-CBI affects the bee development. Larvae were obtained and exposed to the insecticide as aforementioned (sections Insect collection and Exposure to insecticide) and their development evaluated. Briefly, 48 control and 48 λ-CBI exposed larvae were followed to evaluate the larval weigh in the last instar, the ratio of molt to pupae and adult emergence.
Statistical analysis
The means of the data obtained by quantifying the pixels marked for lipids, proteins, and polysaccharides for each of the five control and treatment larvae and the larval weight were analyzed for normality and homogeneity using the Shapiro-Wilk and Levene tests, respectively, followed by analysis of variance and Tukey’s test, at a significance level of 5%. The data on the means of the lipid droplet diameters were analyzed using Generalized Lianear Model with Gaussian distribution, and differences were assessed using the Kruskal-Wallis and Conover-Iman tests, at a significance level of 5%, adjusted by the Bonferroni method. All analyses were performed using the R software system (https://cran.r-project.org/).
Results
Midgut
The epithelium of the midgut in the larvae of A. mellifera control group presented a single layer of cuboidal digestive cells with spherical nuclei and predominance of decondensed chromatin (Figure 1A). The apical surface of the epithelium exhibited a well-developed brush border, and the basal region was onto a thin basement membrane, where nests of regenerative cells were also found (Figure 1A).
Light micrographs of the midgut of Apis mellifera larvae. (A) Control showing epithelium (ep) with apical surface with well-developed brush border (bb) toward the lumen (L), nucleus with the predominance of decondensed chromatin (n), and nest of regenerative cells (rc). (B–D) Epithelium (ep) of larvae treated with lambda-cyhalothrin-based insecticide showing apical protrusion (arrowhead) toward the lumen (L), nucleus with condensed chromatin (n), disorganized cytoplasm (black arrow) of digestive cells (dc), numerous apocrine secretory vesicles (white arrow) in the brush border (bb), and regenerative cells (rc). Note. fb = fat body. Scale bars = 20 µm.
In larvae chronically exposed to λ-CBI, histopathological alterations were observed in the digestive and regenerative cells, characterized by intense cytoplasm vacuolization (Figure 1B), apical protrusions toward the lumen, nuclei with increased condensed chromatin (nuclear pyknosis; Figure 1C), and apocrine secretion (Figure 1D).
To verify the cytoplasm vacuolization in the midgut of larvae fed on λ-CBI, the ultrastructural analyses reveal the occurrence of many autophagic vacuoles containing mainly mitochondria with damaged cristae (Figure 2), which were not found in the cells of control larvae.
Transmission electron micrograph of the midgut of Apis mellifera larvae fed on lambda-cyhalothrin-based insecticide showing digestive cell with microvilli (mv) and cytoplasm rich in autophagic vacuoles (av) containing damaged mitochondria (m).
The periodic acid-Schiff (PAS) test on the midgut epithelium of A. mellifera larvae from the control group revealed a uniform distribution of polysaccharides and glycoconjugates in the cytoplasm, with a strong positive reaction at the brush border (Figure 3A). In the larvae treated with λ-CBI, the epithelium presented a strong reaction in the cytoplasm and weaker, irregular reactions at the brush border (Figure 3B).
Light micrographs of the midgut of Apis mellifera larvae submitted to histochemical tests. (A) Epithelium (ep) of the control and (B) treated larvae, showing stronger periodic acid-Schiff-positive reaction in the cytoplasm and weaker reaction in the brush border (bb) in the bee treated with lambda-cyhalothrin-based insecticide. (C) Epithelium (ep) of the control and (D) treated larvae, showing positive reaction for proteins in the control and negative areas (arrow) in the treated larvae. Note. L = lumen; fb = fat body; arrowhead = cell protrusion. Scale bars = 20 µm. and
The protein detection test in larvae from the control group revealed a uniform distribution of these compounds throughout the cytoplasm of the digestive cells, with a weak reaction at the brush border (Figure 3C). The midgut epithelium of larvae fed on insecticide exhibited irregular positive reactions in the cytoplasm, indicating areas of vacuolization and damage to cell integrity, and weaker reaction at the border, compared to the control group (Figure 3D).
Fat body
The fat body of A. mellifera larvae from the control group was formed by ovoid trophocytes with irregular nuclei rich in decondensed chromatin and cytoplasm with evident lipid droplets (Figure 4A). Occasionally, urocytes were also found, ovoid and bigger than the trophocytes, with a central nucleus rich in decondensed chromatin and abundant urate deposits of varying sizes in the cytoplasm (Figure 4A). Scattered among the fat body cells, oenocytes were observed, spherical with irregular borders, a central nucleus rich in decondensed chromatin, evident nucleoli, and uniformly basophilic cytoplasm (Figure 4C).
Light micrographs of the fat body of Apis mellifera larvae. (A) Trophocytes (tr) of control larvae showing cytoplasm with abundant lipid droplets (ld) of varied sizes and well-defined boundaries (arrowhead) and the nucleus (n) rich in decondensed chromatin. Insert: Urocyte (uc). (B) Trophocytes (tr) of larvae exposed to lambda-cyhalothrin-based insecticide showing cytoplasm with small lipid droplets and less defined boundaries (arrowhead) and nucleus (n). Insert: detail of a urocyte (uc). (C) Oenocytes (oe) from the control group and (D) those treated with λ-cyhalothrin-based insecticide among the trophocytes (tr), showing basophilic cytoplasm (cy) and nucleus rich in decondensed chromatin (n), with evident nucleoli (arrows). Scale bars = 20 µm.
Larvae of A. mellifera fed on λ-CBI presented no histopathological alterations in trophocytes, urocytes, or oenocytes (Figure 4B, D). In both the control group and those exposed to λ-CBI, transmission electron microscopy revealed trophocytes with cytoplasm rich in lipid droplets and mitochondria and nuclei with prevalence of decondensed chromatin (see online supplementary material Figures S1A, B).
The PAS test on larvae from the control group revealed a positive reaction for polysaccharides (Figure 5A) in a large part of the cytoplasmic region of the trophocyte, without lipid droplets. In the trophocytes of larvae fed on λ-CBI, this reaction was stronger, compared to the control (Figure 5B), with increased pixel density (F1,30 = 4.475, p < 0.05), which indicates the accumulation of polysaccharides in these cells (Figure 6C).
Light micrographs of the fat body of Apis mellifera larvae subjected to histochemical tests. (A) Trophocytes (tr) from the control group and (B) treated with lambda-cyhalothrin-based insecticide, showing a strong positive reaction for polysaccharides and smaller lipid droplets (ld) in treated larvae compared to the control. (C) Trophocytes (tr) from the control group and (D) Treated with λ-cyhalothrin-based insecticide showing nucleus (n) positive for proteins and cytoplasm (cy) with a weak positive reaction. Note. Arrowhead = lipid droplet boundary. Scale bars = 20 µm.
Semiquantitative data of cellular chemical components in the trophocytes of the fat body of Apis mellifera larvae control and exposed to lambda-cyhalothrin-based insecticide. (A) Pixel density for lipids (mean ± SD) in the cytoplasm. (B) Diameter of lipid droplets (mean ± SD) in the cytoplasm. (C) Pixel density for polysaccharides (mean ± SD) in the cytoplasm. (D) Pixel density for proteins (mean ± SD) in the cytoplasm. Asterisks—indicates difference by statistical test. ns—indicates no difference by statistical test. (A), (C), and (D)—Tukey’s test. (B)—Kruskal-Wallis test. All tests at 5% significance level.
The test with mercury bromophenol presented no alterations in the distribution of proteins in the larvae fed on λ-CBI compared to the control (Figures 5D), and no difference in the amount between the treatment and the control (F1,30 = 1.154, p = 0.30; Figure 6D).
In the cytoplasm, the pixel density related to the abundance of lipids exhibited no changes compared to the control group (F1,30 = 2.93, p = 0.10; Figure 6A). However, reduction was observed in the diameter of lipid droplets (t = 3.380, df = 1, p < 0.05; Figure 6B).
Honey bee development
The larval weight in Day 8 (last instar) was similar (p > 0.05) in the control (174.8 ± 28 mg) and in the λ-CBI treated (147 ± 35 mg) larvae. The rate of larvae molting for pupae and adult emergence was similar between the control and λ-CBI treated groups.
Discussion
Pyrethroids may have repellent effects, decreasing food consumption in some species (Rieth & Levin, 1988; Kawada et al., 2014; Ceuppens et al., 2015; Vinha et al., 2021), but in our study, food was manually supplied to the honey bee larvae, and we verified that always the brood cells were empty, without food remains, proving that they consumed the estimated insecticide concentration.
The histological alterations observed in the midgut epithelium of A. mellifera larvae fed on λ-CBI include intense cytoplasm vacuolization in the digestive and regenerative cells, apical protrusions of the cytoplasm, and nuclei with condensed chromatin. Similar results were found in the midgut epithelium of insects orally exposed to commercial formulation of insecticides, including adult workers of A. mellifera exposed to λ-CBI (Arthidoro de Castro et al., 2020), spiromesifen (Serra et al., 2021), iprodione (Carneiro et al., 2020), and imidacloprid (Carneiro et al., 2022); workers of the stingless bee Partamona helleri Friese, 1900 (Hymenoptera: Apidae) exposed to fipronil (Farder-Gomes et al., 2021), Anticarsia gemmatalis Hübner; 1818 caterpillars (Lepidoptera: Noctuidae) exposed to esquamocin (Fiaz et al., 2018) and azadirachtin (Farder-Gomes et al., 2022); and Aedes aegypti (Linnaeus, 1762) larvae (Diptera: Culicidae) exposed to novaluron (Fiaz et al., 2021), which indicates that the midgut epithelium presents similar histopathological damage in different species in response to different pesticides.
The intense vacuolization observed in the epithelial cells of the midgut of larvae exposed to λ-CBI was due to autophagic vacuoles, revealed by transmission electron microscopy. Autophagy is a cellular process involving the formation of vesicles that enclose organelles and other cytosolic components that participate in the degradation of lysosomes and the recycling of their components. At low rates, autophagy represents a primary response to metabolic stress to maintain homeostasis by eliminating damaged organelles. However, cells with a high degree of vacuolization may have their functions compromised, and, in this case, autophagy may precede cell death by apoptosis. Thus, the autophagic vacuoles observed in the midgut cells of A. mellifera larvae exposed to λ-CBI may be related to the cellular metabolic response to mitigate the damage caused by the pesticide. The autophagic vacuoles found in the digestive cells of bee larvae exposed to λ-CBI contained predominantly damaged mitochondria, which may have been affected by the insecticide, thus affecting adenosine triphosphate (ATP) synthesis, impairing nutrient absorption, and hindering midgut epithelium renewal.
The apical protrusions, similar to “blebs” or “membrane blebbing,” and pyknotic nuclei observed in the digestive cells of A. mellifera larvae fed on λ-CBI are morphological characteristics associated with the onset stages of cell death by apoptosis (Allen et al., 1997; Häcker, 2000; Van Cruchten & Van Den Broeck, 2002). Apoptosis occurs in a programmed and controlled manner, mediated by a cascade of molecular events, with the cysteine protease enzymes of the caspase family (Elmore, 2007; Hengartner, 2000) as the main effectors. The caspase-mediated cleavage of specific substrates triggers structural modifications, such as nuclear lamina breakdown, which leads to nucleus condensation (pyknosis; Goldman et al., 2002). The cleavage of other cytoskeletal components (Ndozangue-Touriguine et al., 2008), as well as the PAK2 kinase, which regulates cellular morphology through cytoskeletal rearrangements, induces loss of cell shape and function and the formation of apical protrusions (Rudel & Bokoch, 1997), similarly to the characteristics observed in the cells here. Thus, it is plausible to suggest that A. mellifera midgut cells exposed to λ-CBI are undergoing apoptosis to release damaged cells.
The increased apocrine secretion in digestive cells of the midgut epithelium of A. mellifera is observed in the larvae exposed to λ-CBI. The midgut epithelium in insects produces digestive enzymes and proteins of the peritrophic matrix, which protect the epithelial cells against physical and chemical damage (Terra & Ferreira, 2012). There is an increase in the release of apocrine secretory vesicles containing digestive enzymes and other enzymes involved in detoxification when the organism is exposed to xenobiotic agents (Ferreira et al., 1990). Proteins from the cytochrome P450 superfamily may be part of the cargo transported by these vesicles, which contribute to insecticide resistance (Feyereisen, 1999; Li et al., 2007). Thus, the increased apocrine secretion in epithelial cells of A. mellifera larval midgut may be a metabolic response to mitigate the effects of λ-CBI exposure.
The PAS histochemical test revealed a weak reaction for polysaccharides in the brush border and a strong one in the cytoplasm of digestive cells of A. mellifera larvae exposed to λ-CBI. The brush border of the digestive cells is rich in glycocalyx, consisting of glycoproteins and glycolipids. Glycogen reserves can also be found in the cytoplasm of these cells (Serrão & Cruz-Landim, 2000), which explains the affinity for PAS in these cell regions. The brush border, formed by microvilli, plays a role in digestion, nutrient absorption, and ion transport. In some bees, it can increase the apical surface area by up to 230-fold (Serrão & Cruz-Landim, 1996a; Terra et al., 2019). The weak and irregular PAS reactions in the midgut brush border of larvae exposed to λ-CBI may indicate that the glycocalyx functions are compromised by the insecticide. Additionally, the accumulation of neutral polysaccharides in the cytoplasm suggests that these cells may store energy sources to be used in detoxification.
The positive reactions to mercury bromophenol reveal that the whole cytoplasmic region and the brush border of the digestive cells are rich in proteins, which seems to be suppressed in larvae exposed to λ-CBI, characterized by irregular reaction in the cytoplasm, due to an increased number of vacuoles and cell integrity damage. The midgut cells of insects participate in digestion and nutrient absorption, production and secretion of digestive enzymes, defense against xenobiotics (Terra & Ferreira, 2012), and peritrophic matrix proteins (Terra et al., 2019). Thus, the widespread distribution of proteins in the digestive cells of larvae in the control group may be due to these cellular functions, whereas the affected regions of larvae exposed to λ-CBI may have these functions compromised.
The trophocytes, oenocytes, and urocytes of the fat body in A. mellifera larvae fed a diet containing λ-CBI presented no histopathological alterations. The fat body in insect larvae is abundant and helps nutrient storage and the intermediary metabolism (Arrese & Soulages, 2010; Liu et al., 2009). Thus, due to the large number of these cells in the larvae, the residual concentration of λ-CBI used here may not have induced morphological changes in the organ.
Although no morphological variations were observed, the trophocytes of A. mellifera larvae exposed to λ-CBI present a reduced diameter of lipid droplets compared to the control. In insect larvae, trophocytes work in hormonal signaling and regulation, nutrient storage (lipids, carbohydrates, and proteins), and innate immunity through the synthesis of detoxifying enzymes (Roma et al., 2010; Skowronek et al., 2021). These cells harbor a large number of receptors and signaling molecules on their plasma membrane, which integrate multiple signals and regulate the development and behavior of the organism (Liu et al., 2009). The signals received, whether hormonal or nutritional, elicit an organ response in the form of nutrient mobilization and the release of signaling molecules, known as fat body signals (Li et al., 2019; Liu et al., 2009). Most of the glucose consumed with the diet is converted into lipids to be used as an energy source during metamorphosis. However, glucose is also stored as glycogen, which can be readily mobilized for energy demand (Arrese & Soulages, 2010). Thus, despite no significant change being observed in the number of lipids in the trophocytes of larvae exposed to λ-CBI, a nutritional deficiency signaling pathway may have been activated as a result of ineffective nutrient absorption due to damage to the cells of the midgut, which should be verified in further studies. Additionally, it is plausible that there is a high energy demand due to the detoxification process, and the cell may be using its reserves for this metabolic function. Thus, there may have been a mobilization of lipids, which are stored in smaller droplets and are more readily accessible to enzymatic actions via β-oxidation, due to the increased surface area (McClements & Decker, 2000; Osborn & Akoh, 2004).
The energy demand may also explain the polysaccharide increase evidenced by the PAS reaction in the trophocytes of the honey bee larvae fed on λ-CBI. The substrate for ATP production depends on multiple factors, including the substrate/ATP molecule ratio and metabolic by-products. Therefore, glycolysis may be the main pathway used by trophocytes for ATP synthesis to meet their energy demand and avoid higher reactive oxygen species production.
No morphological alterations were observed in the urate cells of A. mellifera larvae exposed to λ-CBI compared to the control group. Urate cells are trophocytes modified to store urate derived from the metabolism of nucleic acids or proteins (Furtado et al., 2013; Skowronek et al., 2021). Due to the increased apoptosis and autophagy in some cells of the midgut of larvae fed on λ-CBI, structural modifications in urate cells would be expected to store the high production of urate as a by-product of the metabolism of these senescent cells, which did not occur, though. Thus, it can be suggested that instead of structural modifications, there is an increased number of trophocytes modified into urate cells to supply this demand, which can be further evaluated.
In the fat body, there are also oenocytes, which did not undergo alterations in the bee larvae exposed to λ-CBI. Oenocytes play a role in synthesizing hydrocarbons, proteins, and lipids in the insect exoskeleton (Arrese & Soulages, 2010; Martins & Ramalho-Ortigão, 2012). However, these cells also participate in the detoxification process in adults of A. aegypti (Diptera; Martins et al., 2011). Assis et al. (2022) reported alterations in the oenocytes of adult solitary bee Tetrapedia diversipes exposed to imidacloprid, including increased cytoplasm vacuolization, nuclear pyknosis, and fragmentation. Thus, despite contributing to detoxification, the oenocytes of A. mellifera larvae did not undergo morphological alterations, possibly due to the greater quantity of trophocytes in the larval fat body. Additionally, it is plausible that these cells are functionally buffered, and when exposed to low concentrations of λ-CBI, they maintain their homeostasis for the critical function in the production of new cuticle during larval molt.
The histochemical reactions for proteins present no difference in the quantity of this component in the trophocytes of the honey bee larvae exposed to λ-CBI compared to the control. Organisms under stress conditions, such as exposure to insecticides, demand a high amount of energy, which may lead to a protein catabolic stimulus and reduce the insect protein levels (Arrese & Soulages, 2010). Associated with this fact, as previously mentioned, the fat body, besides providing storage, functions as the main site where detoxification enzymes are produced (Li et al., 2019). Thus, the levels of catalysis and protein synthesis in A. mellifera larvae exposed to λ-CBI may be similar, which results in quantities equal to those observed in the control group.
In this study, to simulate a realistic field scenario for the insecticide sublethal effects in the non-target honey bee larvae, we use a λ-CBI to control some pests in many crops visited by bees (He et al., 2008). However, commercial insecticides have some co-formulants and are often applied with adjuvants, which increase the spreading and sticking of the active ingredients, as well as facilitate their penetration into the insect body. These co-formulants and adjuvants are classified as inert components by Environmental Protection Agencies. However, concerns have emerged because some studies have pointed out that many of those compounds have lethal and sublethal effects on non-target insects, including bees (Fine et al., 2017; Shannon et al., 2023b; Straw & Brown, 2021).
Despite the histopathological damage observed in the midgut and fat body of λ-CBI-treated larvae, these larvae complete their development without adverse effects on larval weight, pupation, or adult emergence. Although Phan et al. (2024) reported a decrease in the weight of Osmia cornifrons (Radoszkowski, 1887; Megachilidae) larvae exposed to various insecticides (acetamiprid, flonicamid, and sulfoxaflor) and the fungicide dodine, similar negative effects to those here reported were not observed in Osmia cornuta (Latreille, 1805; Megachilidae) exposed to spirotetramat (Sgolastra et al., 2015), in the stingless bee P. helleri exposed to spinosad (Araújo et al., 2019), or in honey bees exposed to thiamethoxam (Friol et al., 2017), clothianidin and pyraclostrobin (Tadei et al., 2019; 2020), or imidacloprid, ethion, and hexaflumuron (Delkash-Roudsari et al., 2022). However, it cannot be ruled out that adult workers emerging from λ-CBI-treated larvae may exhibit certain symptoms, as reported for hygienic and foraging behaviors (Morfin et al., 2019; Tadei et al., 2019) and physiological alterations (Friol et al., 2017; Tadei et al., 2020).
Conclusion
The chronic ingestion of the residual concentration of λ-CBI found in pollen grains, here evaluated with λ-CBI, causes morphological damage and induces apoptosis in the midgut cells, as well as alterations in the nutrient storage pattern in the fat body of A. mellifera larvae. The midgut epithelium and fat body are not targets of the insecticide. However, the induced changes may compromise their functions and cause developmental impairments in this pollinator. Our data reinforce the importance of correct insecticide management and the development of new pesticide technologies to minimize harm to non-target species.
Supplementary material
Supplementary material is available online at Environmental Toxicology and Chemistry.
Data availability
Data will be made available upon request for the corresponding author ([email protected]).
Author contributions
Pedro Henrique Ambrosio Nere (Conceptualization, Formal analysis, Investigation, Validation), Rebecca Rey-Chai Kern (Formal analysis, Investigation), Lenise Silva Carneiro (Data curation, Investigation, Methodology), Barbara Amoroso Soares Lima (Investigation, Methodology, Validation), Diego dos Santos Souza (Methodology, Software), and José E Serrão (Conceptualization, Funding acquisition, Resources, Supervision)
Funding
This research was supported by Brazilian research agencies Coordenaçãode Aperfeiçoamento de Pessoal de Nível Superior (code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (303243/2022-8), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (APQ-02486-22).
Conflicts of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Disclaimer
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.
Acknowledgments
The authors are grateful to the Nucleus of Microscopy and Microanalysis from the UFV for the technical assistance provided and to Enedina Sacramento for the English editing service.
