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  3. Effects of larval exposure to the fungicide pyraclostrobin on the post-embryonic development of Africanized Apis mellifera workers

Effects of larval exposure to the fungicide pyraclostrobin on the post-embryonic development of Africanized Apis mellifera workers

The ingestion of the fungicide in the larval stage can impair the bee development and the health and performance of the colony. In this perspective, studies that consider the effects of fungicides in the different stages of bee development are crucial for a better interpretation of the risks of exposure. Environmental Advances 4 (2021) 100069 Contents lists available at ScienceDirect Environmental Advances journal homepage: www.elsevier.com/locate/envadv Effects of larval exposure to the fungicid

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  1. Environmental Advances 4 (2021) 100069 Contents lists available at ScienceDirect Environmental Advances journal homepage: www.elsevier.com/locate/envadv Effects of larval exposure to the fungicide pyraclostrobin on the post-embryonic development of Africanized Apis mellifera workers Caio Eduardo da Costa Domingues a,∗, Rafaela Tadei b, Lais Vieira Bello Inoue a, Elaine Cristina Mathias da Silva-Zacarin b,∗, Osmar Malaspina a a Universidade Estadual Paulista (UNESP) - “Júlio de Mesquita Filho”, Instituto de Biociências (IB), Departamento de Biologia, Centro de Estudos de Insetos Sociais (CEIS), Rio Claro, SP, Brazil b Universidade Federal de São Carlos (UFSCar), Departamento de Biologia (DBio), Laboratório de Ecotoxicologia e Análise de Integridade Ambiental (LEIA), Sorocaba, SP, Brazil a r t i c l e i n f o a b s t r a c t Keywords: Among the more than twenty thousand bee species currently described, Apis mellifera stands out for its high Africanized bee economic relevance due to crop pollination. In recent decades, many studies have registered a decline in bee Late effects populations associating multiple factors, including pesticides. These molecules can arrive through residues present Intestine in the nectar and pollen, which can be ingested by the larvae. However, studies that contemplate the effects of Peritrophic matrix fungicides on larvae and adults are rare. Thus, the objective of the study was to evaluate the effects of the exposure Cell death to pyraclostrobin in the larval phase and verify the late effects in newly emerged bees. The larvae were subjected to repeated exposure (third to the sixth day of the bioassay). The concentrations of pyraclostrobin consumption were 127.58 ng/larva (PT1), 13.16 ng/larva (PT2), and 4.24 ng/larva (PT3), both based on residues found in the field like as pollen and bee bread. The effects on larval mortality, pupation, emergence and survival rates, and median lethal time of newly emerged worker bees were evaluated. We also evaluated cell death and chitin marking of the peritrophic matrix in the intestine of larvae and newly emerged bees. No adverse effects were observed on the post-embryonic development of the larvae and survival time of the newly emerged workers, showing tolerance to the fungicide. However, the intestine epithelium of larvae (PT1 and PT2) and adults (PT1) showed immunostaining for cell death and increased intensity of chitin marking in the peritrophic matrix, indicating a late effect of the pyraclostrobin fungicide. Thus, the ingestion of the fungicide in the larval stage can impair the bee development and the health and performance of the colony. In this perspective, studies that consider the effects of fungicides in the different stages of bee development are crucial for a better interpretation of the risks of exposure. 1. Introduction dominant throughout Brazil and spread the Latin America and the south- western United States of America (Schneider et al., 2004). Although they The economic, agricultural, and ecological importance of Apis are known for their high defense capacity, they are currently entirely mellifera (Linnaeus, 1758) is reported in many studies (Morse and managed and due to their characteristics of a long time in foraging ac- Calderone, 2000; Rader et al., 2009; Calderone, 2012; Rucker et al., tivity, more resistance to diseases, less affected by cool weather, and 2012; Garibaldi et al., 2017; Hung et al., 2018). However, the intensive greater efficiency in resource collection in comparison to European sub- use of pesticides in crops, associated with multiple stressors, has caused species, demonstrate high economic relevance, for pollination of a lot of the decline of their populations in different countries (Neumann and crops and for the value of its bee products (Malaspina and Stort, 1987; Carreck, 2010; Sanchez-Bayo and Goka, 2014; Simon-Delso et al., 2014; Francoy et al., 2009). However, although this species is used as a model Goulson et al., 2015). for pesticide regulation in Brazil (Cham et al., 2017), beekeepers report In Brazil, African bees were introduced and quickly dominated the weakening of the colonies and increasing mortality, due to the intense European subspecies introduced earlier (Kerr, 1967; Sheppard et al., use of pesticides in the country (Pires et al., 2016). 1991; De Jong, 1996). The poly hybrid populations of A. mellifera, Among the great diversity of pesticides used, insecticides are exten- known as the Africanized bees, have adapted and became present and sively studied, emphasizing the class of neonicotinoids (Blacquière et al., ∗ Corresponding author at: UNESP - CEIS, Avenida 24 A, n 1515, Jardim Bela Vista, 13.506-900, Rio Claro, SP, Brazil. E-mail address: cecdomingues@gmail.com (C.E.d.C. Domingues). https://doi.org/10.1016/j.envadv.2021.100069 Received 8 May 2021; Received in revised form 13 May 2021; Accepted 14 May 2021 2666-7657/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
  2. C.E.d.C. Domingues, R. Tadei, L.V.B. Inoue et al. Environmental Advances 4 (2021) 100069 2012; Fairbrother et al., 2014; Lundin et al., 2015). Nonetheless, sev- while the peritrophins are involved in their elasticity and permeability eral studies have related adverse effects in bees after exposure to fungi- (Shao et al., 2001). cides, in which we can highlight changes in the immune (Degrandi- Therefore, the present study aims to fill the gaps in knowledge of Hoffman et al., 2015; Cizelj et al., 2016) and behavioral response the fungicide’s effects on the normal development patterns of newly (Tadei et al., 2019), decreased survival of adult individuals and injuries emerged larvae and adults, using residual concentrations. Thus, we eval- in the midgut (Domingues et al., 2020a, 2020b; Carneiro et al., 2020; uated and compared the sublethal effects of exposure to the fungicide Batista et al., 2020), flight impairment (Liao et al., 2019), and weak- pyraclostrobin in the larval phase using biological parameters through- ening of the colony (Yoder et al., 2013). Despite the many studies con- out post-embryonic development. We also diagnosed cell death in the ducted, Cullen et al. (2019) highlight that there are still gaps in knowl- midgut epithelium and evaluated chitin marking in the peritrophic ma- edge that must be addressed regarding the effects of fungicides on bees, trix of exposed larvae and adults. ensuring better management in agricultural systems and reducing the risk of exposure. 2. Materials and methods Until the present moment, studies that contemplate the effects of fungicide exposure on A. mellifera larvae are still minimal (Tadei et al., 2.1. Obtaining the larvae of Africanized A. mellifera and maintaining 2019; Tadei et al., 2020) when compared to other classes of pesti- them in the laboratory cides (Gregorc and Bowen, 2000; Cruz et al., 2010; Zhu et al., 2014; Silva et al., 2015; Tavares et al., 2015; Tavares et al., 2017; Friol et al., The bioassays were conducted using three non-parental colonies 2017; Dai et al., 2018; Tavares et al., 2019). This scenario may represent with known physiological status and without visible symptoms of dis- a risk for the colony since fungicide residues found in pollen (Pettis et al., eases and parasites selected from the apiary located in the rural area of 2013; Zioga et al., 2020) and in bee bread (Kubik et al., 1999; Tong et al., Piedade - São Paulo (23°37′5.506" S; 47°29′7.926" W). All colonies 2018) can be stored in the alveoli, exposing the larvae by contact and were previously monitored monthly, and no treatment was applied be- consumption of the food containing these residues (Wu et al., 2011). fore their use in the bioassays. Initially, a clean frame of honeycomb wax Given this reality of exposure, the intestine proves to be an organ was added inside each of the three colonies to induce the queen’s ovipo- of great relevance since it directly participates in the route of absorp- sition and thus obtain sufficient quantities of first instar larvae. After the tion and metabolization of food after its ingestion (Serrão and Cruz- three-day embryonic development of the larvae, the brood combs were Landim, 1996; Silva-Zacarin et al., 2011). The digestive system of bees collected from the three colonies, between 8:00 - 9:00 AM, with temper- is divided into three well-defined regions: anterior, middle, and poste- atures equal or superior to 15 °C, and transported to the Ecotoxicology rior intestine (Cruz-Landim and Rodrigues, 1967). As the region where and Environmental Integrity Analysis Laboratory (LEIA in Portuguese) nutrient digestion and absorption occurs, the midgut is extensively ana- of the Universidade Federal de São Carlos in Sorocaba - São Paulo. All lyzed in toxicological studies with A. mellifera larvae (Cruz et al., 2010; experimental procedures used were performed according to the protocol Gregorc and Ellis, 2011) and adults (Catae et al., 2014; Batista et al., OECD n° 239 (2016). 2020; Domingues et al., 2020a). In the laboratory, the first instar larvae were individually transferred It is important to highlight that larvae organs exhibit characteris- from brood combs to polystyrene grafting cells (domes) (1 × 1 × 1 cm), tics and adaptations specific to this phase and, during metamorphosis, previously sterilized, containing 20 μL of artificial diet A (50% sugar undergo intense tissue remodeling until the emergence of the adult indi- solution containing D-glucose, D-fructose, yeast extract and 50% royal vidual (Cruz-Landim, 2009). Tadei et al. (2019) described late effects on jelly, w/w) using brushes (n° 0) (Aupinel et al., 2005). The domes were the behavior of adult bees (newly emerged) after exposure in the larval arranged in cell culture plates with 48 wells, with each well previously phase to pesticides clothianidin and pyraclostrobin, administered iso- filled with half of a piece of dental cotton soaked in 500 𝜇L of sterilizing lated and in combination. Such changes could not be observed if adult solution containing 15% w/v glycerol and 0.2% w/v methylbenzetho- bees were not studied after larval exposure. Still, some studies demon- nium chloride solution (MBC). After the transfer, the viability of each strate the greater tolerance of larvae to pesticides (Tavares et al., 2015; larva was verified by observation in a cold light stereomicroscope (Leica Friol et al., 2017; Tavares et al., 2019; Tadei et al., 2020), but the mech- EZ4 HD), where the dead larvae or submerged in the food were replaced anisms of this greater tolerance are still unknown. by others from a reserve plate. Currently, the influence of the fat body on responses to pesticides The plates containing the larvae in the domes were placed inside is believed to be due to its functions of regulating the chemical com- a hermetic desiccator (Nalgene® 5317-0070, 178 × 305 × 305 mm) position of hemolymph, metabolizing xenobiotics, and a large amount containing a tray with a potassium sulfate (K2 SO4 ) saturated aqueous of this tissue in the larval phase (Cruz-Landim, 1985; Abdalla and solution to maintain the relative humidity of 95 ± 5%. The hermetic Domingues, 2015). In addition to the fat body, a hypothesis to try to desiccator was kept inside an incubator at 34 ± 2 °C, under dark condi- elucidate this greater tolerance may be related to the presence of the per- tions. A tray containing saturated sodium chloride solution (NaCl) was itrophic matrix (MP). This extracellular matrix internally lines the bees’ added to maintain the humidity inside the incubator. intestine. It may have structural and functional differences in the larva and adult individual, being an efficient barrier to pesticides in the larva, 2.2. Composition and elaboration of the larval diet given that it accumulates feces throughout the larval development and defecates only in the transition from larva to pupa (Chapman, 2013). The three types of larval diets (A, B, and C) (Aupinel et al., 2005) The MP is a semipermeable extracellular layer composed of chitin were prepared using D-glucose (99.5%, G8270, Sigma-Aldrich), D- fibrils associated with glycoproteins. It forms an envelope around the fructose (99%, F0127, Sigma-Aldrich), yeast extract (70161, Sigma- feces present in the insects’ midgut lumen, separating the luminal con- Aldrich), and pure royal jelly in natura (APACAME). The concentration tent into two functional compartments: the endo and ectoperitrophic of each of these components varied according to larval development spaces (Lehane and Billingsley, 1996). The MP plays an important role stages due to different nutritional requirements, as shown in the supple- in the digestion and absorption of nutrients since its permeability and mentary material (table S1 in supplementary material) (Aupinel et al., porosity allow the selective movement of molecules, such as digestive 2005). enzymes, in the endoperitrophic space (Hegedus et al., 2009). The MP The larval diet components were diluted in an autoclaved aqueous also limits pathogens and protects cellular microvilli from abrasion dur- solution (distilled water) and subsequently filtered with a syringe using ing digestion (Lehane, 1997; Tellam et al., 1999). The balance between a milipore filter (non-pyrogeninc 0.20 𝜇m - Sterile-R). The sugary so- synthesis and degradation of the MP of insects, which occurs through lution and royal jelly were mixed with gentle agitation when feeding the action of chitinases and chitin-synthases, determines their thickness, and supplied individually using a micropipette for viscous liquids. The 2
  3. C.E.d.C. Domingues, R. Tadei, L.V.B. Inoue et al. Environmental Advances 4 (2021) 100069 type of diet, the amount of food provided, and the feeding frequency on the total consumption of the larval diet during the repeated exposure are described in Table S2 (supplementary material). The larvae were (D3 to D6) and based on the concentration of the fungicide in the diet not fed on the second day of the bioassay due to the period required for provided (table S3 in supplementary material). acclimatization under laboratory conditions (Aupinel et al., 2005). After the repeated exposure, on D7, when pupation begins, the hu- midity inside the hermetic desiccator and the incubator was adjusted 2.3. Pyraclostrobin and chosen concentrations to 80%. On D15, the pupae domes (n = 20) were moved to bee cages (plastic pots, 250 mL, 9 cm x 7 cm) with feeders containing syrup (50% The standard analytical pyraclostrobin fungicide, purchased from sucrose + 50% water, v/v), organized according to the experimental Sigma-Aldrich (CAS Number 175013-18-0, 99.9% purity), was used in group/colony and kept in the incubator with a relative humidity of 70 the larval bioassays. Firstly, a stock solution of 1,000 ng a.i./mL was ± 10%. prepared using distilled water and acetone, in the proportion of 60% - 40%, which was diluted in series until reaching the working concentra- 2.6. Evaluation of the biological effects of repeated exposure tions of the present study, necessary to oral exposure bioassays. Since pyraclostrobin is not entirely soluble in water, acetone was chosen as an The larval mortality of the experimental groups was determined for organic solvent, adding a group of this solvent as a control for acetone the repeated exposure (D3 to D6). From D7 to D14, the pupation rate (item 2.4). and the cumulative emergency rate for the period from D15-D22 were The choice of concentrations was based on pyraclostrobin residues quantified. The survival rate of emerging bees was assessed until the found in the pollen collected by forager bees, in the range of 2,787.1 death of the last individual to verify the effects of larval exposure on ± 1,890.1 (27,000, maximum) ng/g (Pettis et al., 2013), as well as in adult bees. samples of bee bread collected from the colony (2,170 ng/g - 319 ng/g) (Yoder et al., 2013), also based on in vitro experiments with larvae (169 2.7. Obtaining and processing the intestines of larvae and adults 𝜇g/L) (Tadei et al., 2019). Five pre-defecating fifth instar larvae and five newly emerged adults 2.4. Experimental design (up to 48 hours) were collected from each experimental group, anes- thetized at 4 °C for one minute, and dissected at room temperature At the beginning of the repeated exposure bioassays, the larvae were using a stereomicroscope (Leica EZ4 HD) to remove the intestines. divided into the following experimental groups: control (CT), acetone The organs were individually fixed in a fixative solution for 24 hours control (AC), pyraclostrobin 911.25 ng/mL (PT1), pyraclostrobin 94.06 following the methodology used by Domingues et al. (2020a) and ng/mL (PT2), pyraclostrobin 30.25 ng/mL (PT3), and dimethoate (DMT, Domingues et al. (2020b) and dehydrated in increasing concentrations intake of 7.420 ng/larva) as a reference chemist established for A. mel- of ethanol at 4 °C, as described by Silva-Zacarin et al. (2012). Subse- lifera (OECD, 2016), totaling six experimental groups. quently, the organs were immersed in an alcohol-xylol solution (1:1) for The bioassay consisted of 48 larvae per experimental group, dis- one hour and immersed in pure xylol for three hours. The organs were tributed in six plates, with 16 larvae for each of the three selected then soaked in liquid paraffin (60 ± 2 °C), with three changes of one colonies (A, B, and C), as shown in the supplementary material (Fig- hour each, and added to ultrapure paraffin (Paraplast) at room temper- ure S1). This procedure was performed in triplicate, i.e., 144 larvae ature. The blocked intestines were submitted to the microtome to obtain were used per experimental group distributed in 18 plates, fulfilling four sequential histological sections of 8 μm. Eight slides were made for each times the minimum requirements of OECD n° 239 (2016), of 36 larvae of the five individuals of larvae and adults, containing an average of 12 per experimental group (12 larvae x 3 colonies = 36). cuts per slide. Each plate contained all separate experimental groups to vary this organization in each of the 18 plates used in the bioassays and ensure 2.8. Slide processing for immuno-fluorescence the diversity of larvae distribution in the individual domes (Figure S2 in supplementary material). This provides us with a great heterogeneity Initially, the silanized slides (StarFrost®) containing the histological of the plates, guaranteeing greater precision and reliability of the col- sections of the larvae and adult intestines were dewaxed by immersion lected data, avoiding the possible changes that can occur in each plate’s in pure xylol, three baths of five minutes, followed by the alcohol-xylol microenvironment. solution (1:1) for five minutes and alcohol absolute (100%) for 10 min- utes. After deparaffinization, the intestine slides were rehydrated us- 2.5. Repeated exposure for pyraclostrobin concentrations ing decreasing concentrations of ethanol (95%, 80%, 70%, 60%, and 50%) for five minutes in each concentration, as described by Silva- The different pyraclostrobin concentrations were mixed with the lar- Zacarin et al. (2012). With the rehydration completed, the slides were val diet, and the larvae were orally exposed from D3 to D6 of the bioas- immersed in 0.1M citrate buffer (pH 6.0) with 0.05% triton and ir- say after the larvae’s transfer. The larvae of the AC group received the radiated in the microwave (700-750W) for 120 seconds for antigen same volume of acetone as groups PT1, PT2, and PT3, not exceeding 2% permeabilization-recovery. Finally, the slides were washed five times, of the diet’s final volume from D3 to D6, as determined by the OECD n° one minute each, with phosphate-buffered saline (PBS - pH 7.4). 239 (2016). The CT group received the larval diet (50% sugar solution and 50% royal jelly, w/w) without the fungicide and acetone solvent. 2.8.1. Detection of cell death by the TUNEL method The concentrations of pyraclostrobin used in the larval diet were cal- Slides containing the histological sections of the intestines of three culated according to the total pollen consumption of 5.4 mg during the individuals per experimental group were used to detect cell death by larval feeding phase (Simpson, 1955; Babendreier et al., 2004; Simon- the TUNEL method (Terminal deoxynucleotidyl transferase-mediated Delso et al., 2017; Tadei et al., 2019) and based on the value of the pyr- dUTP nick end labeling), using the "In Situ Cell Death Kit, Fluorescein" aclostrobin fungicide found in pollen. Thus, the selected concentrations (Roche, Sigma-Aldrich - REF 11684795910) following the instructions presented the following consumption per larva (intake): PT1 - 911.25 in the manufacturer’s manual. After the reaction, the slides were washed ng/mL (four-day intake: 127.58 ng/larva) representing the worst sce- three times with PBS (pH 7.4) at room temperature and mounted with nario found in pollen (27,000 ng/g), PT2 - 94.06 ng/mL (four-day in- the Fluoromount-G, with DAPI (4′,6-diamidino-2-phenylindole) (Invit- take: 13.16 ng/larva), and PT3 - 30.27 ng/mL (four-day intake: 4.24 rogen, Thermo Fisher Scientific, ref 00-4959-52). We added a positive ng/larva) representing frequent concentrations to be found in the field, control, incubated with DNase-I (3000 U/mL to 3U/mL in Tris-HCl 2,787 ng/g and 897 ng/g respectively. The intake was calculated based buffer, pH 7.5 - 10mM MgCl2 , 1mg/mL bovine serum albumin) for 10 3
  4. C.E.d.C. Domingues, R. Tadei, L.V.B. Inoue et al. Environmental Advances 4 (2021) 100069 minutes at room temperature to induce breaks in DNA, and a negative control, without the TUNEL solution. The analyses were conducted in a laser scanning confocal microscope (LEICA TCS-SP8) according to the configurations described by Domingues et al. (2017), with 36 histologi- cal sections from each experimental group analyzed qualitatively for the presence of fragmentation marks in the DNA of the intestines of larvae and adults of Africanized A. mellifera. 2.8.2. Marking of the peritrophic matrix (WGA-FITC) Slides containing the histological sections of the intestines of three individuals per experimental group were used to determine the stabil- ity of the peritrophic matrix by detecting chitin using Triticum vulgaris (wheat) lectin conjugated to FITC (Sigma-Aldrich, L4895) - WGA/FITC (Wheat Germ Agglutinin/Fluorescein isothiocyanate), as described by Oliveira et al. (2019). The WGA-FITC binds to the chitin N-acetyl- Fig. 1. The survival pattern of newly emerged workers of Africanized A. mellif- glucosamine (NAG) residues, and the analysis of the fluorescence sig- era exposed in larval phase to pyraclostrobin fungicide. There is no difference nal intensity indicates the amount of chitin in the peritrophic matrix between the experimental groups (P > 0.05). n = 65 bees per treatment. (Malta et al., 2016). The slides were individually incubated with 100 μL of the WGA-FITC working solution (1:100) and covered with plastic coverslips for three hours at 28 °C to quantify the intestines of larvae and ing the bioassay (P < 0.0001) according to the protocol of the OECD n° adults. After incubation, the slides were washed with PBS (3x), mounted 239 (2016). with Fluoromount-G, with DAPI, and photo-documented in a laser scan- The survival of newly emerged bees from the PT1, PT2, and PT3 ning confocal microscope (LEICA TCS-SP8), using the 20x magnifica- groups showed no significant difference (P = 0.1281) compared to the tion, according to the following configurations: Laser intensity in 3%, control groups (Figure 1). A similar result was found for the median Smart Gain 1100.0 V (brightness), Smart Offset -30% (background), Pin- lethal time (TL50) (Table 2). hole 0.50 AU, and Zoom 2.0 (larvae) and 3.0 (adults). Thirty images from each experimental group (n = 3 individuals, ten 3.2. TUNEL reaction images per individual) were analyzed, selecting ten regions of interest in each image, 15 μm wide and 15 μm high, to cover the entire length of Figs. 2 and 3 show the diagnosis of cell death, by DNA fragmenta- the organ in the images, totaling 300 analyses per group. The quantifi- tion, performed by the TUNEL method in the intestines of the larvae and cation of the WGA’s marking intensity conjugated with FITC was per- adults of Africanized A. mellifera, respectively. No positive immunola- formed using the ImageJ software, analyzing the intensity histogram belling was observed in the intestinal epithelium for the bees in groups (unit = pixels) for green. CT, AC, and PT3 (Fig. 2A-B). However, bees from groups PT1 and PT2 (higher concentrations) demonstrated positive immunolabelling in the 2.9. Statistical analysis intestinal epithelium (Fig. 2C-E). It is important to highlight that, in the regions of cell differentiation around the regenerative cell nests, there The data were analyzed using the software R (version 3.6.1; R was no positive immunolabelling in any of the experimental groups and Foundation for Statistical Computing, Vienna, AT, 2019). The data re- the nuclei of the immature cells in the regenerative cell nests, which garding the biological effects of repeated exposure to pyraclostrobin also proved negative for the TUNEL reaction. Positive markings of cells were analyzed using generalized linear models with quasibinomial and already eliminated and located in the intestine lumen were observed in quasipoisson distribution, followed by a post-test with a P-value ad- all experimental groups. justed by Tukey (Lenth, 2019). The suitability of the model was as- Regarding adult individuals, the bees from the CT, AC, PT2, and PT3 sessed by analyzing residues using half-normal plots (Demétrio et al., groups presented no positive immunolabelling for the diagnosis of cell 2014) from the "hnp" package (Moral et al., 2017). Survival analysis was death by the TUNEL method. Only the bees in the group exposed to the performed using the Cox’s regression model and the "survival" package highest concentration of the fungicide, PT1, showed positive marking, (Therneau, 2020; Therneau and Grambsch, 2000). The "ecotox" package although less pronounced than in the larval phase (Fig. 3C). As with was used to analyze the median lethal time (Hlina et al., 2018). the larvae, positive immunolabelling was not observed in the region of The analysis of the fluorescence signal intensity was performed by regenerative cell nests (Fig. 3), and positive immunolabelling of cells the Kruskal-Wallis non-parametric statistical test, followed by the post- eliminated for the lumen was observed in all experimental groups. test of Dunn’s multiple comparisons using the GraphPad Prism 9.0.0. (121) software (GraphPad Prism Software, Inc). The results were pre- 3.3. Quantification of WGA-FITC sented with mean ± standard error, and the level of significance adopted was P < 0.05. The chitin marking of the peritrophic (ectoperitrophic) matrix was visualized throughout the intestine of Africanized A. mellifera larvae and 3. Results adults. The quantification of chitin marking by WGA-FITC (Fig. 4A) re- vealed an increase in the intensity of the intestines of bee larvae in the 3.1. Biological effects of repeated exposure to pyraclostrobin PT1 group (mean = 206.49) with statistical difference (P < 0.0001) in relation to the other groups, CT (mean = 143.63), AC (mean = 141.52), Repeated exposure to the pyraclostrobin fungicide during the larval PT2 (mean = 170.19), and PT3 (mean = 152.40) (Fig. 4B). The intestines phase did not affect post-embryonic development (mortality, pupation, of the larvae of the PT2 group were statistically different (P < 0.001) and emergence) when compared to control groups (P > 0.05), as shown from the control groups and PT3 group (P < 0.005) (Fig. 4B). The larvae in Table 1. Although without statistical difference, it was possible to ob- of the CT, AC, and PT3 groups are statistically similar to each other (P serve a lower emergence rate in the PT1 group. Larval mortality caused > 0.05). by dimethoate (DMT), a reference insecticide used in the present study, Regarding the newly emerged workers (Fig. 5A-B), the bees in the presented an average of 84.72 ± 4.62% of exposed individuals, validat- PT1 group (mean = 161.78) showed a significant difference (P < 0.0001) 4
  5. C.E.d.C. Domingues, R. Tadei, L.V.B. Inoue et al. Environmental Advances 4 (2021) 100069 Table 1 Relative mean rate (mean ± standard error) of larval mortality, pupation rate and emer- gence rate of Africanized A. mellifera workers repeated exposure to pyraclostrobin fungi- cide during the larval phase. 1 There is no difference between the experimental groups (P > 0.05). ∗ Asterisk indicate statistical differences between the experimental groups (P < 0.05). Experimental Groups Larval mortality (%) Pupation rate (%)1 Emrgence rate (%)1 CT 10.15 ± 4.17 79.76 ± 5.10 77.28 ± 7.58 AC 7.89 ± 3.17 80.73 ± 4.13 84.04 ± 7.21 PT1 7.91 ± 2.32 77.47 ± 3.84 66.20 ± 10.58 PT2 15.64 ± 3.64 65.60 ± 6.73 81.84 ± 4.91 PT3 7.63 ± 3.09 72.37 ± 4.99 72.11 ± 10.39 DMT 84.72 ± 4.62∗ - - Fig. 2. Detection of cell death (DNA fragmen- tation) by the TUNEL reaction in midgut of lar- vae of Africanized A. mellifera. A - Intestine ep- ithelium of larvae of the CT group staining with DAPI. B - Negative control of the TUNEL reac- tion. C - Intestinal epithelium of larvae from the PT1 group with positive immunostaining. D - Positive immunostaining with emphasized in a nucleus of an individual from the PT1 group. E - Intestinal epithelium of larvae from the PT2 group with positive immunostaining. Legend: asterisks = cells in the lumen with positive immunostaining, ec = cell being elimi- nated into the lumen, ep = epithelium, lu = lu- men, n = nuclei, rc = nest of regenerative cells, white arrow = nucleus in the intestinal ep- ithelium positive for marking by the TUNEL method. to all other CT groups (mean = 134.41), AC (mean = 137.70), PT2 the knowledge gaps regarding fungicides, mainly analyzing the post- (mean = 138.56), and PT3 (mean = 136.73) (Fig. 5B). The other exper- embryonic development. imental groups, CT, AC, PT2, and PT3 are statistically similar to each The absence of effects of the pyraclostrobin fungicide concentrations other (P > 0.05) (Fig. 5B). on larval mortality and pupation and emergence rates may be related to the detoxification systems present in A. mellifera (Berenbaum and 4. Discussion Johnson, 2015). The detoxification process may have generated greater energy expenditure as a compensatory response to the fungicide, conse- The results presented here reinforce the importance of analyzing the quently maintaining the development until the emergence of the adults. late effects of the pyraclostrobin fungicide after exposure in the larval In this sense, knowing that during pupation, bees use the energy re- phase since late responses were evidenced through the TUNEL reaction serve accumulated in the larval phase to complement their develop- and the quantification of the peritrophic matrix (ectoperitrophic) using ment (Bishop, 1961; Cruz-Landim, 2009), the deviation in energy de- the WGA-FITC technique, although we found no immediate biological mand to respond to the pyraclostrobin can compromise the changes effects. Chmiel et al. (2020) highlight the notable absence of studies induced during metamorphosis, reducing the performance of the bees with sublethal effects using fungicides compared to other pesticides, that will emerge. In a prolonged exposure scenario, it could compro- such as insecticides. Therefore, the present study is relevant to reduce mise the population dynamics of the colonies (Domingues et al., 2020a). 5
  6. C.E.d.C. Domingues, R. Tadei, L.V.B. Inoue et al. Environmental Advances 4 (2021) 100069 Fig. 3. Detection of cell death (DNA fragmen- tation) by the TUNEL reaction in midgut of adults of Africanized A. mellifera. A - Intes- tine epithelium of adult bees of the CT group staining with DAPI. B - Negative control of the TUNEL reaction. C - Intestinal epithelium of adult bees from the PT1 group with positive im- munostaining. D - Intestinal epithelium of PT2 group larvae negative for TUNEL reaction. Leg- end: ep = epithelium, lu = lumen, n = nuclei, rc = nest of regenerative cells, white arrow = nucleus in the intestinal epithelium positive for marking by the TUNEL method. Fig. 4. Marking intensity of chitin fluores- cence in the peritrophic (ectoperitrophic) ma- trix of the intestine of larvae of Africanized A. mellifera exposed to different concentrations of the pyraclostrobin fungicide in the larval phase. A - Intestine marked with WGA-FITC and nucleus in blue (DAPI staining). B - Flu- orescence intensity in pixel units. Legend: lu = lumen, n = nucleus in blue (DAPI stain- ing), rc = nest of regenerative cells, white ar- row = marked with WGA-FITC. ∗ Asterisks indi- cate statistical differences in relation to control groups (P < 0.05). Mussen et al. (2004) suggest that, even in residual concentrations, fungi- gence rates (Friol et al., 2017). However, in all these studies, the authors cides can interfere in the post-embryonic development of A. mellifera. observed important late effects on the development of other biomarkers Some studies highlight the cytotoxicity of realistic concentrations of such as behavioral, histopathological, immunohistochemical, and ultra- fungicides in the hepato-nephrocitic system (Domingues et al., 2017), structural. midgut (Carneiro et al., 2020; Tadei et al., 2020), and Malpighi tubules Regarding the survival and median lethal time of newly emerged (Batista et al., 2020) of newly emerged workers. bees, our results are consistent with the findings of Tadei et al. (2020), Corroborating with our findings, studies using pyraclostrobin (active who showed that repeated oral exposure to pyraclostrobin (4.93 ng/mL) ingredient) (Tadei et al., 2019) and a commercial formulation (Comet®) in the larval phase did not affect the survival of the adult workers of (Tadei et al., 2020) also presented no effects under the same parameters Africanized A. mellifera. Domingues et al. (2020a), in a comparative analyzed in the present study. Like fungicides, studies using field con- study with adult individuals (newly emerged and foragers), found that centration of the neonicotinoid insecticide thiamethoxam (0.001 ng/𝜇L) oral exposure to three concentrations of pyraclostrobin (0.125 ng/mL, demonstrated no effects on the larval mortality and pupation and emer- 0.025 ng/mL, and 0.005 ng/mL) changed the pattern of survival only of 6
  7. C.E.d.C. Domingues, R. Tadei, L.V.B. Inoue et al. Environmental Advances 4 (2021) 100069 Fig. 5. Marking intensity of chitin fluores- cence in the peritrophic (ectoperitrophic) ma- trix of the intestine of newly emerged workers of Africanized A. mellifera exposed to different concentrations of the pyraclostrobin fungicide in the larval phase. A - Intestine marked with WGA-FITC and nucleus in blue (DAPI staining). B - Fluorescence intensity in pixel units. Leg- end: lu = lumen, n = nucleus in blue (DAPI staining), rc = nest of regenerative cells, white arrow = marked with WGA-FITC. ∗ Asterisk in- dicate statistical differences between the exper- imental groups (P < 0.05). Table 2 ostasis of digestion and nutrient absorption of the individual since they Effects of pyraclostrobin fungicide on the mean lethal time, in are responsible for renewing the epithelium, which progressively de- days, of Africanized A. mellifera workers. TL50: mean lethal time, creases with age (Martins et al., 2006). Domingues et al. (2020a) high- CL: confidence limites with 95% of probability, 𝜒 2 : Chi-square, light the greater sensitivity of the intestinal epithelium of foragers to P: probability. 1 There is no difference between the experimental pyraclostrobin. groups (P > 0.05). The higher intensity of chitin marking of the peritrophic matrix Experimental groups TL50 (CL)1 𝜒2 P identified for the larvae (PT1 and PT2) and adults (PT1) suggests CT 8.61 (7.75 - 9.45) 27.7 0.010 an increase in the amount of synthesis of the peritrophic matrix AC 9.92 (8.72 - 11.05) 42.0 < 0.001 components (ectoperitrophic) caused by the fungicide. According to PT1 10.53 (9.33 - 11.61) 33.0 < 0.001 Shao et al. (2001), the balance between the synthesis and degradation PT2 8.96 (8.07 - 9.76) 11.7 0.387 of the peritrophic matrix of insects, which occurs through the action of PT3 10.19 (9.37 - 11.04) 24.1 < 0.001 chitinases and chitin-synthase, determines their thickness, and the per- itrophins are involved in its elasticity and permeability. According to Oliveira et al. (2019), there are no differences in permeability across the midgut. In this sense, the greater synthesis of peritrophic matrix foragers and showed no effect on the longevity of newly emerged bees. components, evidenced by the intense marking, would block a large In the same sense, Carneiro et al. (2020) found no immediate effects amount of absorption of the fungicide, thereby mitigating the adverse after exposure to the fungicide iprodione (LD50 = 2 mg/kg). effects. In Drosophila melanogaster (Meigen, 1830), protective effects of However, there were responses at the cellular level in the in- the peritrophic matrix against pathogens and toxins have already been testinal epithelium of bees from groups PT1 (larvae and adults) and described through genetic evidence (Kuraishi et al., 2011). PT2 (larvae). The TUNEL diagnosis was positive for digestive cells, Oliveira et al. (2019) demonstrated that the concentration of chitin in which they perform important functions such as digestion and ab- in the matrix of A. mellifera midgut was significantly higher in the an- sorption of nutrients, excretory capacity, and synthesis of the compo- terior region than in the posterior region, when using the peritrophic nents of the peritrophic matrix (Serrão and Cruz-Landim, 1996; Silva- matrix marking with WGA, also higher in foragers than in nurse work- Zacarin et al., 2011; Terra and Ferreira, 2012). Thus, lesions in these ers. Based on the above, we can infer that the larvae had a protective cells can reduce the viability of the organ. Our findings corroborate response to the fungicide, although studies on the peritrophic matrix those of Tadei et al. (2020), who detected positive markings for the in bee larvae are limited. Studies on the peritrophic matrix of insect commercial formulation and the active ingredient of pyraclostrobin. larvae focus on species considered to be agricultural pests and aim to Batista et al. (2020) and Carneiro et al. (2020) also highlighted changes understand the structure and functioning of the peritrophic matrix for in the digestive cells of Africanized A. mellifera workers when using pi- the synthesis of new bioinsecticides and new pest control technologies coxystrobin and iprodione, respectively. (Hegedus et al., 2009; Sandoval-Mojica and Scharf, 2016; Wang et al., The positive markings for DNA fragmentation in newly emerged bees 2016). From this perspective, this study provides relevant information from the PT1 group suggest no complete metabolism and elimination of on how late effects should be considered and how exposure can alter pyraclostrobin along the metamorphosis. It is possible that the fungi- the post-embryonic development pattern of A. mellifera bees. cide was stored in the predominant tissue of the larvae and pre-pupae, the fat body, due to its little polar or lipophilic characteristic. Accord- ing to Bishop (1961), this tissue can represent 60% of the larvae’s body 5. Conclusions weight and has direct contact with the hemolymph. In other words, pyr- aclostrobin can reach the tissue quickly and remain due to the affinity Our results showed that, after exposure to the fungicide pyra- characteristics of its molecule. Silva et al. (2015) identified the presence clostrobin in the larval phase, the intestinal epithelium of larvae and of anomalies in larvae and pupae after acute exposure on the fourth day adults indicated cell death due to fragmentation in the DNA of digestive of larval development to fipronil. The authors suggest that the insecti- cells and an increase in the staining intensity of the peritrophic matrix. cide caused changes in the metabolism of the fat body cells, compromis- These findings demonstrate that, although pyraclostrobin did not cause ing the normal development of Africanized A. mellifera. mortality, the energy response of the larvae can generate less healthy Although digestive cells showed evidence of DNA fragmentation, the emerging bees, thereby reducing the strength of the colony, given that nest of regenerative cells of larvae and adults was not affected. Con- these bees will take care of the activities internally and will later be re- sequently, the intestinal epithelium’s integrity was maintained, which sponsible for the identification and collection of resources. In this sense, corroborates the absence of effects on mortality and survival described it is important to study the late effects of fungicides on larvae since this here. The cells that make up the nest are essential to maintain the home- gap is still high compared to insecticides. It is also worth mentioning the 7
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We would like to thanks the beekeeper Edson Sampaio for main- Domingues, C.E.C., Abdalla, F.C., Balsamo, P.J., Pereira, B.V.R., Hausen, M.A., Costa, M.J., taining hives and collecting bees. The PPGBMA (UFSCar, Sorocaba) Silva-Zacarin, E.C.M, 2017. Thiamethoxam and picoxystrobin reduce the survival and overload the hepato-nephrocitic system of the Africanized honeybee. Chemosphere by the use of Laser Scanning Confocal Microscopy and DBio (De- 186, 994–1005. doi:10.1016/j.chemosphere.2017.07.133. partment of Biology at UFSCar, Sorocaba) by infrastructure for the Domingues, C.E.C., Inoue, L.V.B., Silva-Zacarin, E.C.M., Malaspina, O, 2020a. Foragers confocal microscopy. We also thanks the “Brazilian National Council of Africanized honeybee are more sensitive to fungicide pyraclostrobin than newly emerged bees. Environ. Pollut. 266, 1–12. doi:10.1016/j.envpol.2020.115267. for Scientific and Technological Development” (CNPq) [grant number Domingues, C.E.C., Inoue, L.V.B., Silva-Zacarin, E.C.M., Malaspina, O, 2020b. Fungi- 400540/21097-3]". This work was supported by São Paulo Research cide pyraclostrobin affects midgut morphophysiology and reduces survival of Brazil- Foundation (FAPESP) [grant numbers 2016/15743-7, 2017/21097-3]. ian native stingless bee Melipona scutellaris. Ecotoxicol. Environ. Saf. 206, 1–19. doi:10.1016/j.ecoenv.2020.111395. Fairbrother, A., Purdy, J., Anderson, T., Fell, R., 2014. Risks of neonicotinoid insecticides Supplementary materials to honeybees. Environ. Toxicol. Chem 33, 719–731. doi:10.1002/etc.2527. Francoy, T.M., Wittmann, D., Steinhage, V., Drauschke, M., Muller, S., Cunha, D.R., Nascimento, A.M., Figueiredo, V.L.C., Simões, Z.L.P., De Jong, D., Arias, M.C, Supplementary material associated with this article can be found, in Gonçalves, L.S., 2009. 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