Glyphosate affects Tetragonisca angustula (Latreille, 1811) (Hymenoptera: Apidae) worker’s locomotion, behavior and biology

doi:10.21203/rs.3.rs-2129592/v1

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Glyphosate affects Tetragonisca angustula (Latreille, 1811) (Hymenoptera: Apidae) worker’s locomotion, behavior and biology

https://doi.org/10.21203/rs.3.rs-2129592/v1

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published 29 Apr, 2023

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Declining bee populations diminish pollination services, damaging plant, and agricultural biodiversity. One of the causes of this decline is the use of pesticides. Pesticides with glyphosate as an active ingredient are among the most used pesticides worldwide, being the most used in Brazil. This study determined the 24 and 48 hours LD50 (median lethal dose) of the herbicide glyphosate by ingestion, identified sublethal doses, and investigated its effects on the locomotion and behavior of Tetragonisca angustula workers. The LD50 found indicates that glyphosate is highly toxic to T. angustula. The doses applied, including concentrations found in nature and recommended dosage, caused death, motor changes (decreased speed and tremors), excessive self-cleaning, and disorientation (return to light and stop). These results suggest that this herbicide can negatively affect the pollination activity of T. angustula. Toxicity and sublethal effects evaluation of pesticides on bees contributes to a better understanding of the harmful effects on hives and to adopt strategies to reduce intoxication.

stingless bee

excessive self-cleaning

locomotion

acute toxicity

tremors

More than 20,000 bee species are known worldwide (Michener 2007; Catalog of Life 2020), with 1,576 described in Brazil (Silveira et al. 2002). Among them, 400 are stingless bees (Catalogue of Life 2020), pollinating 40 to 90% of plants in the Brazilian forests and other tropical and subtropical regions (Kerr et al. 1996; Slaa et al. 2006).

The stingless bee Tetragonisca angustula (Latreille, 1811), known as Jataí, is a trigoniform meliponine of the Meliponini tribe (Michener 2007). They are distributed in the Neotropical region and found in all Brazilian biomes (Nogueira-Neto, 1997). This species is critical for agroecosystems as it pollinates and adapts to different environments, produces honey, wax, and propolis, and is easy to handle (Vossler et al. 2018).

Stingless bees are exposed to various threats, especially in agricultural systems, due to the indiscriminate use of pesticides. In bees, they alter morphology (Schenk et al. 2001), morphogenetics (Solomon and Hooker 1989), physiology, immunity (Sánchez-Bayo et al. 2016), behavior, communication, cognition (Cox and Wilson 1984; Bendahou et al. 1999; Decourtye et al. 1999; Bortolotti et al. 2003; Stanley et al. 2016), foraging efficiency, (Stanley et al. 2016) and motor functions (Jacob et al. 2019). They further cause the death and decline of bee populations (Faita et al. 2018).

The decline in bee populations is notorious worldwide (Kerr et al. 2005; Lopes et al. 2005; Aizen and Harder 2009; Engelsdorp et al. 2008, 2009). It reduces agricultural productivity, decreasing the pollination of native and cultivated plant species (Pigozzo and Viana 2010), and when it affects native species, it intensifies the extinction of plants and unbalances ecosystems (Absy and Kerr 1977).

Herbicides with glyphosate as an active ingredient are among the most used pesticides worldwide (Jones et al. 2010), being the most used in Brazil, with 173 thousand tons sold in 2017 (ANVISA 2018). At recommended doses of 1.4mgL^-1 to 7.6mgL^-1 (Herbert et al. 2014), it kills weeds and grasses that compete with food crops by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (Galli and Montezuma 2005).

The action of glyphosate should only occur on plants, microorganisms, and fungi (Duke et al. 1989), although research showed it is toxic and has adverse effects on vertebrates and invertebrates (ANVISA 2018). There are reports of its action on mammals (Romano 2007), amphibians (Relyea 2005a), fish (Relyea 2005b), earthworms (Springett and Gray 1992), snails (Tate et al. 1997), bees (Toledo and Guillen 2014), wasps, neuropterans, (Hassan et al. 1994) and crustaceans (Dutra et al. 2011).

One of the ways to assess a pesticide lethality is through the LD50 (median lethal dose), a dose capable of causing 50% mortality in animals within 24 to 48 hours, evaluated by protocols such as the Organization for Economic Cooperation and Development - OECD (1998). Doses below LD50 are considered sublethal (Pereira 2010). Studying the LD50 and sublethal dosages makes it possible to determine the toxicity level and the effects of pesticides, such as glyphosate, on non-target organisms, such as bees.

Atkins et al. (1981) evaluated the effects of 399 products on bees and classified them according to their acute toxicity. Products with LD50 < 2µg/bee were considered highly toxic, products with LD50 equal to 2 to 11µg/bee were moderately toxic, and relatively non-toxic those with LD50 > 11µg/bee.

Bees become contaminated with pesticides by contact during spraying, by consuming pesticide droplets in the environment, or through colleting and ingesting contaminated water, nectar, and pollen (Jay 1986). The analysis of the sublethal effects of the recommended concentration for spraying (0.0076gL^-1) (Herbert et al. 2014) and the highest concentration ever found in nature (0.0026gL^-1) of glyphosate (Relyea 2005a) allows us to assess the effects on bees near the plantations that use this chemical.

Toxicological studies are the first step toward understanding the effects of pesticides on bee colonies and contributing to the conservation of these pollinators (Guedes et al. 2016). This study determined the LD50 and sublethal doses of glyphosate and evaluated the toxicity and effects of this herbicide on the locomotion and behavior of T. angustula workers. The results will help elucidate the impacts of glyphosate on T. angustula, besides providing information on toxicity to further studies and support actions against its anthropic threats.

2.1. Bee sampling

We prepared solutions and collected bees at the Instituto Multidisciplinar em Saúde, of Universidade Federal da Bahia (IMS/UFBA), and on private property, lethality, locomotion, and behavior tests were carried out, both in Vitória da Conquista, Bahia, Brazil. A total of 470 T. angustula bees were collected when they went out to forage between 8 and 10 a.m. from 5 healthy nests in a wooden box kept in the meliponary of the IMS/UFBA. The colonies were approximately 1.5 years old and had 4,000 individuals, including adults and offspring. The specimens were placed in individual pots at the Zoology Laboratory of the IMS/UFBA and kept at room temperature, between 22 and 25°C.

This research is following Law 11,794 of October 8, 2008 (Brazil 2008), which regulates animal experimentation, and with Normative Instruction 03, of September 1, 2014 (Brazil 2014), from SISBIO (Sistema de Autorização e Informação em Biodiversidade).

2.2. Glyphosate lethality test

Glyphosate, in the Roundup® commercial form, was purchased from Plantbem Comércio e Representações LTDA in Vitória da Conquista - Bahia, at a concentration of 445gL^-1 (370gL^-1 of acid equivalent). The methodology for determining the sublethal dose was based on Boff et al. (2018).

In the laboratory, bees isolated in individual pots, capped with holes for ventilation, were divided into 12 groups of 10 bees treated with different doses of glyphosate in duplicate, and 20 bees for the control group, totaling 260 individuals tested.

The control solution used was honey with 50% water. Diluting 1/3 of the control solution with 2/3 of different concentrations of glyphosate constituted the exposure solution. Each bee received 5µL of this solution, considering feeding experiences with eusocial bees (Mayack and Naug 2009).

After a pilot test to determine dosages, the final doses per bee of glyphosate per individual pot were: 50, 25, 10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025 and 0.01µg/bee.

Deaths were noted for 24 and 48 hours after the bees ingested 5µL of the solution. Dose, time, and proportion of survivors were related, determining the death ratio at different dosages and the LD50 of 24h and 48h. LD50s were determined by observation, verifying which dosage killed 50% of the bees, and by statistical analysis.

2.3. Locomotion activity and behavior

Tests of locomotor and behavioral activity used five groups of 10 bees with concentrations of 0.25µg/bee, 0.025µg/bee, and 0.0025µg/bee (all three determined in the lethality test), 0.038µg/bee and 0.013µg/bee. The 0.038µg/bee dose corresponds to sublethal concentrations of 0.0076gL^-1 (recommended dose of the herbicide) and the 0.013µg/bee corresponds to 0.0026gL^-1 (highest concentration found in nature). The control group had 60 more bees, totaling 210 individuals.

Under laboratory conditions, the locomotion test simulates the time and orientation to return to the hive, using a dark 50 x 10 x 10 centimeters lane, with a glass cover placed on the ray allowing visualization of the bees. The ray emitter was positioned vertically with a fluorescent lamp on top for the bees to move by positive phototaxis (Lambin et al. 2001). A transparent tape covered the hole to prevent the bees from reaching the light (Fig. 1).

Each bee was fed 5µL of the solution, then released into the lane through an opening at the bottom, noting the time to travel the 50cm distance for each specimen at 0 (no dose), 1, 4, and 24 hours after ingesting the solution.

During travel, the number of bees that showed excessive self-cleaning behavior, tremors, did not complete the route after 180 seconds in the lane, died, remained still, or turned against the light was recorded. The still behavior only counted when the bees did not complete the route. Thus, if the bee stopped for a while yet completed the course, it was not considered still behavior.

2.4. Statistical analysis

The lethality test was analyzed using Logistic Regression of doses and death numbers relationship in the R program version 4.1.1 (R Core Team 2021). The LD50 was determined by observation and later by Probit using the Finney Probit Analysis Calculator version 2021 (Mekapogu 2021).

Normality was verified by inspecting the data distribution on the Normal probability plot and confirmed with the Shapiro-Wilk test. Non-parametric Kruskal – Wallis test was used for the locomotion test, as the data did not present a normal distribution followed by Dunn's post-test when analyzes were significant in BioEstat 5.0 (Callegari-Jacques 2003).

Student's T-test was used for paired samples to verify if there was a difference between behaviors before and after glyphosate application.

If data did not follow normality, analyzes used the Wilcoxon test for paired samples in the R program version 4.1.1 (R Core Team 2021) (Callegari-Jacques 2003).

All analyzes used a 5% significance level.

Results show that T. angustula is sensitive to all doses of glyphosate tested. The mortality observed after 24 and 48 hours at each dose in the lethality test showed that deaths increase with increasing pesticide dose (Fig. 2A and 2B). No bee in the control group died during the analyzed period.

Similar results with glyphosate were observed in other studies using T. angustula and A. mellifera (Toledo and Guillén 2014; Lunardi 2018, respectively). Yet, at the 50µg/bee dose, the highest tested, the number of deaths was lower than that recorded at the 25, 10, and 5µg/bee doses, showing that the effects cannot always be linear (Suchail et al. 2000), not only as a function of the dose but also of the bee's metabolism.

Observational LD50 was 0.25µg/bee during 24 hours observation. By probit, LD50 was 0.205µg/bee, with a 95% confidence interval from 0.09µg/bee to 0.47µg/bee. In 48 hours, the observational LD50 was 0.025µg/bee and by probit, 0.015µg/bee, with a 95% confidence interval ranging from 0.005µg/bee to 0.04µg/bee. As classified by Atkins et al. (1981), the LD50 obtained indicates that glyphosate is a highly toxic product for T. angustula, which can cause great damage to bee populations if used close to them.

The LD50 of the present work differed from the one described by Lunardi (2018), of 273.93µg/bee by ingestion and 255.73µg/bee by contact with Apis mellifera in 24 hours. Different genetic susceptibility of bee species may explain this difference, as differences between the techniques used, besides environmental conditions (Moraes et al. 2000; Pereira 2010).

Glyphosate had an LD50 similar to insecticides applied orally and topically on A. mellifera, such as Chlorpyrifos (LD50 = 0.25µg/bee, oral) (Suchail et al. 2000) and Malathion (0.27µg/bee, topical) (Smart and Stevenson 1982). These demonstrate that insecticides and herbicides can have the same lethal effect on bees and that hives close to agricultural environments can be affected by those agrochemicals.

If a bee ingests 5µL of glyphosate at a concentration of 0.0026gL-1, as found in nature and used in this research, it will ingest 0.013µg/bee of glyphosate. A value like our probit 48h-DL50 of 0.015µg/bee, lying within the confidence interval. So, bee mortality may be occurring in nature.

At the beginning of the tests, bees were in the same condition as shown when comparing the pre-ingestion tests (hour 0) at all doses, with no statistical difference between locomotion times or behavioral changes.

Glyphosate increased the locomotion time of bees at the doses tested. Comparisons of the times for bees to travel 50cm before the ingestion of the solutions (0h) did not reveal a significant difference (H = 10.6400, GL = 6, p = 0.1002) between the groups of bees that would receive glyphosate and the control group (Fig. 3A).

One hour after glyphosate ingestion, there was a significant difference (H = 136.0303, GL = 6, p < 0.0001) between the tested groups and the control group, except at the dose of 0.0025µg/bee (Fig. 3B). After 4 hours and 24 hours of ingestion of the solutions, there was a significant difference (H = 140.7984, GL = 6, p < 0.0001; H = 122.8370, GL = 6, p < 0.0001, respectively) between the tested groups and the control group.

Similar effects on locomotion were found with glyphosate in Melipona capixaba at a concentration of 17.5gL^-1, decreasing the survival of individuals and impairing flight (Gomes 2017). In A. mellifera, concentrations of 100gL-1 caused 100% mortality (Serpa, 2017), while 0.01gL^-1 in the field, altered hive return time, interfering with the species’ cognition (Balbuena et al. 2015).

For other products, there are also reports of effects like those caused by glyphosate in the present work. For Melipona quadrifasciata (Tomé et al. 2015), Melipona scutellaris (Costa et al. 2015)d angustula (Quiroga-Murcia et al. 2017; Jacob et al. 2019), neonicotinoid insecticides changed behaviors (Quiroga-Murcia et al. 2017), impaired motor functions, and locomotion, and changed average speed and distance traveled (Jacob et al. 2019).

The results differed from those found by Souza (2021) in Scaptotrigona aff. xanthotricha, in which glyphosate, in a concentration of 1.375L/100L of water, reduced the running time of the bees. The increase in locomotion time observed in this work can also occur in the field, decreasing bee speed and foraging efficiency, adversely affecting pollination services (Jacob et al. 2009).

Locomotion time increase had higher statistical significance at higher doses, indicating that higher dosage causes more intense effects and longer travel time, especially near 24h. Similar to the results of Lunardi (2018) testing glyphosate in A. mellifera. Those findings may indicate a slow metabolization of glyphosate by bees with a higher risk of intoxication (Yue et al. 2018).

Control solutions at 0, 1, 4 and 24h had no statistical significance (H = 6.1812, GL = 3, p = 0.1031) (Fig. 4A). There was a statistical significance between different times for each dose. Doses of 0.0025µg/bee, 0.013µg/bee, 0.025µg/bee, 0.038µg/bee and 0.25µg/bee, showed a statistical significance (H = 38.7700, GL = 3, p < 0, 0001; H = 58.2975, GL = 3, p < 0.0001; H = 54.3401, GL = 3, p < 0.0001; H = 71.7682, GL = 3, p < 0.0001 and H = 59.3940, GL = 3, p < 0.0001, respectively) between 0h (before receiving the dose) and the other hours (1, 4, and 24h after receiving the dosage. The difference was greater at 4 and 24h after application (Fig. 4B, C, D, E and F).

Hundred fifty bees that received glyphosate, but 67 did not complete the route (44.6%), of which 19 returned to the light (12.6%), 21 stayed in the lane (14%) and 27 died (18%) (Table 1).

Table 1

Bees that did not complete the course. N = 30 for each dose at each time.
Time Course	Dose (µg/bee)	Did not complete the route
Time Course	Dose (µg/bee)	Returned to the light	Stayed in the lane	Died	Total
1h	0.25	2 (6.6%)	1 (3.3%)	-	3 (10%)
4h	0.013	-	2 (6.6%)	-	2 (6.6%)
	0.025	1 (3.3%)	1 (3.3%)	-	2 (6.6%)
	0.038	3 (10%)	2 (6.6%)	-	5 (16.6%)
	0.25	5 (16.6%)	6 (20%)	1 (3.3%)	12 (40%)
24h	0.0025	1 (3.3%)	1 (3.3%)	1 (3.3%)	3 (10%)
	0.013	2 (6.6%)	2 (6.6%)	2 (6.6%)	6 (20%)
	0.025	1 (3.3%)	2 (6.6%)	2 (6.6%)	5 (16.6%)
	0.038	3 (10%)	3 (10%)	5 (16.6%)	11 (36.6%)
	0.25	1 (3.3%)	1 (3.3%)	16 (53%)	18 (60%)
Total		19	21	27	67
Source: Own elaboration.

In addition, among the 150 bees, 34 (22.6%) presented excessive self-cleaning behavior and 42 (28%) presented tremors, and 5 performed both. Of the 34 who showed excessive self-cleaning, 29 completed the route, and among the 42 who had tremors, 33 finished the course (Table 2).

Table 2

Excessive self-cleaning behavior and tremors observed during the course. N = 30 for each concentration at each time.
Time course	Dose (µg/bee)	Did not complete the route		Completed the route
Time course	Dose (µg/bee)	Excessive self-cleaning	Tremors	Excessive self-cleaning	Tremors
1h	0.25	-	1	6	3
4h	0.0025	-	-	-	1
	0.013	-	-	1	-
	0.025	-	2	1	2
	0.038	3	3	4	5
	0.25	-	-	4	5
24h	0.0025	1	-	-	1
	0.013	1	1	4	3
	0.025	-	-	3	4
	0.038	-	2	3	4
	0.25	-	-	3	5
Total		5	9	29	33
Source: Own elaboration.

T-test and Wilcoxon testes showed statistical significance before and after glyphosate ingestion on all behaviors analyzed (excessive self-cleaning, turning back to light, tremors and standing still) (Fig. 5), indicating that the pesticide causes the observed changes. Analysis of the control group and before ingestion (0h) tests did not show those behaviors, but they were present at 0.25µg/bee at the one-hour observation.

In the natural environment, these effects affect the ability of adults to perform their functions, such as foraging, and make entry into the hive difficult, as they are attacked and rejected by guard bees, damaging the colony (Johansen and Mayer 1990; Freitas and Pinheiro 2010).

In nature, bees use the sun as a reference for orientation (Bomfim et al. 2017), and in a dark environment, they present positive phototaxis (Lambin et al. 2001). Comparing the lane light with the sunlight, the movement against the light indicates a change in orientation behavior and cognition, which can mean an inability to return to the colony in the wild.

Studies with deltamethrin (insecticide) also caused disorientation. Sublethal doses affected flight muscles and coordination and made it difficult for A. mellifera to return to the hive, suggesting a loss of integration of the visual pattern concerning orientation by the sun (Vandame et al. 1995).

A. mellifera bees exposed to permethrin (insecticide) lost their ability to orient and presented severe behavioral disturbances, such as excessive self-cleaning, similar to the observed in the present study. These disturbances affect foraging ability and impact colony development and survival (Cox and Wilson 1984).

The neonicotinoids, carbamates, pyrethroids, and organophosphates alter sodium transport and compete with acetylcholine. These collapse the nervous system causing poisoning symptoms such as hyperexcitation, convulsions, hypersensitivity, tremors, uncoordinated movements, and paralysis (Soderlund et al. 2002; Faria 2009; Silva et al. 2016). All those combined affect bees’ mobility.

Glyphosate action mechanism in bees is not understood yet, but T. angustula tremors and inability to move may indicate a neurotoxic action, generating a loss of motor functions, like the ones caused by insecticides. As such, Chaves (2020) showed that glyphosate was toxic affecting T. angustula antennations and trophallaxis frequency.

Mortality and all observed sublethal effects are detrimental to bee survival, and those sublethal effects are a problem since experiments that confer licenses for pesticide use only analyze acute toxicity to bees (Cham et al. 2020). Also, current official protocols used to determine pesticide toxicity consider only its effects on A. mellifera (OECD 1998; Thompson 2003). Researchers have described more than 400 species of stingless bees, and it is necessary to standardize the studies using representatives of the native fauna as model organisms.

It is necessary to minimize the harmful effects and reduce the use of pesticides in Brazilian agricultural ecosystems, demonstrating the need to change regulatory policies for the registration, release, and monitoring of pesticides. It is necessary to consider the safety of pollinators concerning lethal effects, but also long-term sublethal toxic ones, considering herbicides and not just insecticides.

Colonies' honey quality can change when contaminated nectar transfer occurs onto the hive and by trophallaxis. Honey quality affects all colony members, altering oviposition and brood rearing, decreasing hive vigor, and leading to death (Free 1980; Sánchez-Bayo et al. 2016).

In A. mellifera, glyphosate also causes metabolic changes (20 mg/bee) (Helmer et al. 2015), reduces sucrose sensitivity and memory, and impairs learning and the odor-reward association (5.0mgL^-1e 2.5mgL^-1) (Herbert et al. 2014). It also reduces hive population, causes frequent absence of queens, and death of pupae promotes changes in the hypopharyngeal glands cells responsible for royal jelly production (0.0075mgL^-1) (Faita et al. 2018), and affects the composition of the beneficial intestinal microbiota of bees (10mgL^-1 and 5mgL^-1), increasing the susceptibility to problems by pathogens (Motta et al. 2018).

All these behavioral and metabolic changes can lead to hive abandonment in response to the disturbance (Almeida 2008) in an attempt to survive. However, the relationship between the abandonment of nests and pesticides is unknown, and there are no guarantees that bees will restore a new nest, which may cause colony losses.

This study analyzed acute effects in adults. Further investigations are needed to understand if bees can recover after 48 hours and at different development stages to determine larval mortality rate as proposed by Zhu et al. (2014) and glyphosate's long-term effects on colony behavior and survival.

In conclusion, the results suggest that glyphosate can negatively affect the pollination activity of T. angustula. Toxicity and sublethal effects evaluation of pesticides on bees contributes to a better understanding of the harmful effects on hives and to adopt strategies to reduce intoxication.

FUNDING AND COMPETING INTERESTS

No funds, grants, or other support was received. The authors have no relevant financial or non-financial interests to disclose.

FUNDS

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

COMPETING INTERESTS

The authors have no relevant financial or non-financial interests to disclose.

AUTHOR CONTRIBUTIONS

Prado, Isabela Sousa made experimental planning, data collection, analysis and writing of data. Silva, Laís Alves contributed to the data analysis. Rocha, Agda Alves and Gonzalez, Vinícius Cunha did the experimental planning, data analysis and revision of the manuscript. All authors reviewed and approved the final manuscript.

ETHICS APPROVAL

This is a study with invertebrates. The Comissão de Ética no Uso de Animais – CEUA-IMST/CAT-UFBA has confirmed that no ethical approval is required.

Data availability: Registered data and custom analysis scripts are available upon request.

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Glyphosate affects Tetragonisca angustula (Latreille, 1811) (Hymenoptera: Apidae) worker’s locomotion, behavior and biology

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1. Introduction

2. Methodology

2.1. Bee sampling

2.2. Glyphosate lethality test

2.3. Locomotion activity and behavior

2.4. Statistical analysis

3. Results And Discussion

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