Pervasive sublethal effects of agrochemicals as contributing factors to insect decline

Lautaro Gandara, Richard Jacoby, François Laurent, Matteo Spatuzzi, Nikolaos Vlachopoulos, Noa O Borst, Gülina Ekmen, Clement M Potel, Martin Garrido-Rodriguez, Antonia L Böhmert, Natalia Misunou, Bartosz J Bartmanski, Xueying C Li, Dominik Kutra, Jean-Karim Hériché, Christian Tischer, Maria Zimmermann-Kogadeeva, Victoria Ingham, Mikhail M Savitski, Jean-Baptiste Masson, Michael Zimmermann, Justin Crocker

Preprint posted on 14 January 2024

Insect decline disentangled

Selected by Roberto Amadio



The global insect population is declining at a rapid and alarming rate (Wagner et al. 2020). The reduction of their habitats, global warming and the extensive use of pesticides are believed to be the main factors responsible for this general and widespread decline. However, the root, relevance and scope of this problem has rarely been documented systematically. The authors of this preprint use a library of 1024 insecticides and pesticides – at sub-lethal doses on D. melanogaster larvae – in order to gain a systematic understanding of the contribution of these ‘agrochemicals’ to the global decline of insects.


Key findings of this preprint

  1. Set up of a high throughput screen to monitor the effect of pesticides

Using D. Melanogaster as a model system, the authors focused on the effects of pesticides on larvae. They measured acute (16h) and long term (10 days) lethality as well as behavioural shifts in larvae movements. The concentrations of agrochemical compounds used were 200 uM, 20 uM (both often used for many pesticides) and 2 uM (concentration often detected in the soil). At the lower concentration of 2 uM, only some chemicals caused a high degree of acute toxicity, though long-term toxicity and a behavioural shift were widely detected across the compound library even in non-insecticide pesticides (Fig. 1).


Figure 1 – a,b,c) Schematic representation of the screening workflow, compounds used and parameters analyzed. g,h,i,j,k) Representation of acute, long term toxicity and a behavioral shift for each agrochemical compound tested. Non gray colored dots represent compound for which a toxic of behavioral effect was observed.


  1. Measuring phosphoproteomics changes upon acute exposure of common agrochemical compounds

To quantitatively measure the impact of sub-lethal common agrochemical pesticides, larvae were exposed to 0.2 uM of either Chlorpyrifos, 1,2-dibromoethane, Cyhalothrin, Dodine or Glyphosate. Subsequently, any phosphoproteomic changes were recorded and analysed. Changing the phosphorylation status of proteins is one of the fastest ways in which an organism can adapt to changing environmental conditions. All tested compounds caused behavioural shifts and phosphoproteomic dysregulation in larvae upon acute exposure (Fig. 2). Particularly enriched hits were related to muscular physiology, indicating a potential important stress response axis.


Figure 2 – Volcano plots show the deregulation of the phosphoproteome (upper panel) and proteome (lower panel) by agrochemical compounds exposure. Red dots indicate upregulation while blue dots downregulation.


  1. Increasing the variables of the assay: temperature and compound combinations

The high throughput assay developed by the authors allowed them to study the interaction of pesticides with factors that may also cause insect decline, such as rising global temperatures. As such, they selected a number of compounds and re-tested them at 25°C vs 29°C. Strikingly, an increase of just 4°C strongly impacted the lethality and behavioural effects of many pesticides, indicating that global warming might worsen the effect of agrochemical compounds on insects. Additionally, when the author tested several combinations of chemicals normally present in soil samples, they found some synergistic effects, raising concerns about the safety of agrochemical mixes.


  1. Validation in other species and natural isolates

To corroborate their findings, the authors also analysed the sensitivity of natural isolates and other insect species to pesticides. Natural isolates showed higher resistance, especially to organophosphates, which might be explained by an adaptive response to chemical exposure. When focusing on other species, in particular the mosquito Anopheles stephensi and the butterfly Vanessa cardui, they also found sub-lethal effects of agrochemicals affecting these species’ behaviour, highlighting the relevance of the findings described in this preprint for different insects.



Why I chose this preprint

Designing conservation plans and gaining a deep understanding of the global effects of pesticides on insect biodiversity and survival is a major challenge of our time. This preprint offers a great resource to interpretate and expand our knowledge on agrochemical compounds and their effects on insects. By having also generated a database, The Agrotoxin Database (, this preprint also offers an important resource freely available to the community.



Questions to the authors

  • Would it be possible to use the assay you developed to also test the sub-lethal effects of agrochemicals directly on adult insect populations rather than larvae?
  • What do you think is the most useful way in which your database could be used?




·         Wagner, D. L. Insect Declines in the Anthropocene. Annu Rev Entomol 65, 457–480 (2020).



Tags: climate change, drosophila, insects, pesticides

Posted on: 7 February 2024 , updated on: 9 February 2024


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Author's response

Lautaro Gandara shared

Thanks for your interest in our work!

Q1: Would it be possible to use the assay you developed to also test the sub-lethal effects of agrochemicals directly on adult insect populations rather than larvae?

Yes, in principle similar assays could be used to test the effects of these molecules on adult flies. However, doing so at this scale would pose substantial technical challenges. The delivery of these molecules to larvae is relatively straightforward, as they can live for a short time (<24 h) in liquid environments where pesticides can be added at the desired concentration. In contrast, administering drugs to adult flies requires slightly more complicated methods. Besides, adults can also regurgitate “toxic food”, introducing potential noise to assay outcomes.
Moreover, the behavioral repertoire of adult flies is significantly more complex than larval behavior. There are methods (e.g. Berman, 2014) for characterizing the full range of behaviors in adult flies confined to 2D arenas—which is similar to what we have done with larvae in this study. But in the case of adults, controlling the baseline state becomes a more difficult task, as adult behaviors are extremely sensitive to factors like the circadian clock, temperature, humidity, etc. Thus, these assays are not well-suited for extensive screenings, as the one reported here.
Having said that, I would also like to emphasize that the reasons for doing the screen on larvae rather than adult flies were not exclusively technical. From a biological perspective, the larva constitutes the primary feeding stage—adult Drosophila flies do eat, but the main food intake across the life cycle occurs during the larval stage. As a result, in natural settings larvae are necessarily more exposed to pesticides than adults, and therefore testing the effects of these molecules at the larval stage holds greater ecological relevance.

Q2: What do you think is the most useful way in which your database could be used?

We expect that this database will prove particularly useful for non-experts in the field. As you pointed out, designing effective conservation plans constitutes a significant challenge of our time. In order to revert—or at least stop—the global decline in insect populations, we need to quantitatively understand the contribution of each underlying cause, as policies that only target minor drivers are unlikely to succeed. Through its dedicated website, our database provides easy access to our results, helping journalists, science communicators, concerned citizens, and policymakers appreciate the impact of these molecules in an insect model system. We hope this information will contribute to the rational design of environmental policies aimed at mitigating human impact on insect populations while simultaneously preserving food supplies.
From a more technical standpoint, the idea is that the volume of information hosted in the database will keep growing in the coming years, as we are currently analyzing the effects of many of these molecules in other arthropod systems. Thus, eventually it could become a valuable tool for ecologists, ethologists, arthropod physiologists, and all researchers interested in the interactions between animals and xenobiotics.

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