Over the past half century, a number of problems have emerged due to agriculture and poor nature conservation practices, to which little attention has been paid. Stressors like disease-causing pathogens and parasites, the use of plant protection products, climate change and their interactions are considered as factors affecting pollinator populations (Hristov et al., 2020; Insolia et al., 2022; Piot et al., 2022). Chemical pesticides used in intensive agriculture often get the most attention in this regard. The consistent and sometimes excessive use of these has exacerbated the problem of pesticide residues accumulating in the environment (Sharma et al., 2019), where they come into contact with non-target organisms like pollinators (Shafeeque, Ahmad, and Kamal, n.d.; Nicholls and Altieri, 2013). Traces of chemical pesticides used in agriculture have been detected in various beekeeping products as well as in bees themselves (Kasiotis et al., 2014; Kiljanek et al., 2017; Böhme et al., 2018). The bee declines have been observed in America, Europe, and many other parts of the world, especially in areas where intensive farming practices are used (Dirzo et al., 2014; Potts et al., 2010; Potts et al., 2016; van Engelsdorp and Meixner, 2010; Osterman et al., 2021).

The majority of honeybee colony losses occur during the winter, but it is difficult to pinpoint the specific cause of the mortality, and it is likely the case that poor bee health often results not from single stressors, but rather combinations of stressors to which bees are exposed (Engelsdorp et al., 2009; Goulson et al., 2015; Insolia et al., 2022; van). While the parasitic varroa mite Varroa destructor is considered the most harmful pest to honeybees, partly because of vectoring other viruses (EPILOBEE Consortium et al., 2016), there are also other diseases which should not remain understudied. The two microsporidian pathogens, Nosema apis and N. ceranae (according Tokarev et al. (2020) Vairimorpha apis and V. ceranae) cause nosemosis, a serious but mostly not deadly disease (Botías et al., 2013). Nosema ceranae, originally a parasite of the Asian honeybee which nowadays causes serious problems to European honeybees all over the world and has largely replaced the Nosema apis, an original parasite of European honeybee (Paxton et al., 2007). Both pathogenic species infect the epithelical cells of the honeybee midgut, causing energetic stress (Mayack and Naug 2009; Zheng et al., 2014). Honeybee nosemosis is a disease that often has no clear clinical symptoms and there is no specific treatment against it (Galajda et al., 2021). Partly because of this, nosemosis is considered to be among the factors seriously affecting the overwintering success of honeybees (Fries, Ekbohm, and Villumstad, 1984).

The interaction of the disease with other stress factors, such as pesticide residues found in bee-collected pollen and nectar (Kasiotis et al., 2014; Karise et al., 2017; Kiljanek et al., 2017; Böhme et al., 2018; Raimets et al., 2020) may be detrimental to honeybees (Goblirsch, 2018). Several pesticides can affect a bee’s ability to cope with various diseases. For example, simultaneous exposure to glyphosate and difenoconazole, in Nosema-infected honeybees, was demonstrated to induce additional physiological stress (Almasri et al., 2021). Furthermore, in an Australian field experiment, combined exposure to thiamethoxam and N. apis substantially increased mortality, as well as reduced immunocompetence, in honeybees (Grassl et al., 2018). Overall, pathogen-pesticide interactions, either overlapping or sequential, can reduce honeybee survival and weaken colonies (Alaux et al., 2010; Sánchez-Bayo et al., 2016; Grassl et al., 2018; Almasri et al., 2021). Negative co-effects of Nosema ceranae infection and pesticides are previously observed over different life stages of worker honeybees (Glavinic et al., 2019; Tesovnik et al., 2020).

Fast and reliable methods for measuring non-invasive endpoints in living bees are needed. Until now, most studies have focused on the effects of one or multiple stress factors on cellular processes, survival and humoral immune response (Hartfelder et al., 2013; Havard, Laurent, and Chauzat, 2019; Trytek et al., 2022). Other studies focus on behavioural changes, brood development, number of mites or spores counted or estimated (Henry et al., 2017; Bird et al., 2021). However, it is difficult to measure the severity of these interactions on living bees. These methods are usually invasive and the bee will be sacrificed during the experiment. One of the methods allows measurements of living and intact insects might be respirometry, which allows to observe changes in metabolic rate in real-time and in living organisms (Muljar et al., 2012; Mänd and Karise, 2015; Karise et al., 2016; Ploomi et al., 2018; Castro et al., 2021). The traditional survival experiments may hide some important toxicological aspects like the process of recovery from toxicosis (El-Seedi et al., 2022) allowing also repeated measurement of treated individuals.

Nosemosis does not cause rapid death of colonies (Martín-Hernández et al., 2011) and is detectable throughout several seasons (Naudi et al., 2021). However, in combination with other stressors, the severity increases (Straub et al., 2020). The two Nosema species differ in their virulence (Martín-Hernández et al., 2011; Naudi et al., 2021), but both affect the bee gut by destroying the epithelial cells (Adl et al., 2005). We hypothesise that this could cause a nutritional deficiency that could be detectable with respirometry measurements. For instance the effects of neurotoxic insecticides are usually easily detectable with respirometry (Kestler, 1991; Muljar et al., 2012; Vinha et al., 2021). We also expect that the combination of the low dose of a neurotoxic insecticide and the pathogen might severely affect the overall metabolic rate of honeybees.

Our results suggest that N. apis as a stressor does not significantly affect the respiration rate on individual level in honeybees. Still, the N. ceranae had a greater impact compared to N. apis. Different studies also found the same difference between the two pathogens (Higes et al., 2010; Goblirsch, 2018; Sinpoo et al., 2018). N. ceranae is described as more energy demanding on the host, indicated by higher feed consumption (Martín-Hernández et al., 2011).

Dimethoate has been demonstrated to be lethal to honeybees when administered at concentrations of 0.2 µg per bee (Baskar, Sudha, and Tamilselvan, 2016). Sublethal concentration of dimethoate may increase the bumblebee’s food consumption (Cornement et al., 2017), it is possible that similar increase took place in our study. However, as the feed consumption was not monitored, the consumed amount of feed is based on average nutritional needs of 0.0003 µg per day.

Different stressors can result in different responses in honeybees, including altered metabolism (Even, Devaud, and Barron, 2012). In the present study, the absence of any significant effect may have resulted from high variability within the data accompanied with relatively low spore loads (Figure 2.). Such variability can be due to the individual physiological characteristics of bees, as each individual has a different ability to withstand stress. In addition to the treatments, further stress is induced by handling the bees when putting them in the analyser and limiting their movement. To reduce the impact of handling stress, it could be helpful to extend the period of analysis, during which the individuals could get used to new and more unpleasant conditions (Mänd et al., 2005). In the case of acclimated bees, the variability in the dataset may somewhat decrease (Even, Devaud, and Barron, 2012). However, based on our experiences, honeybees do not tolerate long detainment in the analyser.

Individual stress factors (N. apis, N. ceranae and dimethoate) had no individual impact on honeybee mortality. The authors suggest that this was because the bees used in the experiments were mainly adult bees. The adult honeybees can be more resistant to stress induced by pathogens (Roberts and Hughes, 2014). The selective breeding throughout the years has also resulted in more tolerant bees that are more capable to maintain close to normal functionality even when infected with Nosema spp (Kurze et al., 2016).

Spore counts have often been used to describe the severity of the disease (Botías et al., 2013; Vidau et al., 2014; Kurze et al., 2016). It is proven that 10–33 000 spores per bee is sufficient to induce infection (Fries et al., 2013). In an experiment carried out in Sweden, where mixture of N. apis and N. ceranae spores were used. There the number of spores used to infect honey bees ranged from 10 to 10,000 per bee. Regardless of the mixed pathogen spore ratio used, the number of spores equalized by day 12 of the experiment and infection occurred in both cases (Forsgren and Fries, 2010). From the collected and analyzed bees, after the experiment, we conclude that the amount of spores used in this work were sufficient to induce infection, cause stress and alter the metabolic rate. Natural resistance to nosemosis of the bees used in our experiment is unknown. It is demonstrated that in artificial laboratory conditions can result in a longer lifespan of bees (Human et al., 2013). In this regard, the physical capacity to tolerate stress in laboratory conditions can be greater than in the field conditions.

The combined effect of multiple stressors, in the form of N. apis and N. ceranae, resulted in a significantly higher stress response, expressed by higher honeybee mortality. Control groups, both with and without dimethoate, and also infection with solely N. apis, resulted in 50% of the specimens still alive at the end of the experiment (25 days). Approximately half of the bees (53%) were alive on day 20. This is similar to the results of Vidau (2011), where specimens were infected only with N. ceranae, but the additionally exposure to fipronil and thiacloprid caused the mortality of 82% and 71% respectively (Vidau et al., 2011). In our study, N. ceranae and the overlapping exposure to dimethoate, resulted both in the 60% mortality of individuals on the day 20 of observation (Figure 2). But with N. apis, our results clearly demonstrate the additive stress from the dimethoate—already at Day15, the mortality increased rapidly being similar to the mortality level honeybees of N. ceranae treatment groups. Given that the 24 h LD₅₀ of dimethoate for honey bee workers is 0.126 µg/bee, dimethoate alone in the concentrations chosen in our experiment, is unlikely to cause significant mortality.

The maximum amount of dimethoate that one bee can take per day in this experiment remained significantly lower, as mentioned above. The negative interaction of different pesticides and nosemosis has been proven in various researches. The interaction is often manifested in the fact that a sublethal dose of pesticide causes faster growth of Nosema spores in the nosemosis honeybees (Alaux et al., 2010; Vidau et al., 2011; Aufauvre et al., 2012; Pettis et al., 2012). According to the present study, the combined effects of stressors results in shorter lifespan of worker bees even at very low doses, which hardly impact the test results at shorter time scale as shown by our respirometry experiment. Therefore it is important to pay attention to the honeybee colony health. Beekeepers should be aware about combined effects of stressors and try to avoid adding any synthetic pesticides as parasite or disease treatment. Even if it would be hard to avoid agricultural pesticides, avoiding synthetic veterinary medicines in beehives, is something, every beekeeper can do to protect their honeybees.