Effects of commercial beverages on the neurobehavioral motility of Caenorhabditis elegans

Chemical reagents and nematodes

NaCl, KCl, NaClO, NaOH, CaCl₂, MgSO₄, K₂HPO₄, KH₂PO₄, cholesterol, bacterial peptone, agar, tryptone, yeast extract, PI were all obtained from Sigma-Aldrich; the wild-type N2 C. elegans used in this study was originally obtained from the Caenorhabditis Genetic Center (Minneapolis, MN, USA). NGM medium, s-basal solution and k-medium solution were used in the experiment.

Test substance

Eighteen beverages were purchased from the supermarket (Walmart), which were divided into seven categories, namely functional beverage, tea beverage, plant protein beverage, fruit juice beverage, dairy beverage, carbonated beverage and coffee beverage (Table 1). Samples were separated and stored at 4 °C.

C. elegans maintenance

Nematodes were maintained on the nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 °C as described (Brenner, 1974). Gravid nematodes were washed off the plates into centrifuge tubes, and lysed with a bleaching mixture (0.45 mol/L NaOH, 2% HOCl). Age synchronous populations of L1- or L4-larvae were obtained as described (Donkin & Dusenbery, 1993).

Grouping and processing

A liquid culture system was used in the experiment; four gradient doses were set for each beverage: ultrahigh dose group (500 µL/mL), high dose group (250 µL/mL), middle dose group (125 µL/mL), low dose group (62.5 µL/mL); k-medium group (0 µL/mL) was set as blank control. Four parallel wells were set as repeats.

Beverage character detection

The pH value, specific gravity and osmotic pressure of 18 kinds of beverage were determined. The osmotic pressure of beverage samples was determined using an ice point osmotic pressure gauge (OM819.C, Löser Messtechnik, Berlin, Germany), the pH value was determined using a digital acidometer (Leici PHS-33; Leico, Shanghai, China), and the specific gravity was determined by taking one mL of the sample with a pipette and weighing it with a balance (Mettler Toledo, ML204). The instrument instruction manuals were consulted for specific operations, and the values of the measurement results were recorded. For more information, see Table 1.

Acute toxicity test

A 200 µL aliquot of test beverages was added into each well of a 96-well plate, which was subsequently loaded with 30 nematodes for each treatment. After exposure to 0 µL/mL, 62.5 µL/mL, 125 µL/mL, 250 µL/mL, and 500 µL/mL concentrations of beverages for 24 h, nematodes were analyzed under the dissecting microscope (Zeiss, Oberkochen, Germany). Nematodes were judged to be dead if they did not respond to stimulation of platinum wire picker. The red fluorescence of nematodes after adding PI staining was used as an auxiliary judgment standard of nematode death.

Body bending frequency

After the nematodes were cultured in 96-well plate for 24 h, nematodes were sucked out of the liquid well, and transferred to NGM medium plate without food (Escherichia coli OP50), the number of body bend in 20 s was recorded. One bend of body was defined as a change in the direction of the nematodes corresponding to the posterior bulb of the pharynx along the y axis, assuming that nematode was traveling along the x axis. Twenty nematodes were counted for each treatment, three replicates were performed.

Head thrashing frequency

After the nematodes were cultured in 96-well plate for 24 h, the nematodes were sucked out of the liquid well, and transferred to NGM medium plate containing no food (Escherichia coli OP50), 60 µL M9 buffer solution was dripped on the NGM plate. After one minute recovery, the number of head thrash was recorded within 20 s. One head thrash was defined as a change in the direction of bending at the mid body. Twenty nematodes were observed for each treatment, three replicates were performed.

Pharyngeal pumping rate

The pharyngeal pumping rate reflected the food intake ability of C. elegans. After the nematode was cultured in 96 well plate for 24 h, nematodes were sucked out of the liquid well and put onto a plate covered with OP50. The plate was placed under a fluorescent stereoscope and magnified enough to observe the movement of pharyngeal pump clearly. Each nematode was videotaped for 30 s. Twenty nematodes were recorded for each treatment, assays were performed in triplicate.

Statistical analysis

All experimental data were expressed as mean ± SD. WPS was used to establish the database and SPSS 21.0 software was used for statistical analysis. Univariate analysis of variance was used for comparison between groups, and the significance level was set as P < 0.05. For standardized comparison, the movement frequency of the beverages-treated nematodes was divided by the frequency of those in control groups to obtain the corresponding fold.

Sample character information and lethal effect of the 18 kinds of drinks on nematodes

Eighteen kinds of beverages from the supermarket were randomly selected and grouped into seven categories namely functional beverage (sports functional drink and fatigue relieving functional drink), tea beverage (black tea beverage, green tea beverage and herbal tea drink), plant protein beverage (almond milk, coconut drink, milk tea beverage), fruit juice beverage (mixed and single juice), dairy beverage (prepared milk beverage A-D), carbonated drinks (brown, colorless and orange carbonated beverages), and coffee drink (coffee). The pH value, specific gravity and osmotic pressure of each tested drink were also examined (Table 1).

To determine the possible adverse effects of 18 beverages on nematodes, we first examined the acute toxicity of the tested beverages in C. elegans, through exposure of synchronized L4 stage worm to five concentrations (0 µL/mL, 62.5 µL/mL, 125 µL/mL, 250 µL/mL, 500 µL/mL) of tested beverages. LC50 is the most important parameter to evaluate the acute toxicity of chemicals in toxicological studies. Our preliminary experiments demonstrated that all tested 18 kinds of beverages at concentrations of 0 µL/mL, 62.5 µL/mL, 125 µL/mL, 250 µL/mL, 500 µL/mL did not affect the survival of nematodes after 24-h exposure (Table S1). This indicates that LC50 of all the 18 kinds of tested beverage/drink were higher than 500 µL/mL.

Effects of the eighteen tested beverages on neurobehavior in C. elegans

We further sought to examine the biological effect of the tested beverages on animal’s locomotion ability at these five selected concentrations (0 µL/mL, 62.5 µL/mL, 125 µL/mL, 250 µL/mL, 500 µL/mL). Given that head thrashing and body bending of nematodes are considered neurobehaviors in C. elegans, we then asked whether exposure to the eighteen tested beverages has effect on the nematode head thrashing and body bending. Remarkably, the data presented in Figs. 1A and 1B demonstrate that the eighteen kinds of tested beverages have varied effects on the locomotion ability in C. elegans. Seven out of the 18 beverages (mixed juice, single juice, brown carbonated beverage, colorless carbonated beverage, fatigue relieving functional drink, prepared milk beverage C and D) reduced the frequency of head thrash, while four of them (sports functional drink, green tea, milk tea, prepared milk beverage A) showed the opposite effect of increased head thrash rate. Meanwhile, eight out of all the beverages (mixed juice, single juice, brown carbonated beverage, colorless carbonated beverage, fatigue relieving functional drink, green tea, coffee, prepared milk beverage C) inhibited the frequency of body bending, while nine of them (sports functional drink, black tea, herbal tea, almond milk, coconut drink, milk tea beverage, prepared milk beverage A, B and D) accelerated the body bending frequency (Figs. 1A and 1B; Tables S1, S3).

Furthermore, we examined the effects of the tested beverages on pharyngeal pumping, and our results showed that after exposure of the L4 nematodes larvae to tested beverages for 24 h, the frequency of nematodes pharyngeal pumping in most groups was affected. The eight beverages including mixed juice, single juice, brown carbonated beverage, colorless carbonated beverage, orange carbonated beverage, fatigue relieving functional drink, green tea, black tea, herbal tea, coffee, prepared milk beverage D reduced the pharyngeal pumping frequency, while the five beverages (almond milk, coconut drink, milk tea beverage, prepared milk beverage A and B) enhanced the rate of pharyngeal pumping in worms (Fig. 1C, Table S4).

Locomotive behaviors are promoted by sports functional beverage and herbal tea drink

The changes in motor behavior of nematodes after beverage treatment can indirectly reflect the changes of nervous system. The evaluation indexes of nematode movement behavior mainly include average movement rate, head thrash, body bend, forward thrash, backward thrash and Omega/U thrash, among which head thrash and body bend frequencies are the most common indexes (Williams & Dusenbery, 1990; Tsalik & Hobert, 2003; Wang & Wang, 2008b). According to the experimental results, the sports functional beverage can accelerate nematode head thrashing from 60.07 ± 7.27 in the control group to 89.60 ± 19.53 (500 µL/mL), 82.00 ± 17.16(250 µL/mL), 67.87 ± 21.55 (125 µL/mL), and body bending from 4.53 ± 1.33 in the control to 6.17 ± 1.97 (500 µL/mL), 6.43 ± 2.05 (250 µL/mL), 6.50 ± 2.69 (125 µL/mL) as well as 5.10 ± 1.45 (62.5 µL/mL), respectively (P < 0.05) (Figs. 2A, 2B). With reference to the nutritional composition table on the packaging bottle (Table S5), this kind of beverage takes the “vitamin group combination” as the market demand and adds four vitamins such as vitamin C, vitamin B3, vitamin B6 and vitamin B12, which can reduce the oxidative damage of the body, and participate in the body energy and protein metabolism, which in turn may enhance the movement ability of the body. The results obtained in herbal tea drink (HT) treated nematodes were similar to those in sports functional beverage which also promotes the body bend frequency from 6.53 ± 1.36 in the control group to 7.33 ± 1.63 (500 µL/mL), 7.53 ± 1.25 (250 µL/mL), 6.67 ± 2.02 (125 µL/mL) and 7.47 ± 1.63 (62.5 µL/mL), increased a degree by 14∼15% (P < 0.05) (Fig. 2C), this is consistent with the results of Lin et al. (2020). In their study, when day 17 adult nematodes were treated with HT at concentrations of 10 mg/mL, 30 mg/mL and 50 mg/mL, the number of free movement nematodes increased significantly, and the number of individuals who could not complete body movement after stimulation decreased, indicating that HT can enhance the motility of nematodes.

Three plant beverages as well as dairy products A and B accelerate pharyngeal pumping rate in C. elegans

There is correlation and interaction between the movement and feeding behaviors of nematodes. The pharynx is the main feeding organ of worm. Presentation of food triggers sensory inputs that drive an increased pumping that draws bacteria into the gut (Dillon et al., 2016). The feeding behavior of nematodes requires rapid head muscle swing to complete, and it also needs body movement to shorten the distance from food. McKay et al. (2004) measured nematode feeding after incubating wild-type N2 and a variety of Eat defects mutants in pumping stimulant (5-HT) and pumping inhibitor (arecoline) as well as exposure to environmental toxicants (cadmium, chlorpyrifos), thereby confirming that the pharynx of C. elegans is a favorable model to study neuromuscular function and effects of chemicals on neuromuscular activity (i.e., eating), clarifying the authoritative position of pharyngeal pump in neurotoxicity evaluation. In this study, the effect of 18 kinds of beverages on the feeding behavior of nematodes was measured by the rate of pharyngeal pump movement.

According to the experimental results, the vibration frequency of the pharyngeal pump of nematodes was significantly accelerated after the worms were treated with three plant protein beverages (almond milk, coconut milk and milk tea) and dairy products A and B, while a decrease in the rate of pharyngeal pump was seen after the worms were treated with other tested beverages. As shown in Fig. 3A, almond milk increased the pharyngeal pump vibration frequency of nematodes by 14%∼26%, from 41.40 ± 7.18 in the control group to 51.80 ± 14.29 (500 µL/mL), 52.00 ± 7.24 (250 µL/mL) and 47.18 ± 9.59(125 µL/mL), respectively (P < 0.05); coconut milk accelerated pharyngeal pumping by 17%∼31%, from 41.40 ± 7.18 in the control group to 48.26 ± 7.92 (125 µL/mL) and 54.05 ± 7.31 (62.5 µL/mL), respectively (P < 0.05, Fig. 3B); the same change to milk tea which increased the frequency of pharyngeal pump by 18%∼46%, from 41.40 ± 7.18 in the control group to 48.70 ± 7.8 (500 µL/mL), 52.50 ± 7.29 (250 µL/mL), 60.30 ± 7.89(125 µL/mL) and 57.85 ± 9.18 (62.5 µL/mL), respectively (Fig. 3C, P < 0.05). At the same time, prepared milk drinks A and B showed positive effect on the nematodes’ pharyngeal pump by increasing the vibration frequency by 10%∼12% and 11%∼18% respectively (P < 0.05). In particular, prepared milk beverage A increased the frequency of pharyngeal pumping from 50.70 ± 9.81 in the control group to 55.75 ± 6.45 (250 µL/mL) and 56.70 ± 7.78 (62.5 µL/mL), whereas prepared milk beverage B increased the frequency of pharyngeal pumping from 50.70 ± 9.81 in the control group to 57.90 ± 8.83 (500 µL/mL), 56.40 ± 8.02 (125 µL/mL) and 59.75 ± 6.34 (62.5 µL/mL) (P < 0.05, Figs. 3D, 3E). It has been reported that nematodes respond to changes in metabolic and nutritional status through a variety of behavioral adaptations, including changes in food intake and food hunting, and the pharyngeal pump feeding behavior of nematodes involves both endogenous and exogenous neuromodulation (Dillon et al., 2016). Nematodes exhibit slow forward and backward movement in the presence of food, and generate a search state after being removed from food (Wakabayashi, Kitagawa & Shingai, 2004; Cermak et al., 2020). According to the data obtained in the current work, the plant protein beverage and dairy products A and B induced the acceleration of pharyngeal pumping and body bending frequency at the same time in nematodes, while fruit juice, carbonated drink, fatigue relieving functional drink, tea and coffee all showed the characteristics of weakened feeding and body movement, which may be related to the food search process of nematodes, in other words, plant protein beverages as well as dairy products A and B treated nematodes were more likely to exercise actively to find and eat food. Furthermore, according to previous research, carbonated drinks can cause nerve and muscle function damage by disrupting the body’s redox balance, and the motor procedures of nematodes, such as locomotion and feeding, are coupled by neuromodulators, which may be the cause of nematode feeding and movement weakness after exposure treatment to certain beverages (El-Terras et al., 2016; Cermak et al., 2020; Zhu et al., 2022). In the future, researchers may explore the possible causes through molecular mechanism research .

Carbonated beverage inhibit motility and feeding in nematodes

It had been reported that drinking carbonated beverages for long periods of time can induce oxidative stress in brain tissue of Wistar rats, reduce the expression of antioxidant markers, and reduce the expression of acetylcholinesterase (AChE) in serum and brain tissue (El-Terras et al., 2016). According to Figs. 4A, 4B, 4C, 4D, the brown carbonated drink and colorless carbonated drink reduce the head thrash frequency and body bend frequency of nematodes by 23%∼30% and 12%∼32%, 15%∼25% and 21%∼38%, respectively. The brown carbonated drink reduced head thrashing from 79.60 ± 15.15 in the control group to 60.80 ± 10.50 (500 µL/mL), 55.33 ± 6.44 (250 µL/mL), 60.93 ± 9.06 (125 µL/mL), 54.97 ± 10.71 (62.5 µL/mL), and colorless carbonated drink reduced frequency from 79.60 ± 15.15 in the control group to 69.93 ± 19.65 (500 µL/mL), 59.33 ± 4.43 (125 µL/mL), 53.60 ± 6.49 (62.5 µL/mL). Body bend frequency was inhibited from 6.17 ± 1.90 in the control to 5.17 ± 1.18 (500 µL/mL), 5.20 ± 1.21 (250 µL/mL), 5.07 ± 1.55 (125 µL/mL), 4.60 ± 1.38 (62.5 µL/mL) by brown carbonated drink , and from 6.17 ± 1.90 in control to 4.83 ± 1.97 (500 µL/mL), 4.70 ± 1.66 (125 µL/mL), 3.80 ± 1.40 (62.5 µL/mL) by colorless carbonated drink. In addition, all three types of carbonated beverages can reduce pharyngeal pump vibration. The brown carbonated beverage reduced the vibration frequency of the pharyngeal pump by 13% from 65.50 ± 6.92 in the control group to 56.74 ± 12.06 (125 µL/mL) and 54.65 ± 15.64 (62.5 µL/mL) (P < 0.05, Fig. 4E); colorless carbonated beverages reduced the frequency by 11%∼13% from 65.50 ± 6.92 in the control group to 56.75 ± 5.67 (250 µL/mL) and 58.05 ± 10.99 (125 µL/mL) (P < 0.05, Fig. 4F), and orange carbonated beverage reduced the vibration frequency by 10%∼14% from 65.50 ± 6.92 in the control group to 56.10 ± 8.63 (500 µL/mL), 58.95 ± 8.43 (250 µL/mL) and 56.20 ± 12.86 (62.5 µL/mL) (P < 0.05, Fig. 4G). Previous research has linked REDOX imbalance in nematodes to muscle mass and function loss. Nematode muscle function is primarily manifested as pharyngeal pump (Zhu et al., 2022). Carbonated drinks are known to cause oxidative stress and after the expression of certain genes related to brain activity (El-Terras et al., 2016), which could explain why nematode’s pharyngeal pump vibration frequency was inhibited in this study.