Energy balance of food in a detrito-bryophagous groundhopper (Orthoptera: Tetrigidae)

Insect

Tetrix subulata is one of the most widespread groundhopper species in Europe (Holst, 1986). The body length of adults ranges from 10 to 14 mm, and females are usually larger than males (Steenman, Lehmann & Lehmann, 2013; Steenman, Lehmann & Lehmann, 2015; Lehmann et al., 2018). This species is active from March to the end of October in Central Europe (Holst, 1986, Kočárek, Holuša & Vidlička, 2005), when nymphs hatch in summer (August), molt to adulthood in autumn, hibernate and reproduce in spring. The adult season is split into the autumn dispersal-related cohort (Lehmann et al., 2018) and the reproducing spring cohort (Steenman, Lehmann & Lehmann, 2015). The groundhopper usually prefers damp places, and it is often found near rivers in moist habitats (Baur, Baur & Roesti, 2006). The diet of T. subulata includes detritus, mosses (e.g., genera Brachythecium, Bryum, Calliergonella), algae, and small amounts of other substrates (Ingrisch & Kohler, 1998; Hochkirch et al., 2000; Kuřavová & Kočárek, 2017).

Experimental design

Randomly selected adults of T. subulata (brachypronotal or macroptonotal, brachypronotal dominated in a 4:1 ratio) of the spring cohort after the hibernation were collected by sweeping in flooded depressions of meadow near the city of Ostrava, Czech Republic (49°51′40.4″N, 18°11′19.5″E), from 20 Apr to 28 May 2014. Specimens were transported in plastic boxes to the laboratory at the Department of Biology and Ecology, University of Ostrava. The energy balance of food was evaluated using the gravimetric ingestion method (Waldbauer, 1968; Kogan & Parra, 1981; McEvoy, 1985). This experiment consists of the following parts: the acclimatization of specimens, food deprivation, feeding and calorimetric analysis of samples.

Acclimatization

Specimens were acclimated in eight insectaria in laboratory conditions for three days (approximately 40 individuals per insectarium (30 × 15 × 20 cm) with a sex ratio of 1:1). The insectaria were ventilated by covering their tops with textile membranes with 1 × one mm pores. Each insectarium contained a soil depth of seven cm (a mixture of detritus fragments), planted mosses, including Brachythecium rutabulum (Hedw.) B.S.G. and Calliergonella cuspidata (Hedw.) Loeske as well as the grass (Festuca spp.). The upper layer of the substrate was covered with 70% bare soil, 20% mosses and 10% grass. The soil layer in the insectaria was kept slightly wetted with water. Both substrate and all plants used in insectaria were collected from the same locality as the specimens.

Food deprivation

The acclimated specimens were placed in plastic boxes (25 × 25 × 25 cm) to empty their digestive tracts. The boxes had double bottoms. The inner bottom was composed of a perforated textile membrane with 1 × one mm pores, which allowed feces to pass through. Each box contained a wet inert fabric (cotton wool) to provide drinking water. Food deprivation lasted 24 h for each specimen. The sufficiency of food deprivation (i.e., the rate of food passage through the digestive tract) was tested experimentally in a microclimate chamber (Snijders Imago 500, Tilburg, The Netherlands) under laboratory conditions (temperature 25 °C, humidity 80%, photoperiod 12 h light and 12 h dark). The mean rate of food passage through the alimentary tract was approximately 5 h for detritus and approximately 7 h for the moss C. cuspidata. The last fecal pellet was recorded 22 h after the last consumption of detritus and moss.

Feeding and feces collection

The starved specimens were weighed (analytical balances Kern EG 420; Balingen, Germany) to an accuracy of 10⁻³ g, and 20 individuals of the same sex were placed in boxes with one type of food: “feeding group with detritus” (a mixture of soil particles and decaying organic matter), “feeding group with Calliergonella cuspidata” (only phyllodes), and “feeding group with Brachythecium rutabulum” (only phyllodes). The food was collected from the same locality as specimens of T. subulata on 18 Apr 2014. Detritus was collected from the upper layer of decomposing phyto-organic matter found between the moss cushions. Phyllodes of the mosses C. cuspidata and B. rutabulum were manually collected. The diet was adjusted before it was served, the detritus and mosses were air-dried to a constant weight under laboratory conditions (temperature 23 °C, 48% humidity), and the dry matter was weighed (scale Sartorius AG, Göttingen, Germany) to an accuracy of 10⁻⁵ g due to the exact characteristics of the served food. The detritus and the mosses (in dried form) were analyzed for their carbon, hydrogen, nitrogen, sulfur, phosphorus, and silicon contents using the Leco CHN628 analyzer (Leco 628S, Saint Joseph, Michigan); the calorific values and the proportion of ash in the organic matrix were analyzed by the adiabatic calorimeter IKA C4000 (Staufen, Germany) (Table 1). Before serving, the detritus and the mosses were again wetted (moss was submerged in water for 20 min, and detritus was wetted with water to achieve a 2:1 ratio). Wet phyllodes of mosses and wet detritus were placed separately into boxes. All boxes, specimens and served food were controlled twice a day (in 12-h periods). Defecated feces fell individually down into a collection container (the lower of the container bottoms). The feces were collected continuously twice a day (in 12-h periods) during the 30 day feeding period. The collected feces were frozen at −18 °C (Beko freezer, CN 237231, Gaesti, Romania).

Calorimetric analysis

The collected feces were analyzed with an adiabatic calorimeter (IKA C4000, Staufen, Germany), identical to the method used by Hadley & Bliss (1964), White (1978) and Köhler, Brodhun & Schäller (1987). Benzoic acid (C₆H₅COOH, 26 kJ/g) was used for calibration. The calorific value of a sample was expressed as calories per gram of ash-free sample. The ash residues were recorded for all samples.

A total of 240 individuals (20 males and 20 females in each feeding group with two replications) were used in this experiment. Acclimation, food deprivation, feeding, and fecal collection were conducted in a climate chamber (Snijders Imago 500, Tiburg, The Netherlands) with temperature held at 25 °C and humidity at 70% during the day (12 h of light) and 23 °C and 80%, respectively, during the night (12 h of dark). These conditions were constant over the experiment.

Data analyses

The experiment was evaluated using the gravimetric method that relies on ingestion and egestion (White & Watson, 1972), where Ingestion = Assimilation + Egestion. The assimilated energy was determined by the difference between the initial (calorific value of ingested food component) and the final (calorific value of egested feces) dry matter using the following equations:

The calorific value of assimilated food (CV_diet) was calculated for the group of 20 individuals using the following formula: ${\displaystyle {CV}_{diet}{=CV}_{digested}{-CV}_{feces},\left[J∕g\right],}$ and i is the type of food (detritus, moss species).

The real calorific value of assimilated food (RCV_diet) was calculated for each specimen using the following formula, and it is the calorific value that the specimen gains from food: ${\displaystyle {RCV}_{dieti}{=CV}_{diet}{-AD}_{dieti},\left[J\right],}$ where AD_diet is the approximate digestibility of food and i is the type of food (detritus, moss). The approximate digestibility of food components in the groundhopper T. subulata was calculated by Kuřavová & Kočárek (2017) according to the following formula: ${\displaystyle AD=\left(WCF-WF∕WCF\right)\times 100,}$ where WCF is the weight (mg) of the consumed food and WF is the weight (mg) of the feces.

The energy food budget (E_fb) was calculated for each specimen using the following formula: ${\displaystyle E_{fbi}{=RCV}_{dieti}\times 4.187,\left[J\right]}$ where 4.187 is the conversion factor used to convert the calorific value to joules, and i is the type of food (detritus, moss species).

The overall experimental design was a 2 × 3 factorial design (2 sexes and 3 levels of food source) with replicate measures at each level of explanatory variables. Sex, food source and their interaction were entered into the models as fixed effects, and replicates were entered as random effects. For data analysis, we used repeated-measures nonparametric ANOVA (ligned rank transformation ANOVA). It is a robust statistical tool for the analysis of multiple factorial designs with non-normal residuals. Before using ANOVA itself the data were transformed by the “art” function (ARTool package) (Wobbrock et al., 2011). This function first aligns the data for each effect (main or interaction) and then assigns averaged ranks (Mansouri, 1998). The post hoc comparison of the main effect for food source was conducted by the “emmeans” package with Bonferroni corrected p-values (Russell, 2019). All analyses were performed using the statistical software R (Ver. 3.1.3, Vienna, Austria) (R Core Team, 2015). The level of probability was considered significant at a P-value < 0.05.