Effects of Taurine as Dietary Supplement on the Biological and Demographic Parameters of Nezara viridula (Heteroptera: Pentatomidae)

Abstract

The southern green stink bug, Nezara viridula (L.) (Heteroptera: Pentatomidae), is a widely distributed pest of many economically important crops. Because of its economic impact, multiple examples of rearing methods and diets for N. viridula have been published. However, rearing this pest year-round consistently in all-vegetable diets has been challenging. Preliminary observations have shown that supplementing N. viridula diet with insect components improves the survival and reproduction of this insect. We hypothesized that taurine could be the nutrient present in insect components that was providing the benefits. Treatments consisting of three different watering regimes: 1) Reverse osmosis (RO) water only (W), 2) 2% taurine solution only (T), and 3) a choice between RO water and 2% taurine solution (T&W) were compared for their effects on life cycle and demographic parameters of N. viridula. Both taurine-containing treatments (T and T&W) resulted in a significant increase in nymphal and premating adult survival and egg viability as compared with treatment ‘W’. Taurine supplementation did not have significant effect on fecundity and development time significantly increased in the ‘T’ treatment compared with W and W&T treatments. However, there were significant improvements in demographic parameters showing an increase in fitness levels after taurine supplementation. These results suggest that taurine is an important nutrient for N. viridula, which has been deficient in traditional diets consisting exclusively of vegetable components. Adoption of this new information will help to improve the survival of N. viridula in culture to facilitate this study to develop new methods for its control.

Nezara viridula (L.) (Heteroptera: Pentatomidae), the southern green stink bug is a polyphagous insect distributed in the Americas, Europe, Asia, Africa, New Zealand, and Australia subtropical areas (Squitier 2020) being a major pest of numerous economically important crops (Hirose et al. 2006). N. viridula has been reared in the laboratory using diets that consisted mostly of vegetables with diverse degrees of success (Panizzi et al. 2000, Noda and Kamano 2002, Fortes et al. 2006, Cantón and Bonning 2020). Continuous laboratory rearing of N. viridula is a challenging process and colonies tend to become weak during winter months requiring intensive maintenance practices (Rojas and Morales-Ramos 2014). Substantial improvements in N. viridula colony development have been made when vegetable-based diets were supplemented with frozen pupae of Tenebrio molitor L., provided after high incidences of cannibalism were observed in cultures (Rojas unpublished). Preliminary studies in which taurine were incorporated into the colony’s water supply showed a positive effect in the general health of the colony. Consequently, we hypothesize that taurine could be one of the nutrients required by N. viridula and not provided in sufficient amounts by a pure vegetable diet due to the scarcity of taurine in plant tissue (Huxtable 1992, Spitze et al. 2003).

Taurine is a sulphonic amino acid compound, largely studied in mammals due to its multiple benefits including cellular volume regulation, providing a substrate for the formation of bile salts, antioxidation, and modulation of free calcium concentration inside the cells (Sculler-Levis and Park 2003, Ripps and Sheen 2012). Taurine does not form polypeptide chains and does not combine to form part of proteins; however, it is the most abundant amino acid in the brain, retinal tissue, leucocytes, skeletal muscle, and heart of mammals (Sculler-Levis and Park 2003, Ripps and Sheen 2012). Taurine has been isolated from the nervous system of insects of various genera including Locusta (Orthoptera: Acrididae), Apis (Hymenoptera: Apidae), and Drosophila (Diptera: Drosophilidae) (Bicker 1992). This amino acid has also been found in the pupal brain of Mamestra configurata Walker (Lepiodptera: Noctuidae) (Bodnaryk 1981) and in the flight muscles of Tenebrio molitor, L. (Coleopterea: Tenebrionidae) (Finke 2002), Blatella orientalis (Blattodea: Blattelidae, L.), and Schistocerca gregaria, Forsskål, (Orthoptera: Acrididae) (Whitton et al. 1987). Taurine fed to the Japanese carpenter ant, Camponotus japonicus Mayr, (Hymenoptera: Formicidae,) showed a significant positive effect on its eusociality and colony health in general (Kim and Lee 2019). The objective of this study was to determine the effects of supplementing the diet with taurine on the life cycle of N. viridula and their potential impact on fitness.

Materials and Methods

The colony of N. viridula used in this study was originally collected from a cotton field located in Stoneville, MS, during summertime 2014 and has been maintained in culture at USDA ARS SEA NBCL in Stoneville, MS, using a combination of natural products and artificial formulations as reported by Rojas and Morales-Ramos (2014). Treatments consisting of three different watering regimes: 1) reverse-Osmosis (RO) water only (W), 2) in house made, 2% aqueous taurine solution only (T), and 3) a choice between RO water and 2% taurine solution (T&W) were provided to determine the effects of taurine supplementation on the life cycle of N. viridula. The taurine solution was prepared using 2 g of taurine powder (TAUR100, Bulk supplements, Henderson, NV) dissolved in 98 ml of RO water. Three different experiments were designed to separately evaluate the impact of taurine supplementation on immature survival (experiment 1), development time (experiment 2), and fecundity (experiment 3). These experiments were designed to compare the levels of indicated biological parameters under the three different treatments. The values of the biological parameters were later used to calculate the demographic parameters to use the intrinsic rate of increase (r) and doubling time (DT) as measures of fitness (Roff 1992). All the experiments were conducted under controlled environmental conditions of 26 ± 1°C, 55 ± 5% RH, and a photoperiod of 16:8 (L:D) h inside an environmental chamber (Percival I66VLC20, Percival Scientific Inc., Perry, IA 50220).

Experiment 1, Immature Survival

To test the effect of taurine on survival and development of N. viridula from first to fifth instar, 4,500 mg of eggs were weighed, and evenly divided in nine groups. Groups of eggs were maintained in 145 mm × 20 mm vented Petri dishes. The egg groups were divided in three treatments and three repetitions. Hatching nymphs were provided with two 55 mm × 10 mm diameter Petri dish covers lined with two cotton rounds moistened with either reverse osmosis (RO) water or 2% taurine solution. Treatments consisted of 1) only reverse osmosis (RO) water (no-choice treatment ‘W’ also considered the control), 2) with only 2% taurine solution (in RO water) (no-choice treatment ‘T’), and 3) a combination of treatments ‘W’ and ‘T’ where one of the dishes contained RO water only and the other a 2% taurine solution (choice treatment ‘T&W’). Treatment cotton pads were placed next to each group of eggs so the hatching first instars could drink from them. The covers of the Petri dishes holding the eggs were secured using a piece of tape across the cover and bottom through the center of the dish. First instars conglomerated in a single group on the moisten cotton pads and did not move until they molted to the second stadium. The egg groups were placed in an environmental chamber set at the conditions described above until the eclosed nymphs reached second instar.

To eliminate damage to the nymphs and prevent escapes, each dish containing an egg group with moisten cotton pads was separately placed inside Lexan rearing boxes (320 mm × 260 mm × 100 mm) with 10 windows (27 mm dia.) on the sides and four windows (65 mm dia.) on the top all covered with nylon screen (mesh 500 µm) and the inside bottom of each box was lined with a paper towel. Petri dishes containing the nymphal groups were opened while inside the experimental boxes to prevent any losses due to escapees. An artificial diet consisting of 33% sunflower kernels, 5% soybean flour, 5% peanut flour, 3% of a vitamin solution (Vanderzant F8045, Bio-Serv, Flemington, NJ), and 16% of a mix of dry vegetables that included broccoli, kale, and green beans (M. G. Rojas, unpublished) and raw jumbo peanuts were used as food sources. In total, four Petri dish covers with moistened (with the corresponding treatment) cotton rounds were provided to each box with its corresponding treatment. Fresh food was added every 3 d, and cotton rounds were moistened daily with its corresponding treatment. Experimental boxes were placed in an environmental chamber with conditions described above. Fifth instar nymphs were collected daily, and their numbers were separately recorded by treatment. The egg masses were retrieved to count the hatched eggs under a stereo microscope and recorded by treatment. The number of hatched eggs was used to determine the initial number of alive first instars from each of the egg groups.

Experiment 2, Development Time

Six hundred mg of N. viridula eggs, obtained from one single day of oviposition from the stock colony, were used to determine developmental time from egg to adulthood. Taurine supplementation treatments were as described in experiment 1. Eggs were randomly divided in nine groups, three repetition groups per treatment, and placed in paper-lined vented Petri dishes with their respective taurine treatment as described in experiment 1. Eggs were allowed to hatch inside the Petri dishes and nymphs were kept in the dishes until they reached the second instars. Groups of second instars were transferred to boxes as described in Experiment 1 with their corresponding treatment (‘T’, ‘W’, and ‘T&W’). Diet and raw peanuts were provided every 3 d as described above. Boxes were placed in an environmental chamber at the conditions described above and maintained until nymphs reached the fifth instar. Fifth instars of similar age were transferred to clean Lexan boxes previously prepared according to their corresponding treatment as described above. Boxes with fifth instars were fed with same diet and two moisten large cotton balls with corresponding aqueous treatment held in a plastic weighing boat (two weighing boats per box) were provided. Cotton balls were watered daily with their corresponding treatment. Cotton balls were replaced every four days. Boxes with fifth instars were also maintained in the same environmental chamber until every one of the nymphs reached the adult stage. The date of emergence and sex of every individual was recorded for each of the treatments and repetitions. Development time was calculated for every individual by the number of days between the oviposition date and adult emergence date.

Experiment 3, Fecundity

Adults resulting from experiment 2 were grouped according to emergence date and treatment. Adults were maintained for 10 d until mating started. Adults dying during this period were taken into account to calculate adult mortality for the life table analysis. These groups of surviving adults of similar age were observed daily for mating events, which facilitate the selection of breeding pairs. In total, 54 mating pairs from same emergence date, per corresponding treatment were divided in three repetitions of 18 mating pairs per treatment (a total of 162 pairs for the whole experiment) were transferred to clean boxes and provided with food as described above for experiment 2. In addition, adult groups were provided with oviposition substrate consisting of three sheets of crumbled paper towel sheets (23 × 23 cm) (Tork multifold hand towel 42-04-83, Essity Professional Hygiene North America LLC, Philadelphia, PA). Adult groups were maintained in an environmental chamber at the same conditions described above for a period of 45 d. Daily observations were done to record adult deaths and to search the oviposition substrate for egg masses. Collected egg masses were examined to count individual eggs, placed in the vented Petri dishes, and maintained at the same conditions for 5 d. Egg masses were reexamined after the eggs hatched to count the number of eggs that failed to hatch.

Data Analysis

Data from immature survival, egg viability, and premating adult mortality were analyzed as proportions. Nymphal survival from first instar to adult was determined for each of the egg groups from experiment 1 by dividing the ending number of emerging adults by the number of hatched eggs, which was equivalent to the number of alive first instars at the start of the experiment. Egg viability was calculated as the number of hatching eggs divided by the total number of eggs. Premating adult mortality was calculated as number of adults dying within 24 h after emergence divided by the total number of adults that mated successfully.

Contingency analysis was used to compare categorical data among the treatments and analysis of means (ANOM) for proportions (Nelson et al. 2005) was used to compare proportion levels of categorical data among the treatments. In ANOM an overall mean of proportion (p) is calculated for all the treatments combined as:

$${\displaystyle \begin{array}{l}{\displaystyle pMean=\frac{{\displaystyle \sum ^{}}(p_i\times n_i)}{N}}\end{array}}$$

Where ‘p_i’ is the proportion for treatment ‘i’, ‘n_i’ is the number of observations for treatment ‘i’, and N is the total number of observations of all treatments. A difference level (DL) is calculated for each of the treatments as:

$${\displaystyle \begin{array}{l}{\displaystyle DL=m(\alpha ,I,N)\times \sqrt{pMean\times (1-pMean)}\times \sqrt{\frac{N-n_i}{N\times n_i}}}\end{array}}$$

Where ‘m(α, I, N)’ is a critical value from tables for error level ‘α’, number of treatments ‘I’, and number of observations ‘N’. Using DL values for each treatment the upper difference limit (UDL) and lower difference limit (LDL) are calculated as UDL = pMean + DL and LDL = pMean—DL (Nelson et al. 2005). The expected values for ‘p’ should be between ‘LDL’ and ‘UDL’. Values below ‘LDL’ will be considered significantly lower than expected and values exceeding ‘UDL’ will be considered significantly higher than expected.

The Z test was used to confirm significant differences between paired treatments. Standard deviations for the survival proportions were calculated as the square root of (p × q)/(N−1), where ‘p’ is the proportion alive, ‘q’ is the proportion dead, and N is the total number of observations (Zar 1999).

Development time and fecundity were analyzed using general linear mixed model (GLMM). Comparisons of these two variables among the treatments were done using the Tukey-Kramer HSD test for least square means. Adult age in days was included in the model as a numerical variable for the analysis of fecundity expressed as eggs oviposited per female per day. JMP version 16.2 software was used to perform all statistical analyses (SAS Institute 2021).

Life and fertility table analysis was used to calculate demographic parameters using the data of egg viability, immature survival, development time, and age-dependent fecundity from each of the three treatments and each of the three repetitions. This produced a set of three parameter calculations per treatment. Net reproductive rate (R_o), defined as fertile female progeny per female (FFP/F); generation time (G), as the time in days between reproductive cycles; and doubling time (DT), as the time in days required to double the population in size were calculated according to Carey (1993) as:

$${\displaystyle \begin{array}{l}{\displaystyle R_o={\displaystyle \underset{x=0}{{\displaystyle \sum ^w}}}{l}_x{m}_x}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle G=\frac{{\displaystyle \sum _{x=0}^w}x{l}_x{m}_x}{R_o}}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle DT=\frac{\mathrm{l}\mathrm{n}(2)}{r_m}}\end{array}}$$

Where ‘x’ is the age in days, ‘w’ is the oldest age, ‘l_x’ is the proportion surviving to age ‘x’, and ‘m_x’ is the number of female progeny produced per female of age ‘x’. The multiplication of ‘l_xm_x’ is defined as the fecundity function.

The intrinsic rate of increase (r_m), defined as a comparative measure of fitness by Roff (1992) was calculated by iteration using the Euler-Lotka equation (Lotka 1907, 1913), which takes the form:

$${\displaystyle \begin{array}{l}{\displaystyle 1={\displaystyle \underset{x=0}{{\displaystyle \sum ^w}}}{e}^{-r_{m}x}{l}_x{m}_x}\end{array}}$$

Because there were three full repetitions of life and fertility tables, it was possible to analyze the demographic parameters using GLMM and to compare them among treatments using the Tukey-Kramer HSD test for least square means.

Results

Experiment 1, Immature Survival

Results of the contingency analysis of the combined data showed significant differences in the proportion of surviving nymphs among the treatments (χ² = 250.88; df = 2; N = 7,684; P < 0.0001). ANOM results showed that survival proportions of treatments ‘T’ and ‘T&W’ (0.428 and 0.467, respectively) significantly exceeded their UDL values (0.409 and 0.406, respectively) indicating a significant improvement in nymph survival. Conversely, the survival proportion of the control ‘W’ (0.27) was below its LDL value (0.37) showing a significant decrease in nymph survival (Fig. 1). The Z test showed significant differences between the control ‘W’ and both treatments ‘T’ and ‘T&W’ (|Z| = 11.64 and 15.07, respectively) and significant differences between treatments ‘T’ and ‘T&W’, where the latter had significantly higher immature survival (|Z| = 2.8) (Fig. 1).

Fig. 1.

Survival proportions of N. viridula nymphs from the first to the fifth instar with three different taurine treatments. Treatment T = 2% taurine solution only; T&W = a choice between RO water and 2% taurine solution; and W = RO water only. UDL = upper difference level, LDL = lower difference level. Circles represent survival proportions and brackets represent standard deviation. Proportions with the same letter are not significantly different after Z test at α = 0.05.

Experiment 2, Development Time

N. viridula completed development significantly faster in the control ‘W’ (37.85 ± 4.21 d mean ± SD) and treatment ‘T&W’ (37.97 ± 3.38 d) compared with treatment ‘T’ (39.89 ± 3.71 d) (F = 103.6; df₁ = 2, df₂ = 3,206; P < 0.0001). There were no significant differences in development time between the control ‘W’ and treatment ‘T&W’ (Fig. 2). There was not any significant difference in progeny sex ratio among the treatments, but sex ratio was slightly male biased in all the treatments. Mortality of newly emerged adults before mating occurred (premating mortality) was significantly higher in the control ‘W’ (0.263 ± 0.016) than in the two treatments (|Z| = 14.2 and 8.25 for ‘T’ and ‘T&W’, respectively) and it was above the UDL (0.153) value (Fig. 3). Premating adult mortality in treatment ‘T’ (0.04 ± 0.006) was significantly lower than in treatment ‘T&W’ (0.121 ± 0.009) (|Z| = 7.21) and its value was below the LDL (0.106) (Fig. 3).

Fig. 2.

Development time in days from egg to adult of N. viridula under three different taurine treatments. Treatment T = 2% taurine solution only; T&W = a choice between RO water and 2% taurine solution; and W = RO water only. Circles represent means and brackets represent standard error of the mean. Means with the same letter are not significantly different after Tukey-Kramer HSD test at α = 0.05.

Fig. 3.

Premating adult mortality of N. viridula with three different taurine treatments. Treatment T = 2% taurine solution only; T&W = a choice between RO water and 2% taurine solution; and W = RO water only. UDL = upper difference level, LDL = lower difference level. Circles represent survival proportions and brackets represent standard deviation. Proportions with the same letter are not significantly different after Z test at α = 0.05.

Experiment 3, Fecundity

The taurine supplementation treatments had no significant effect on fecundity. There were no significant differences in the mean number of eggs oviposited per female per day (105.7 ± 99.77, 119.73 ± 133.86, and 105.91 ± 96.24 for treatments ‘T’, ‘T&W’, and the control ‘W’, respectively). However, there were significant differences in egg viability among the treatments. The control ‘W’ showed significantly lower egg viability (0.53 ± 0.005) than treatments ‘T’ (0.711 ± 0.004) (|Z| = 26.18) and ‘T&W (0.795 ± 0.004) (|Z| = 40.68) (Fig. 4). The viability value in the control ‘W’ was below the LDL (0.673), while both treatments ‘T’ and ‘T&W’ had viability values above the UDL (0.693) (Fig. 4). However, egg viability was significantly higher in treatment ‘T&W’ than in treatment ‘T’ (|Z| = 14.01).

Fig. 4.

Egg viability of N. viridula with three different taurine treatments. Treatment T = 2% taurine solution only; T&W = a choice between RO water and 2% taurine solution; and W = RO water only. UDL = upper difference level, LDL = lower difference level. Circles represent survival proportions and brackets represent standard deviation. Proportions with the same letter are not significantly different after Z test at α = 0.05.

The life and fertility table analysis revealed important differences in fitness among N. viridula groups under different taurine supplementation treatments. The net reproductive rate (R_o), which results from the cumulative l_xm_x values throughout the life cycle (Fig. 5), was significantly impacted showing benefits of the taurine containing treatments (25.81 ± 4.01 and 32.05 ± 2.8 FFP/F for ‘T’ and ‘T&W’, respectively) over the control ‘W’ (9.45 ± 2.81 FFP/F) with only water (F = 44.5; df₁ = 2, df₂ = 6; P = 0.0003) (Table 1). Similar results were observed for doubling time (DT), where treatments ‘T’ and ‘T&W’ showed a significantly shorter DT (13.62 ± 0.3 and 11.59 ± 0.52 d, respectively) than the control ‘W’ (19.77 ± 2.56 d) (F = 23.57; df₁ = 2, df₂ = 6; P = 0.0014) (Table 1). Generation time (G) was also significantly affected by taurine treatment (F = 29.62; df₁ = 2, df₂ = 6; P = 0.0089), but in this case treatment ‘T&W’ had a significantly shorter G (57.77 ± 1.4 d) than treatment ‘T’ (63.7 ± 2.35 d) and the control ‘W’ (62.53 ± 0.46) (Table 1). The intrinsic rate of increase (r_m) was the most impacted of the demographic parameters by the taurine treatments, which were all significantly different (F = 44.5; df₁ = 2, df₂ = 6; P = 0.0003). Treatment ‘T&W’ had the highest r_m value (0.0599 ± 0.0019) followed by treatment ‘T’ (0.0509 ± 0.0011) and the control ‘W’ had the lowest value (0.035 ± 0.0047).

Fig. 5.

Cumulative l_xm_x values and net reproductive rates (R_o) of N. viridula under three different taurine treatments. Treatment T = 2% taurine solution only; T&W = a choice between RO water and 2% taurine solution; and W = RO water only.

Table 1.

Demographic parameters of N. viridula under three different treatments of taurine supplementation

Demographic parameters	T	T&W	W	F	P
rm	0.051 ± 0.001b	0.06 ± 0.003a	0.035 ± 0.005c	44.5	0.0003
Ro	25.81 ± 4.01a	32.05 ± 4.85a	9.46 ± 2.8b	25.78	0.0011
DT	13.62 ± 0.3b	11.59 ± 0.52b	19.77 ± 2.56a	23.57	0.0014
G	63.7 ± 2.36a	57.77 ± 1.4b	62.53 ± 0.46a	11.46	0.0089

r_m = intrinsic rate of increase; R_o = net reproductive rate (number of reproductive females produced per female); DT = doubling time (number of days required to double the population in size); G = generation time (length in days of a generation).

Treatments: T = taurine solution only; W = water only; T&W = choice between taurine solution and water.

Mean ± standard deviation. Means with the same letter within rows are not significantly different after Tukey-Kramer HSD test at α = 0.05; df₁ = 2, df₂ = 6.

Discussion

Results indicate that providing N. viridula nymphs with taurine in solution significantly improves overall survival of immature and early adult stages and egg viability. Furthermore, these improvements are even higher when taurine solution is presented as a choice in the presence of source of pure water. Immature survival and egg viability were significantly higher in the choice ‘T&W’ treatment than in the pure taurine solution treatment ‘T’. Also, N. viridula with the nonchoice taurine solution treatment showed a significantly longer development time than those in the control and the choice treatment. It is possible that excessive consumption of taurine may undermine its potential nutritional benefits. Consumption levels of taurine may be self-regulated by N. viridula in the presence of a source of water. On the other hand, the nonchoice taurine solution treatment ‘T’ provided significant improvements in reducing premating adult mortality compared with the choice treatment ‘T&W’. This appears to be a special case for newly emerged adults where taurine requirements may be higher. Life and fertility table analysis showed an overall improvement on demographic parameter levels with taurine supplementation.

The importance of taurine in vertebrate physiology is well understood (Schuller-Levis and Park 2003, Ripps and Shen 2012). The role of this amino acid in insect physiology has not been studied at the same level of detail as it has in vertebrates. Relatively high concentrations of taurine (26 µmol/g) have been detected in the flight muscle of S. gregaria and flight triggers an increase in its concentration (Whitton et al. 1987). Bicker (1992) determined the distribution of taurine in the central nervous system of the honeybee, Apis mellifera L., and concluded that this amino acid may play an important role in the development of insect central nervous system. This conclusion is supported by extreme changes in taurine concentrations in the nervous system of the bertha army worm, M. configurata, occurring during metamorphosis Bodnaryk 1981).

The importance of taurine in the development of the central nervous system in insects can explain the increase in survival observed in N. viridula with taurine supplementation. However, it does not necessarily explain the increase in egg viability observed in this study. The high level of improvement observed in life cycle and demographic parameters of N. viridula after taurine supplementation, suggests that deficiency of this amino acid may be an important factor on the decline of cultured colonies of this species. It is likely that taurine may be important in other insect physiological processes not yet studied. More research is required to determine the full impact of taurine in insect biology and nutrition.

Author contributions

MGR (Conceptualization, data curation, investigation, methodology, supervision, resources, writing-original draft, writing-review & editing), and JAM-R (data curation, formal analysis, investigation, methodology, project administration, validation, visualization, writing-original draft, writing-review & editing).