Nutritional Ecology of the Southern Green Stink Bug Nezara viridula (Hemiptera: Pentatomidae) on Selected Varieties of Cowpea and Tomato

Cowpea, Vigna unguiculata (L.) Walp. (Fabales: Fabaceae), and tomato, Solanum lycopersicum L. (Solanales: Solanaceae), are two economically important crops in the southern United States. The southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae) is a highly polyphagous pest that attacks a wide range of crops including cowpea and tomato causing a reduction in yield and produce quality. Considering the damage and the associated losses in cowpea and tomato production by this and other pests, as well as the demand for reduced use of pesticides, there is a need to cultivate varieties that are resistant to N. viridula. One key indicator of resistance is host nutritional availability and adequacy. This study evaluated the effect of two selected varieties of cowpea and tomato on the growth and development of N. viridula under laboratory conditions as a first step towards the assessment of nutritional adequacy of both crops. We carried out feeding experiments to compare food utilization and suitability by various life stages of N. viridula. The food substrates tested included fresh immature cowpea pods, fresh cowpea seeds, dry cowpea seeds and newly ripened tomato fruits. Overall, mean nymphal mortality was less when reared on cowpea (63.7±2.9%) than tomato (83.0±1.8%). Nymphs required less time to complete development on cowpea (27.8±2.8d) than on tomato (45.0±3.9d). Fresh body weight at adult emergence was greater on cowpea (132.3±12.1mg) than tomato (83.9±1.5mg). Consumption index (CI) and growth rate (GR) were higher on cowpea (CI: 22.0 ±1.3; GR: 2.1±0.3) than tomato (CI: 8.7±1.4; GR: 0.9±0.1). Collectively, these results suggest that cowpea provided a more suitable nutritional substrate for nymphal development. Many small vegetable growers cultivate both crops.


Introduction
Southern green stink bug, Nezara viridula (L.), is one of several polyphagous pentatomid insect pests that attacks a wide variety of crops that include cowpea and tomato [1][2][3][4][5][6]. In the United States the southern green stink bug is an important pest that feeds primarily on seeds, fresh pods and immature fruits and sporadically on the tender shoots of several crop species [7][8][9][10]. Feeding damage often results in deterioration of fruit and seed quality and significant yield loss in many crops [11][12][13]. In the United States, damage by N. viridula and other stinkbugs in cotton alone is estimated to cause losses of $9.4 million annually [14].
Nymphs and adults of stink bugs feed by puncturing plant tissues with their piercing sucking mouthparts (stylets) removing the cell contents [4,5]. Feeding results in seed damage that may lead to distorted development of pods [15]. Feeding may also result in premature fruit drop, delay in crop maturity and reduced seed quality or quantity [15]. Secondary damage may also occur when many toxicogenic fungi and bacteria are transmitted during feeding [15,16]. Damaged fruits and pods typically have hard brownish or black spots.
Stink bugs are generally multivoltine and highly polyphagous; they attack a wide variety of important food crops [17]. Host switching is common in N. viridula, which potentially benefits them by helping meet the nutritional requirements of the developmental stages [18,5,19]. Consumption of a diet by insects and their growth, development and survival on a particular diet is affected by factors that may include morphological attributes and chemical composition of the diet [20][21][22][23]. There are many reports on the growth and development of N. viridula on soybean (8) and on other crop hosts (21). The performance of N. viridula on soybean varies on different phenological stages and plant parts (especially on seeds and pods) on which they are feed [24]. Apparently, N. viridula has higher survival, fitness and fecundity when fed on pods from plants at full seed stage [24]. However, to the best of our knowledge, there are no studies on the utilization and performance of N. viridula on cowpea and tomato, two important hosts of the pest. Changes in nutrient composition of the plant plays a vital role in food choice and performance responses by phytophagous insects [25][26][27]. In this study, we investigated the nutritional ecology of N. viridula on cowpea and tomato.
Nutritional ecology underscores the importance of nutritional factors to our understanding of the behavioral strategies of herbivorous insects directed toward optimization of nutrient acquisition, utilization and allocation [28]. As noted by Scriber and Slansky [29], nutritional ecology can be a valuable tool that may be used to understand feeding, habitat selection and reproduction by an insect that would potentially help in the selection of insect resistant crop varieties. Nutritional ecology utilizes quantitative nutritional approaches which measure the amount of food consumed, digested, assimilated, excreted, metabolized and converted into biomass. Analysis of these metrics reveals the responses of arthropods to different foods, including the effect on growth [30]. Accurate measurements on consumption, utilization, and allocation of food using gravimetric methods reinforce this [31]. Despite their importance, to date no quantitative food utilization studies are available for the two crops in this study, nor generally among most Pentatomidae [30]. In this study, we compared food utilization indices for N. viridula on cowpea seeds and tomato fruit as an index of food suitability which will serve as a prelude to making decisions on their resistance to this and other stink bugs. The use of resistant varieties is a key component of many integrated pest management programs and has proved to be a reliable alternative or companion to insecticide use on many crops thus reducing their frequency or mitigating the negative environmental impact associated with frequent pesticide application.

Source of Insects
Feral N. viridula adults were collected from the Research farm at Tuskegee University to start a culture that served as the source of insects for the laboratory experiments. The culture was maintained in mesh cages measuring 30.2cm x 30.2cm × 30.2cm (BioQuip Product, Rancho Dominguez, California). Paper towels were suspended from the roof along the inner side of the cages to serve as oviposition substrates and moist cotton balls were placed in Petri-dishes on the floor of the cage as a source of water, especially for the first and second nymphal stages that hardly or barely feed. Egg masses were collected daily and held in 500 ml cups to hatch under laboratory conditions (27.5±1°C, 60 ± 10% RH 12L: 12D photoperiod). Lightly moistened cotton wool was provided in the cups with egg masses to maintain high humidity. The adults and their progeny were reared on a mixture of fresh green beans, peanuts and other fruits as described by [32]. The food source was replaced every 2 days or sooner if it became moldy or desiccated. ) were used in this study. These varieties are among the most popular varieties in many southern states. MS is an erect cowpea variety with a brown and smooth seed coat with silvery-green pods. It is resistant to fusarium wilt and root knot nematodes [33]. Pink Purple Hull (PPH) is a semi-spreading variety with a wrinkled cream-colored seed coat and a distinctively purple-colored pod hull. MAR has a uniform smooth shape with large internal locules. It is resistant to the fungi Verticillium dahliae Kleb and Fusarium oxysporum Schltdl [34]. German Johnson (GJ) is an heirloom tomato variety with an indeterminate habit and large fruits that have a rough and undulating surface; it is slightly disease resistant [34]. The seeds of each variety were planted in the greenhouse in 5-gallon pots filled with Metro-Mix (potting soil). We watered the plants daily, and labelled newly formed fruits with the dates of their formation. Young cowpea pods and newly ripened tomato fruits were the food substrates used in the study. The insects did not feed on unripe tomato fruits.

Food Consumption and Utilization
Food consumption and utilization by N. viridula were determined using third instar (N3) nymphs from the laboratory culture, immature pods (10-12d) of the two cowpea varieties and ripened fruits (N3 did not feed on unripe tomato fruits) of the two tomato varieties. The first nymphal stage (N1) does not feed, and the second nymphal stage (N2) does not feed as actively as the third nymphal stage. In addition, handling the N1 and N2 generally lead to high mortality; this advised our decision to exclude these instars from the tests. Twenty N3 nymphs were used for each variety. A weighing scale (Mettler ME54TE, Toledo, OH, USA) with sensitivity 0.0001g was used to weigh the insects and the food substrates daily. Each food substrate and a set of 40 insects were weighed and dried at 75°C to constant weight. Food consumption was calculated on both dry food (using dry weight of food eaten and fresh weight of insects) and a fresh food basis (using fresh weight of food eaten and fresh weight of insect) using the method described by Waldbauer [31] and modified by Baker et al. [35].
The following indices were calculated: a. Consumption index (CI), the consumption rate corrected for final body weight: CI=F/TA, where F is dry weight of food ingested, T is duration of feeding period (in days) and A is the mean dry weight of insect; b. Growth Rate (GR), biomass gained per day=WT/TA, where WT=dry weight gained; c. Efficiency of conversion of ingested food to body mass or Conversion of Ingested food (ECI), a measure of the ability to convert ingested food into biomass: ECI=(WT/F) X100; d. Relative growth rate (RGR), the amount of growth attained (mg dry matter) per unit body weight (mg dry matter) per unit time=(Insect wet weight gain) (Insect wet weight at the beginning of the trail) (Time); e. Relative Consumption rate (RCR), food ingested per unit nymphal mass per day: (Dry weight of food eaten) (Insect weight at the beginning of the trail) (Time); f. Growth Index (G I)=no surviving nymphs/initial no of nymphs [36].

Nymphal Development on Cowpea Seeds, Pods and Tomato Fruit
Egg masses were collected on the day of oviposition and placed in plastic containers as described in the previous section. First instars (N1) were meticulously removed and placed individually in Petri-dishes (9.0 × 1.5cm) with paper towels and moisten cotton balls. Fresh cowpea pods (12-day old after bloom initiation) were placed individually in the Petri-dishes. Each insect was caged separately, as a replication, and forty insects were used for each food substrate or variety. The insects were weighed after each molt until they molted to adult; other data was collected on survival as well as development time. Petri-dishes were cleaned when necessary and the food source replenished ad libitum. A similar experiment was conducted using fresh seeds, dry seeds and ripened tomato fruits.

Statistical Analysis
Differences in fresh body weight and developmental time were compared by t-test. All data were analyzed using SAS 9.4 at a 0.05 level of significance. Tukey HSD was performed to separate means if significant effects existed.

Discussion
Nutritional ecology underscores the importance of nutritional factors in the behavioral patterns of insect herbivores [28] that are always seeking to optimize food utilization to enhance survival, growth, and reproduction. The quantification of food utilization by insects therefore provides us an insight on the suitability of various foods.
Several indices are used to understand this. Development time and mortality are used to compute the growth index which provides a good estimate of food suitability and its conversion into energy by the insect. Growth Index (GI) was low for both tomato varieties and Mississippi Silver (MS) cowpea possibly due to high mortalities experienced on these substrates, the same varieties that had relatively longer development rates. Development on MS cowpea and both tomato varieties was poor with high mortalities on all Pentatomidae) on Selected Varieties of Cowpea and Tomato food that was tested except the pods of Pink Purple Hull (PPH). This is a clear indication that only PPH pods are suitable food substrate. Longer nymphal developmental time, lower body weight at adult emergence, higher nymphal mortality and lower growth index on tomato indicate that tomato suggest a less suitable food source compared to cowpea.
Extended juvenile development time is usually indicative of a suboptimal food substrate, as is low female weight gain especially in the adult. Only the fresh pods of PPH gave relatively high body weight (Table 2). To better understand how growth and development are impacted by the nutritional status of each food substrate we need to examine the results of the various nutritional indices of each tested food substrate. All of these indices culminate into producing the relative growth rate, or RGR. The highest CI value was obtained on green cowpea pods of PPH which would be indicative of the ability of the insect to convert much of the food it consumed to biomass. This produced the highest weight and survival, predictably leading to a higher state of fitness.
With herbivores, Mulatu et al. [38] found that the presence of allelochemicals in tomato slows the growth of potato tuber moth, Phthorimaea operculella. We also known that host plant properties reportedly influence growth, development and survival of juveniles with direct implication on adult fitness [39,40]. Steroidal glycoalkaloids α-tomatine, rutin, chlorogenic acid and tomatin are the major constitutive allelochemicals in tomatoes that interfere with growth and development of insect pests such as Heliothis zea [41]. The presence of these chemicals may account for the low consumption index (consumption rate corrected for final body weight of these insects) on the two varieties of tomato in our current study.
In the current study, fifth instars required a longer time to complete development on both tomato and cowpea. Previous studies have demonstrated hemipterans require longer duration to complete fifth instars compared to earlier stages [42,43]. The longer time to complete development suggests that the insects must feed for a longer time to have enough energy resources to develop structures and maximum reproductive potential. Fifth instars of southern green stink bug have previously been reported to cause a greater damage to seeds than any other stage [44]. Females required a longer developmental time probably because they need greater amount and higher quality of nutrients for reproduction than do males [45,46]. Third and fourth instars that fed on pods of MS required a longer developmental time, a phenomenon usually associated with sub-optimal diets. This may be the reason for the low consumption index (CI) (cf. PPH). On other hand, it has also been suggested that an increase in consumption rate reduces immature development [47]. The results from both cowpea varieties would seem to indicate that they may also not be the best cowpea varieties for this insect given the relatively high nymphal mortalities that occurred on each of them. More susceptible varieties are known, including "Early Scarlet" which only has recently received research interest.
In addition, an increase in consumption rate of the nymphs that fed PPH might account for their high relative growth rate. MS recorded a higher efficiency of conversion of ingested (ECI) food indicating that the nymphs were more efficient at the conversion of ingested food to biomass. Studies have shown that high ECI is associated with low food conversion [48]. Two explanations have been proposed for this correlation [49]. First, when juvenile consume less, the food slowly passes through the digestive system and it is completely converted and used, and secondly insects eat less because they can convert it easily. In contrast, MAR tomato variety displayed a relatively high CI and ECI compared to GJ suggesting that the insects were able to convert MAR (as cf. to GJ) more efficiently into biomass.
Nymphs on fresh cowpea seeds required less time to develop compared to those on dry seeds or immature pods. This is an indication of the higher nutritional suitability of fresh seeds over dry seeds for growth and development. Panizzi and Slansky (unpublished data) indicated that development of N. viridula was shorter on immature seeds compared to pods and dry seeds of soybeans. Other studies have shown that the pod walls contain sclerotic cells in addition to parenchyma and other plant tissues that hinder the nymphs of herbivorous insects from having a normal feeding activity [50].
A similar study showed that the mortality of young nymphs of N. viridula was high on pods of the legume Sesbania vesicaria Jacq. but most nymphs survived on exposed seeds. The high mortality was probably because young nymphs find it difficult to reach the seeds in the pods because of air space that separates the seeds from the pod wall (Panizzi and Slansky, unpublished data). Stink bugs that feed on seeds of leguminous crops normally insert their stylets through the pod wall to have access to the seeds which are packages of highly concentrated nutrients [51]. Longer developmental time was required on dry seeds than fresh seeds most probably because the insects took a longer time to digest dry seeds than is required for fresh seeds. This might explain why mortality on dry seeds was higher compared to fresh seeds, especially since this insect is not known to be a dry seed feeder in nature.
The differences in developmental time, mortality and weight gain by nymphs on the two cowpea varieties suggest differences in the level and availability of physical plant traits that make the food substrate unsuitable. It appears that seeds of PPH meet these criteria more than MS seeds. In contrast, there was a high mortality on the pods of MS compared to PPH. PPH pod walls are thin and can be penetrated more easily. It has been suggested that tissue hardness could hinder sucking insects by preventing easy access to feeding sites [52]. The pod walls of MS are thicker than those of PPH thus making it more difficult to penetrate [51]. Biochemical analyses are needed to determine the major factors that may be impeding the development of N. viridula in these varieties.

Conclusion
These results indicate that cowpea is more suitable host plant for Nezara viridula than is tomato as indicated by the various metrics examined in this study. The nymphs of N. Viridula required less time to develop and performed better on cowpea than tomato. In fact, consumption indices and growth rates were significantly higher on cowpea. So while the Pentatomidae are known for their wide diet breadth that contributes to their success and adaptive radiation, we are reminded that "all plants are not created equal" and this has direct application to food suitability. From a more applied perspective, one can see the possible use of cowpea as a trap crop to protect tomato from damage by N. viridula. It is unclear whether the reverse situation (to deter infestation of cowpea by inter-planting it with tomato) is even possible, or desirable, especially given that tomato is considered of higher value than cowpea that is generally seen as a crop of the poor. An intercropping study of both crops would probably provide some insight into these questions. Biochemical analyses would also help to determine the range of factors in these varieties that are responsible for the differences observed in various indices measured in this study.