Development of a Physiological Age-Grading System for Nezara viridula (Hemiptera: Pentatomidae)

Abstract

The southern green stink bug (SGSB), Nezara viridula (L.), is an important agricultural pest in the United States. Limited information is available on the morphology of the female’s reproductive system in relation to morphological changes associated with the number of eggs produced and egg masses oviposited. The ability to assess reproductive health and reproductive status based on ovarian morphology (i.e., physiological age-grading) can be an important tool for evaluating field populations and laboratory colonies intended for the application of different management strategies and experimental trials. Thus, the goal of this study was to develop a physiological age-grading system for SGSB. Females aged from 0 to 79 d chronologically randomly selected from laboratory colonies and dissected to assess ovarian morphology. Specific morphological differences in ovarian structures including differentiation of the ovarioles, deposition of yolk in the most proximal follicle, quantity and appearance of follicular relics, expansion of the lateral oviducts, and number of developing follicles per ovariole were related to chronological age, the number of eggs produced and number of egg masses. Based on specific combinations of these morphological characteristics, the continuum of ovarian development was divided into three nulliparous (i.e., ‘no eggs’; N1, N2, and N3) and three parous stages (i.e., ‘with eggs’; P1, P2, and P3). Direct relationships were noted between number of eggs produced and physiological age with over 7-fold higher number of eggs and 14-fold higher number of egg masses associated with the P2 and P3 stages, respectively.

The southern green stink bug (SGSB), Nezara viridula (L.) (Pentatomidae: Hemiptera), is a worldwide pest with a distribution that includes tropical, subtropical, and warm temperate regions on four continents (Esquivel et al. 2018). While the actual date it was introduced in the United States is unknown, it was reported from Texas in 1880 and Florida in 1883 and is believed to be of Ethiopian origin (Esquivel et al. 2018). SGSB attacks a wide range of crops, as well as various native and ornamental plant species. Crop species include peanuts, peaches, sorghum, soybeans, corn, tomato, and cotton, among others. It has been reported that it prefers leguminous plants in the Family Fabaceae but can feed and develop on fruit and nut trees, as well as other noncultivated trees (Esquivel et al. 2018). All plant parts are likely to be fed upon but growing shoots and developing fruit are preferred. Currently, it has been reported from >25 states in the United States.

The life cycle of the SGSB is similar to other species of Pentatomidae (Esquivel et al. 2018). Eggs are deposited in masses of 30 to 130 eggs which take about 3 d to hatch depending on temperature. Five instars have been reported. Time to reach the adult stage is about 30 d dependent on temperature and food source. There is a 7- to 10-d preoviposition period. It is known to have a reproductive diapause with induction mediated by short day lengths with corresponding diapause termination coinciding to a return to longer day lengths (Musolin and Numata 2003). The later instars are more suspectable to entering a diapause phase. Some evidence indicates that length of time exposed to cold temperatures may also play a role for terminating diapause.

Management methods are varied and include broad-spectrum insecticides, classical biological control, as well as various cultural practices such as the use of trapping crops (Esquivel et al. 2018). Broad-spectrum insecticides employed include organophosphates, pyrethroids, and neonicotinoids. While there has been considerable research into the use of classical biological control for the management of SGSB, limited success has been observed. Most of the biological control efforts have concentrated on the use of host-specific (not completely host-specific, but highly preferred) egg parasitoids. The use of trap crops has seen some success (Esquivel et al. 2018). Researchers have demonstrated that several plants species enhance the action of parasitoids by enticing individuals into the trap crops and hence in closer proximity to the crop. Other cultural practices include varying planting dates, tillage, crop rotation, etc. Such practices have limited efficacy alone but offer some enhanced control when used in conjunction with other management practices.

The reproductive process in SGSB is well described with supplemental information on reproductive physiology, impact of nutrition, various plant hosts, morphology, among others in the literature. Probably the first reported detailed description of the SGSB reproductive systems was by Maluf (1933) followed by Pendergast (1957) with further refinement by Kiritana (1963), Banerjee and Chatterjee (1985), and Esquivel (2009). Several authors have attempted to delineate the continuum of ovarian development into distinct stages to aid in defining the current status of reproduction in field populations of the SGSB (Kiritani (1963), Banerjee and Chatterjee (1985), Adams (2000), Esquivel (2009), and Fortes et al. (2011)). Most of these use changes in morphology of the female reproductive system to characterize reproductive status. Interestingly, Fortes et al. (2011) uses morphology and the presence of proteins associated with reproduction in the hemolymph to determine reproductive condition in SGSB.

However, there are discrepancies and differences among the several proposed reproductive stage methodologies. The information presented here further delineates the continuum of ovarian development in SGSB and demonstrates the relationship between physiological stage, eggs produced, number of egg masses, and age of adult.

Materials and Methods

Colony Origin and Maintenance

Southern green stink bug, N. viridula, were collected from Tift County, GA and a colony established at the National Biological Control Laboratory (NBCL), Stoneville, MS during 2017. Insects were maintained in 30.5 × 30.5 cm screened cages, smaller plastic boxes (32.0 × 25.4 × 11.4 cm), or 15 cm diameter Petri dishes under 14: 10 (L:D) h cycle at ambient humidity, and 28^o C. The colony was maintained on a diet of raw peanuts (Arachis hypogaea L.), fresh green beans (Phaseolus vulgaris L.), and cabbage (Brassica oleracea L.). Food was replaced every other day or at a minimum of three times per week.

Experimental Design

One female and two male newly emerged adults were randomly selected and placed into a vented 15 cm diameter petri dish. A 15 cm diameter filter paper was placed in the bottom of each dish along with one green bean, a small piece of cabbage, and a 35 × 10 mm petri dish bottom containing peanuts. Food was exchanged as needed. The dishes were placed in a Percival incubator model I-30VL (Perry, IA) at the same conditions described earlier. In total, 72 replicates were set-up over time providing with ages ranging from 0 to 79 d. The reproductive systems of all 72 females were examined. The replicates were checked daily for eggs and, if present, removed, counted, and recorded for each replicate. Observed occurrence of mating was also recorded daily. Males were replaced with similar aged individuals if they died. If the female died, that replicate was removed from the experiment. No females were kept without a potential mate to examine changes in the reproductive system with no mating. Approximately, every 2 d, females from the paired set-up described earlier, were selected at random to examine the reproductive system.

Dissections

Females were pinned through the thorax to beeswax filled well plates dorsal side up. The wings and pronotum were removed using fine tipped Vannas Spring scissors (Fine Science Tools, Foster City, CA). The female was then covered with pH 7.4 phosphate buffered saline (P. No. P4417, Sigma–Aldrich, Saint Louis, MO) to keep internal tissues moist and maintain the correct osmotic conditions to avoid cell rupture and tissue degradation. The dorsal abdominal cuticle was removed by cutting along the lateral margins thereby exposing the internal organs. The alimentary tract was removed and discarded. Then the reproductive system was carefully excised from the abdomen while keeping it as intact as possible and placed on a microscope slide in a small drop of phosphate buffered saline. A stereo microscope (Model No. M165C, Leica Microsystems, Buffalo Grove, IL) was used to determine the following:

1. Number of eggs

2. Number of eggs present in oviducts

3. Number of egg masses

4. Number of ovarioles per ovary

5. Ovarioles differentiated into distinct follicles

6. Number of follicles per ovariole

7. Yolk present in follicles

8. Follicular relics presence/color/appearance

9. % expansion of proximal region of spermathecal complex was determined by observation. Expansion is quite obvious and delineating different amounts of expansion was done by trained people (see Fig. 1)

10. Sperm present in spermathecal bulb

11. Expansion of the lateral oviducts as determined by comparison to females which had ovulated to those that had not. Expansion is quite obvious with a distinct lengthening of the lateral oviducts in those females which had ovulated.

Fig. 1.

Micrograph of the spermatheca complex in Nezara viridula illustrating the expansion of the proximal region. a) no expansion (0%), b) mid-level expansion (50%), c) fully expanded (100%). (pr—proximal region, sd—spermathecal duct, sg—spermathecal gland).

In some instances, in order to obtain higher magnifications and resolution, a Keyence DHX-5000 Series Digital Microscope was used at magnifications ranging from 500× to 1,500× (Keyence Corporation of America, Itasca, IL).

Statistical Analysis

Analysis of Variance (ANOVA), assumption tests including Levene Test of Homogeneity of Variances and Brown–Forsythe Test of Homogeneity of Variances, and the post-hoc test Newman–Keuls were all performed using Statistica version 13 (TIBCO Software Inc. 2017). Unless noted otherwise significant differences occurred at P ≤ 0.05. ANOVA was performed to determine significant differences between parous stages and mean number of eggs produced, number of egg masses, and physiological age versus adult chronological age. Product–Moment and Partial Correlations analysis was also performed to determine the relationship between Physiological Age and age of adults in days. An exponential curve was also fitted using the least squares method to the physiological age versus adult age in days using Statistica version 13. This fitted exponential curve was mainly produced to show the overall trend between physiological age and adult age.

Results and Discussion

The reproductive system in female SGSB is similar to that observed in other pentatomids (Maluf 1933, Kiritani 1963, Banerjee and Chatterjee 1985, Esquivel 2009, Fortes et al. 2011). It is composed of two ovaries each containing seven tubular ovarioles per ovary (Fig. 2). The ovaries are located dorsally in the abdominal cavity somewhat lateral to the medial line. The ovarioles combine proximally into the calyx region which then combine forming the lateral oviducts. The lateral oviducts from the two ovaries combine into the common oviduct. As the follicles (= ova plus surrounding epithelium tissue) pass through the lumen of the ovariole the resulting eggs often accumulate in the lateral and common oviducts until enough eggs are present for egg mass oviposition. Mean number (±±SE) of eggs per egg mass for the entire reproductive period was 62.9 ± 4.7 (n = 54) with significantly (F = 8.39, n = 2, P = 0.0007) lower number of eggs per egg mass occurring for the early reproductively active period ‘P1’ (37.1 ± 11.6) when compared with later periods P2 and P3 (70.2 ± 4.4 and 78.6 ± 5.3, respectively). This is well within the range reported by other authors (Esquivel et al. 2018). Mean number (±SE) of eggs found mainly in the lateral and, to some extent, in the common oviducts was 28.6 ± 4.7 (n = 54) with a minimum of 0.0 to a maximum of 149.0. Eggs are apparently often held in the oviducts until a high enough number are accumulated for oviposition of an egg mass. Apparently, the maximum number of eggs ovulated within a short time frame is 14; one from each ovariole, hence, the need to hold the eggs. The accumulation of eggs in the lateral oviducts often cause the calyx and oviducts to expand (Figs. 3c,e and 4). Ovarioles, in latter preoviposition and reproductive females, contain a series of maturing follicles destined to become eggs after passing through the lumen of the ovariole into the calyx, then into the lateral oviducts and subsequently into the common oviduct; a process known as ovulation. The most mature follicles are located proximally in the ovarioles. Ovarioles are composed of two distinct regions; the more distal enlarged somewhat bulbous area commonly referred to as the germarium (Fig. 5). The germarium contains trophic tissue, oogonia, and oocytes which give rise to the developing follicles (Chapman 1998, Perez-Mendoza et al. 2004). The tubular portion of the ovariole is known as the vitellarium which houses the maturing follicles. The follicles are surrounded by epithelium tissue known as follicular epithelium.

Fig. 2.

Micrograph of the entire reproductive system of female SGSB (cx—calyx, co—common oviduct, f—follicle, fr—follicular relic, lo—lateral oviduct, ov—ovary, ovl—ovariole).

Fig. 3.

Micrographs of SGSB ovaries illustrating the three physiological parous stages including P1 (a, b), P2 (c, d), and P3 (e, f). Note main differences between the three stages are based on the quantity and appearance of the follicular relics where P1 females may or may not have eggs present in the lateral and common oviducts, follicular relics not completely surrounding ovariole base, and follicular relics of a pale to light yellow coloration, P2 females follicular relics surround the ovariole base and have a pale to bright yellow coloration, and P3 females where the follicular relics may or may not surround the ovariole base, bright to darkened yellow coloration, and there are distinct darkened particles in the follicular relics. There are no distinct darkened particles within the follicular relics for either the P1 or P2 stages.

Fig. 4.

Micrograph of the SGSB ovary in the parous stage where degeneration or senescence of follicles is evidenced by lack of maturing follicles, clear areas in much of the ovariole, lack of a distinct germarium, and blackened areas with the ovariole. Reasons for the degeneration include age and possible disease (unpublished data).

Fig. 5.

Micrograph of a single ovariole of Nezara viridula depicting major structures (fe—follicular epithelium, f—follicle, gm—germarium, nc—nutritive cord, vt—vitellarium).

The female reproductive system in SGSB is both meroistic and telotrophic. The follicles are meroistic since specialized nurse cells are present (Chapman 1998, Perez-Mendoza et al. 2004) and maintained in the germarium. In the more primitive insect orders (e.g., Odonata, Plecoptera), the ovarioles are typically panoistic and do not contain specialized nurse cells. In the SGSB, the ovarioles are telotrophic and contain specialized nurse cells which transport nutrients to the follicles. These reside in the germarium and are connected to each follicle via a nutritive cord (Fig. 5; Chapman 1998). In other insect species, the ovarioles are polytrophic where specialized nurse cells coincide with each follicle as observed in Hydrellia pakistanae Deonier (Diptera: Ephydridae) (Lenz et al. 2007). In the case of species with polytrophic ovarioles, the nurse cells and the follicular epithelium are cast-off when the follicle is ovulated and accumulate in the ovariole base contributing to the formation of follicular relics (Tyndale-Biscoe 1984, Hayes and Wall 1999). While often difficult to detect, the ovarioles are surrounded by a thin layer of tissue known as the ovariole sheath. In the SGSB, as the follicles pass through the lumen of the ovarioles, the surrounding follicular epithelium of the follicles is sloughed off and accumulates at the base of the ovarioles and are known as follicular relics (Fig. 2). Follicular relics can range in color from a pale to dark yellow to brown in coloration and their quantity and appearance is often used to delineate physiological ages (Tyndale-Biscoe 1984, Grodowitz and Brewer 1987, Grodowitz et al. 1997, Hayes and Wall 1999, Perez-Mendoza 2004, Lenz et al. 2007, Eisenberg et al. 2018, Pratt et al. 2018).

In the SGSB, sperm are stored in the spermathecal bulb which is part of the spermathecal complex as described in detail by Maluf (1933) and even in more detail using Scanning Electron Microscopy by Candan et al. (2015). The spermathecal complex connects to the reproductive system about midway down the length of the common oviduct via the spermathecal duct (Fig. 1a–c). Eggs are fertilized in the common oviduct as they pass the spermathecal duct connection. The proximal region of the spermathecal complex can expand and appears related to mating though we have never observed sperm in the proximal region, only in the spermathecal bulb located distally and covered by glandular tissue known as the spermathecal gland. Within the proximal region is a hardened tube, presumably hardened by sclerotization based on coloration. Expansion of the proximal region does not always guarantee that sperm are present. Only about 30% of parous females contained sperm in the spermathecal bulb even with a high degree of expansion of the proximal region (Fig. 6). Interestingly, what causes the expansion of the proximal region is not known. It is apparently filled with a somewhat hardened jelly-like substance (Maluf 1933). More research is needed to determine the composition of the material causing the expansion and where it arises from (e.g., male or female). While no sperm has been detected in the proximal region, the percent of the expansion is highly related to the reproductive state of the female. For individuals in the preoviposition state or nulliparous stage, the mean percent expansion was only 1.0% ± 1.4 (SE, n = 18) and sperm were not detected in any individual. In comparison, females in the parous state had a mean expansion of 60.7% ± 4.6 (SE, n = 54); a 60-fold increase and was statistically different from the nulliparous stage (F = 54.47, df = 1, P < 0.0001).

Fig. 6.

Average percent expansion of the proximal region of the spermathecal complex in Nezara viridula for both nulliparous and parous females. Note that the average % expansion of the proximal region in the parous stage exceeds 60% compared with nulliparous females who show <5% expansion on average. Significant differences in % expansion was noted between nulliparous and parous females with bars containing different letters significant at P ≤ 0.05 (sd—spermathecal duct, sg—spermathecal gland, pr—proximal region).

The continuum of ovarian development was divided into distinct stages or physiological ages based on several characteristics associated with the reproductive system (Table 1). For the nulliparous stages (i.e., preoviposition period), characteristics used included differentiation of the ovarioles into distinct follicles, presence or absence of yolk, expansion of the lateral oviducts, and lack of follicular relics (Table 1). Three stages were identified and termed N1, N2, and N3. For the N1 stage, ovarioles were highly tracheated, containing no follicular relics, and the ovarioles were simple tubes with no differentiation into distinct follicles (Fig. 7a). The N2 stage was characterized by having the ovarioles differentiated into distinct follicles, no follicular relics and the most proximal follicle with only limited yolk deposition (Fig. 7b). The only difference between the N2 and N3 stage occurred in the deposition of yolk in the most proximal follicle. In the N3 stage, the most proximal follicle was completely expanded and filled with yolk and ready to be ovulated (Fig. 7c). In all nulliparous stages, the lateral oviducts were not expanded.

Table 1.

Characteristics associated with each physiological age for N. viridula

Characteristics	Nulliparous			Parous
	N1	N2	N3	P1	P2	P3
Ovarioles differentiated	No	Yes	Yes	Yes	Yes	Yes
Follicles with yolk	No	Yes/no	Yes	Yes	Yes	Yes
Follicular relics present	No	No	No	Yes	Yes	Yes
Follicular relic color	N/A	N/A	N/A	Pale yellow	Yellow to dark brownish yellow	Dark brownish yellow
Follicular relics surrounding ovariole base	N/A	N/A	N/A	No	Yes	Yes/no
Dark particles present in follicular relics	N/A	N/A	N/A	No	No	Yes
Eggs present in oviducts	No	No	No	Yes/no	Yes/no	Yes/no
Lateral oviducts expanded	No	No	No	Yes/no	Yes/no	Yes/no

Fig. 7.

Micrographs of the ovaries of nulliparous individuals illustrating the three physiological stages including N1 (a) where the ovarioles are simple tubes with no differentiation into distinct follicles, N2 (b) where the ovarioles are differentiated into distinct follicles but the most proximal follicle is not fully expanded with yolk, and N3 which is similar to the N2 stage but the most proximal follicle is fully expanded with yolk and nearly ready for oviposition.

The active reproductive period was characterized by three parous stages (i.e., P1, P2, and P3) mainly based on the presence of eggs in the lateral oviducts and more importantly presence and appearance of follicular relics. For the P1 stage, eggs may or may not be present in the lateral and common oviducts. In addition, follicular relics are pale yellow and do not surround the ovariole base (Fig. 3a and b). Determination of whether follicular relics are surrounding the ovariole base can be difficult. So, if there are eggs in the lateral or common oviducts and limited follicular relics then that female would be characterized as P1. Once follicles are ovulated, follicular epithelium tissues are sloughed off and will be deposited as follicular relics though with only a small number of ovulations follicular relics will be small and have a very pale yellow color. Clear areas in the follicular relics are another indication that the follicular relics are not surrounding the ovariole base. The P2 stage differs in that follicular relics surround the ovariole base and have a pale to bright yellow coloration (Fig. 3c and d). There are no distinct darkened particles within the follicular relics for either the P1 or P2 stages. The P3 stage differs in that the follicular relics may or may not surround the ovariole base and there are distinct darkened particles in the follicular relics (Fig. 3e and f). Increased ovulations do not always reflect in an increase in the quantity of follicular relics. Follicular relics can flush out of the ovariole base by the action of ovulation so quantity of follicular relics cannot be used to associate number of eggs produced to changes in reproductive system morphology (Tyndale-Biscoe 1984, Grodowitz and Brewer 1987, Grodowitz et al. 1997, Hayes and Wall 1999, Lenz et al. 2007, Eisenberg et al. 2018). Why darkened particles form in the follicular relics is also out for speculation. It has been suggested that with continued ovulations, as the follicles pass through the small lumen of the ovariole, the follicular relics become compressed thus leading to the formation of the darkened particles. Number of eggs per egg mass differs significantly among the three parous stages (F = 8.39, df = 2, 51, P = 0.0007). Mean number (±SE) of eggs per egg mass for the P1 stage averaged only 37.1 ± 11.6 (n = 16) when compared with the P2 and P3 stage, where the number of eggs per mass were 70.2 ± 4.4 (n = 22) and 78.6 ± 5.3 (n = 16), respectively. The P1 stage differed significantly in eggs per egg mass from both the P2 and P3 stages; the number of eggs per egg mass for P2 and P3 did not differ.

Another ovarian condition in SGSB, where the ovaries appear to be in a degenerative or senescing state (Fig. 4), is characterized by blackened areas within the ovarioles typically with none or only limited number of maturing follicles. This condition often shows no appreciative differentiation of the follicles and the germarium reduced in size with clear areas occurring within. Eggs may be present within the oviducts. In this study, the degenerative state may have been caused by a microsporidia infection based on RNA analysis (unpublished data). In other species, senescence is caused by the decline in the reproductive state and cessation of oogenesis due to changes in nutrition or ‘old age.’ For example, Pratt et al. (2018) found large numbers of female Oxyops vitiosa Pascoe (Coleoptera: Curculionidae) with ovaries containing reduced follicles, clear areas within the ovarioles, and in most cases distinct follicular relics. These individuals apparently had stopped reproduction because of a lack of suitable nutritional sources which occur during dry conditions when the primary food source (i.e., young leaf material of Melaleuca quinquenervia (Cavanilles) Blake (Myrtaceae)) is not available. This condition was never observed in SGSB. Others have also noted such conditions in Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae) (Grodowitz and Brewer 1987) and Neochetina eichhorniae Warner (Coleoptera: Curculionidae) (Grodowitz el al. 1997).

While considerable overlap was found, there was a significant relationship between the parous stages and mean number (+SE) of eggs produced on a per female basis (i.e., a combination of oviposited and ovulated eggs) with higher number of eggs produced for the P2 and P3 physiological stages (F = 44.396, df = 2, 40, P < 0.001). The mean number of eggs produced for the P1 stage is 71.2 ± 13.5 (n = 16) with a range of 5–188 eggs (Fig. 8). In contrast, the P2 and P3 stages produced mean number (± SE) of eggs ranging from 312.7 ± 32.9 to 544.0 ± 42.5, respectively. The P2 stage had a range from 134 to 893 eggs; the P3 stage exhibited a range from 203 to 791 eggs.

Fig. 8.

Total number of eggs produced (a) (both those that have been ovulated and those that have been oviposited) and number of egg masses (b) in relation to the three parous physiological stages. Higher number of eggs produced, and egg masses occur in the latter physiological stages. Bars with different letters are significantly different at P ≤ 0.05.

Similar results were obtained with the number of egg masses per female; again, higher number of egg masses occurred for the latter physiological stages although considerable overlap was observed between the stages as with number of eggs produced. For the P1 stage, the mean number (± SE) of egg masses per female averaged 0.5 ± 0.16 (n = 16) ranging from 0 to 2. While some individuals had not oviposited an egg mass in the P1 stage, ovulated eggs being held in the oviducts attributed to the P1 classification. Significantly higher numbers (F = 32.284, df = 2, 51, P < 0.001) occurred for the P2 and P3 stages, respectively. The P2 stage exhibited a mean number of egg masses per female of 4.3 ± 0.47 (n = 22) ranging from 2 to 12 while the P3 stage averaged 7.1 + 0.77 (n = 16) with a range from 2 to 16.

Granted, physiological age-grading is not a perfect system to determine number of eggs produced or egg masses oviposited. This is evident from the considerable overlap between the parous stages. However, results suggest these are valid categories based on the statistical analyses, which show significant differences in all the fecundity parameters among parous stages. Similar overlap between parous stages have been observed for other insect species including A. grandis grandis (Grodowitz and Brewer 1987) and N. eichhorniae (Grodowitz et al. 1997). Such variation may be due to seasonal conditions. These include changes in follicular relic deposition due to repeated ovulations flushing out the relics and causing changes in morphologies not indicative for a specific stage. In addition, short-term changes in reproductive status caused by nutritional deficiencies can also cause deviations in morphologies allowing for misidentifications. Finally, higher number of replications may also limit variation.

Nonetheless, physiological grading presents an opportunity to more accurately determine the reproductive status of adult female N. viridula. In addition to providing a conservative estimate of number of eggs produced and egg masses oviposited, this system allows for an estimation of adult age using a fitted exponential curve based on the least squares method (Fig. 9) noting that the study was not specifically designed to quantify this relationship. For example, the mean (±SE) age of females in the N3 stage was 5.6 ± 0.5 d (n = 5) when compared with the P3 stage which has a mean age of 45.8 ± 3.4 d (n = 16). It is important to note that the data used to calculate ages in relation to physiological stages is comparatively small and is highly dependent on temperature and nutrition. However, using these data may provide a rough estimate of the age of an individual based on physiological stage.

Fig. 9.

Age (days) of adult in relation to the different physiological ages. Means followed by the same letter are not statistically different at the P ≤ 0.05.

The physiological observations presented here can provide coarse estimates of the chronological ages of any individual sampled in a field setting. For instance, collection of an abundance of females with N1–N3 status may warrant more aggressive insecticide applications to address the adults before oviposition occurs. Alternatively, an abundance of females with P3 status may indicate a senescing adult population and may also influence applications. Thus, incorporating physiological age assessments into a pest management scheme may allow for more accurate predictions of timely control measures. However, while this study provided females with adequate nutrition and constant temperature, such does not occur in the field. Such variability in environmental conditions can cause differences in reproductive development and hence may impact the determination of physiological age. However, since physiological age, as described herein, is totally dependent on morphological changes in the reproductive system related to egg production such changes in environmental conditions should have minimum effect. The major impact will be correctly determining chronological age based on physiological stage since changes in environmental conditions may hinder or even stop reproductive development, but the individual will still continue to age.

In conclusion, the physiological age-grading system discussed in this paper is based on characteristics of the female reproductive system; characteristics that have been used for other insect species with varying success. These include A. grandis grandis (Grodowitz and Brewer 1987), N. eichhorniae (Grodowitz et al. 1997), Sitophilus oryzae (L.) (Perez-Mendoza 2004), H. pakistanae (Lenz et al. 2007), and Cyrtobagous salviniae Calder and Sands (Eisenberg et al. 2018). This study builds upon research describing the female reproductive system as well as previous attempts to delineate reproductive age of female N. viridula (Kiritani 1963, Banerjee and Chatterjee 1985, Esquivel 2009, Fortes et al. 2011). Changes in the morphology of the female reproductive system through the preoviposition period and the production of large number of eggs and egg masses provides a methodology to characterize reproductive health in both field and laboratory colonies. Morphologies used included differentiation of the ovarioles, absent, partial, or full yolk deposition, eggs present in the oviducts and the quantity and appearance of follicular relics, among others. This includes evidence of past reproductive performance as well as the ability to speculate on future reproductive capabilities ultimately leading to the development of reproductive life tables. Such information can aid in the determination of when to initiate management protocols. In addition, the use of such a system can often be used in conjunction with developmental studies especially in the evaluation of different diets.