Age-dependence of Lygus hesperus diapause characters

D.W. Spurgeon USDA-ARS, Western Integrated Cropping systems Research Unit, Shafter, CA

Dale.Spurgeon@ars.usda.gov 661-746-8001

Background and Justification The western tarnished plant bug (Lygus hesperus) is a key pest of cotton and other crops in the San Joaquin Valley. Currently, most management efforts in cotton involve the use of conventional pesticides. Development of alternative, ecologically-based management strategies will require improved understanding of lygus ecology. Of particular interest is overwintering ecology and how this aspect of lygus biology influences their seasonal movement among a wide array of cultivated and non-cultivated hosts. If the dynamics of lygus overwintering ecology are known, ecologically-based tactics, such as use of the naturally occurring fungal pathogen, Beauveria bassiana, might be designed to reduce lygus populations in non-cropping habitats or non-susceptible crop hosts. These population reductions would then reduce the numbers of lygus to subsequently migrate to cotton and other susceptible crop hosts. A key aspect of lygus overwintering ecology that is poorly known is the adult diapause. It is widely accepted that Lygus hesperus spends at least a portion of the non-cropping winter season in an adult diapause. Adult diapause is normally characterized by a period of reduced metabolic activity and reproductive development, and extended host-free longevity. These characteristics of diapause enhance survival of adult lygus during periods of unfavorable environmental conditions. Although photoperiod is known to induce the adult diapause in L. hesperus (Beards and Strong 1966, Leigh 1966, Strong et al. 1970), some inconsistencies exist in reports of these photoperiodic effects. Striking among the various reports are the inconsistencies in the morphological characters used to distinguish diapause, and the ranges of ages at which diapause was assessed. There is a need for a standardized suite of diapause-identifying characters, and for knowledge of the temporal patterns of development of those characters. This information would facilitate the meaningful comparison of results from different experiments and investigators, and would simplify the interpretation of experimental data. Objectives The objective of the project was to define a character set to reliably distinguish between the physiological states of diapause and reproduction, and to determine the most appropriate adult age at which these physiological states should be assessed. Procedures Eggs were obtained by exposing pods of green bean overnight to sexually mature adult lygus in our laboratory colony. Bean pods containing eggs were held in environmental chambers at 80°F. Half of the beans were held under a day length of 14 hours, whereas the other half were held under 10-hour days. On the day of egg hatch, nymphs were transferred individually to labeled plastic vials provided with a short section of green bean pod. The nymphs in vials were returned to their respective environmental chamber for development to adults. Green bean sections were replaced three times weekly, and the date on which each nymph became an adult was recorded.

Each group of eggs resulted in a cohort of 50–100 nymphs. The experiment was repeated three times, resulting in three cohorts of nymphs at each day length. Adults were dissected at ages of 3, 7, 10, 14, and 17 days. At the time of dissection, detailed notes were recorded to describe the amount, color, and configuration of fat bodies, the extent of ovary development in females, and testis color, seminal vesicle condition, and size and condition of medial and lateral accessory glands in males. The characters evaluated were selected based on the results of preliminary studies (Spurgeon 2009). Fat body quantities were classified as sparse (Fig. 1a), abundant (Fig. 1b), or hypertrophied (Fig. 1c,d). Fat body colors were recorded as aqua, light aqua, or white, and the presence of multiple color classifications within an individual bug was possible. Fat body configurations were recorded as sheet like (Fig. 1a), globular (Fig. 1b), columnar adjacent to the abdominal mid-line (Fig. 1c,d), and the various combinations of these characters. Male testes were recorded as having a fatty sheath (T, Fig. 2c) or not (T, Fig. 2a), and male seminal vesicles (SV, Fig. 2b), which are the sperm storage organs, were recorded as being empty, filling, filled, or distended. Male lateral accessory glands were recorded as being small and undeveloped (LAG, Fig. 2c) or large with opaque bases and tips (LAG, Fig. 2a,b). Characteristics of the basal and distal (tip) sections of the medial accessory glands were recorded separately. Tips of the medial accessory glands were recorded as being empty (MAG, Fig. 2c), or as having colored contents (MAG, Fig. 2a,b). Bases of the medial accessory glands were recorded as being empty, having some colored material (Fig. 2c), filled (Fig. 2a) or distended (Fig. 2b). Female ovaries were classified according to their contents. The ovaries were classified as undeveloped (Fig. 2d), or as containing oocytes with yolk (OOC, Fig. 2e), eggs (EGG, Fig. 2e), or follicular relics (RL, Fig. 2f). The follicular relics are an indication of previous oviposition regardless of the current contents of the ovaries. Fat body characteristics (amount, locations, and colors) were examined for associations using contingency tables. Similar analyses were conducted to examine the data for relationships between the conditions of different male reproductive organs (testes, seminal vesicles, and medial and lateral accessory glands), between the conditions of male accessory glands and fat body quantities, and between female ovary classes and fat body quantities. Finally, the proportions of bugs exhibiting the characters of diapause (males, hypertrophied fat bodies, no development of medial accessory glands; females, hypertrophied fat bodies, no ovary development) were calculated for each combination of photoperiod and age of dissection. Results Analyses of associations among the fat body characteristics indicated that fat body color and configuration were strongly associated with fat body quantity. In general, aqua fat bodies were associated with sparse fat bodies. White fat bodies were most often associated with hypertrophy. However, more than one fat body color was often observed within a single individual. Fat bodies classified as abundant often contained both white and light aqua fat. While these

associations were strong, numerous exceptions were observed, including individual bugs with all three fat body colors. Associations between fat body quantity and configurations exhibited the same types of relationship as the association between fat body quantity and color. The presence of a fatty sheath on the testes was associated with hypertrophied and abundant fat bodies. However, this condition was merely a reflection of the extent and type of fat present, and was not indicative of the extent of development of other male sexual organs. Condition of the seminal vesicles was closely related to bug age, but was not related to fat body development. The conditions of the medial and lateral accessory glands of males were closely related; when one type of gland was undeveloped, the other gland type was also undeveloped. Male accessory gland development was also related to fat body quantity. Undeveloped accessory glands were associated with hypertrophied fat bodies, and fully developed accessory glands were generally associated with the less developed fat bodies. Partially developed accessory glands were observed for all fat body quantity classes. Ovary development was also closely related to fat body quantity as the presence of undeveloped ovaries generally occurred in conjunction with fat body hypertrophy. As in the case of the male accessory glands, conditions of partial or complete ovary development were observed in association with all fat body quantity classes. When the proportions of bugs exhibiting characters of diapause were tabulated, no bugs of either sex were classified as diapausing under the 14-h photoperiod. For males reared under the 10-h photoperiod, the incidence of diapause increased from 17% at 3 days to 45 and 41% at 7 and 10 days, respectively. The proportion of males in diapause subsequently decreased to 17% by day 17. The estimated proportions of females in diapause were more stable with age, ranging from 40% at day 17 to 52% at day 7. Discussion Based on associational analyses and observations of apparent fat body maturity, the most appropriate criteron for use in assessing male diapause is a combination of fat body and medial accessory gland development. The reason medial accessory gland condition is preferred over lateral gland development is because the colored contents of developed medial glands make them easier to rate objectively. We propose that diapausing male lygus be identified by the presence of hypertrophied fat bodies and an absence of medial accessory gland development. The most appropriate diapause criterion for females is a combination of fat body and ovary development. We propose that diapausing females be identified by the presence of hypertrophied fat bodies and an absence of ovary development. The most appropriate age for assessment of the occurrence of diapause, under the temperature conditions we used, appears to be 10 days. Fat bodies do not appear to be completely developed before this time, and the sharp decrease in male diapause observed at 14 and 17 days (based on partially developed accessory glands) suggests some bugs are terminating the diapause by that time under the conditions of our study. Our assessments of the frequency of occurrence of diapause contrast starkly with previous reports (Beards and Strong 1966, Leigh 1966, Strong et al. 1970). These earlier investigators

reported that all lygus enter the diapause state under 10-h days. However, those previous reports also included a classification of “intermediate diapause” that allowed some reproductive development, and there is no biological justification for such a classification. Although the incidence of diapause that we observed was much lower than those previously reported, conclusions regarding the influence of photoperiod on diapause occurrence should be deferred until we confirm that colony-reared insects respond to photoperiod in a manner similar to that of field-collected insects. An experiment to compare these two populations is currently underway. The observations gathered during this study should make assessment of diapause in lygus more standardized among investigators. Such standardization will allow for meaningful comparisons of results of studies conducted in different geographical regions. Our findings should also pave the way for more sophisticated studies of the genetics and endocrinology of diapause in lygus, and should contribute to a more comprehensive understanding of lygus overwintering ecology. References Beards, G.W., and F. E. Strong. 1966. Photoperiod in relation to diapause in Lygus hesperus Knight. Hilgardia 37: 345-362. Leigh, T.F. 1966. A reproductive diapause in Lygus hesperus Knight. J. Econ. Entomol. 59: 1280-1281. Spurgeon, D. W. 2009. Observations of diapause characters in the western tarnished plant bug, Lygus hesperus. pp. 807-812. In Proc. Beltwide Cotton Conf., National Cotton Council, Memphis, TN. Strong, F.E., J.A. Sheldahl, P.R. Hughs, and E.M.K. Hussein. 1970. Reproductive biology of Lygus hesperus Knight. Hilgardia 40: 105-147.

Fig. 1. Fat bodies of Lygus hesperus adults: a) 7-d-old reproductive male, b) 3-d-old reproductive female, c) 3-d-old diapausing female, d) 10-d-old diapausing male.

Fig. 2. Development of reproductive organs in adult L. hesperus: a) 3-d-old reproductive male, b) 7-d-old reproductive male, c) 10-d-old diapausing male, d) 3-d-old diapausing female, e) 3-d-old reproductive female, f) 10-d-old reproductive female. Labels are: T, testes; LAG, lateral accessory glands; MAG, medial accessory glands; SV, seminal vesicle; OV, ovary; OOC, oocyte; EGG, chorionated egg; RL, follicular relic.