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Overwintering survival, starvation resistance, and post-diapause reproductive performance of Nezara viridula (L.) (Hemiptera: Pentatomidae) and its parasitoid Trichopoda giacomellii Blanchard (Diptera: Tachinidae)
Biological Control 30 (2004) 141–148 www.elsevier.com/locate/ybcon
Overwintering survival, starvation resistance, and post-diapause reproductive performance of Nezara viridula (L.) (Hemiptera: Pentatomidae) and its parasitoid Trichopoda giacomellii Blanchard (Diptera: Tachinidae) Marc Coombs* CSIRO Entomology, Long Pocket Laboratories, 120 Meiers Road, Indooroopilly, Queensland 4068, Australia Received 11 December 2002; accepted 8 October 2003
Abstract The interaction between diapause in Nezara viridula and parasitism by the introduced Trichopoda giacomellii was studied at Moree in northern NSW, Australia. N. viridula adults were shown to enter diapause progressively over a 6–8 week period commencing in mid-May. Fifty percent of unparasitised N. viridula males and 60% of females survived the winter period to re-emerge in early August. This population survived in the field until late spring and had an average post-diapause fecundity of 146 eggs per female. No parasitised N. viridula adults survived the winter period. Parasitoid larvae underwent a protracted development with pupariation occurring in mid-winter. Twenty-two percent of parasitised N. viridula adults produced parasitoid puparia. The remaining 78% of N. viridula adults died prior to parasitoid larvae completing development. Host starvation was shown not to influence parasitoid development success. Emergence of T. giacomellii adults from these puparia coincided with the re-appearance of N. viridula adults in the field during early to late spring. Those T. giacomellii present as puparia in the soil during May emerged during mid-winter in asynchrony with N. viridula. Clearly, aspects of the ecological requirements and life-history of T. giacomellii do not match those of its host N. viridula. Implications for the success or otherwise of T. giacomellii as a biological control agent for N. viridula in Australia are discussed. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Diapause; Reproductive performance; Starvation resistance; New associations; Nezara viridula; Parasitism; Trichopoda giacomellii
1. Introduction Nezara viridula (L.) is a cosmopolitan pest of fruit, vegetable, and field crops (Todd, 1989). The native geographic range of N. viridula is thought to include Ethiopia, southern Europe, and the Mediterranean region (Hokkanen, 1986; Jones, 1988). Other species in the genus occur in Africa and Asia (Freeman, 1940). In Australia, N. viridula is a pest of most legume crops, curcubits, potatoes, tomatoes, passion fruit, sorghum, sunflower, tobacco, maize, crucifers, spinach, grapes, citrus, rice, and macadamia nuts (Hely et al., 1982;
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Waterhouse and Norris, 1987). In northern Victoria, central New South Wales, and southern Queensland N. viridula is a serious pest of soybeans and pecans (Clarke, 1992; Coombs, 2000; Seymour and Sands, 1993). In the Americas, tachinids of the genus Trichopoda Berthold attack primarily hemipterous insects of the families Pentatomidae and Coreidae (Arnaud, 1978; Guimaraes, 1977; Liljesthrom, 1980). Two species, Trichopoda pennipes (F.) and Trichopoda pilipes (F.) are important parasitoids of N. viridula in the southern United States and have been successfully established in Hawaii (Davis, 1964) and more recently, though unintentionally, in Italy (Colazza et al., 1996). Both species have an apparently broad host range which also includes species of Coreidae, Scutelleridae, Largidae, Mantidae, and Acrididiae (Arnaud, 1978). Though previous,
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unsuccessful, attempts were made in Australia to establish T. pennipes and T. pilipes (see Waterhouse and Norris, 1987), neither species would now be considered for introduction because of their lack of specificity. Introductions of other agents for control of N. viridula in Australia include several species of egg parasitoids (primarily Scelionidae), of which Trissolcus basalis (Wollaston) has contributed to the control of N. viridula in south-eastern Australia (Waterhouse and Norris, 1987; Waterhouse and Sands, 2001). In certain regions of eastern Australia, particularly those that produce soybeans and nut crops, N. viridula has remained a significant pest (Clarke and Walter, 1993; Seymour and Sands, 1993). Recently, Trichopoda giacomellii Blanchard, a species native to Argentina was established in Australia as a biological control agent for N. viridula (Coombs and Sands, 2000). In Argentina, T. giacomellii has been shown to regulate populations of N. viridula in soybeans in conjunction with T. basalis (Liljesthrom and Bernstein, 1990). T. giacomellii [ ¼ Eutrichopodopsis nitens Blanchard (Liljesthrom, 1992)] is also an important parasitoid of N. viridula in Brazil (Ferreira et al., 1991). Live specimens of T. giacomellii were imported to Australia from La Plata, Argentina (34°520 S, 57°550 W) (Sands and Coombs, 1999). Climate matching (Sutherst and Maywald, 1985) indicated the suitability of eastern mainland Australia for survival of T. giacomellii (D.P.A. Sands, unpublished data). Release and establishment studies for T. giacomellii in Australia were centred on a 1400 acre pecan plantation located at Moree, New South Wales (29°290 S, 149°530 E) (Coombs and Sands, 2000). Since its establishment in Australia, T. giacomellii has had a sustained impact on the abundance of N. viridula, reducing peak abundances to 15–35% of preestablishment densities for the years 1999–2002 (Coombs, 2003; Coombs and Sands, 2000). Although field and laboratory studies have shown T. giacomellii to be well adapted to N. viridula (Coombs, 1997, 2003; Coombs and Khan, 1998; Sands and Coombs, 1999) aspects of the interaction between T. giacomellii and Australian populations of N. viridula remain undetermined. In particular, host diapause and parasitism by T. giacomellii has not been investigated. Data from establishment sites in eastern Australia (Coombs and Sands, 2000) showed that N. viridula adults are subject to parasitism by T. giacomellii prior to entering overwintering sites during May and June. Parasitism of N. viridula by T. giacomellii at this time requires that the developing parasitoid have a suitable mechanism to survive the winter period and subsequently synchronize its emergence with N. viridula the following spring. In Argentina, Liljesthrom (1997) reported that at least part of the T. giacomellii population entered winter as larvae within diapausing N. viridula adults. These larvae will presumably be subject to declining host quality
as the non-feeding N. viridula adult depletes its reservoir of stored lipids. Accumulation of lipids prior to, and depletion during overwintering has been reported for several Pentatomidae, including N. viridula (Banerjee and Chatterjee, 1985; Panizzi and Hirose, 1995). How host starvation affects T. giacomellii development is not known. Liljesthrom (1997) also showed that those T. giacomellii present in the soil as puparia during autumn emerged as adults in midwinter in total asynchrony with N. viridula, indicating that aspects of the life-history of T. giacomellii may be poorly matched with those of N. viridula. N. viridula was first reported in Argentina in 1919 (La Porta and de Crouzel, 1984). Indigenous hosts of T. giacomellii in Argentina include species of the genera Acrosternum, Acledra, and Edesia (Liljesthrom, 1980), whereas parasitism of N. viridula represented a new association (sensu Hokkanen and Pimentel, 1984, 1989). The role of indigenous hosts in maintaining populations of T. giacomellii in Argentina has not been investigated and these species will necessarily be absent in countries where T. giacomellii is being considered for introduction. Understanding the interaction between the overwintering phenology of N. viridula and that of T. giacomellii will be critical in assessing the likely effectiveness of T. giacomellii in countries where it has been newly introduced as a biological control agent. The degree of seasonal synchrony between host and parasitoid is clearly an important determinant of the permanence of host–parasitoid relations and the efficacy of biological control. This study was undertaken to determine the seasonal phenology of diapause and post-diapause reproductive performance of N. viridula, the impact of T. giacomellii parasitism on host (N. viridula) over-wintering survival, and the subsequent phenology of T. giacomellii development. In addition, it aimed to determine what effect reduced host nutrition (starvation) had on the development success of T. giacomellii.
2. Materials and methods 2.1. Study site The study was conducted at the 700 ha. ÔTrawallaÕ pecan orchard located 35 km east of Moree (29°290 S, 149°530 E) New South Wales, Australia. During spring and summer, the orchard understory supports growth of various weeds that are suitable for development of N. viridula including dock (Rumex spp.), marshmallow (Malva parviflora [L.]), and variegated thistle (Silybum marianum [L.]) (Coombs, 2000). Adjacent vegetation is dominated by castor oil (Ricinus communis L.) and various native genera including Eucalyptus, Casuarina, and Callistemon.
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2.2. Insect material Cultures of N. viridula and of T. giacomellii were maintained at the ÔTrawallaÕ pecan orchard at Moree, NSW. Adults of N. viridula were held in mesh screened cages (n ¼ 30–50 per cage) measuring 30 30 30 cm in dimension and provided with green bean pods, segments of corn cob, and shelled raw peanuts as a food source. Bean pods and corn were renewed three times per week and peanuts once per week. Adults of T. giacomellii (n ¼ 10–15 pairs per cage) were held in mesh screened cages (n ¼ 3) measuring 1 1 1.4 m in dimension and provided with raisins, sugar cubes, and moistened cloth pads as a food source. Adult bugs were introduced daily (n ¼ 30–50 per day) to each of the cages holding T. giacomellii adults for parasitism. The exposed bugs were checked routinely (every 1–2 h) during daylight hours and those with 2–3 parasitoid eggs attached to the thorax and abdomen were removed and held in mesh cages as described above. Sex ratio of the exposed bugs was not controlled for, but approximated 1:1. Parasitised bugs were fed as described above to enable larval parasitoid development. T. giacomellii puparia were collected from the floor of the cage and stored in finely sieved moist soil until emergence. All insect material was held at a temperature of 25 1 °C, 40–50% RH, and a 14:10 L:D photoperiod provided by overhead fluorescent (daylight) tubing. Rearing methods used for N. viridula and T. giacomellii in this study followed those previously developed for these species by Coombs (1997), Coombs and Khan (1998), and Sands and Coombs (1999). 2.3. Determining the seasonal phenology of N. viridula diapause Adults were monitored on the host plants R. communis and M. parviflora during autumn and spring, and from diapause sites (beneath the bark of Eucalyptus spp.) during winter to provide profiles of seasonal occurrence. Samples were recorded as either the number of adult bugs per plant (n ¼ 20) (R. communis and Eucalyptus spp.) or bugs per 200 1 m sweeps (M. parviflora). Eucalypt trees were examined from ground level to a height of approximately 2 m by removing loose and flaking bark and examining the undersurface for N. viridula adults. Sweeps were made using a standard insect net with 35 cm hoop diameter. To assess seasonal changes in reproductive activity, the proportion of adults collected as mating pairs was recorded for each sample date. In some instances mating pairs may have been dislodged during sweep net capture and hence numbers recorded may be an underestimation of actual numbers. In addition, a sub-sample of females (n ¼ 20–25) was collected at monthly intervals and dissected under a stereomicroscope. The ovaries were ex-
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amined and females classified as being either reproductive when they contained mature and/or developing oocytes or non-reproductive when ovaries lacked differentiated oocytes. Distension of the spermatheca was considered evidence of insemination. The condition of the fat body was rated following the criteria of Banerjee and Chatterjee (1985). The fat body was determined as being replete, when filling all or nearly all of the abdominal cavity, partial, when the fat body was restricted to approximately half the abdominal cavity around the inside of the body wall, or depleted, when restricted to the dorso-lateral corners of the abdomen. 2.4. Overwintering survival and post-diapause reproductive performance of parasitised and unparasitised N. viridula Adult N. viridula were collected from the field in April (n ¼ 40) and May (n ¼ 140). Those collected during April were exposed to parasitism by T. giacomellii in the laboratory as described above. All were parasitised within 24 h and subsequently held in a single 30 30 30 cm cage inside a mesh screened field cage (2 2 2.5 m in dimension) under ambient conditions of light and temperature. Those collected during May were either parasitised as described above and held as single male/female pairs (n ¼ 33) in 325 ml plastic containers or remained unparasitised and similarly held as single male/female pairs (n ¼ 37) to act as a control. All parasitised and unparasitised pairs were held in the field cage under ambient conditions of light and temperature. All bugs were provided with a diapause site (roll of paper towel) and food (green beans and peanuts) that was replaced weekly. Trichopoda giacomellii puparia (n ¼ 23) derived from parasitised bugs from the April collection date were lightly buried in moist sand to a depth of 10 cm in 325 ml plastic pots and retained in the field cage. In all cases puparia were manually removed from emergence cages prior to burying. Prior laboratory studies have shown that there are no detrimental effects to pupal survival or adult emergence success when allowing T. giacomellii to pupate on the cage floor in the absence of a soil substrate or subsequently when manually buried (Coombs, 1997; Coombs and Khan, 1998). Each plastic pot was checked once weekly for emergence of adult parasitoids. These puparia were used to determine the emergence phenology of T. giacomellii adults that entered the winter period as puparia in the soil. For the parasitised and unparasitised pairs, weekly measures of reproductive performance (number of eggs laid per pair) and survival were recorded. Statistical analysis of paired comparisons (longevity) was made using StudentÕs t test (Zar, 1984). Observations of adult activity (feeding, mating, and sheltering) were noted. Parasitoid development times (days) from oviposition to
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pupariation was recorded for each pair. Allocation of a puparia to either the male or female host of a given pair was based on gender-specific tissue damage to the hostsÕ abdomen. Emergence of final instar T. giacomellii larvae does not necessarily cause host mortality, though death of male hosts occurs sooner than for female hosts (Coombs and Khan, 1998). Puparia were lightly buried in moist sand to a depth of 10 cm in 325 ml plastic pots and retained in the field cage. Each plastic pot was checked once weekly for emergence of adult parasitoids. These puparia were used to determine the emergence phenology of T. giacomellii adults that had entered the winter period as larvae within diapausing N. viridula adults. Daily maximum and minimum temperature (°C) and relative humidity (% RH) were recorded via a weather station located adjacent to the field cage. 2.5. Host starvation and parasitoid development success To assess the effect of host starvation on parasitoid development success, newly parasitised bugs (1–2 weeks of age) were allocated to one of four feeding regimes (n ¼ 15 males and 15 females per treatment). Treatments were food available seven days per week and changed three times per week as per the normal laboratory rearing routine (termed fully fed), food available every third day for 24 h only (limited food), food available every sixth day for 24 h only (very limited food), and fully starved. In those treatments where bugs were fed, food consisted of a single bean pod and one shelled raw peanut. In all four treatments free water was available at all times via a soaked cotton wool roll suspended through the roof of each holding container. Each bug received three parasitoid eggs as per the methods described above. All parasitised adult bugs were held under constant temperature and relative humidity as described for general culture maintenance. Date of pupariation and fresh weight of the puparia (mg) was recorded. Chi-square analysis, ANOVA, and the Tukey test (for multiple pairwise comparisons) were employed to determine the significance of differences in proportions of individuals pupating, mean development times, and puparial weights for the four feeding regimes (Zar, 1984).
3. Results
Fig. 1. (A) Seasonal abundance (no. adults/plant) (line) and proportion (vertical bars) of N. viridula adults collected as mating pairs on weed hosts and (B) numbers of adults present in overwintering sites (beneath the bark of eucalypts) from May to November 1998 at Moree, NSW.
species during May with numbers of adults greatest during June and August (Fig. 1B). Relatively few adults were recovered in diapause sites by mid-autumn (September). Adults were first recorded on weed hosts during the second half of August with numbers peaking in late September. Numbers of adults declined thereafter and were absent by late November. Mating pairs were recovered coincident with adults first appearing on weed hosts in August. The proportion of adults collected as mating pairs was greatest (0.48) during late September, declining thereafter and none were evident by early November. Dissection of female bugs revealed that the proportion of adults reproducing declined rapidly from April through June and none were reproductive during July and August (Table 1). Mated females, as evidenced by distension of the spermatheca, were present during April and September/October only. The condition of the fat body was assessed as being full (replete) during June only.
3.1. Overwintering phenology of N. viridula The numbers of N. viridula adults recovered on weed hosts declined during May and June and none were detected by early July (Fig. 1A). Similarly, the proportion of adults collected as mating pairs declined during May and none were present during June. Overwintering adults were first detected beneath the bark of Eucalyptus
3.2. Overwintering survival and post-diapause reproductive performance of parasitised and non-parasitised N. viridula Both parasitised and unparasitised females continued laying eggs during May and early June. No oviposition occurred during July or August. Adults were not
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Table 1 Reproductive status of N. viridula females during April (mid-autumn) to October (late spring) Month
N
Proportion reproductive
Proportion mated
Fat body
April May June July August September October
22 20 25 25 21 20 22
0.82 0.2 0.08 0 0 0.6 1.0
0.32 0 0 0 0 0.1 0.86
Partial Partial Replete Partial Partial Depleted Depleted
observed to feed and all had assumed a russet colouration by mid-winter (July/August). No parasitised adults survived beyond August. Reproductive activity of unparasitised adults (n ¼ 84) resumed in early September and oviposition was greatest during late October to early November (Fig. 2A). All adults had reverted to their summer green colouration by mid-September. Approximately 60% of unparasitised females and 50% of unparasitised males survived the winter period to commence reproductive activity in mid-August. Fecundity (mean + SD) of post-diapause females averaged 146 82 eggs per female. Mortality of unparasitised
bugs was greatest during late autumn (June) and early spring (October). For both parasitised and unparasitised bugs, mortality of males was more rapid than females (Fig. 2B). Mean (SE) longevity (as weeks after parasitism) of parasitised male bugs (4.3 0.3 weeks) was significantly less (t ¼ 4:05, df ¼ 83, P < 0:001) than for female bugs (6.3 0.4 weeks). However, mean longevity of unparasitised males (13.6 1.2 weeks) did not differ significantly (t ¼ 1:01, df ¼ 83, P > 0:25) from that of unparasitised females (15.4 1.3 weeks). Ambient temperatures (mean SD) were lowest during July with an average daily minimum of 3.9 3.8 °C and average daily maximum of 16.1 3.3 °C. Ambient temperatures (mean SD) were greatest during November with an average daily minimum of 13.3 4.8 °C and average daily maximum of 29.6 6.0 °C. Relative humidity (% RH SD) ranged from 56 18% (November) to 80 7% (July) over the course of the study. 3.3. Phenology of T. giacomellii adult emergence Emergence of T. giacomellii adults commenced in early July and continued until early October (Fig. 3). T. giacomellii present in the soil as puparia during autumn (May/June) emerged as adults in winter (July/August) and early spring (September), whereas those that entered winter as larvae in diapausing N. viridula adults emerged in late winter (August) and early spring (September/ October).
Fig. 2. (A) Pre- and post-diapause oviposition profiles for parasitised (d) and unparasitised (s) N. viridula females and (B) survival curves for parasitised (closed symbols) and unparasitised (open symbols) N. viridula males () and females (s) held in a field cage at Moree, NSW.
Fig. 3. Timing of emergence of T. giacomellii adults that entered winter as either puparia in soil (columns) or as larvae in diapausing N. viridula adults (–d–).
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3.4. Host starvation and parasitoid development success Parasitoid success (as numbers of puparia produced) was independent of feeding regime (X 2 ¼ 5:2, df ¼ 3, P > 0:1). Percent success to pupariation was 76% (n ¼ 23=30) under the fully fed regime; 50% (n ¼ 15=30) for the limited food; 66% (n ¼ 20=30) for the very limited and 57% (n ¼ 17=30) for the starved regime. No differences in percent success to pupariation were detected between male or female hosts. Analysis of variance indicated significant differences in mean development time (F ¼ 8:7, df ¼ 3; 71, P < 0:001) and puparial weight (F ¼ 3:6, df ¼ 3; 71, P < 0:02) between treatments. Pairwise comparisons of means (Tukey test) identified that parasitoid development time (mean SD) was significantly (P < 0:05) less in hosts which were either fully fed (n ¼ 23) (19.1 2.0 days) or those provided with limited food (n ¼ 15) (20.5 2.8 days) than in those hosts subjected to either the very limited (n ¼ 20) (22.7 2.6 days) or the starved (n ¼ 17) (21.7 2.5 days) feeding regimes. Parasitoid puparia derived from fully fed hosts were significantly heavier (P < 0:05) than those of all other treatments. Means (SD) were 480 50 mg (fully fed), 411 95 mg (limited); 420 107 mg (very limited), and 401 81 mg (starved).
4. Discussion The N. viridula population present at Moree, NSW was shown to enter a non-reproductive diapause progressively over a 6–8 week period beginning in early May as evidenced by declining reproductive effort (mating, oviposition, and egg maturation), declining numbers of adults on field hosts and was coincident with the appearance of adults in over-wintering sites. All adults had entered diapause sites by early July, were assessed to be non-reproductive, had accumulated increased amounts of body fat and had assumed a darker (russet) body colouration. No adult feeding was observed during diapause and cumulative adult mortality was 50–60% by the end of the diapause phase in early August. Numbers of adults in over-wintering sites declined from August onwards and was coincident with the re-appearance of adults on field hosts (see Fig. 1). Kiritani (1971) reported on the over-wintering ecology of N. viridula populations in southern Japan, showing adults moved into winter hibernacula over a 12 week period and that overwintering mortality averaged 56%. Recovery of adults, including mating pairs, from the field in August and September during this study indicated the resumption of reproductive activity. This was confirmed by renewed oviposition from adults which had survived the winter while held within a field cage (see Fig. 2A). This post-diapause population was indi-
cated to persist in the field until early November confirming prior light trap and host-plant studies which had identified a post-diapause population present from August until late October or early November (Coombs, 2000). Post-diapause adults were shown to have an average fecundity of 146 eggs per female with oviposition peaking during late October to early November. Prior estimates of oviposition for N. viridula (based on laboratory studies) indicated an average lifetime fecundity of 908 eggs per female (range 311–1811) (Coombs and Khan, 1998). No prior studies have examined survival or subsequent reproductive performance of an overwintering N. viridula population in Australia, though the identity of overwintering sites and numbers of N. viridula adults present had previously been determined (Coombs, 2000). Numbers of N. viridula adults surviving the winter period and their subsequent reproductive capacity is likely to have a significant bearing on the rate at which damaging populations develop in subsequent generations. As N. viridula adults are able to feed on a wide variety of non-crop hosts at this time (Coombs, 2000; Velasco et al., 1995) it is the progeny of the postdiapause population that are responsible for increasing pest pressure in a variety of crops. Prior studies (Coombs, 2003; Coombs and Sands, 2000) demonstrated that N. viridula is subject to parasitism by T. giacomellii during autumn and that 40–50% of adults enter diapause parasitised. Results from the present study demonstrated that such adults are subject to high mortality rates with none indicated to survive beyond early August (see Fig. 2B). Parasitism success for T. giacomellii was shown to be low, with only 22% of parasitised N. viridula adults producing a parasitoid puparia. The remaining 78% of parasitised adults died prematurely, suggesting significant parasitoid induced mortality. In contrast, 55–60% of unparasitised adults survived beyond the date at which all parasitised bugs had died (see Fig. 2B). The low parasitism success rate observed in the study contrasts with those observed in laboratory studies, where up to 85% of parasitised adults typically produce parasitoid puparia (Coombs and Khan, 1998). Further, a proportion (5–20%) of N. viridula adults is normally able to survive the immediate effects of parasitism and live from 6 to 10 weeks beyond emergence of the mature parasitoid, though no eggs are produced. Results from the starvation experiment suggest that lack of feeding by N. viridula adults following parasitism does not significantly affect parasitoid development success as 55–60% of parasitised N. viridula adults produced puparia when maintained on either a restricted diet or were fully starved. Clearly, added stresses are imposed when parasitised N. viridula adults are exposed to the lower temperatures experienced during ambient winter conditions resulting in significant numbers of adults dying prior to the parasitoid being able to complete development. Liljesthrom (1996) cal-
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culated mortality thresholds for T. giacomellii pupae indicating zero survival at temperatures of 5 °C and below. Though average daily minimum air temperatures fell below this level during July (3.9 °C) and August (4.3 °C) soil temperatures were not recorded, but presumably did not reach these minima given the emergence of T. giacomellii adults during July. The effects of low temperatures on the interaction between larval parasitoid development and host survival and has not been determined for T. giacomellii and N. viridula. Results from the field cage study indicated that the majority (70%) of T. giacomellii present in the soil as puparia during autumn (April/May) emerged during mid-winter (July) when the N. viridula population was entirely confined to overwintering sites (compare Figs. 2 and 3). The remaining puparia (30%) emerged during spring coincident with the appearance of N. viridula. Those T. giacomellii present as larvae within parasitised N. viridula completed a protracted development to subsequently emerge during early spring coincident with the re-appearance of N. viridula. Obviously, no N. viridula adults would be available for parasitism during winter months and that portion of the T. giacomellii population emerging as adults would necessarily die without making any reproductive contribution to the next generation. These results confirm the findings of Liljesthrom (1997) who demonstrated a similar asynchrony in the appearance of T. giacomellii with respect to the seasonal availability of N. viridula in Argentina. Beard (1940) also noted that part of the adult autumn generation of T. pennipes emerged after its host (Anasa tristis DeGeer) population had entered hibernation. Beard (1940) identified this attribute as a significant deficiency in the ability of T. pennipes to limit A. tristis abundance. Winter emergence of T. giacomellii adults suggests that one or more indigenous hosts for T. giacomellii are seasonally available for parasitism at this time of year in its native range. The role of indigenous hosts, Acrosternum, Acledra, and Edesia (Liljesthrom, 1980) in maintaining populations of T. giacomellii in Argentina has not been determined and presumably one or more of these alternate hosts remains active throughout winter. The known alternative field hosts for T. giacomellii in Australia, Plautia affinis Dallas and Glaucias amyoti (White), also diapause during winter (Coombs, 2003). In southern Brazil, Panizzi and Oliveira (1999) reported that N. viridula adults remain active (feeding and reproducing) during June to August with parasitism by T. giacomellii at this time ranging from 85 to 100%. This suggests that the T. giacomellii/N. viridula association may be better suited to warmer climates. Despite the failure of T. giacomellii to closely synchronize its seasonal development with N. viridula, postrelease studies have shown that T. giacomellii has been able to establish sustainable populations at release sites in northern New South Wales with summer parasitism
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rates of 60–70% being achieved during each of four consecutive years (Coombs, 2003; Coombs and Sands, 2000). Clearly, that portion of the T. giacomellii population emerging during spring in synchrony with N. viridula is sufficient to bring about the observed level of control. Beard (1940) argued that the impact of T. pennipes on A. tristis would be significantly greater had its seasonal synchrony been more closely aligned. Presumably, T. giacomellii would similarly be able to exert greater impact on N. viridula populations with closer seasonal synchrony. Though predictions of habitat range for T. giacomellii based on climatic modeling (Sutherst and Maywald, 1985) indicated the suitability of much of eastern Australia for T. giacomellii (D.P.A. Sands, unpublished data) the model makes no assumption about its interaction with N. viridula. The effectiveness of T. giacomellii as a control agent for N. viridula may be limited in more temperate climates where the adverse (cool) season is longer or more severe. Conversely, T. giacomellii may be better suited to the lower latitudes where N. viridula is reported to remain active throughout the year (Panizzi and Oliveira, 1999; Waterhouse and Norris, 1987). The relationship between T. giacomellii and N. viridula, both in Argentina and Australia, represents a new species association as defined by Hokkanen and Pimentel (1984, 1989) who postulated that new associations avoid the predator/ prey stability typical of old (evolved) associations and are thereby more effective as biological control agents. In Argentina, studies by La Porta (1990), Liljesthrom (1997), and Liljesthrom and Bernstein (1990) have demonstrated the effectiveness of T. giacomellii as a biological control agent for N. viridula. Evidence to date suggests that this success will be repeated in Australia (Coombs, 2003; Coombs and Sands, 2000).
Acknowledgments This study was supported by funds from the Horticulture Australia Limited (HAL), the Australian Centre for International Agricultural Research (ACIAR), and Stahmann Farms Inc. The generous support provided by Deanne Stahmann and Michael Crouch and the technical assistance of Shama Khan, Colleen Crouch, and Kerry McNamara are gratefully acknowledged.
References Arnaud, P.H., 1978. A host-parasite catalogue of North American Tachinidae (Diptera). U.S. Department of Agriculture, Miscellaneous Publication 1319. Banerjee, T.C., Chatterjee, M., 1985. Seasonal changes in feeding, fat body and ovarian conditions of Nezara viridula (Hemiptera: Pentatomidae). Insect Sci. Appl. 6, 633–635.
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Beard, R.L., 1940. The biology of Anasa tristis DeGeer with particular reference to the tachinid parasite, Trichopoda pennipes Fabr. Conn. Agric. Exp. Sta. Bull. 440, 597–679. Clarke, A.R., 1992. Current distribution and pest status of Nezara viridula (L.) (Hemiptera: Pentatomidae) in Australia. J. Aust. Entomol. Soc. 31, 289–297. Clarke, A.R., Walter, G.H., 1993. Biological control of green vegetable bug (Nezara viridula (L.)) in eastern Australia: current status and perspectives. In: Corey, S.A., Dall, D.J., Milne, W.M. (Eds.), Pest Control and Sustainable Agriculture. CSIRO, Australia, pp. 223– 225. Colazza, S., Gianagiuliani, G., Bin, F., 1996. Fortuitous introduction and successful establishment of Trichopoda pennipes F., adult parasitoid of Nezara viridula (L.). Biol. Control 6, 409–411. Coombs, M., 1997. Influence of adult food deprivation and body size on fecundity and longevity of Trichopoda giacomellii: a South American parasitoid of Nezara viridula. Biol. Control 8, 119–123. Coombs, M., 2000. Seasonal phenology, natural enemies and evaluation of mowing as a control measure for Nezara viridula (L.) (Hemiptera: Pentatomidae) in Australian pecans. Environ. Entomol. 29, 1027–1033. Coombs, M., 2003. Post-release evaluation of Trichopoda giacomellii (Diptera: Tachinidae) for efficacy and non-target effects. In: Van Driesche, R.G. (Ed.), Proceedings of 1st International Symposium on Biological Control of Arthropods. USDA Forest Service, FHTET-03-05, pp. 399–406. Coombs, M., Khan, S.A., 1998. Fecundity and longevity of green vegetable bug, Nezara viridula following parasitism by Trichopoda giacomellii. Biol. Control 12, 215–222. Coombs, M., Sands, D.P.A., 2000. Establishment in Australia of Trichopoda giacomellii Blanchard (Diptera: Tachinidae) a biological control agent for Nezara viridula (L.) (Hemiptera: Pentatomidae). Aust. J. Entomol. 39, 219–222. Davis, C.J., 1964. The introduction, propagation, liberation and establishment of parasites to control Nezara viridula variety smaragdula (Fabricius) in Hawaii (Heteroptera: Pentatomidae). Proc. Hawaiian Entomol. Soc. 18, 369–375. Ferreira, B.S.C., Thomazina, M.J., Zamataro, C.E., 1991. Effect of parasitism by Eutrichopodopsis nitens Blanchard on the longevity and reproduction of Nezara viridula (L.). Pesqu. Agropecu. Bras. 16, 837–842. Freeman, P., 1940. A contribution to the study of the genus Nezara Amyot and Serville (Hemiptera: Pentatomidae). Trans. R. Entomol. Soc. Lond. 90, 351–374. Guimaraes, J.H., 1977. Host-parasite and parasite-host catalogue of South American Tachinidae (Diptera). Arq. Zool. 28, 1–131. Hely, P.C., Pasfield, G., Gellatley, G.G., 1982. Insect Pests of Fruit and Vegetables in NSW. Inkata Press, Melbourne. Hokkanen, H., 1986. Polymorphism, parasites and the native area of Nezara viridula (L.) (Hemiptera: Pentatomidae). Ann. Entomol. Fenn. 52, 28–31. Hokkanen, H., Pimentel, D., 1984. New approach for selecting biological control agents. Can. Entomol. 8, 1109–1121. Hokkanen, H., Pimentel, D., 1989. New associations in biological control agent: theory and practice. Can. Entomol. 10, 829–840. Jones, W.A., 1988. World review of the parasitoids of the southern stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae). Ann. Entomol. Soc. Am. 81, 262–273.
Kiritani, K., 1971. Distribution and abundance of the southern green stink bug, Nezara viridula.. Trop. Agric. Res. Ser. 5, 235–248. La Porta, N.C., 1990. Evaluation of field parasitism by Trichopoda giacomellii (Blanch.) Guimaraes, 1971 (Diptera, Tachinidae), on Nezara viridula (L.) 1758. (Hemiptera: Pentatomidae). Rev. Chil. Entomol. 18, 83–87. La Porta, N.C., de Crouzel, I.S., 1984. Estudios basicos para el control biologico de Nezara viridula (L., 1758) (Hemiptera, Pentatomidae) en la Argentina. Rev. Soc. Entomol. Argentina 43, 119–143. Liljesthrom, G., 1980. Nota sobre Trichopoda giacomellii (Blanchard 1966) (Diptera: Tachinidae). Neotropica 26, 233–236. Liljesthrom, G., 1992. Revision de las especies de los generos Trichopoda Berthold, Trichopodopsis Townsend y Eutrichopodopsis Blanchard, descriptas para La Republica Argentina. Rev. Soc. Entomol. Argentina 50, 51–71. Liljesthrom, G., 1996. Estimation of threshold temperature and heat unit accumulation required for pupal development of Trichopoda giacomellii (Diptera: Tachinidae). Acta Entomol. Chil. 20, 19–22. Liljesthrom, G., 1997. Persistence of Trichopoda giacomellii (Diptera: Tachinidae) during the hibernating period of the host, Nezara viridula (Hemiptera: Pentatomidae) in the northeast of Buenos Aires province. Rev. Soc. Entomol. Argentina 56, 133–136. Liljesthrom, G., Bernstein, C., 1990. Density dependence and regulation in the system Nezara viridula (L.) (Hemiptera: Pentatomidae), host and Trichopoda giacomellii (Blanchard) (Diptera: Tachinidae), parasitoid. Oecologia 84, 45–52. Panizzi, A.R., Hirose, E., 1995. Seasonal body weight, lipid content, and impact of starvation and water stress on adult survivorship and longevity of Nezara viridula and Euschistus heros. Entomol. Exp. Appl. 76, 247–253. Panizzi, A.R., Oliveira, E.D.M., 1999. Seasonal occurrence of tachinid parasitism on stink bugs with different overwintering strategies. Ann. Soc. Entomol. Bras. 28, 169–172. Sands, D.P.A., Coombs, M., 1999. Evaluation of the Argentinian parasitoid, Trichopoda giacomellii (Diptera: Tachinidae), for biological control of Nezara viridula (Hemiptera: Pentatomidae) in Australia. Biol. Control 15, 19–24. Seymour, J.E., Sands, D.P.A., 1993. Green vegetable bug (Nezara viridula (L.)) (Hemiptera: Pentatomidae) in Australia pecans. In: Corey, S.A., Dall, D.J., Milne, W.M. (Eds.), Pest Control and Sustainable Agriculture. CSIRO, Australia, pp. 226–228. Sutherst, R.W., Maywald, G.F., 1985. A computerised system for matching climates in ecology. Agric. Ecosyst. Environ. 13, 281– 299. Todd, J.W., 1989. Ecology and behaviour of Nezara viridula. Annu. Rev. Entomol. 34, 273–292. Velasco, L.R.I., Walter, G.H., Harris, V.E., 1995. Voltinism and host plant use by Nezara viridula (L.) (Hemiptera: Pentatomidae) in Southeastern Queensland. J. Aust. Entomol. Soc. 34, 193–203. Waterhouse, D.F., Norris, K.R., 1987. Biological Control: Pacific Prospects. Inkata Press, Melbourne. Waterhouse, D.F., Sands, D.P.A., 2001. Classical Biological Control of Arthropods in Australia. Australian Center for International Agricultural Research Monograph No. 77, 560 pp. Zar, J.H., 1984. Biostatistical Analysis, second ed. Prentice-Hall International, Engelwood Cliffs, NJ.
Overwintering survival, starvation resistance, and post-diapause reproductive performance of Nezara viridula (L.) (Hemiptera: Pentatomidae) and its parasitoid Trichopoda giacomellii Blanchard (Diptera: Tachinidae) Marc Coombs* CSIRO Entomology, Long Pocket Laboratories, 120 Meiers Road, Indooroopilly, Queensland 4068, Australia Received 11 December 2002; accepted 8 October 2003
Abstract The interaction between diapause in Nezara viridula and parasitism by the introduced Trichopoda giacomellii was studied at Moree in northern NSW, Australia. N. viridula adults were shown to enter diapause progressively over a 6–8 week period commencing in mid-May. Fifty percent of unparasitised N. viridula males and 60% of females survived the winter period to re-emerge in early August. This population survived in the field until late spring and had an average post-diapause fecundity of 146 eggs per female. No parasitised N. viridula adults survived the winter period. Parasitoid larvae underwent a protracted development with pupariation occurring in mid-winter. Twenty-two percent of parasitised N. viridula adults produced parasitoid puparia. The remaining 78% of N. viridula adults died prior to parasitoid larvae completing development. Host starvation was shown not to influence parasitoid development success. Emergence of T. giacomellii adults from these puparia coincided with the re-appearance of N. viridula adults in the field during early to late spring. Those T. giacomellii present as puparia in the soil during May emerged during mid-winter in asynchrony with N. viridula. Clearly, aspects of the ecological requirements and life-history of T. giacomellii do not match those of its host N. viridula. Implications for the success or otherwise of T. giacomellii as a biological control agent for N. viridula in Australia are discussed. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Diapause; Reproductive performance; Starvation resistance; New associations; Nezara viridula; Parasitism; Trichopoda giacomellii
1. Introduction Nezara viridula (L.) is a cosmopolitan pest of fruit, vegetable, and field crops (Todd, 1989). The native geographic range of N. viridula is thought to include Ethiopia, southern Europe, and the Mediterranean region (Hokkanen, 1986; Jones, 1988). Other species in the genus occur in Africa and Asia (Freeman, 1940). In Australia, N. viridula is a pest of most legume crops, curcubits, potatoes, tomatoes, passion fruit, sorghum, sunflower, tobacco, maize, crucifers, spinach, grapes, citrus, rice, and macadamia nuts (Hely et al., 1982;
* Fax: +61-7-3214-2885. E-mail address: [email protected].
1049-9644/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2003.10.001
Waterhouse and Norris, 1987). In northern Victoria, central New South Wales, and southern Queensland N. viridula is a serious pest of soybeans and pecans (Clarke, 1992; Coombs, 2000; Seymour and Sands, 1993). In the Americas, tachinids of the genus Trichopoda Berthold attack primarily hemipterous insects of the families Pentatomidae and Coreidae (Arnaud, 1978; Guimaraes, 1977; Liljesthrom, 1980). Two species, Trichopoda pennipes (F.) and Trichopoda pilipes (F.) are important parasitoids of N. viridula in the southern United States and have been successfully established in Hawaii (Davis, 1964) and more recently, though unintentionally, in Italy (Colazza et al., 1996). Both species have an apparently broad host range which also includes species of Coreidae, Scutelleridae, Largidae, Mantidae, and Acrididiae (Arnaud, 1978). Though previous,
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unsuccessful, attempts were made in Australia to establish T. pennipes and T. pilipes (see Waterhouse and Norris, 1987), neither species would now be considered for introduction because of their lack of specificity. Introductions of other agents for control of N. viridula in Australia include several species of egg parasitoids (primarily Scelionidae), of which Trissolcus basalis (Wollaston) has contributed to the control of N. viridula in south-eastern Australia (Waterhouse and Norris, 1987; Waterhouse and Sands, 2001). In certain regions of eastern Australia, particularly those that produce soybeans and nut crops, N. viridula has remained a significant pest (Clarke and Walter, 1993; Seymour and Sands, 1993). Recently, Trichopoda giacomellii Blanchard, a species native to Argentina was established in Australia as a biological control agent for N. viridula (Coombs and Sands, 2000). In Argentina, T. giacomellii has been shown to regulate populations of N. viridula in soybeans in conjunction with T. basalis (Liljesthrom and Bernstein, 1990). T. giacomellii [ ¼ Eutrichopodopsis nitens Blanchard (Liljesthrom, 1992)] is also an important parasitoid of N. viridula in Brazil (Ferreira et al., 1991). Live specimens of T. giacomellii were imported to Australia from La Plata, Argentina (34°520 S, 57°550 W) (Sands and Coombs, 1999). Climate matching (Sutherst and Maywald, 1985) indicated the suitability of eastern mainland Australia for survival of T. giacomellii (D.P.A. Sands, unpublished data). Release and establishment studies for T. giacomellii in Australia were centred on a 1400 acre pecan plantation located at Moree, New South Wales (29°290 S, 149°530 E) (Coombs and Sands, 2000). Since its establishment in Australia, T. giacomellii has had a sustained impact on the abundance of N. viridula, reducing peak abundances to 15–35% of preestablishment densities for the years 1999–2002 (Coombs, 2003; Coombs and Sands, 2000). Although field and laboratory studies have shown T. giacomellii to be well adapted to N. viridula (Coombs, 1997, 2003; Coombs and Khan, 1998; Sands and Coombs, 1999) aspects of the interaction between T. giacomellii and Australian populations of N. viridula remain undetermined. In particular, host diapause and parasitism by T. giacomellii has not been investigated. Data from establishment sites in eastern Australia (Coombs and Sands, 2000) showed that N. viridula adults are subject to parasitism by T. giacomellii prior to entering overwintering sites during May and June. Parasitism of N. viridula by T. giacomellii at this time requires that the developing parasitoid have a suitable mechanism to survive the winter period and subsequently synchronize its emergence with N. viridula the following spring. In Argentina, Liljesthrom (1997) reported that at least part of the T. giacomellii population entered winter as larvae within diapausing N. viridula adults. These larvae will presumably be subject to declining host quality
as the non-feeding N. viridula adult depletes its reservoir of stored lipids. Accumulation of lipids prior to, and depletion during overwintering has been reported for several Pentatomidae, including N. viridula (Banerjee and Chatterjee, 1985; Panizzi and Hirose, 1995). How host starvation affects T. giacomellii development is not known. Liljesthrom (1997) also showed that those T. giacomellii present in the soil as puparia during autumn emerged as adults in midwinter in total asynchrony with N. viridula, indicating that aspects of the life-history of T. giacomellii may be poorly matched with those of N. viridula. N. viridula was first reported in Argentina in 1919 (La Porta and de Crouzel, 1984). Indigenous hosts of T. giacomellii in Argentina include species of the genera Acrosternum, Acledra, and Edesia (Liljesthrom, 1980), whereas parasitism of N. viridula represented a new association (sensu Hokkanen and Pimentel, 1984, 1989). The role of indigenous hosts in maintaining populations of T. giacomellii in Argentina has not been investigated and these species will necessarily be absent in countries where T. giacomellii is being considered for introduction. Understanding the interaction between the overwintering phenology of N. viridula and that of T. giacomellii will be critical in assessing the likely effectiveness of T. giacomellii in countries where it has been newly introduced as a biological control agent. The degree of seasonal synchrony between host and parasitoid is clearly an important determinant of the permanence of host–parasitoid relations and the efficacy of biological control. This study was undertaken to determine the seasonal phenology of diapause and post-diapause reproductive performance of N. viridula, the impact of T. giacomellii parasitism on host (N. viridula) over-wintering survival, and the subsequent phenology of T. giacomellii development. In addition, it aimed to determine what effect reduced host nutrition (starvation) had on the development success of T. giacomellii.
2. Materials and methods 2.1. Study site The study was conducted at the 700 ha. ÔTrawallaÕ pecan orchard located 35 km east of Moree (29°290 S, 149°530 E) New South Wales, Australia. During spring and summer, the orchard understory supports growth of various weeds that are suitable for development of N. viridula including dock (Rumex spp.), marshmallow (Malva parviflora [L.]), and variegated thistle (Silybum marianum [L.]) (Coombs, 2000). Adjacent vegetation is dominated by castor oil (Ricinus communis L.) and various native genera including Eucalyptus, Casuarina, and Callistemon.
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2.2. Insect material Cultures of N. viridula and of T. giacomellii were maintained at the ÔTrawallaÕ pecan orchard at Moree, NSW. Adults of N. viridula were held in mesh screened cages (n ¼ 30–50 per cage) measuring 30 30 30 cm in dimension and provided with green bean pods, segments of corn cob, and shelled raw peanuts as a food source. Bean pods and corn were renewed three times per week and peanuts once per week. Adults of T. giacomellii (n ¼ 10–15 pairs per cage) were held in mesh screened cages (n ¼ 3) measuring 1 1 1.4 m in dimension and provided with raisins, sugar cubes, and moistened cloth pads as a food source. Adult bugs were introduced daily (n ¼ 30–50 per day) to each of the cages holding T. giacomellii adults for parasitism. The exposed bugs were checked routinely (every 1–2 h) during daylight hours and those with 2–3 parasitoid eggs attached to the thorax and abdomen were removed and held in mesh cages as described above. Sex ratio of the exposed bugs was not controlled for, but approximated 1:1. Parasitised bugs were fed as described above to enable larval parasitoid development. T. giacomellii puparia were collected from the floor of the cage and stored in finely sieved moist soil until emergence. All insect material was held at a temperature of 25 1 °C, 40–50% RH, and a 14:10 L:D photoperiod provided by overhead fluorescent (daylight) tubing. Rearing methods used for N. viridula and T. giacomellii in this study followed those previously developed for these species by Coombs (1997), Coombs and Khan (1998), and Sands and Coombs (1999). 2.3. Determining the seasonal phenology of N. viridula diapause Adults were monitored on the host plants R. communis and M. parviflora during autumn and spring, and from diapause sites (beneath the bark of Eucalyptus spp.) during winter to provide profiles of seasonal occurrence. Samples were recorded as either the number of adult bugs per plant (n ¼ 20) (R. communis and Eucalyptus spp.) or bugs per 200 1 m sweeps (M. parviflora). Eucalypt trees were examined from ground level to a height of approximately 2 m by removing loose and flaking bark and examining the undersurface for N. viridula adults. Sweeps were made using a standard insect net with 35 cm hoop diameter. To assess seasonal changes in reproductive activity, the proportion of adults collected as mating pairs was recorded for each sample date. In some instances mating pairs may have been dislodged during sweep net capture and hence numbers recorded may be an underestimation of actual numbers. In addition, a sub-sample of females (n ¼ 20–25) was collected at monthly intervals and dissected under a stereomicroscope. The ovaries were ex-
143
amined and females classified as being either reproductive when they contained mature and/or developing oocytes or non-reproductive when ovaries lacked differentiated oocytes. Distension of the spermatheca was considered evidence of insemination. The condition of the fat body was rated following the criteria of Banerjee and Chatterjee (1985). The fat body was determined as being replete, when filling all or nearly all of the abdominal cavity, partial, when the fat body was restricted to approximately half the abdominal cavity around the inside of the body wall, or depleted, when restricted to the dorso-lateral corners of the abdomen. 2.4. Overwintering survival and post-diapause reproductive performance of parasitised and unparasitised N. viridula Adult N. viridula were collected from the field in April (n ¼ 40) and May (n ¼ 140). Those collected during April were exposed to parasitism by T. giacomellii in the laboratory as described above. All were parasitised within 24 h and subsequently held in a single 30 30 30 cm cage inside a mesh screened field cage (2 2 2.5 m in dimension) under ambient conditions of light and temperature. Those collected during May were either parasitised as described above and held as single male/female pairs (n ¼ 33) in 325 ml plastic containers or remained unparasitised and similarly held as single male/female pairs (n ¼ 37) to act as a control. All parasitised and unparasitised pairs were held in the field cage under ambient conditions of light and temperature. All bugs were provided with a diapause site (roll of paper towel) and food (green beans and peanuts) that was replaced weekly. Trichopoda giacomellii puparia (n ¼ 23) derived from parasitised bugs from the April collection date were lightly buried in moist sand to a depth of 10 cm in 325 ml plastic pots and retained in the field cage. In all cases puparia were manually removed from emergence cages prior to burying. Prior laboratory studies have shown that there are no detrimental effects to pupal survival or adult emergence success when allowing T. giacomellii to pupate on the cage floor in the absence of a soil substrate or subsequently when manually buried (Coombs, 1997; Coombs and Khan, 1998). Each plastic pot was checked once weekly for emergence of adult parasitoids. These puparia were used to determine the emergence phenology of T. giacomellii adults that entered the winter period as puparia in the soil. For the parasitised and unparasitised pairs, weekly measures of reproductive performance (number of eggs laid per pair) and survival were recorded. Statistical analysis of paired comparisons (longevity) was made using StudentÕs t test (Zar, 1984). Observations of adult activity (feeding, mating, and sheltering) were noted. Parasitoid development times (days) from oviposition to
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pupariation was recorded for each pair. Allocation of a puparia to either the male or female host of a given pair was based on gender-specific tissue damage to the hostsÕ abdomen. Emergence of final instar T. giacomellii larvae does not necessarily cause host mortality, though death of male hosts occurs sooner than for female hosts (Coombs and Khan, 1998). Puparia were lightly buried in moist sand to a depth of 10 cm in 325 ml plastic pots and retained in the field cage. Each plastic pot was checked once weekly for emergence of adult parasitoids. These puparia were used to determine the emergence phenology of T. giacomellii adults that had entered the winter period as larvae within diapausing N. viridula adults. Daily maximum and minimum temperature (°C) and relative humidity (% RH) were recorded via a weather station located adjacent to the field cage. 2.5. Host starvation and parasitoid development success To assess the effect of host starvation on parasitoid development success, newly parasitised bugs (1–2 weeks of age) were allocated to one of four feeding regimes (n ¼ 15 males and 15 females per treatment). Treatments were food available seven days per week and changed three times per week as per the normal laboratory rearing routine (termed fully fed), food available every third day for 24 h only (limited food), food available every sixth day for 24 h only (very limited food), and fully starved. In those treatments where bugs were fed, food consisted of a single bean pod and one shelled raw peanut. In all four treatments free water was available at all times via a soaked cotton wool roll suspended through the roof of each holding container. Each bug received three parasitoid eggs as per the methods described above. All parasitised adult bugs were held under constant temperature and relative humidity as described for general culture maintenance. Date of pupariation and fresh weight of the puparia (mg) was recorded. Chi-square analysis, ANOVA, and the Tukey test (for multiple pairwise comparisons) were employed to determine the significance of differences in proportions of individuals pupating, mean development times, and puparial weights for the four feeding regimes (Zar, 1984).
3. Results
Fig. 1. (A) Seasonal abundance (no. adults/plant) (line) and proportion (vertical bars) of N. viridula adults collected as mating pairs on weed hosts and (B) numbers of adults present in overwintering sites (beneath the bark of eucalypts) from May to November 1998 at Moree, NSW.
species during May with numbers of adults greatest during June and August (Fig. 1B). Relatively few adults were recovered in diapause sites by mid-autumn (September). Adults were first recorded on weed hosts during the second half of August with numbers peaking in late September. Numbers of adults declined thereafter and were absent by late November. Mating pairs were recovered coincident with adults first appearing on weed hosts in August. The proportion of adults collected as mating pairs was greatest (0.48) during late September, declining thereafter and none were evident by early November. Dissection of female bugs revealed that the proportion of adults reproducing declined rapidly from April through June and none were reproductive during July and August (Table 1). Mated females, as evidenced by distension of the spermatheca, were present during April and September/October only. The condition of the fat body was assessed as being full (replete) during June only.
3.1. Overwintering phenology of N. viridula The numbers of N. viridula adults recovered on weed hosts declined during May and June and none were detected by early July (Fig. 1A). Similarly, the proportion of adults collected as mating pairs declined during May and none were present during June. Overwintering adults were first detected beneath the bark of Eucalyptus
3.2. Overwintering survival and post-diapause reproductive performance of parasitised and non-parasitised N. viridula Both parasitised and unparasitised females continued laying eggs during May and early June. No oviposition occurred during July or August. Adults were not
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Table 1 Reproductive status of N. viridula females during April (mid-autumn) to October (late spring) Month
N
Proportion reproductive
Proportion mated
Fat body
April May June July August September October
22 20 25 25 21 20 22
0.82 0.2 0.08 0 0 0.6 1.0
0.32 0 0 0 0 0.1 0.86
Partial Partial Replete Partial Partial Depleted Depleted
observed to feed and all had assumed a russet colouration by mid-winter (July/August). No parasitised adults survived beyond August. Reproductive activity of unparasitised adults (n ¼ 84) resumed in early September and oviposition was greatest during late October to early November (Fig. 2A). All adults had reverted to their summer green colouration by mid-September. Approximately 60% of unparasitised females and 50% of unparasitised males survived the winter period to commence reproductive activity in mid-August. Fecundity (mean + SD) of post-diapause females averaged 146 82 eggs per female. Mortality of unparasitised
bugs was greatest during late autumn (June) and early spring (October). For both parasitised and unparasitised bugs, mortality of males was more rapid than females (Fig. 2B). Mean (SE) longevity (as weeks after parasitism) of parasitised male bugs (4.3 0.3 weeks) was significantly less (t ¼ 4:05, df ¼ 83, P < 0:001) than for female bugs (6.3 0.4 weeks). However, mean longevity of unparasitised males (13.6 1.2 weeks) did not differ significantly (t ¼ 1:01, df ¼ 83, P > 0:25) from that of unparasitised females (15.4 1.3 weeks). Ambient temperatures (mean SD) were lowest during July with an average daily minimum of 3.9 3.8 °C and average daily maximum of 16.1 3.3 °C. Ambient temperatures (mean SD) were greatest during November with an average daily minimum of 13.3 4.8 °C and average daily maximum of 29.6 6.0 °C. Relative humidity (% RH SD) ranged from 56 18% (November) to 80 7% (July) over the course of the study. 3.3. Phenology of T. giacomellii adult emergence Emergence of T. giacomellii adults commenced in early July and continued until early October (Fig. 3). T. giacomellii present in the soil as puparia during autumn (May/June) emerged as adults in winter (July/August) and early spring (September), whereas those that entered winter as larvae in diapausing N. viridula adults emerged in late winter (August) and early spring (September/ October).
Fig. 2. (A) Pre- and post-diapause oviposition profiles for parasitised (d) and unparasitised (s) N. viridula females and (B) survival curves for parasitised (closed symbols) and unparasitised (open symbols) N. viridula males () and females (s) held in a field cage at Moree, NSW.
Fig. 3. Timing of emergence of T. giacomellii adults that entered winter as either puparia in soil (columns) or as larvae in diapausing N. viridula adults (–d–).
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3.4. Host starvation and parasitoid development success Parasitoid success (as numbers of puparia produced) was independent of feeding regime (X 2 ¼ 5:2, df ¼ 3, P > 0:1). Percent success to pupariation was 76% (n ¼ 23=30) under the fully fed regime; 50% (n ¼ 15=30) for the limited food; 66% (n ¼ 20=30) for the very limited and 57% (n ¼ 17=30) for the starved regime. No differences in percent success to pupariation were detected between male or female hosts. Analysis of variance indicated significant differences in mean development time (F ¼ 8:7, df ¼ 3; 71, P < 0:001) and puparial weight (F ¼ 3:6, df ¼ 3; 71, P < 0:02) between treatments. Pairwise comparisons of means (Tukey test) identified that parasitoid development time (mean SD) was significantly (P < 0:05) less in hosts which were either fully fed (n ¼ 23) (19.1 2.0 days) or those provided with limited food (n ¼ 15) (20.5 2.8 days) than in those hosts subjected to either the very limited (n ¼ 20) (22.7 2.6 days) or the starved (n ¼ 17) (21.7 2.5 days) feeding regimes. Parasitoid puparia derived from fully fed hosts were significantly heavier (P < 0:05) than those of all other treatments. Means (SD) were 480 50 mg (fully fed), 411 95 mg (limited); 420 107 mg (very limited), and 401 81 mg (starved).
4. Discussion The N. viridula population present at Moree, NSW was shown to enter a non-reproductive diapause progressively over a 6–8 week period beginning in early May as evidenced by declining reproductive effort (mating, oviposition, and egg maturation), declining numbers of adults on field hosts and was coincident with the appearance of adults in over-wintering sites. All adults had entered diapause sites by early July, were assessed to be non-reproductive, had accumulated increased amounts of body fat and had assumed a darker (russet) body colouration. No adult feeding was observed during diapause and cumulative adult mortality was 50–60% by the end of the diapause phase in early August. Numbers of adults in over-wintering sites declined from August onwards and was coincident with the re-appearance of adults on field hosts (see Fig. 1). Kiritani (1971) reported on the over-wintering ecology of N. viridula populations in southern Japan, showing adults moved into winter hibernacula over a 12 week period and that overwintering mortality averaged 56%. Recovery of adults, including mating pairs, from the field in August and September during this study indicated the resumption of reproductive activity. This was confirmed by renewed oviposition from adults which had survived the winter while held within a field cage (see Fig. 2A). This post-diapause population was indi-
cated to persist in the field until early November confirming prior light trap and host-plant studies which had identified a post-diapause population present from August until late October or early November (Coombs, 2000). Post-diapause adults were shown to have an average fecundity of 146 eggs per female with oviposition peaking during late October to early November. Prior estimates of oviposition for N. viridula (based on laboratory studies) indicated an average lifetime fecundity of 908 eggs per female (range 311–1811) (Coombs and Khan, 1998). No prior studies have examined survival or subsequent reproductive performance of an overwintering N. viridula population in Australia, though the identity of overwintering sites and numbers of N. viridula adults present had previously been determined (Coombs, 2000). Numbers of N. viridula adults surviving the winter period and their subsequent reproductive capacity is likely to have a significant bearing on the rate at which damaging populations develop in subsequent generations. As N. viridula adults are able to feed on a wide variety of non-crop hosts at this time (Coombs, 2000; Velasco et al., 1995) it is the progeny of the postdiapause population that are responsible for increasing pest pressure in a variety of crops. Prior studies (Coombs, 2003; Coombs and Sands, 2000) demonstrated that N. viridula is subject to parasitism by T. giacomellii during autumn and that 40–50% of adults enter diapause parasitised. Results from the present study demonstrated that such adults are subject to high mortality rates with none indicated to survive beyond early August (see Fig. 2B). Parasitism success for T. giacomellii was shown to be low, with only 22% of parasitised N. viridula adults producing a parasitoid puparia. The remaining 78% of parasitised adults died prematurely, suggesting significant parasitoid induced mortality. In contrast, 55–60% of unparasitised adults survived beyond the date at which all parasitised bugs had died (see Fig. 2B). The low parasitism success rate observed in the study contrasts with those observed in laboratory studies, where up to 85% of parasitised adults typically produce parasitoid puparia (Coombs and Khan, 1998). Further, a proportion (5–20%) of N. viridula adults is normally able to survive the immediate effects of parasitism and live from 6 to 10 weeks beyond emergence of the mature parasitoid, though no eggs are produced. Results from the starvation experiment suggest that lack of feeding by N. viridula adults following parasitism does not significantly affect parasitoid development success as 55–60% of parasitised N. viridula adults produced puparia when maintained on either a restricted diet or were fully starved. Clearly, added stresses are imposed when parasitised N. viridula adults are exposed to the lower temperatures experienced during ambient winter conditions resulting in significant numbers of adults dying prior to the parasitoid being able to complete development. Liljesthrom (1996) cal-
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culated mortality thresholds for T. giacomellii pupae indicating zero survival at temperatures of 5 °C and below. Though average daily minimum air temperatures fell below this level during July (3.9 °C) and August (4.3 °C) soil temperatures were not recorded, but presumably did not reach these minima given the emergence of T. giacomellii adults during July. The effects of low temperatures on the interaction between larval parasitoid development and host survival and has not been determined for T. giacomellii and N. viridula. Results from the field cage study indicated that the majority (70%) of T. giacomellii present in the soil as puparia during autumn (April/May) emerged during mid-winter (July) when the N. viridula population was entirely confined to overwintering sites (compare Figs. 2 and 3). The remaining puparia (30%) emerged during spring coincident with the appearance of N. viridula. Those T. giacomellii present as larvae within parasitised N. viridula completed a protracted development to subsequently emerge during early spring coincident with the re-appearance of N. viridula. Obviously, no N. viridula adults would be available for parasitism during winter months and that portion of the T. giacomellii population emerging as adults would necessarily die without making any reproductive contribution to the next generation. These results confirm the findings of Liljesthrom (1997) who demonstrated a similar asynchrony in the appearance of T. giacomellii with respect to the seasonal availability of N. viridula in Argentina. Beard (1940) also noted that part of the adult autumn generation of T. pennipes emerged after its host (Anasa tristis DeGeer) population had entered hibernation. Beard (1940) identified this attribute as a significant deficiency in the ability of T. pennipes to limit A. tristis abundance. Winter emergence of T. giacomellii adults suggests that one or more indigenous hosts for T. giacomellii are seasonally available for parasitism at this time of year in its native range. The role of indigenous hosts, Acrosternum, Acledra, and Edesia (Liljesthrom, 1980) in maintaining populations of T. giacomellii in Argentina has not been determined and presumably one or more of these alternate hosts remains active throughout winter. The known alternative field hosts for T. giacomellii in Australia, Plautia affinis Dallas and Glaucias amyoti (White), also diapause during winter (Coombs, 2003). In southern Brazil, Panizzi and Oliveira (1999) reported that N. viridula adults remain active (feeding and reproducing) during June to August with parasitism by T. giacomellii at this time ranging from 85 to 100%. This suggests that the T. giacomellii/N. viridula association may be better suited to warmer climates. Despite the failure of T. giacomellii to closely synchronize its seasonal development with N. viridula, postrelease studies have shown that T. giacomellii has been able to establish sustainable populations at release sites in northern New South Wales with summer parasitism
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rates of 60–70% being achieved during each of four consecutive years (Coombs, 2003; Coombs and Sands, 2000). Clearly, that portion of the T. giacomellii population emerging during spring in synchrony with N. viridula is sufficient to bring about the observed level of control. Beard (1940) argued that the impact of T. pennipes on A. tristis would be significantly greater had its seasonal synchrony been more closely aligned. Presumably, T. giacomellii would similarly be able to exert greater impact on N. viridula populations with closer seasonal synchrony. Though predictions of habitat range for T. giacomellii based on climatic modeling (Sutherst and Maywald, 1985) indicated the suitability of much of eastern Australia for T. giacomellii (D.P.A. Sands, unpublished data) the model makes no assumption about its interaction with N. viridula. The effectiveness of T. giacomellii as a control agent for N. viridula may be limited in more temperate climates where the adverse (cool) season is longer or more severe. Conversely, T. giacomellii may be better suited to the lower latitudes where N. viridula is reported to remain active throughout the year (Panizzi and Oliveira, 1999; Waterhouse and Norris, 1987). The relationship between T. giacomellii and N. viridula, both in Argentina and Australia, represents a new species association as defined by Hokkanen and Pimentel (1984, 1989) who postulated that new associations avoid the predator/ prey stability typical of old (evolved) associations and are thereby more effective as biological control agents. In Argentina, studies by La Porta (1990), Liljesthrom (1997), and Liljesthrom and Bernstein (1990) have demonstrated the effectiveness of T. giacomellii as a biological control agent for N. viridula. Evidence to date suggests that this success will be repeated in Australia (Coombs, 2003; Coombs and Sands, 2000).
Acknowledgments This study was supported by funds from the Horticulture Australia Limited (HAL), the Australian Centre for International Agricultural Research (ACIAR), and Stahmann Farms Inc. The generous support provided by Deanne Stahmann and Michael Crouch and the technical assistance of Shama Khan, Colleen Crouch, and Kerry McNamara are gratefully acknowledged.
References Arnaud, P.H., 1978. A host-parasite catalogue of North American Tachinidae (Diptera). U.S. Department of Agriculture, Miscellaneous Publication 1319. Banerjee, T.C., Chatterjee, M., 1985. Seasonal changes in feeding, fat body and ovarian conditions of Nezara viridula (Hemiptera: Pentatomidae). Insect Sci. Appl. 6, 633–635.
148
M. Coombs / Biological Control 30 (2004) 141–148
Beard, R.L., 1940. The biology of Anasa tristis DeGeer with particular reference to the tachinid parasite, Trichopoda pennipes Fabr. Conn. Agric. Exp. Sta. Bull. 440, 597–679. Clarke, A.R., 1992. Current distribution and pest status of Nezara viridula (L.) (Hemiptera: Pentatomidae) in Australia. J. Aust. Entomol. Soc. 31, 289–297. Clarke, A.R., Walter, G.H., 1993. Biological control of green vegetable bug (Nezara viridula (L.)) in eastern Australia: current status and perspectives. In: Corey, S.A., Dall, D.J., Milne, W.M. (Eds.), Pest Control and Sustainable Agriculture. CSIRO, Australia, pp. 223– 225. Colazza, S., Gianagiuliani, G., Bin, F., 1996. Fortuitous introduction and successful establishment of Trichopoda pennipes F., adult parasitoid of Nezara viridula (L.). Biol. Control 6, 409–411. Coombs, M., 1997. Influence of adult food deprivation and body size on fecundity and longevity of Trichopoda giacomellii: a South American parasitoid of Nezara viridula. Biol. Control 8, 119–123. Coombs, M., 2000. Seasonal phenology, natural enemies and evaluation of mowing as a control measure for Nezara viridula (L.) (Hemiptera: Pentatomidae) in Australian pecans. Environ. Entomol. 29, 1027–1033. Coombs, M., 2003. Post-release evaluation of Trichopoda giacomellii (Diptera: Tachinidae) for efficacy and non-target effects. In: Van Driesche, R.G. (Ed.), Proceedings of 1st International Symposium on Biological Control of Arthropods. USDA Forest Service, FHTET-03-05, pp. 399–406. Coombs, M., Khan, S.A., 1998. Fecundity and longevity of green vegetable bug, Nezara viridula following parasitism by Trichopoda giacomellii. Biol. Control 12, 215–222. Coombs, M., Sands, D.P.A., 2000. Establishment in Australia of Trichopoda giacomellii Blanchard (Diptera: Tachinidae) a biological control agent for Nezara viridula (L.) (Hemiptera: Pentatomidae). Aust. J. Entomol. 39, 219–222. Davis, C.J., 1964. The introduction, propagation, liberation and establishment of parasites to control Nezara viridula variety smaragdula (Fabricius) in Hawaii (Heteroptera: Pentatomidae). Proc. Hawaiian Entomol. Soc. 18, 369–375. Ferreira, B.S.C., Thomazina, M.J., Zamataro, C.E., 1991. Effect of parasitism by Eutrichopodopsis nitens Blanchard on the longevity and reproduction of Nezara viridula (L.). Pesqu. Agropecu. Bras. 16, 837–842. Freeman, P., 1940. A contribution to the study of the genus Nezara Amyot and Serville (Hemiptera: Pentatomidae). Trans. R. Entomol. Soc. Lond. 90, 351–374. Guimaraes, J.H., 1977. Host-parasite and parasite-host catalogue of South American Tachinidae (Diptera). Arq. Zool. 28, 1–131. Hely, P.C., Pasfield, G., Gellatley, G.G., 1982. Insect Pests of Fruit and Vegetables in NSW. Inkata Press, Melbourne. Hokkanen, H., 1986. Polymorphism, parasites and the native area of Nezara viridula (L.) (Hemiptera: Pentatomidae). Ann. Entomol. Fenn. 52, 28–31. Hokkanen, H., Pimentel, D., 1984. New approach for selecting biological control agents. Can. Entomol. 8, 1109–1121. Hokkanen, H., Pimentel, D., 1989. New associations in biological control agent: theory and practice. Can. Entomol. 10, 829–840. Jones, W.A., 1988. World review of the parasitoids of the southern stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae). Ann. Entomol. Soc. Am. 81, 262–273.
Kiritani, K., 1971. Distribution and abundance of the southern green stink bug, Nezara viridula.. Trop. Agric. Res. Ser. 5, 235–248. La Porta, N.C., 1990. Evaluation of field parasitism by Trichopoda giacomellii (Blanch.) Guimaraes, 1971 (Diptera, Tachinidae), on Nezara viridula (L.) 1758. (Hemiptera: Pentatomidae). Rev. Chil. Entomol. 18, 83–87. La Porta, N.C., de Crouzel, I.S., 1984. Estudios basicos para el control biologico de Nezara viridula (L., 1758) (Hemiptera, Pentatomidae) en la Argentina. Rev. Soc. Entomol. Argentina 43, 119–143. Liljesthrom, G., 1980. Nota sobre Trichopoda giacomellii (Blanchard 1966) (Diptera: Tachinidae). Neotropica 26, 233–236. Liljesthrom, G., 1992. Revision de las especies de los generos Trichopoda Berthold, Trichopodopsis Townsend y Eutrichopodopsis Blanchard, descriptas para La Republica Argentina. Rev. Soc. Entomol. Argentina 50, 51–71. Liljesthrom, G., 1996. Estimation of threshold temperature and heat unit accumulation required for pupal development of Trichopoda giacomellii (Diptera: Tachinidae). Acta Entomol. Chil. 20, 19–22. Liljesthrom, G., 1997. Persistence of Trichopoda giacomellii (Diptera: Tachinidae) during the hibernating period of the host, Nezara viridula (Hemiptera: Pentatomidae) in the northeast of Buenos Aires province. Rev. Soc. Entomol. Argentina 56, 133–136. Liljesthrom, G., Bernstein, C., 1990. Density dependence and regulation in the system Nezara viridula (L.) (Hemiptera: Pentatomidae), host and Trichopoda giacomellii (Blanchard) (Diptera: Tachinidae), parasitoid. Oecologia 84, 45–52. Panizzi, A.R., Hirose, E., 1995. Seasonal body weight, lipid content, and impact of starvation and water stress on adult survivorship and longevity of Nezara viridula and Euschistus heros. Entomol. Exp. Appl. 76, 247–253. Panizzi, A.R., Oliveira, E.D.M., 1999. Seasonal occurrence of tachinid parasitism on stink bugs with different overwintering strategies. Ann. Soc. Entomol. Bras. 28, 169–172. Sands, D.P.A., Coombs, M., 1999. Evaluation of the Argentinian parasitoid, Trichopoda giacomellii (Diptera: Tachinidae), for biological control of Nezara viridula (Hemiptera: Pentatomidae) in Australia. Biol. Control 15, 19–24. Seymour, J.E., Sands, D.P.A., 1993. Green vegetable bug (Nezara viridula (L.)) (Hemiptera: Pentatomidae) in Australia pecans. In: Corey, S.A., Dall, D.J., Milne, W.M. (Eds.), Pest Control and Sustainable Agriculture. CSIRO, Australia, pp. 226–228. Sutherst, R.W., Maywald, G.F., 1985. A computerised system for matching climates in ecology. Agric. Ecosyst. Environ. 13, 281– 299. Todd, J.W., 1989. Ecology and behaviour of Nezara viridula. Annu. Rev. Entomol. 34, 273–292. Velasco, L.R.I., Walter, G.H., Harris, V.E., 1995. Voltinism and host plant use by Nezara viridula (L.) (Hemiptera: Pentatomidae) in Southeastern Queensland. J. Aust. Entomol. Soc. 34, 193–203. Waterhouse, D.F., Norris, K.R., 1987. Biological Control: Pacific Prospects. Inkata Press, Melbourne. Waterhouse, D.F., Sands, D.P.A., 2001. Classical Biological Control of Arthropods in Australia. Australian Center for International Agricultural Research Monograph No. 77, 560 pp. Zar, J.H., 1984. Biostatistical Analysis, second ed. Prentice-Hall International, Engelwood Cliffs, NJ.