Temperature-dependent development of Anagyrus pseudococci (Hymenoptera: Encyrtidae) as a parasitoid of the vine mealybug, Planococcus ficus (Homoptera: Pseudococcidae)

Biological Control 31 (2004) 123–132 www.elsevier.com/locate/ybcon

Temperature-dependent development of Anagyrus pseudococci (Hymenoptera: Encyrtidae) as a parasitoid of the vine mealybug, Planococcus Wcus (Homoptera: Pseudococcidae) Kent M. Daane,¤ Raksha D. Malakar-Kuenen, and Vaughn M. Walton1 Center for Biological Control, Division of Insect Biology (ESPM), University of California, Berkeley, CA 94720, USA Received 31 October 2003; accepted 19 April 2004 Available online 21 July 2004

Abstract The inXuence of temperature on Anagyrus pseudococci (Girault) development and overwintering was investigated to improve biological control of the vine mealybug, Planococcus Wcus (Signoret), in California vineyards. At a constant 32 °C, egg development required 2 days, larval development ranged from 0.7 § 0.1 (second instar) to 1.9 § 0.1 (Wfth instar) days, and pupal development was 3.9 § 0.1 days. Under eight constant temperatures (12, 14, 17, 22, 27, 32, 34, and 36 °C), A. pseudococci completed development (egg to adult eclosion) from 14 to 34 °C. Development times ranged from 79.1 § 1.0 days (14 °C) to 10.2 § 0.3 days (34 °C). We determined optimal, maximum and minimum development temperatures to be 24.7, 36.0 and 11.6 °C, respectively, and the thermal constant is 223.5 degree-days. We compared these laboratory-derived temperature relationships to A. pseudococci Weld-monitored populations from March through November. Laboratory-data suggests there are seven to eight A. pseudococci generations during this period, two generations to each vine mealybug generation. Overwintering studies show that A. pseudococci emergence was concentrated over a 15 day period in early May, regardless of when vine mealybugs were exposed (October 2001 to March 2002). Results suggest that cues other than temperature are used to synchronize overwintered A. pseudococci adult emergence with Weld availability of vine mealybug.  2004 Published by Elsevier Inc. Keywords: Anagyrus pseudococci; Pseudococcus Wcus; Vine mealybug; Grape; Temperature-dependent development; Degree day; Biological control

1. Introduction Anagyrus pseudococci (Girault) (Hymenoptera: Encrytidae) is well-known as a parasitoid of the citrus mealybug, Planococcus citri (Risso) (Noyes and Hayat, 1994). A polyphagous parasitoid, it also attacks distantly related species such as Pseudococcus comstocki (Kuwana), Phenacoccus herreni Cox and Williams, Dysmicoccus brevipes (Cockerell), and Maconellicoccus hirsutus Green (Noyes and Hayat, 1994). Due in part to its wide host and geographic range, A. pseudococci has been often used for biological control of pseudococcids. In ¤

Corresponding author. Fax: 1-559-646-6593. E-mail address: [email protected] (K.M. Daane). 1 Present address: Department of Entomology and Nematology, University of Stellenbosch, 7599, South Africa. 1049-9644/$ - see front matter  2004 Published by Elsevier Inc. doi:10.1016/j.biocontrol.2004.04.010

California, A. pseudococci was Wrst introduced from South America in 1934 to control the citrus mealybug (Compere, 1939); currently, it is being used to suppress a new invasive pest, the vine mealybug, Planococcus Wcus (Signoret) (Homoptera: Pseudococcidae) (Daane et al., 2004). The vine mealybug was accidentally introduced into California’s Coachella Valley, a southern table grape region, in the early 1990s (Gill, 1994), and soon spread throughout California’s grape-growing regions (Daane et al., 2004). Similar to other vineyard mealybug species, the vine mealybug excretes honeydew, which acts as a substrate for sooty mold, and infests grape bunches, both of which lower crop quality (Geiger and Daane, 2001; Godfrey et al., 2002). More unusual is that uncontrolled vine mealybug populations can result in defoliation and, with repeated annual infestations, vine death

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(personal observation). Vine mealybug also vectors viral diseases (Engelbrecht and Kasdorf, 1990) and, therefore, can be an economic pest even at low densities. Insecticide programs include applications of organophosphates combined with either or both a systemic chloronicotinyl and an insect growth regulator. Costs for this program can be prohibitively expensive for some grape producers. Moreover, insecticide controls for mealybugs are often incomplete and can disrupt natural enemies (Meyerdirk et al., 1982; Walton and Pringle, 1999). Biological control programs have been quite successful against other mealybug pest species (Dean et al., 1979; Meyerdirk et al., 1988; Neuenschwander and Herren, 1988) and we are studying biological control approaches for use in combination with more selective insecticides, or as a sustainable replacement for insecticides targeting the vine mealybug. Anagyrus pseudococci was the most commonly reared parasitoid from vine mealybug in California vineyards (Malakar-Kuenen et al., 2001), and has been reported as a vine mealybug parasitoid in Israel (Berlinger, 1977), Italy (Duso, 1989), Argentina (Trjapitzin and Trjapitzin, 2002), and South Africa (Walton, 2003). Moreover, A. pseudococci is being commercially produced and released for vine mealybug control, with little information available on A. pseudococci eYcacy against this pest. While A. pseudococci has been well-studied as a parasitoid of the citrus mealybug (Avidov et al., 1967; Islam and Copland, 1997, 2000; Islam and Jahan, 1993a,b; Rosen and Rössler, 1966; Tingle and Copland, 1988a,b, 1989), there are no comparable studies with the vine mealybug. And, while the vine and citrus mealybugs are morphologically similar (Blumberg et al., 1995), Cox (1989) showed A. pseudococci had variable parasitism on diVerent host species. Therefore, we conducted a series of studies with A. pseudococci reared on vine mealybug to improve eVectiveness of biological control in California vineyards, with the objectives of this study to determine the impact of temperature on development and overwintering.

ally in gelatin capsules. Emerged adults were sexed and 25 pairs of male and female A. pseudococci were placed in parasitoid rearing containers, which consisted of sleeve cages (45 £ 45 £ 45 cm) provisioned with four infested squash, each with 400–800 immature vine mealybug, and dilute honey (2:1 honey and water). Adult parasitoids were harvested after 4–6 weeks. We reintroduced Weld-collected A. pseudococci to the colony two to three times per year to reduce inbreeding. Individuals used in the described experiments were from the F412 generations. Subsequent mealybug and parasitoid generations were derived from these cultures. All colonies were held at 22 § 2 °C, with 12:12 (L:D) photoperiod. The insectary is located at the Kearney Agricultural Center, near Parlier, CA. 2.2. Larval development

2. Materials and methods

We determined the development time of A. pseudococci immature stages at 32 § 1 °C, with a 12:12 (L:D) photoperiod. Sprouted potatoes, Solanum tuberosum L., were each inoculated with »200 third instar mealybugs and then held at 25 § 2 °C for 2 days to provide time for the mealybugs to settle and begin feeding. The inoculated potatoes were placed in an oviposition cage, which was a plastic container (2.2 L) with two screened windows (3 cm in diameter) on its lid for ventilation. Approximately, 25 pairs of female and male A. pseudococci were introduced for a 4 h oviposition period and the squash was then moved to the 32 °C temperature cabinet. Thereafter, 10 parasitized mealybugs were removed every 24 h and dissected to determine the A. pseudococci development stage. To select parasitized mealybugs during the initial days after oviposition, when the mealybugs were still mobile and did not show outward signs of parasitism, we searched for the small (00.5 mm) black scar tissue that is a residual mark from the oviposition event. Selected mealybugs were dissected by opening the internal cavity with a horizontal slit near each oviposition scar. The numbers of oviposition scars and A. pseudococci eggs (or egg casings) were recorded and the larval stage was determined, using descriptions provided by Rosen and Rössler (1966).

2.1. Insect and plant materials

2.3. Temperature-dependent development

Laboratory cultures of vine mealybug and A. pseudococci were derived from Weld-collected material in vineyards near Del Rey, California. Vine mealybugs were reared on butternut squash, Cucurbita moschata L., which was cleaned in a 0.5% bleach solution to reduce mold growth, and then triple rinsed. Each squash was inoculated with 5–10 gravid female mealybugs, which resulted in an initial infestation level of 600–1000 mealybugs. For the A. pseudococci culture, mummiWed vine mealybugs were Weld-collected and then placed individu-

The eVect of constant rearing temperatures on A. pseudococci development time was tested at 12, 14, 17, 22, 27, 32, 34, and 36 °C. Sprouting potatoes were each inoculated with »100 second instar to pre-ovipositional adult mealybugs. After inoculation, mealybugs were provided a 2 day period to settle and begin feeding. The infested potatoes were placed into oviposition cages, as described previously. Mated female A. pseudococci were added for a 4 h oviposition period, removed, and the infested potatoes were randomly assigned to temperature

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treatments. After mealybugs began to mummify and could be removed from the potato without disrupting parasitoid survival, we isolated individuals in gelatin capsules to accurately record development time from egg to adult emergence. For mid-range temperatures (22, 27, and 32 °C), we also recorded parasitoid gender and the mealybug development stage. We used Wve temperature cabinets, which maintained temperatures (T) at T § 1 °C, with a 16:8 (L:D) photoperiod. Temperature cabinets were randomly assigned to each treatment, with four replicates (a single potato) for the 22, 27, 32, 34, and 36 °C treatments, which were run sequentially, and eight replicates for the 12, 14, and 17 °C treatments, which were run in two concurrent trials of three and Wve replicates each. 2.4. Temperature-dependent adult egg deposition Initial observations indicated that A. pseudococci larval development may occur at lower temperatures than adult egg deposition. For this reason, we tested for the low temperature threshold for adult activity, as determined by the parasitoid’s ability to oviposit at 12, 14, 16, and 18 °C (T § 1 °C). Squash were inoculated with 20–30 mealybugs (third instar to pre-ovipositional stages), which were clustered under an inverted vial (25 dram) that was held in place with modeling clay. Concurrently, adult female A. pseudococci were collected from the insectary colony and placed in glass vials (25 dram, 5 per vial). While still separated, the inoculated squash and parasitoids were randomly assigned to one of the four temperature treatments. Host and parasitoids remained separate for 1 day, which provided time for both to acclimatize to treatment temperatures. After which, Wve female A. pseudococci were added to each group of isolated mealybugs, with the transfer occurring inside the temperature cabinet. After 6 h, the parasitoids were removed and the mealybugs were held at 25 § 2 °C for 4 weeks, after which the number of parasitized mealybugs was recorded. There were Wve squash (replicates) per treatment, with temperature cabinets randomly assigned to each treatment and temperatures maintained at T § 1 °C, with a 16:8 (L:D) photoperiod. 2.5. Seasonal parasitoid egg deposition To compare laboratory results of A. pseudococci development to Weld populations, we followed A. pseudococci activity in a San Joaquin Valley vineyard, located near Del Rey, CA. Every 2–4 weeks, during the 2001 and 2002 growing seasons, 10 vines were randomly selected and searched for mealybugs at development stages susceptible to A. pseudococci. The collected mealybugs (range 40–305 mealybugs per collection) were placed individually in gelatin capsules and held at 25 § 2 °C. After 2 months, percentage parasitism was recorded.

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2.6. Temperature and parasitoid overwintering To determine if temperature plays a role in A. pseudococci overwintering and spring emergence patterns, we placed newly parasitized mealybugs at ambient temperatures from late fall 2001 through spring 2002. For each inoculation date tested, sprouting potatoes were inoculated with »100 mealybugs (third instar to pre-ovipositional stages) and then placed in a plastic container (2.2 L) with an organdy-screened lid for ventilation. After 1 day, the mealybugs were exposed to A. pseudococci for a 24 h oviposition period at 22 § 2 °C. Six containers were prepared on each inoculation date, with three containers randomly selected to remain in the Kearney Agricultural Center insectary (control treatment) and three placed outside the insectary at ambient air temperatures (the containers were sheltered from direct sunlight, rain, and wind). The mealybugs were checked weekly until there were outward signs of parasitism, thereafter, the containers were checked daily and all adult parasitoids found were collected and their gender determined. The experiment was repeated each month with inoculations dates of 18 October 2001, 15 November 2001, 12 December 2001, 23 January 2002, 28 February 2002, and 15 March 2002. Weather data during the experiment were obtained from a California Irrigation Management Information System (CIMIS) weather station, with data downloaded from the University of California Statewide IPM Program website (www.ipm.ucdavis.edu). 2.7. Statistics Results are presented herein as means per treatment (§ SEM). Treatment eVects were analyzed using analysis of variance (ANOVA), with treatment means separated using Tukey’s HSD test (three or more treatments) or a t test (two-way comparisons) at P 0 0.05. We used regression analysis to describe the relationship between temperature and A. pseudococci developmental rate (egg to adult emergence). The mean development time per temperature treatment, rather than the mean of each replicate, was used for regression analysis. An initial viewing of development time (days¡1) graphed against temperature (see Fig. 1) suggested that a nonlinear model would best Wt the entire data set (including the temperature extremes). We used a nonlinear model described by Wang et al. (1982):

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Fig. 1. The rate of development (§ SEM) from egg to adult eclosion of A. pseudococci plotted against seven diVerent constant temperatures (14, 17, 22, 27, 32, 34, and 36 °C). The data were Wt to a nonlinear regression model (solid line, R2 D 0.997) that provided estimates of optimal (T0) and maximum (TH) temperatures as 24.7 and 36.0 °C, respectively. Mid-range temperatures (between 14 and 32 °C) were Wt to a linear regression model (dashed line, y D ¡ 0.0535 C 0.0045x, r2 D 0.99) that was used to determine the lower temperature threshold (Tlow) as 11.6 °C.

where Rt is the developmental rate at a given temperature T (°C), H is the value of the upper asymptote of the curve, rm is the exponential increase rate, T0 is the optimum temperature for development, TL and TH are, respectively, the minimum and maximum temperature boundaries, and B is the boundary width at the upper and lower temperatures. Because this nonlinear model predicted an unrealistically low temperature boundary (TL), we determined the lower temperature threshold using data within the mid-range temperature treatments (14–32 °C) and a linear equation (Davidson, 1944): Rt D a C bT,

(2)

where the development rate (Rt) is a linear function of temperature, T (°C), and a and b are regression parameters Wtted to the data. The low development threshold, Tlow, is calculated as Tlow D ¡ a/b, and the thermal constant (k) from birth to adult, in required degree-days (DD), is calculated as k D 1/b (Liu and Meng, 1999).

3. Results and discussion 3.1. Larval development After 1 day, a black scar was visible surrounding the area where A. pseudococci’s ovipositor penetrated the mealybug integument, typically on the dorsum, near

the lateral margin. An encyrtiform egg with an aeroscopic plate and stalk was associated with each oviposition scar. Only 2 of 100 mealybugs dissected had more than one oviposition scar and egg, and in each of the two mealybugs with multiple oviposition scars, only one live A. pseudococci larva was found. A. pseudococci is reported to be a solitary parasitoid (Islam and Copland, 2000). After 2 days, an encyrtiform larva hatched. First and second instar development times (at 32 °C) were 1.3 § 0.1 and 0.7 § 0.1 days, respectively. Both the Wrst and second instars are transparent to pale white, with light-brown and conspicuously scleritized mouthparts, and are attached to the host integument via the egg stalk. During this initial A. pseudococci development, the mealybugs were mobile, although they appeared sluggish. After 5 days, when most A. pseudococci had developed to the third instar, the mealybugs did not move when probed. The third and fourth instars are white in color, robust and hymenoptiform in shape, with well-developed mandibles. Third and fourth instars each required a 1.1 § 0.1 day development period. While the third instars were still attached to the host integument via the egg stalk, this connection had largely broken by the fourth instar. Fifth instars were observed 6–8 days after oviposition and had a 1.9 § 0.1 day development period; they occupied most of the host body cavity, with only the host integument remaining intact. We often observed shed portions of the mouthparts of the previous instars near the posterior of the Wfth instar, as well as a dark brown fecal pellet at the anterior end of the mummiWed mealybug. Pupae required the longest development time (3.9 § 0.1 days) and were found 8–11 days after oviposition. Adult emergence began 12 days after oviposition and continued for the next 2 days. Development of A. pseudococci immature stages reared on vine mealybug was similar to that reported by Rosen and Rössler (1966) for citrus mealybug, with encyrtiform larvae in the Wrst and second instars and hymenoptiform larvae in the third through Wfth instars. Disparate larval forms are found in other Anagyrus species parasitic on mealybugs. For example, A. mangicola has three larval instars (Cross and Moore, 1992), while A. kamali Moursi is reported to have six larval instars (Moursi, 1948). 3.2. Temperature-dependent development One replicate each in the 27 and 32 °C temperature treatments were lost due to a malfunction in the temperature cabinets, and are not included in the data analysis. There were no signiWcant diVerences in the development times of female and male A. pseudococci at any temperature treatment (Table 1), therefore, male and female development times are combined. A. pseudococci developed from egg to adult at temperatures between 14 and

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Table 1 The percentage of male and female A. pseudococci emerging from diVerent developmental stages of the vine mealybug reared at 22, 27, and 32 °C T (°C)

A. pseudococci gender1

Mealybug development stage1

n

Second instar

Third instar

Adult

22

Male Female Total2

3.8 § 1.3 0 3.8 § 1.3 a

19.8 § 11.9 9.0 § 2.3 28.7 § 11.2 b

38.8 § 11.5 28.6 § 6.4 67.4 § 11.8 c

99 62 161

27

Male Female Total2

1.4 § 1.4 0 1.4 § 1.4 a

24.9 § 3.7 1.2 § 1.2 27.5 § 4.4 b

30.5 § 3.2 41.0 § 5.3 72.5 § 4.4 c

80 60 140

32

Male Female Total2

2.0 § 2.0 0 2.0 § 2.0 a

33.7 § 3.9 10.6 § 1.6 44.3 § 2.4 b

7.5 § 1.5 46.1 § 3.0 53.6 § 4.5 c

81 106 187

1 A two-way contingency table of parasitoid gender versus mealybug development stage was submitted to Pearson’s chi square test for each temperature: 22 °C:
2 D 3.958, df D 2, P D 0.138; 27 °C:
2 D 31.695, df D 2, P 0 0.001; 32 °C:
2 D 76.136, df D 2, P 0 0.001. 2 For percentage parasitism by stage (total), means followed by the same letter within each row are not signiWcantly diVerent (Tukey’s HSD test, P 0 0.05).

34 °C, but failed to develop at the lowest (12 °C) or highest (36 °C) temperatures tested. At those constant temperatures where development was complete, the mean development time decreased as temperatures increased until the temperature was held at or above 34 °C (Fig. 1). Development times ranged from 79.3 § 3.3 days (at 14 °C) to 10.5 § 0.7 days (at 34 °C). Using data from all temperatures tested, the nonlinear model provides an excellent Wt (Fig. 1; R2 D 0.992, F D 165.82, df D 1, 6, P D 0.058) and provides T0 and TH temperatures as 24.7 and 36.0 °C, respectively, and rm as 0.18. Development is primarily linear throughout the temperature range that A. pseudococci survived, with little gradation downwards as the upper temperature threshold is neared, but rather a distinct decline when development and survival ceased. Parasitoid development, as a function of temperature, often shows an abrupt decline when critical temperatures are reached for the host. In this case, the upper threshold for A. pseudococci (TH D 36.0 °C) was similar to that estimated for vine mealybug in South Africa (TH D 36.0 °C) (Walton, 2003). At temperatures between 14 and 32 °C, the development rate was a positive linear function of temperature (y D ¡ 0.0535 C 0.0045x, r2 D 0.99, F D 913.1, df D 1, 5, P 0 0.001). A. pseudococci development (egg to adult) required 78.5 § 1.1, 53.3 § 1.8, 22.7 § 0.7, 14.8 § 0.1, and 11.1 § 0.02 days at 14, 17, 22, 27, and 32 °C, respectively. The linear model of temperature-dependent development, using only the mid-range values, is indicated by the dashed line on Fig. 1. The linear model provides a lower temperature threshold (Tlow) of 11.6 °C, and the thermal constant as 223.5 DD. Temperature-dependent development reported herein is similar to that reported by other researchers who studied A. pseudococci development on citrus mealybug (Avidov et al., 1967; Islam and Jahan, 1993a; Tingle and Copland, 1988a), although our determined thermal constant is lower than that

reported by Avidov et al. (1967), who found A. pseudococci required 297 and 298 DD for females and males, respectively. A. pseudococci development rate was also within a similar range (§ 3 days at 25–31 °C) to that reported for other Anagyrus species (Chandler et al., 1980; Mani and Krishnamoorthy, 1992; Nechols and Kikuchi, 1985; Sagarra and Vincent, 1999). However, these reported development times may be diYcult to compare between trials because the precise developmental stage of the mealybug host was often diVerent or not reported, and a single temperature was often evaluated. Constant temperature studies can also overestimate mortality at lower and higher temperatures, resulting in underestimates of Tlow and TH temperature. For example, high temperature thresholds were investigated by Tingle and Copland (1988a), who report that A. pseudococci can develop at a constant 35 °C, but above that the parasitoids died unless they were exposed to periods of lower temperatures. However, A. pseudococci survived 40 °C for 12 h, when the alternating temperature was 26 °C for another 12 h. Clearly, A. pseudococci is Xourishing in the San Joaquin Valley vineyards, where recorded temperatures were often below the 11.6 °C or above 36 °C thresholds for brief periods throughout the season (Fig. 2). At mid-range temperatures (22, 27, and 32 °C), signiWcantly more A. pseudococci were reared from adult mealybugs (64.8 § 5.3%), than third instars (33.0 § 3.0%) or second instars (2.1 § 0.9%) (F D 553.39, df D 2, 27, P 0 0.001, n D 488). Results were similar for individual temperatures, with signiWcantly more A. pseudococci reared from adult mealybugs (22 °C: F D 11.51, df D 2, 9, P D 0.003; 27 °C: F D 103.5, df D 2,6, P 0 0.001; 32 °C: F D 74.30, df D 2, 6, P 0 0.001; Table 1). There was no signiWcant diVerence in the number of female (45.0 § 3.6%) and male (55.0 § 3.6%) A. pseudococci reared (t test D 1.40, df D 1, 9, P D 0.19, n D 488). However, A. pseudococci gender was signiWcantly inXuenced

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Fig. 2. Seasonal maximum (䊊) and minimum (䊉) ambient air temperatures for (A) 2001 and (B) 2002, which corresponds to the period of Weld study. On both graphs, the 10-year-average for maximum and minimum temperatures are indicated by a solid line. A. pseudococci maximum (36 °C) and minimum (11.6 °C) development temperatures are indicated by the horizontal dashed lines. Ambient air temperatures were recorded by a California Irrigation Management Information System weather station, with data and degree-days downloaded from the University of California Statewide IPM Program website (www.ipm.ucdavis.edu).

by host development stage (Pearson’s chi square,
2 D 63.646, df D 2, P 0 0.001). From second instar vine mealybug we reared exclusively male A. pseudococci, third instar mealybugs produced 74.1 § 7.1% male and 25. 9 § 7.1% female A. pseudococci, and from adult (without an ovisac) mealybugs we reared 37.8 § 7.6 male and 62.1 § 7.6% female A. pseudococci. With each temperature tested individually, this pattern was evident at 27 and 32 °C, but not at 22 °C (Table 1).

While the temperature-dependent development study was not a host-stage preference test, an over-abundance and equal number of second, third and pre-ovipositional adult mealybugs were available for oviposition and yet most female parasitoids were reared from the larger mealybugs (Table 1). These observations are supported by previous research that showed A. pseudococci prefers to oviposit in larger citrus mealybug stages, which may convert to higher adult fecundity (Avidov et al., 1967;

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Islam and Copland, 1997). Similarly, for other Anagyrus species parasitic on mealybugs, ovipositional preference for larger hosts appears to be the norm rather than the exception. For example, A. indicus on the spherical mealybug, Nipaecoccus vastador (Maskell), A. kamali on the pink hibiscus mealybug, M. hirsutus Green, and A. mangicola on the mango mealybug, all exhibit oviposition preference for larger mealybugs (Bokonon-Ganta et al., 1995; Nechols and Kikuchi, 1985; Sagarra and Vincent, 1999; respectively). In parasitoids with arrhenotokous parthenogenesis, host size has been shown to be a component of a parasitoid’s decision to deposit female or male eggs, with female eggs laid in the larger hosts because they oVer higher food quality. 3.3. Temperature-dependent adult egg deposition After a 6 h exposure period, there were no A. pseudococci progeny produced in the 12 and 14 °C temperature treatments. At 16 and 18 °C, there were 4.1 § 0.7, and 8.6 § 4.9 eggs deposited per female per 6 h period, respectively. Our results suggest that winter and spring temperatures below 14 °C will reduce or eliminate oviposition. These results provide valuable information for augmentative biological control programs that propose earlyspring releases of A. pseudococci. In the San Joaquin Valley, air temperatures in mid-April were often below 14 °C (Fig. 2), which would limit released A. pseudococci impact. Lower temperatures have also been reported to adversely impact A. pseudococci lifetime fecundity as well (Mani and Krishnamoorthy, 1992; Sagarra et al., 2000). Tingle and Copland (1989) looked at A. pseudococci adult oviposition at temperatures ranging from 18 to 34 °C, and found that adult A. pseudococci successfully deposited eggs at all temperatures in that range. However, more eggs were deposited at the higher temperatures. They also report that A. pseudococci survived for longer periods at lower temperatures, as would be expected. Nevertheless, while parasitoids released in early spring would survive long enough to bridge periods of cooler temperatures, the increased time needed for A. pseudococci to deposit her full compliment of eggs may reduce lifetime fecundity as longer-lived adult parasitoids may be at an increased risk of predation before their full complement of eggs are deposited (Heimpel et al., 1998). 3.4. Seasonal parasitoid egg deposition Anagyrus pseudococci egg deposition during the growing season, as indicated by the percentage parasitism, began in mid- to late-May in both 2001 and 2002 (Fig. 3). The late-spring A. pseudococci emergence corresponded with movement of vine mealybug from protected locations on the vine, such as under the bark, to more exposed locations, such as the leaves (MalakarKuenen et al., 2001). This initial adult emergence period

Fig. 3. Seasonal Weld activity of A. pseudococci in 2001 (䊉) and 2002 (䊊) as measured by percentage parasitism of Weld collected vine mealybug from an infested vineyard, Del Rey, CA. Arrows indicate new generations in 2001 and 2002 as predicted by the temperature model’s thermal constant of 223.5 DD for each A. pseudococci generation. We used a bioWx of 111.7 DD from the Wrst appearance of adult A. pseudococci emergence (4 May 2001 and 28 May 2002), which is one-half the thermal constant.

was followed by a sharp increase in parasitism in earlyJuly, to about 35% (2001) and 60% (2002) (Fig. 3). In both 2001 and 2002, there was a mid-season decrease in late-July, followed by a second peak to about 65% (2001) and 55% (2002) in August. We believe the rapid rise in percentage parasitism, seen in both years, reXects multiple parasitoid generations to each mealybug generation, rather than a higher fecundity of the parasitoid compared to the host. This is corroborated by adding to the graph the laboratory-derived data on A. pseudococci temperature-dependent development, which has been combined with Weld temperature data for both 2001 and 2002. On Fig. 3, arrows indicate new A. pseudococci generations as predicted by the temperature model of 223.5 DD per generation. We used a bioWx of 115.9 DD from the Wrst appearance of adult A. pseudococci emergence (4 May 2001 and 28 May 2002), which is one-half the thermal constant (k), because the oviposition date of the collected mealy bugs and the development stage of the overwintered A. pseudococci were not known. The accumulated degree-days were calculated using a single sine model (Zalom et al., 1983). There are a predicted 7–8 A. pseudococci generations to 3–4 vine mealybug generations from May to November (Fig. 3), indicating the parasitoid can develop twice as quickly as the mealybug. The faster development, as compared with its host, allows for a more rapid increase during the periods of favorable temperatures. 3.5. Temperature and parasitoid overwintering Under ambient winter temperatures, emergence of adult A. pseudococci in the spring was relatively

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Fig. 4. Average (§ SEM) emergence periods for A. pseudococci on vine mealybug that were exposed to A. pseudococci at diVerent periods, from October through March, and then stored either inside at 22 § 2 °C or outside at ambient air temperatures.

well-synchronized, regardless of when (October–March) vine mealybugs were exposed to A. pseudococci (Fig. 4). For each exposure period, between treatment comparisons show that A. pseudococci stored outside had a signiWcantly greater development time (days) and accumulated degree-days than material stored in the insectary (Table 2). Within treatment comparisons show the development time of A. pseudococci stored in the insectary ranged between 20.89 and 24.58 days and varied signiWcant among exposure periods (F D 25.94, df D 5, 12, P 0 0.001; Table 2). However, the diVerence can be largely explained by temperature variation in the insectary room, which was about 1.5 and 1 °C cooler in December and January, respectively, than in February and March. When this temperature diVerence is taken

into account by determining the day-degrees for insectary-stored material, which ranged from 221.6 to 238.8 DD, there was no diVerence among exposure periods (F D 468.6, df D 5, 12, P D 0.076). Development of A. pseudococci stored outside was signiWcantly diVerent among exposure periods in both development time (F D 2396, df D 5, 12, P 0 0.001; Table 2) and accumulated degree-days (F D 67.24, df D 5, 12, P 0 0.001; Table 2). As expected, there was a considerable diVerence in development time, which ranged from 59.9 to 194.6 days, in order to synchronize overwintering A. pseudococci adults with host availability in the spring. There was also considerable diVerence in accumulated degree-days, which ranged from 291.0 to 469.9 DD, or a 25.5 to 102.7% increase over the laboratory-derived thermal constant. These results suggest that cues other than a simple accumulation of degree-days are required to stimulate development and emergence of overwintered A. pseudococci, as has been found for other insect species in previous investigations (Tauber et al., 1983). There might also have been an overestimation of accumulated degree-days using the single sine model (Raworth, 1994), when lower temperature thresholds were reached for short periods each day. We believe temperature relationships may be an important component of A. pseudococci eVectiveness in California’s San Joaquin Valley, where seasonal air temperatures can range from less than ¡5 °C in winter to more than 45 °C in summer. These results provide valuable information to improve augmentative biological control programs that propose early spring releases of A. pseudococci to attack overwintered vine mealybug. For example, air temperatures in mid-April were often below 16 °C, which would limit released A. pseudococci impact because of reduced oviposition. Still, seasonal

Table 2 Development time (egg to adult) in (A) days and (B) accumulated degree-days (§ SEM) of A. pseudococci reared either in the insectary at 22 § 2 °C, or outside at ambient air temperatures at the Kearney Agricultural Center, Parlier, CA, for each of Wve diVerent exposure periods Exposure period2

Insectary

Outside

t test

P value

(A) Development time in days1 18 October 2001 15 November 2001 19 December 2001 23 January 2002 28 February 2002 15 March 2002

23.05 § 0.34 a 22.16 § 0.24 ac 24.58 § 0.16 b 22.28 § 0.13 ac 21.71 § 0.64 ac 20.89 § 0.08 c

194.6 § 1.3 a 173.5 § 0.8 b 143.8 § 1.6 c 112.1 § 0.3 d 76.5 § 1.4 e 59.9 § 0.2 f

¡126.4 ¡189.3 ¡73.35 ¡359.2 ¡34.30 ¡150.5

00.001 00.001 00.001 00.001 00.001 00.001

469.9 § 3.4 a 380.0 § 4.5 b 377.3 § 9.6 b 392.7 § 0.9 b 337.7 § 9.8 c 291.0 § 2.5 d

¡49.96 ¡31.03 ¡12.47 ¡153.1 ¡6.446 ¡22.96

00.001 00.001 0.005 00.001 0.008 0.001

(B) Development time in accumulated degree-day1 18 October 2001 230.5 § 3.3 ab 15 November 2001 221.6 § 2.4 a 19 December 2001 230.3 § 1.4 ab 23 January 2002 233.3 § 1.2 ab 28 February 2002 238.8 § 7.0 b 15 March 2002 229.8 § 0.9 ab

1 Within columns, means followed by the same letter are not signiWcantly diVerent (Tukey’s HSD test, P 0 0.05); between columns and within the same row (same exposure period) treatment means are separated by a t test (n D 3). 2 Ambient air temperatures recorded by a California Irrigation Management Information System weather station, with data and degree-days downloaded from the University of California Statewide IPM Program website (www.ipm.ucdavis.edu).

K.M. Daane et al. / Biological Control 31 (2004) 123–132

temperatures in the San Joaquin Valley are conducive to A. pseudococci survival and development for most of the growing season, providing a good climatic match of parasitoid to environmental conditions. Furthermore, most of California’s grape-growing regions have milder climates than that found in the San Joaquin Valley, suggesting that A. pseudococci can be successfully manipulated throughout the state. We also showed that A. pseudococci has two generations to each vine mealybug generation, which allows for a more rapid population increase provided that susceptible mealybug host stages are available. We are currently investigating host size preference and gender allocation for A. pseudococci on vine mealybug to provide additional information for improved augmentation programs.

Acknowledgments We thank Glenn Yokota for his assistance with laboratory and Weld experiments; Walter Bentley for information on P. Wcus Weld biology and vineyard management; and the Del Rey Packing Company and Chooljian Brothers for use of their vineyards. Research funding was provided by the California Table Grape Commission, California Raisin Marketing Board, University of California Statewide Integrated Pest Management Program, and the Western Region Sustainable Agriculture Research and Education Program and is gratefully appreciated. Voucher specimens are deposited at the Kearney Agricultural Center, Parlier, CA, where the research was conducted.

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