Seasonal Abundance and Synchrony Between Laricobius osakensis ...

ARTHROPOD BIOLOGY

Seasonal Abundance and Synchrony Between Laricobius osakensis (Coleoptera: Derodontidae) and Its Prey, Adelges tsugae (Hemiptera: Adelgidae), in Japan L. C. VIEIRA,1,2 A. B. LAMB,3 S. SHIYAKE,4 S. M. SALOM,1

AND

L. T. KOK1

Ann. Entomol. Soc. Am. 106(2): 249Ð257 (2013); DOI: http://dx.doi.org/10.1603/AN12075

ABSTRACT Laricobius osakensis Montgomery & Shiyake, native to Japan, is being measured as a potential biological control agent of the hemlock woolly adelgid, Adelges tsugae Annand, in the eastern United States. Adelges tsugae, a destructive pest threatening the hemlock ecosystems in the eastern United States, was introduced from Japan. This is the Þrst detailed study of the life history of L. osakensis, A. tsugae, and their interaction in Japan. The seasonal abundance of L. osakensis and A. tsugae was assessed in a Þeld study conducted from December of 2007 to November of 2008 in Hyogo, Nara, and Osaka Prefectures (Japan). The sistentes generation of A. tsugae in Japan matured 2 mo later than that reported in British Columbia, Canada, but 1 and 2 mo earlier than in Virginia and in Connecticut, respectively. Timing of the sexuparae generation occurrence in Japan was similar to that observed in Connecticut and Virginia, but this generation has not been observed in British Columbia. Phenological differences among the various A. tsugae population in the eastern U.S. states and Japan can be explained by temperature differences at the locations. Aestival diapause of the predator L. osakensis coincided with diapausing Þrst-instar A. tsugae sistentes. The start of L. osakensis adult activity, oviposition, and larval development was synchronized closely with sistentes development resumption, sistentes adults close to oviposition, and sistentes adults with eggs, respectively. These results indicate good synchrony between L. osakensis and suitable prey stages of A. tsugae in the native habitat of both species. KEY WORDS Laricobius osakensis, Adelges tsugae, phenology, synchrony, Japan

The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae) is a destructive invasive pest threatening the hemlock ecosystems in the eastern United States (Orwig et al. 2002). This pest is native to all hemlock (Tsuga spp.) ranges, with the exception of the eastern United States (Havill et al. 2006). In the native range it is not usually considered a pest, possibly because of a combination of host resistance and a complex of natural enemies that keep A. tsugae populations at noninjurious levels (McClure and Cheah 1999). Classical biological control is considered the most promising option for controlling A. tsugae in a forest setting (Cheah et al. 2004, Onken and Reardon 2011). The biological control program for A. tsugae has focused on building a predator community in the eastern United States that collectively could keep the pest population below damaging levels. So far three pred1 216 Price Hall, Department of Entomology, Virginia Tech, Blacksburg, VA 24061Ð 0319. 2 Corresponding author, e-mail: [email protected]. 3 205 Ellington Plant Sciences Bldg., Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 379964560. 4 Osaka Museum of Natural History, Nagai Park, Osaka 546-0034, Japan.

atorsÑSasajiscymnus tsugae Sasaji (Coleoptera: Coccinellidae), Scymnus sinuanodulus Yu & Yao (Coleoptera: Coccinellidade), and Laricobius nigrinus Fender (Coleoptera: Derodontidae)Ñ have been introduced in the eastern United States for the biological control of A. tsugae (McDonald et al. 2008). The promising results from the initial releases of Laricobius nigrinus (Mausel et al. 2010) and the determination that A. tsugae present in the eastern United States originated from Japan (Havill et al. 2006) have led to a strong interest in studying L. osakensis Montgomery & Shiyake, a predator found in association with A. tsugae in Japan (Montgomery et al. 2011). Results from laboratory studies to date have been encouraging. Laricobius osakensis is a highly specialized predator of A. tsugae and adjusts its rate of feeding and oviposition to changes in prey density (Vieira et al. 2011, 2012). The life history of A. tsugae has been studied previously in British Columbia, Canada (Zilahi-Balogh et al. 2003a), where it is native, and in the introduced range in Connecticut (McClure 1989), Virginia (Gray and Salom 1996), and northern Georgia (Joseph et al. 2011). It is similar in the three introduced locations in the eastern United States, except for its faster development rate in northern Georgia. However, differ-

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ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA

ences were found between this pestÕs life history in British Columbia and the three locations in the introduced range. In all locations, A. tsugae completes two asexual generations (sistentes and progredientes) on hemlock per year. The sistentes generation goes through a summer aestivation period, breaks diapause, and develops through fall and winter. The progredientes generation, progeny of the sistentes generation, develops much faster (because of warmer temperatures) than the sistentes generation and is present in the spring. A third generation, the sexuparae, present in Connecticut and Virginia, is absent in British Columbia. This generation was not measured in northern Georgia because the study focused only on adelgid life stages suitable for predators (Joseph et al. 2011). Sexuparae adults (alate) leave hemlock to Þnd its primary spruce (Picea) host. In Japan, A. tsugae has been reported on Picea torano (Inouye 1945), but in North America it has been unable to develop successfully in any of the Picea species present (McClure 1987). The phenology of the L. nigrinus life cycle also was studied in British Columbia (Zilahi-Balogh et al. 2003a). The adults Þrst appear in October when A. tsugae sistentes resume development. Oviposition starts in January and February as A. tsugae sistentes adults begin to oviposit, reaching a peak in March. Larvae are also present when A. tsugae progredientes eggs are present. After developing larvae feed on progredientes eggs, mature larvae migrate to the soil to pupate, and develop to adults. The resulting adults undergo aestival diapause in the soil, coinciding with the diapause of Þrst-instar A. tsugae sistens. Knowledge of prey and predator seasonal abundance and synchrony is of utmost importance in any biological control effort because timing is critical for success (Ascerno 1991). Although an organismÕs phenological adaptations are not static, they do progress in a predictable pattern (Tauber and Tauber 1976). Therefore, these studies can be used to forecast the seasonal activity of both pest and predator (phenology), and determine the best timing for predator releases (synchrony) (Ascerno 1991). This study is the Þrst comprehensive report on the biology of L. osakensis and A. tsugae in Japan. The objectives were to 1) Determine the life history of L. osakensis and A. tsugae, and how both are associated, in Hyogo, Osaka, and Nara Prefectures (Japan); (2) compare the phenology of A. tsugae in Japan with that in Connecticut, Virginia, northern Georgia, and British Columbia; and 3) compare the association of L. osakensis and A. tsugae with that of L. nigrinus and A. tsugae.

Materials and Methods Study Sites. For study site selection, disturbed habitats were preferred over natural forest settings for the study to ensure easy access to sites, ease of sampling, and high infestation levels of A. tsugae were present. However, even in disturbed locations, the number of infested trees was low, limiting the number of trees that could be sampled.

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Every hemlock tree in Kobe Municipal Arboretum (Hyogo Prefecture), Mount Wakakusa (Nara Prefecture), and Myo-on-ji Temple (Osaka Prefecture) was visually inspected to assess the level of infestation. All infested trees in each location were includedÐ9 trees in Hyogo, 3 in Nara, and 1 in Osaka. The trees in Hyogo Prefecture were located along a road, those in Nara Prefecture were on a hilltop in a forest setting, and the tree in Osaka Prefecture was maintained as an ornamental shrub. Seasonal Abundance and Synchrony. To measure the seasonal abundance of L. osakensis and A. tsugae, all three sites were sampled from December of 2007 to November of 2008. For all stages of A. tsugae, and eggs and larvae of L. osakensis, weekly sampling was conducted in December and January, biweekly sampling was conducted in February and March, and monthly sampling was conducted from April to August or November in Nara and Hyogo, respectively. Sampling was missed in June and July in Nara because of weather, limited site access, and lack of time to return to this site. In Osaka, biweekly sampling was conducted in December and January, and monthly sampling was conducted from February to August. At each sampling date, one randomly selected terminal branch tip (2Ð25 cm in length), with sufÞcient A. tsugae density for analysis, was removed from each of the four cardinal directions in each sampled tree (four branches total). Branch tips were placed into Ziploc bags (SC Johnson, Racine, WI) and stored at 4⬚C while life stages were examined and counted. All life stages of A. tsugae, and egg and larval stages of L. osakensis were examined under a dissecting microscope with a calibrated optical scale. Adelges tsugae life stages were determined by the number of exuviae present (McClure 1989). Beat sheet sampling for L. osakensis adults at each tree was conducted weekly from December to February, biweekly from March to June, and monthly from August to November in Hyogo and Nara. Sampling was missed in June, October, and November in Nara, because of inclement weather affecting predator activity or insufÞcient time to return to this site. In Osaka, biweekly sampling was conducted in December, January, March, April, May, June, September, and October, and monthly sampling was conducted in February, July, and August. Each sample involved selecting three infested branches (⬇100 by 50 cm) per tree and beating them three times. To compare the Þndings for A. tsugae in Japan to what was found previously in British Columbia, Connecticut, Virginia, and northern Georgia, the start and end of each A. tsugae stage was determined for this study and from previous studies (McClure 1989, Gray and Salom 1996, Zilahi-Balogh et al. 2003a, Joseph et al. 2011) and temperature data were collected for all studies from weather stations close to the sampling sites (http://www.wunderground.com/). Statistical Analysis. Adelges tsugae were counted on the four branch-tip samples and averaged per centimeter of tree branch at each sample date. Data were tested for differences in mean densities among the

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VIEIRA ET AL.: SEASONAL ABUNDANCE OF L. osakensis and A. tsugae

sampling sites, using the one-way KruskalÐWallis test, chi-square approximation, with site and sampling date as independent variables. Laricobius osakensis adult abundance was calculated as an average per tree at each sample date. Predator egg and larval abundance were calculated as the average number found per centimeter of tree branch at each sample date. For the comparison of L. osakensis abundance among sites, data were summarized per month because sampling dates were different for each site, and tested for differences by using the one-way KruskalÐWallis test, chi-square approximation. Summarized data were plotted against the average temperatures registered for that month at Osaka International Airport, Japan. Relationships between the abundance of L. osakensis life stage, and any A. tsugae life stage were assessed throughout the sampling period by using the SpearmanÕs rank correlation coefÞcient. Densities of speciÞc A. tsugae stages and L. osakensis stages were plotted to illustrate strong correlations. Results Seasonal Abundance of Adelges tsugae. Except for the sistentes Þrst instar, the initial dates of occurrence for each A. tsugae life stage were generally consistent among sites. However, the durations of the various A. tsugae life stages were variable (Fig. 1aÐ c, Fig. 2aÐ c). Adelges tsugae abundance was signiÞcantly different at the different locations for all life stages (␹2 ⫽ 6.0555Ð 32.4683, df ⫽ 2, P ⬍ 0.0001Ð 0.05) with the exception of sistentes adults, sistentes adults with eggs, progredientes Þrst instar, progredientes fourth instar, and progredientes adults with eggs. There was great temporal overlap of life stages, with as many as six different life stages present in samples from one location, as it can be seen on 9 February in Osaka (Figs. 1c, 2c). In Hyogo, the transition from second- to the thirdinstar sistentes occurred in October, and fourth instars were Þrst seen in November (Fig. 1a). At the Þrst sampling periodÑ 6 December 2007ÐA. tsugae was in the fourth instar in all locations, as shown by the higher abundance of this life stage at this sampling date (Fig. 1aÐ c). Sistentes adults were Þrst seen in early December (Fig. 1aÐ b) or mid-December (Fig. 1c), and were present until the end of April (Fig. 1a), mid-May (Fig. 1b), or end of May (Fig. 1c). Peak occurrence of sistentes adults was observed in 21 February (Fig. 1a), 4 March (Fig. 1b), and 20 April (Fig. 1c). Sistentes adults started oviposition by the beginning of January (Fig. 1aÐ b) or February (Fig. 1c). Sistentes adults with eggs were present until the end of April (Fig. 1a) or mid-May (Fig. 1bÐ c). Progedientes Þrst instars were seen Þrst by the end of March (Fig. 2b) or mid-April (Fig. 2a and c). Maturity was reached, as progredientes adults or sexuparae alate adults, by the end of May (Fig. 2c) or mid-June (Fig. 2a). Eggs laid by progredientes adults were seen in mid-June (Fig. 2a). The resulting Þrst-instar sistentes went into aestival diapause by the end of May or mid-June (Fig. 2a and c).

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Seasonal Abundance of Laricobius osakensis. The abundance of L. osakensis adults (␹2 ⫽ 4.3581, df ⫽ 2, P ⫽ 0.1132), larvae (␹2 ⫽ 4.8979, df ⫽ 2; P ⫽ 0.0864), and eggs (␹2 ⫽ 3.9810, df ⫽ 2, P ⫽ 0.1366) was not signiÞcantly different among sites. Adult predators were present from November to April when average temperatures were below 20⬚C (Fig. 3). Males Þrst appeared in November in Hyogo (Fig. 4a) and were present until the beginning of April. In Nara and Osaka, males Þrst were observed in December and were present until March (Fig. 4b) or January (Fig. 4c), respectively. Females were present when sampling began in December in all locations, and were present until March (Fig. 4c) or April (Fig. 4aÐ b). The Þrst L. osakensis eggs were observed by the end of December in Hyogo and Nara (Fig. 4aÐ b), and only in February in Osaka (Fig. 4c). Eggs were present until April (Fig. 4a) or May (Fig. 4bÐ c). Laricobius osakensis larvae Þrst were observed in January (Fig. 4b) or March (Fig. 4a and c), and were present until April (Fig. 4a) or May (Fig. 4bÐ c). Synchrony Between A. tsugae and L. osakensis. Several signiÞcant correlations were found between the abundance of different life stages of L. osakensis and some life stages of A. tsugae throughout sampling. Laricobius osakensis adults, eggs, and larvae showed a negative correlation with Þrst-instar A. tsugae sistentes (Table 1). The adults appeared in November shortly after A. tsugae sistentes resumed development in October (Fig. 1aÐ c, Fig. 4aÐ c). A negative correlation also was found between L. osakensis adults and A. tsugae progredientes adults. All stages of Laricobius osakensis showed a positive correlation with A. tsugae sistentes adults (Table 1). Onset of oviposition by female L. osakensis (Fig. 4aÐ c) began shortly before A. tsugae sistentes adults started oviposition (Fig. 1aÐ c). Abundance of L. osakensis adults, larvae, and eggs dropped with the abundance of A. tsugae sistentes adults (Fig. 5a). Predator eggs and larvae showed a positive correlation with A. tsugae sistentes adults with eggs (Table 1). Abundance of L. osakensis larvae followed very closely that of A. tsugae sistentes adults with eggs (Fig. 5b). Larvae also showed a positive correlation with Þrst- and second-instar progredientes (Table 1). Other signiÞcant correlations only were found among the different L. osakensis life stages, and between a given A. tsugae life stage and the previous life stage, but not with all previous life stages (Table 1). Pupae were not found on the twigs. Discussion Seasonal Abundance of A. tsugae. Greater differences were found when comparing the phenology of A. tsugae in Japan (Fig. 1aÐ c, Fig. 2aÐ c) with A. tsugae in British Columbia (Zilahi-Balogh et al. 2003a), than when compared with A. tsugae in Connecticut (McClure 1989), Virginia (Gray and Salom 1996), or northern Georgia (Joseph et al. 2011) (Table 2). The sistentes generation in Japan matured 2 mo later than in British Columbia, 1 mo earlier than in Virginia and northern Georgia, and 2 mo earlier than in Connect-

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Fig. 1. Seasonal abundance of the sistentes generation of Adelges tsugae between 06 December 2007 and 15 November 2008 in Hyogo (a), Nara (b), and Osaka (c) Prefectures (Japan).

icut. The progredientes generation in Japan (May) matured at the same time as in northern Georgia and a month earlier than the other locations (June). In addition, sexuparae alate adults were observed in Japan. The presence of sexuparae is consistent with reports for Connecticut and Virginia, but in contrast with British Columbia. Differences in A. tsugae phe-

nology among Japan, Connecticut, Virginia, and northern Georgia can be explained by the temperatures registered for those locations during the studies (Fig. 6a). Temperature variation in the year of the study was very similar for Japan (2007 and 2008), Connecticut (1986 and 1987), Virginia (1994 and 1995), and northern Georgia (2004 and 2005). British

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253

Fig. 2. Seasonal abundance of the progredientes and sexuparae generations of Adelges tsugae between 06 December 2007 and 15 November 2008 in Hyogo (a), Nara (b), and Osaka (c) Prefectures (Japan).

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Fig. 3. Seasonal pattern of Laricobius osakensis adults in Japan (line) and mean temperature (⬚C) (bars) on all sampling dates from 6 December 2007 and 15 November 2008. Data for adults were pooled from all sites, as no signiÞcant differences were found.

Columbia (1998 and 1999) showed a lower amplitude of temperatures. The highest temperatures were registered for Japan, followed by northern Georgia, Virginia, Connecticut, and British Columbia. This explains the faster development of A. tsugae in Japan and Virginia, followed by northern Georgia, Connecticut,

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and British Columbia. The absence of sexuparae provides some indication that its A. tsugae population is different from that found in Japan, Connecticut, Virginia, and northern Georgia. This has been conÞrmed by molecular studies that grouped A. tsugae in western North America in one clade, and A. tsugae in Japan and the eastern United States in another clade (Havill et al. 2006). It is likely that the differences found in A. tsugae development in the different locations are consistent across years because the differences in temperatures between locations were similar in 2007Ð 2008, the year of this study (Fig. 6b). There was enormous temporal overlap of A. tsugae life stages in Japan, consistent with what was reported for Connecticut, Virginia, northern Georgia, and British Columbia. Gray and Salom (1996) reported as many as 12 life stages at a single sampling date. In this study only six life stages were found at a single sampling date. The lower number of life stages encountered is likely related to the fact that progedientes and sexuparae nymphs were not differentiated in this study. Seasonal Abundance of L. osakensis. The life cycle of L. osakensis is similar to that described for L. nigrinus (Zilahi-Balogh et al. 2003a). Both predators are univoltine. Laricobius nigrinus adults were reported to be active from September to April, and were not collected on branches from May to August. Laricobius osakensis adults were active from November to April, and were not collected on branches from May to October. Both were mostly active in the winter, and

Fig. 4. Seasonal abundance of Laricobius osakensis adult females, adult males, eggs and larvae between 6 December 2007 and 15 November 2008 in Hyogo (a), Nara (b), and Osaka (c) Prefectures (Japan).

0.4706

a

a

0.4300 0.5916a

LosL

Sis2

0.1893 0.2118 ⫺0.2267 ⫺0.1924

⫺0.4618 ⫺0.3401 ⫺0.3855

a

Sis1 0.1502 0.1181 ⫺0.3354 ⫺0.0824 0.7824a

Sis3 0.5704 0.6679a 0.4431a ⫺0.6062a 0.4059a 0.1594 0.2602

0.3498 0.3315 ⫺0.2547 ⫺0.1465 0.6884a 0.7326a

a

SisA

Sis4

Pro2 ⫺0.2348 0.3156 0.4591a ⫺0.1431 ⫺0.1239 ⫺0.1431 ⫺0.0727 0.0720 0.2725 0.7777a

Pro1 ⫺0.1684 0.2564 0.4790a ⫺0.2445 ⫺0.0426 ⫺0.1588 ⫺0.2368 0.3346 0.5501a

SisO 0.2465 0.7062a 0.5818a ⫺0.4326a 0.2419 ⫺0.0217 0.0125 0.8434a

⫺0.2073 0.3001 0.1319 0.1479 0.1775 0.1479 0.2137 0.1439 0.1831 0.4455a 0.6016a

Pro3 ⫺0.2073 ⫺0.1951 ⫺0.1484 0.1982 ⫺0.2071 ⫺0.2071 ⫺0.1831 ⫺0.2446 ⫺0.1831 ⫺0.1013 ⫺0.0752 ⫺0.0417

Pro4 a

⫺0.4412 ⫺0.1201 ⫺0.1488 0.5729a ⫺0.2115 ⫺0.2292 ⫺0.1533 ⫺0.2891 ⫺0.1715 0.1103 0.2432 0.5320a 0.4433a

ProA

Sexp ⫺0.2992 0.0901 ⫺0.0038 0.2955 ⫺0.0102 ⫺0.0325 0.0335 ⫺0.0615 0.0106 0.2642 0.3994a 0.7217a 0.6615a 0.7064a 0.6615a

ProO ⫺0.2073 ⫺0.1951 ⫺0.1484 0.2663 ⫺0.2071 ⫺0.2071 ⫺0.1831 ⫺0.2446 ⫺0.1831⫺ ⫺0.1013 ⫺0.0752 ⫺0.0417 1.0000a 0.4433a

Highlighted boxes show signiÞcant correlation between L. osakensis and A. tsugae life stages. LosA: L. osakensis adults, LosE: L. osakensis eggs, LosL: L. osakensis larvae, Sis1: first-instar sistentes, Sis2: s-instar sistentes, Sis3: third-instar sistentes, Sis4: fourth-instar sistentes, SisA: sistentes adults, SisO: sistentes adults with eggs, Pro1: first-instar progedientes, Pro2: s-instar progredientes, Pro3: third-instar progredientes, Pro4: fourth-instar progredientes, ProA: progredientes adults, ProO: progredientes adults with eggs, SxpA: Sexuparae alate adults. a Indicates signiÞcant correlation at P ⬍ 0.05.

LosA LosE LosL Sis1 Sis2 Sis3 Sis4 SisA SisO Pro1 Pro2 Pro3 Pro4 ProA ProO

LosE

Correlation between abundance of all L. osakensis life stages and all A. tsugae life stages observed throughout sampling

LosA

Table 1.

March 2013 VIEIRA ET AL.: SEASONAL ABUNDANCE OF L. osakensis and A. tsugae 255

Fig. 5. Variation of the monthly abundance of A. tsugae sistentes adults in relation to abundance of L. osakensis adults, larvae, and eggs (a); and A. tsugae sistentes adults with eggs in relation to L. osakensis larvae (b).

undergo an aestival diapause. Oviposition by L. osakensis and L. nigrinus females started in December and January, respectively, and ended in May. Laricobius osakensis and L. nigrinus larval activity period started in January and March, and ended in June and May, respectively. The earlier start of the larval activity for L. osakensis is a result of starting oviposition earlier and developing faster (Vieira et al. 2012). The absence of pupae in twigs is also consistent with observations of L. nigrinus, and indicates that L. osakensis mature fourth instars also migrate to the soil to pupate (ZilahiBalogh et al. 2003a). Resulting adults go through an aestival diapause, emerging as temperature drops by the end of autumn (Fig. 3). Laricobius osakensis males emerged, and disappeared from the Þeld earlier than females (Fig. 4aÐ c). Synchrony Between A. tsugae and L. osakensis. There was a strong synchrony between A. tsugae and L. osakensis, similar to that found between A. tsugae and L. nigrinus (Zilahi-Balogh et al. 2003a), but the timing in relation to A. tsugae, was slightly different. The L. osakensis adults were detected later than adults of L. nigrinus. They were seen in the Þeld shortly after A. tsugae sistentes resumed development, whereas L. nigrinus adults appeared together with resumption of A. tsugae development. Oviposition by L. osakensis

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Table 2.

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Listing of the month when each A. tsugae life stage was first (start) and last (end) seen in the field

A. tsugae life stage Sistentes Þrst instar Sistentes second instar Sistentes third instar Sistentes fourth instar Sistentes adults Sistentes adults with eggs Progredientes Þrst instar Progredientes second instar Progredientes third instar Progredientes fourth instar Progredientes adults Progredientes with eggs Sexuparae adults

Virginia

Connecticut

Start

End

Start

End

June Oct. Nov Nov Jan Mar. April May June June June June June

Dec. Dec. Mar. Mar. May May June June June June July July July

June Sep. Sep. Sep. Feb. Feb. May May May May June June June

Dec. Feb. Feb. Feb. April June June June June June June July June

British Columbia Start

End

July Dec. Sep. Feb. Sep. Feb. Sep. April Oct. June Jan June Aprilil June May June June June June July June July June July Not present

Northern Georgia Start

Japan

End

Start

End

May Dec. Sep. Feb. Sep. Feb. Sep. Feb. Jan May Jan May Mar. June April June April June April June May June May July Not measured

May Oct. Oct. Nov Dec. Jan Mar. April May June May June May

Oct. May Mar. May May May May May May June July June June

Study in (Japan), McClure (1989); Connecticut, Gray and Salom (1996); Virginia, Joseph et al. (2011); northern Georgia, Zilahi-Balogh et al. (2003a); and British Columbia.

females took place about a week before A. tsugae sistentes adults started laying eggs. Onset of oviposition by L. nigrinus females coincided with the beginning of egg laying by A. tsugae sistentes adults. Both L. osakensis and L. nigrinus larvae were found in association with A. tsugae sistentes with eggs. This observation is consistent with observations in the laboratory for this species (Vieira et al. 2012) and for L. nigrinus larvae (Zilahi-Balogh et al. 2003b), and indicates that the larvae feed predominantly on eggs of A. tsugae. Both predators undergo aestival diapause that

matches the aestival diapause of the Þrst-instar A. tsugae sistentes. Conclusion. Laricobius osakensis appears to be well synchronized with A. tsugae in Japan. Its aestival diapause coincides with that of Þrst-instar A. tsugae sistentes. Onset of adult activity, oviposition, and larval development are closely related to sistentes development resumption, sistentes adults close to oviposition, and sistentes adults with eggs, respectively. In addition, it was possible to see a close match between A. tsugae phenology in Japan and in Connecticut, Vir-

Fig. 6. Average monthly temperatures in Japan, Connecticut, Virginia, northern Georgia, and British Columbia in the year the studies on A. tsugae phenology where conducted (a) and in the year this study was conducted (b).

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ginia, and northern Georgia. Laricobius osakensis is thus adapted to the population introduced into the eastern United States, with very similar phenology to that seen in the introduced areas. Overall, L. osakensis shows great potential as a biological control agent of A. tsugae in the eastern United States, especially in the southern Appalachians where temperatures are more similar to the native range in Japan. Acknowledgments We thank the Kobe Municipal Arboretum (Hyogo Prefecture), the Nara Park Management OfÞce (Nara Prefecture), and the Myo-on-ji Temple (Osaka Prefecture) for the use of their land for the study. Funding was provided by USDA Forest Service Cooperative Agreement 05-CA11244225-039.

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