Appl. Entomol. Zool. 37 (3): 347–355 (2002)

Evaluation of generational percent parasitism on clerkella (: ) larvae in orchards under different management intensity1

Ishizue Adachi Laboratory of Entomology, National Institute of Fruit Tree Science, National Agricultural Research Organization, Tsukuba, Ibaraki 305–8605, Japan (Received 26 December 2001; Accepted 15 March 2002)

Abstract Abundance of live, dead and parasitized larvae of the peach leafminer, Lyonetia clerkella (Linnaeus), was investigated at 2 or 3-day intervals from April to November 1994 and April to October 1995. The proportions of infested leaves were determined by a binomial sampling in peach orchards under different management intensities, and subsequently the infested leaves were sampled to ascertain the fate of each leafminer through individual rearing in the laboratory. The seasonal prevalence showed seven discrete generations in both years. The number of larvae entering each genera- tion was estimated by a stage-frequency analysis. From that estimate, as well as from an estimate of the number of parasitized larvae, the generational percent parasitism was determined for each generation and throughout the year. The yearly result was about 9% parasitism in the insecticide-sprayed orchards and about 19% in the unsprayed or- chards. The relationship between host and parasitoid densities in successive generations showed a tendency toward counterclockwise rotation. Furthermore, the relationship between host density and percent parasitism exhibited a de- layed density-dependent process in a host-parasitoid system.

Key words: Lyonetia clerkella, peach leafminer, parasitism, density relatedness

(Naruse, 1975, 1978a, b, 1981; Fujiwara et al., INTRODUCTION 1978; Shoji and Ueno, 1981; Shoji et al., 1982; The peach leafminer, Lyonetia clerkella (Lin- Sekita, 1987; Naruse and Hirano, 1990). However, naeus), is distributed in Japan, South Korea, Tai- these studies tended not to focus on the parasitoids wan, China, India, and Madagascar (Inoue of L. clerkella. In 1998, 19 parasitoid species be- et al., 1982). This leafminer is a major pest longing to the families Eulophidae, Pteromalidae, in commercial peach orchards in Japan; it causes and Braconidae were first identified on L. clerkella heavy defoliation prior to harvest (Naruse, 1978a; larvae (Adachi, 1998). The ecological role of these Naruse and Hirano, 1990). L. clerkella is a multi- parasitoids remains to be discovered. Recently, a voltine species that has 3 stadia. Females insert peach integrated pest management (IPM) system eggs singly into the underside of leaves. After has been developed that reduced pesticide use in hatching, larvae penetrate into the mesophyll and favor of non-insecticidal control tactics such as the form spiral mines in the upper half of the leaves; employment of sex pheromones (Sugie et al., 1984; thereafter, they form serpentine and/or linear Sato et al., 1986; Gyoutoku et al., 1997). In such a mines, as they grow. Third stadium larvae are dis- system, it is expected that biological control by tinguishable from 1st and 2nd instars by three pairs natural enemies would be an important component of blackish thoracic legs, which are easily observed in pest control. Therefore, within such a system, it through the epidermis of the leaf. Mature 3rd in- would be desirable to evaluate the effectiveness of stars leave their mines and pupate in silken co- parasitoids in terms of quantitative measurement coons under the leaves or on the trunks. (e.g., percent parasitism). The biology of L. clerkella was intensively in- The term “percent parasitism” has commonly vestigated from the late 1970s until the early 1980s been used as the ratio of the number of parasitized

1 Contribution No. 1253, of National Institute of Fruit Tree Science, NARO.

347 348 I. Adachi hosts to the total number of all hosts in a sample were compared between peach orchards under dif- unit at a particular point in time. However, it is er- ferent management intensities. roneous to use the term to imply merely the sample values of parasitism (Van Driesche, 1983). The MATERIALS AND METHODS quantitative impact of parasitoids on their target populations is the necessary focus of such study. Study sites. Research was conducted at a field Therefore, the total losses to parasitism for a stage managed by the National Institute of Fruit Tree over a generation must be measured. Thus, the per- Science, Tsukuba. The field had a total area of cent parasitism should be the parameter that indi- 16 ha, consisting of ca. 50 small orchards sur- cates generational levels of parasitism (Van Dri- rounded by windbreak trees. Several kinds of fruit esche, 1983; Van Driesche et al., 1991). For evalu- trees including peach, Japanese pear, and chestnut ating percent parasitism, a life table analysis is ex- were planted in each of the orchards. A total of tremely useful. Construction of life tables generally four peach orchards (referred to hereafter as the requires accurate estimates of the number of larvae “sprayed orchards”) were managed according to a entering stages that are susceptible to parasitoids, conventional pesticide-application schedule with as well as the number of larvae that die within the organophosphorous insecticides sprayed seven various stages due to parasitoids. A stage-fre- times in a year as illustrated in Fig. 1. Two other quency analysis can be used to obtain these esti- orchards (referred to hereafter as the “unsprayed mates (Bellows and Van Driesche, 1999). orchards”) were not treated with insecticides but In the present study, a two-stage sampling rather with fungicides. Three sprayed and two un- method (Adachi and Yamamura, 2000) was used to sprayed peach orchards were chosen for investiga- estimate the number of L. clerkella larvae at each tion from those six orchards. It should be noted stadium in the field, and the modified KNM that the unsprayed orchards were more than 100 m method (Yamamura, 1998) was used to treat the away from the sprayed orchards. stage-frequency data in each generation. These Sampling. Surveys were carried out in intervals analyses aimed at evaluating the parasitism rate of two to three days from April 27 to November 8 within a generation, i.e., the generational percent in 1994 and April 26 to October 26 in 1995. About parasitism. Subsequently, the levels of parasitism 500 leaves were randomly checked in each of the

Fig. 1. Seasonal prevalence of L. clerkella larvae (A) in pesticide-sprayed peach orchards in 1994, (B) in unsprayed orchards in 1994, (C) in sprayed orchards in 1995, and (D) in unsprayed orchards in 1995. Arrows in the figures indicate the date of pesti- cide spraying. Generational Percent Parasitism on L. clerkella 349 selected peach orchards to ascertain whether or not clerkella larvae was drawn. From the prevalence L. clerkella leafmines had formed. This inspection curve, the number of generations per year and the represented a binomial sampling, yielding esti- duration of each generation were determined; gen- mates of the proportion of infested leaves. Of the erations were basically discrete, as mentioned infested leaves, about 30 to 50 leaves were taken below. randomly from each of the selected orchards on Yamamura (1998) developed a modification of every survey date. After the number of leafmines the Kiritani-Nakasuji-Manly method (the modified was counted, a section containing a single leafmine KNM method) that gave an estimate of the number was excised from the leaf, and placed in a polyeth- of individuals entering a stage based on the fre- ylene bag (5 cmϫ7 cm and 0.04 mm in thickness) quency of two stages, when the stage duration was ϭ with a zipper. At this time, the stadium and the via- known. When Ni the number of individuals enter- ϭ bility of each leafminer larva were checked under a ing the ith stage, di the duration of the ith stage, binocular stereoscopic microscope. The bags were fϭthe probability of survival per unit time, and ϭ kept in the laboratory with screen windows for ci the area under the stage-frequency curve plot- ventilation until L. clerkella adult emergence or ted against time; the estimate of N1 is given by: parasitoid adult emergence. Dead L. clerkella lar- c ln( ˆ) vae, from which parasitoids emerged, were re- ˆ ϭ 1 f N1 . (3) ˆd1 Ϫ garded as having been parasitized. In addition, host (f 1) larvae parasitized by a koinobiont parasitoid species, Pholetesor sp. (Adachi, 1998), were also The fˆ can be obtained by a recurrence formula: considered to be dead larvae. Ϫ(/1 d ) c 1 Data analysis. Means and standard errors of the é 1 d2 ù ˆˆϩ ϭϪ1 æ Ϫ1ö , (4) number per leaf of L. clerkella early-stadium (1st ff()jj1 ê c è () øú ë 2 û and 2nd stadium), 3rd stadium, and dead larvae were calculated with the following equations where j indicates the number of improvements of fˆ. (Adachi and Yamamura, 2000): Development times, i.e., larval durations, of L. clerkella necessary for calculating Eqs. (3) and (4) n 1 l Pˆ i were estimated based on the parameters presented xϭ i x , (1) ik by Naruse and Hirano (1990); the developmental l ååϭϭni i 11k zero (in °C) and the effective accumulative temper- and ature (in degree-day) were 9.1 and 68.0 for early- stadium larvae and 8.7 and 44.4 for 3rd stadium 2 n LlϪ 1 l æ Pˆ i ö larvae, respectively. With respect to seasonal vx()ϭ i xxϪ changes in mean temperatures in Tsukuba based on Lll()Ϫ1 ç n ik ÷ åiϭ1 è i åkϭ1 ø ten-year meteorological data from 1985 to 1994, 2 n n the development times for early-stadium larvae 1 l Pˆ 2 i 1 i ϩ i æ Ϫ ö xik xik , (2) were estimated to be 6 and 5 days in the 1st, 2nd, lL nn()Ϫ1 ç n ÷ ååiϭϭ11ii åkϭ1 è i k ø and 7th generations and in the 3rd to 6th genera- tions, respectively; and those for 3rd stadium lar- where Lϭthe total number of peach orchards in the vae were presumed to be 4 and 3 days in the 1st, field, lϭthe number of orchards selected from the 2nd, and 7th generations and in the 3rd to 6th gen- ϭ field, xik the number of in the kth infested erations, respectively. Finally, the number of L. ϭ leaf in the ith orchard, ni the number of infested clerkella larvae entering the early-stadium stage ˆ ϭ leaves examined in the ith orchard, and Pi the esti- was estimated for each generation. mate of the proportion of infested leaves in the ith Additionally, the number of parasitized L. orchard. The 1st term of the right side of Eq. (2) clerkella larvae in each generation was estimated could be ignored when all of the peach orchards as follows. First, the number of host larvae from were selected from the field, i.e., Lϭl. By plotting which parasitoids emerged was determined for the mean densities (vertical axis) against time each leaf through the individual rearing of (horizontal axis), the seasonal prevalence of L. leafmines in the laboratory with screen windows. 350 I. Adachi

Table 1. Generation times of L. clerkella estimated for 1994 and 1995

Start day of each generation Year 1st 2nd 3rd 4th 5th 6th 7th

1994 Apr. 27 May 25 June 24 July 15 Aug. 5 Aug. 29 Sep. 22 1995 Apr. 26 May 26 June 23 July 17 Aug. 7 Aug. 29 Sep. 26

At the same time, the period of time was noted from the initiation of rearing to the parasitoid emergence, i.e., the periods when there were re- maining host larvae to be parasitized. Next, the mean number of parasitized larvae per leaf in the orchards was estimated by Eq. (1), and then the seasonal prevalence was drawn. This enabled the quantification of the area under the prevalence curve for each generation. Lastly, the number of parasitoids was estimated by dividing the area under the prevalence curve by {the mean period of parasitizationϫ2Ϫ1} (see Appendix) or roughly by {the mean period of parasitizationϫ2} in each gen- eration. The division was performed because, in a large population such as that in these peach or- chards, the parasitized larvae were counted repeat- edly while they were in a parasitized status, such that the area under the curve represented not the actual but rather the cumulative number of para- sitized larvae.

RESULTS

Dynamics and generation time Fig. 2. Seasonal prevalence of dead (pale and dark areas) Figure 1 shows the seasonal prevalence of all and parasitized (dark areas) L. clerkella larvae (A) in pesti- live larvae (1st to 3rd instars) of L. clerkella. cide-sprayed peach orchards in 1994, (B) in unsprayed or- Leafmines were already observed in late April be- chards in 1994, (C) in sprayed orchards in 1995, and (D) in cause overwintering adult females began to lay unsprayed orchards in 1995. their eggs on young leaves immediately after the springtime flush. The trend in population density in the seasonal prevalence curves. The start day of differed between peach orchards under different each generation was significantly coincident be- management intensities. In the sprayed orchards, tween the two years (Table 1). the densities were relatively low during the summer when insecticides were sprayed, and often they in- Occurrence of dead and parasitized larvae creased rapidly after the cessation of pesticide ap- Seasonal occurrence of dead larvae showed plication. In contrast, the densities in the unsprayed seven peaks that roughly reflected the number of orchards remained rather constant throughout the generations (Fig. 2). Although the curves corre- year. Irrespective of the difference in the seasonal sponding to each generation were not always dis- prevalence pattern, seven generations were distin- crete, seven occurrences were identified by taking guishable in both years. Generation times were de- into account both the date when the density low- termined by taking into consideration the troughs ered and the generation times. Figure 2 shows also Generational Percent Parasitism on L. clerkella 351

Table 2. MeanϮSEM period of the time (in days) from collection to adult emergence of parasitoids in each generation of L. clerkella

Generation of L. clerkella

1st 2nd 3rd 4th 5th 6th 7th

14.7Ϯ0.1 15.3Ϯ0.2 13.2Ϯ0.3 11.3Ϯ0.2 11.7Ϯ0.2 12.9Ϯ0.3 18.4Ϯ0.8 (nϭ699) (nϭ408) (nϭ134) (nϭ383) (nϭ285) (nϭ222) (nϭ121a)

a Hibernating individuals are not included.

Table 3. Generational percent parasitism of L. clerkella

Generation Parameter 1st 2nd 3rd 4th 5th 6th 7th All gens.

Sprayed orchards in 1994 Number entering each gen.a 0.273 0.458 0.020 0.123 0.631 0.343 0.578 2.426 Number parasitizeda 0.024 0.053 0.011 0.017 0.031 0.040 0.065 0.241 Parasitism rate (%) 8.8 11.6 55.0 13.8 4.9 11.7 11.2 9.9 Unsprayed orchards in 1994 Number entering each gen. 0.187 1.020 0.653 1.822 0.229 0.035 0.281 4.227 Number parasitized 0.059 0.263 0.100 0.219 0.124 0.021 0.049 0.835 Parasitism rate (%) 31.6 25.8 15.3 12.0 54.1 60.0 17.4 19.8 Sprayed orchards in 1995 Number entering each gen. 0.457 0.270 0.128 0.337 1.014 3.889 1.169 7.264 Number parasitized 0.028 0.049 0.010 0.073 0.114 0.198 0.135 0.607 Parasitism rate (%) 6.1 18.1 7.8 21.7 11.2 5.1 11.5 8.4 Unsprayed orchards in 1995 Number entering each gen. 0.568 0.633 1.037 1.302 1.004 0.465 1.841 6.850 Number parasitized 0.045 0.108 0.083 0.443 0.227 0.203 0.160 1.269 Parasitism rate (%) 7.9 17.1 8.0 34.0 22.6 43.7 8.7 18.5

a The number of larvae per leaf. the mean number of parasitized L. clerkella larvae. bers of dead larvae by {these valuesϫ2Ϫ1}, the The proportion of the area formed by parasitized number of parasitized larvae was estimated (Table larvae (dark areas in Fig. 2) with respect to the area 3). formed by all dead larvae (dark plus pale areas in Fig. 2) was larger in the unsprayed orchards than in Percent parasitism the sprayed ones; this ratio indicated the relative Estimates of the number of L. clerkella larvae importance of parasitism as a mortality factor. The entering the early-stadia stage and the number of proportions throughout the whole year in the parasitized larvae in each generation are summa- sprayed and unsprayed orchards were 0.50 and 0.65 rized in Table 3. From these values, percent para- in 1994, and 0.51 and 0.71 in 1995, respectively. sitism for each generation and for all generations From parasitized L. clerkella larvae, 18 parasitoid throughout the year was calculated. In 1994 and species emerged in 1994 and 1995 (cf. Adachi, 1995, the yearly parasitism rates in the unsprayed 1998). Of these, several species such as orchards were 19.8% and 18.5%, and those in the Chrysocharis nitetis were predominant. sprayed orchards were 9.9% and 8.4%, respec- Table 2 shows the mean periods of time from the tively. initiation of rearing to parasitoid emergence in each generation. By dividing the cumulative num- 352 I. Adachi

Fig. 4. Relationship between the percent parasitism in a generation and the host, L. clerkella, density at the previous generation in unsprayed peach orchards (A) in 1994 (Yϭ 0.09Xϩ0.25, r2ϭ0.08, pϭ0.59) and (B) in 1995 (Yϭ0.29XϪ 0.02, r2ϭ0.44, pϭ0.15).

Fig. 3. Relationship between the number of L. clerkella larvae entering a generation and the number of parasitized lar- DISCUSSION vae in the same generation in unsprayed peach orchards (A) in 1994 and (B) in 1995. Numbers in the figure indicate the order Since L. clerkella is widely distributed in Japan, of the generation. the number of generations subjected to particular temperatures varies by locality. For example, the grows for five or six generations in Yamagata Density relatedness Prefecture (Shoji and Ueno, 1981), six to seven The relationship between the number of larvae generations in both Toyama (Naruse, 1978b) and entering a generation and the number of parasitized Hiroshima Prefectures (Fujiwara et al., 1978), and larvae in the same generation was examined in the nine generations in Kagoshima Prefecture (Miyaji, unsprayed orchards. When the point for each gen- 1991). The seven generations in Tsukuba, as ob- eration was traced in turn, the tendency of a coun- served in the present study, fell within this ex- terclockwise rotation was observed in both years pected range. It is of note in all of these studies that (Fig. 3), suggesting cyclic fluctuations with differ- successive generations hardly overlapped, despite ent phases of L. clerkella and parasitoid popula- repeated generations per year. This may be largely tions. due to a short oviposition period. Discrete genera- When the percent parasitism in a particular gen- tions were similarly observed in Lyonetia pruni- eration was plotted against the host density in the foliella malinella, a congeneric species of the same generation in the unsprayed orchards, the re- peach leafminer, on apple trees (Sekita and Ya- gression line was YϭϪ0.20Xϩ0.43 (r2ϭ0.42, pϭ mada, 1979). The longevity of L. clerkella adult fe- 0.11) in 1994 and YϭϪ0.09Xϩ0.29 (r2ϭ0.09, pϭ males is reported to be 6 to 7 days (Shoji et al., 0.52) in 1995, showing a tendency toward a some- 1982), and the longevity and the preoviposition pe- what inverse density-dependence. In contrast, when riod of L. prunifoliella females were 8.3 and 3.3 the percent parasitism in a generation was plotted days, respectively (Schmitt et al., 1996). against the host density at the previous generation, To evaluate the impact of natural enemies, a life regression lines exhibited density-dependence (Fig. table analysis can be undertaken that estimates 4), although they were not significant at the 5% numbers of entering generations and numbers level (pϭ0.59 in 1994 and pϭ0.15 in 1995). This dying within stages. In general, life tables can be result indicated that delayed density-dependent constructed in one of two ways (Southwood, 1978). processes potentially functioned in a host-para- The first approach employs a horizontal life table; sitoid system. the fate of a real cohort is determined over the course of time for each of a series of stages. The other approach utilizes a vertical life table; the age structure of a population is examined at successive Generational Percent Parasitism on L. clerkella 353 points in time. In the present study, the latter ap- sitoid densities (Fig. 3) and to a delayed density- proach was used because the former required too dependence between host densities and parasitism much time and labor and was therefore not applica- rates (Fig. 4). ble in the context of a wide-area investigation, even Pest insects in deciduous fruit trees are often though a more accurate estimation would have classified into key, sporadic, and secondary pests. been obtained by the former method. In the present The secondary pests include leafminers, aphids, study, the number of larvae entering generations and mites, which feed on foliage and other nonfruit was estimated by stage-frequency analysis using parts of the tree. For managing such secondary only live larvae. This approach may have resulted pests, biological control will continue to play a in an underestimation of L. clerkella recruitment dominant role. There are several ways to utilize because larvae that hatched and died during two parasitoids, including: (1) importation and estab- successive census dates (2 or 3 days) were ex- lishment of exotic parasitoids; (2) augmentation of cluded. More precise estimation of recruitment endemic or exotic parasitoids through mass-cultur- would be gained by adding the number of dead 1st- ing and periodic release; (3) conservation through stadium larvae, although, in the present study, such cultural manipulation and selective pesticide use; larvae were few in number. (4) development of genetically modified strains of The types of host mortality caused by para- parasitoids; and (5) a combination of these tactics sitoids were not restricted to direct parasitism; host (Aliniazee and Croft, 1999). Of these, the conser- feeding, ovipositor piercing and envenomization, vation of endemic parasitoids should be promoted enhanced susceptibility to other factors, and lost in order to control leafminers (Van Driesche et al., natality (i.e., reduced fertility of parasitized adult 1998). The present study demonstrated that the hosts prior to death due to parasitism) were among parasitism rates in the unsprayed orchards were the other causes of mortality (Bellows and Van over two times those of the sprayed orchards. In ad- Driesche, 1999). It has been demonstrated that host dition, the composition of parasitoid species should feeding is nearly ubiquitous in some genera such as be noticed. A parasitoid can affect the attack rates Tetrastichus (Bartlett, 1964). As L. clerkella is at- of other parasitoids through interactions such as tacked by two Tetrastichus species (Adachi, 1998), multi-parasitism and hyper-parasitism. In combina- the leafminer is certainly affected by host feeding. tion with the practical use of parasitoids, it will be However, such losses are difficult to quantify in of continued use to experimentally analyze the field populations; this difficulty could then lead to marginal attack rate (Royama, 1981) of each para- an underestimation of percent parasitism in a sitoid species and the death rates of hosts achieved broader sense that parasitism involves host feeding by contemporaneous parasitoid species (Elkinton et (Van Driesche, 1983). al., 1992). From the perspective of biological control, natu- ral enemies are expected to contribute to reduced ACKNOWLEDGEMENTS and stable pest densities. In other words, a new pest I thank anonymous referees for their invaluable suggestions. density would be expected to be lower than a previ- ous density, and would exhibit fewer fluctuations REFERENCES than a population without natural enemies (Bel- Adachi, I. 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The follow- English summary). ing notations are used: Royama, T. (1981) Evaluation of mortality factors in insect ϭ nt the number of hosts parasitized on the tth life table analysis. Ecol. Monogr. 5: 495–505. day (1ϹtϹT), Sato, R., N. Abe, H. Sugie, M. Kato, K. Mori and Y. Tamaki ϭ (1986) Biological activity of the chiral sex pheromone of l the period of time from parasitization to para- the peach leafminer moth, Lyonetia clerkella Linne. sitoid emergence (constant for simplicity), Appl. Entomol. Zool. 21: 478–480. Nϭthe real number of parasitized hosts through- Schmitt, J. J., M. W. Brown and D. R. Davis (1996) , out a generation (ϭ T ), åtϭ1nt morphology, and biology of Lyonetia prunifoliella (Lepi- ϭ doptera: Lyonetiidae), a leafminer of apple. Ann. Ento- S the total number of parasitized hosts observed mol. Soc. Am. 89: 334–345. throughout the generation, and Sekita, N. (1987) Fat body of overwintering adults and hiber- mϭthe mean period from the observation of par- nation patterns in Lyonetia prunifoliella malinella and asitized hosts to the parasitoid emergence from the Lyonetia clerkella. Tohoku Kontyu 25: 1–3 (in Japanese). hosts. Sekita, N. and M. Yamada (1979) Life history of Lyonetia prunifoliella Hubner subsp. malinella (Matsumura) It is presumed that the parasitoids are not killed (Lepidoptera: Lyonetiidae). Appl. Entomol. Zool. 14: while they develop. 285–292. In this case, the number of hosts that are ob- Generational Percent Parasitism on L. clerkella 355

ϩϪ ϩϪ served to be parasitized on the ith day ist n . Tl1 t Tl1 t åitlϭϪϩ1 i mnltinϭϪϩϬ() , Hence, S is given by TlϩϪ1 t . Note that S i i åtϭ1 åitlϭϪϩ1ni ååtϭ1 itlϭϪϩå 1 åtϭ1 itlϭϪϩ1 corresponds to the dark area in Fig. 2. Because TlϩϪ1 l TlϩϪ1 ϭϬni ln , niϭ0 when iϽ1 or iϾT, S becomes t t åååtϭ1 iϭϭ11t T T TlϩϪ1 T ll()ϩ1 ϭ Ϭ ϭϭϭ nlt nt , SlnlNlnt t . (A1) 2 ååtϭϭ11t ååtϭ1 tϭ1 lϩ1 ϭ . (A2) Next, let us consider the period from the date 2 when a host is observed to be parasitized to the date when a parasitoid emerges from the host. If Consequently, the following relationship is ob- the host that is parasitized on the tth day is ob- tained from Eqs. (A1) and (A2), served on the ith day, a parasitoid will emerge Ϫ ϩ S (l t i) days later. Therefore, m is given by Nϭ . 21mϪ