Effect of Temperature on Development and Survival of Cirrospilus sp

BIOLOGICAL CONTROL

Effect of Temperature on Development and Survival of Cirrospilus sp. near lyncus (Hymenoptera: Eulophidae), Parasitoid of Phyllocnistis citrella (Lepidoptera: Gracillariidae) ´ SCAR TOMA´S, ANTONIO GARRIDO, ALBERTO URBANEJA, ELENA LLA´CER, O AND JOSEP-ANTON JACAS Departament de Proteccio´ Vegetal i Biotecnologia; Institut Valencia` dÕInvestigacions Agra`ries; Ctra. de Montcada a Na`quera km 5; E-46113-Montcada, Spain

Environ. Entomol. 28(2): 339Ð344 (1999)

ABSTRACT Cirrospilus sp. near lyncus is one of the most abundant indigenous parasitoids of the citrus leafminer Phyllocnistis citrella Stainton found in Spain. In this study, data on the inßuence of constant temperatures, ranging from 1 to 408C, on survival and development of the parasitoid are presented. Complete development only occurred between 10 and 358C, but eggs and pupae could survive at lower and higher temperatures, respectively. Upper and lower development thresholds were estimated to occur at 37.8 and 8.88C, respectively. Maximal development rate occurred at 31.88C and thermal constant was 182.0 DD. Therefore, under typical Mediterranean climatic conditions, development of C. sp. near lyncus could continue throughout the year. Because P. citrella populations usually collapse at the beginning of winter, and do not become conspicuous until the end of spring, provision of alternative hosts within citrus orchards would presumably favor the winter permanence of opportunistic C. sp. near lyncus, and thus increase the impact of this parasitoid early in the season, when parasitism on P. citrella is very low. KEY WORDS Cirrospilus sp. near lyncus, Phyllocnistis citrella, biological control

EXCEPT FOR SOME notable exceptions (Parrella 1987), leafminer insects often cause limited damage to their host plants and are therefore considered as secondary pests frequently regulated by their natural enemies (Hespenheide 1991). The citrus leafminer, Phyllocnistis citrella Stainton (Lepidoptera, Gracillariidae) is not an exception to this trend. However, recent spreading of this pest into different parts of the world (CABI 1995) resulted in such explosive outbreaks that at that time it was considered a very serious menace to citrus industry (Hoy and Nguyen 1997). Studies carried out recently have demonstrated that under Mediterranean conditions, leafminer damage on adult trees usually lacks economic importance (Gonza´lez 1997, Granda et al. 1997) and therefore, P. citrella should only be considered an important pest for young and top-grafted trees. Furthermore, leafminer natural enemies can play a very important role in regulating pest populations. In Spain, as well as in other citrus-growing countries, indigenous natural enemies opportunistically recruited onto P. citrella comprise several nonspeciÞc predators, such as lacewings, minute pirate bugs, ants, spiders, and a guild of parasitoids where generalist eulophids prevail. These parasitoids are as follows: Baryscapus sp., Chrysocharis pentheus (Walker), Chrysocharis sp., Cirrospilus sp. near lyncus, Cirrospilus pictus Nees, Cirrospilus vittatus Walker, Diglyphus isaea Walker, Neochrysocharis formosa (Westwood), Pnigalio agraules (Walker), Pnigalio pectinicornis L., Pnigalio soemius (Walker), Ratzebur-

giola cristata (Ratzeburg), Sympiesis gordius (Walker) and Sympiesis gregori Boucek, and an unidentiÞed Pteromalidae species (Pteromalus sp.). In addition, a few exotic species have been introduced (Garrido and Del Busto 1994; Vercher et al. 1995, 1997; Gonza´lez et al. 1996; Garcõ´a et al. 1997; Lacasa et al. 1997; Schauff et al. 1998; Urbaneja et al. 1998a). Field studies aimed at ascertaining the role and importance of these natural enemies in regulating P. citrella populations have pointed out the increasing relevance of C. sp. near lyncus (Urbaneja et al. 1998a). This species, which had not been recorded in Spain before the introduction of the P. citrella, was invariabily found, together with P. pectinicornis, as the most abundant species. Although this parasitoid can be found all over the Mediterranean Basin (FAO 1996, Schauff et al. 1998), almost no data on its biology are available. In a previous study, Urbaneja et al. (1998c) presented laboratory data obtained at 208C: an adult survival of '50 d, a development time of 17 d, and a fecundity of 235.2 eggs per female, and established that this species is arrhenotokous. The objective of the current study was to determine the inßuence of temperature on survival and development of C. sp. near lyncus under controlled laboratory conditions. Materials and Methods The rearing of C. sp. near lyncus included the production of young sour orange plants, Citrus aurantium

0046-225X/99/0339Ð0344$02.00/0 q 1999 Entomological Society of America

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L., and P. citrella forms susceptible for parasitism. Methodology was fully described by Urbaneja et al. (1998b) and is very similar to that developed by Smith and Hoy (1995) for P. citrella parasitoids Ageniaspis citricola Logvinovskaya (Hymenoptera: Encyrtidae) and Cirrospilus ingenuus Gahan [5C. quadrastiatus (Suba Rao & Ramamani)] (Hymenoptera: Eulophidae). The rearings took place in a greenhouse at 25 6 58C and 60 6 10% RH, and production schemes were executed on a weekly basis. No artiÞcial light was supplied. Insects used for the rearings were initially obtained from different citrus orchards located at the Institut Valencia` dÕInvestigacions Agra`ries (IVIA). In addition, Þeld-collected individuals were periodically introduced into the rearings. Voucher specimens were deposited at the Reference Insect Collection of IVIA, and at the British Museum of Natural History (Schauff et al. 1998). Plant Production. Two-year-old potted sour orange trees were pruned to obtain homogeneous young ßushes. The optimal state for introduction of these plants into the P. citrella rearing cages was attained when oldest leaves of the new ßushes measured '4 mm. Rearing of P. citrella. Adult leafminers for the infestation of new plants were obtained from a continuous culture. Groups of 4 plants were introduced weekly into a screened cage (80 by 110 by 120 cm) where the rearing took place. Plants were left undisturbed for 3 wk. Such a long period allowed moths to emerge within the cage so that artiÞcial reinfestation was not necessary. Adult moths were fed a mixture of honey and water (1:3, vol:vol) directly sprayed onto the small trees. When needed, adults were collected and used in our assays. Phyllocnistis citrella larvae for parasitization were reared in another cage (same dimensions as above). In this case, groups of 16 plants were exposed to '300 adult P. citrella. One day later, plants were removed from those cages and kept undisturbed for '7 d. By then, most of the leaves were housing P. citrella 3rd instars, which is the preferred instar both for parasitization and host feeding by C. sp. near lyncus. Rearing of C. sp. near lyncus. Groups of 8 P. citrella infested plants were introduced into a screened cage (same dimensions as above) where 40, presumably mated, 4-d-old C. sp. near lyncus pairs were released and left undisturbed for 11 d. Adult wasps were fed a mixture of honey and water (1:3, vol:vol). At the end of the exposure period, plants were removed and their leaves collected and wrapped with tissue paper and kept in a plastic bag until adult emergence. Emerged parasitoids were collected daily using an aspirator and either used in our assays or released into the rearing cages. Assessment of Development on Seedlings Versus Detached Leaves. To determine the most efÞcient way of assessing development of C. sp. near lyncus, a preliminary assay was carried out on young citrus seedlings. These results were compared with those obtained using detached citrus leaves under the same environmental conditions.

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Five-month-old sour orange seedlings with 1 or 2 young leaves were introduced into the P. citrella continuous rearing cage. One day later, plants were removed and left undisturbed for 1 wk until leafminers attained the 3rd instar. At that moment, plants were transferred to the C. sp. near lyncus rearing cage and left there for 1 d. These were then carefully checked under a stereoscopic binocular microscope and those exhibiting hosts parasitized with 1 single egg were further followed up to pupation. Plants were placed in a climatic chamber at 20 6 18C, 60 6 5% RH, and a photoperiod of 16:8 (L:D) h. Twenty-Þve immature parasitoids were observed daily until pupation. Five days after pupation, leaves were detached and kept individually on a layer of agar (2% weight) in a petri dish (55 mm diameter) until adult emergence. Adults were then sexed. To simplify the methodology, the same study was performed on detached leaves. These were obtained from plants housing P. citrella 3rd instars. Leaves in groups of 12 were deposited on a layer of agar (2% weight) in petri dishes (140 mm diameter). Three presumably mated female parasitoids were released into each dish and left undisturbed. Four hours later, leaves were checked under stereoscopic binocular microscope, and 40 parasitized hosts transferred to a climatic chamber where parasitoid development was checked daily until adult emergence. Eventually, adults were sexed. Development at 1–40&C: Detached Leaves. Further studies at 1, 6, 8, 10, 13, 15, 20, 25, 30, 35, 38, and 408C were performed on detached leaves. Methodology was the same as described above, but after parasitization, petri dishes containing the detached leaves were transferred to the corresponding experimental temperature. To estimate egg mortality occurring at the lowest temperature (18C), apparently healthy eggs that had not eclosed during 14 d of exposure, were transferred to a climatic cabinet at 258C, where parasitoids were allowed to complete their development. Temperature Thresholds and Thermal Constant. Once development times (y) were established for each temperature treatment, development rates (y21) were calculated. These rates were plotted against temperatures and Þtted with modiÞcation 2 of Logan model for nonlinear regression (Lactin et al. 1995; Logan et al. 1976): r~T! 5 er T 2 e@ r Tmax2~Tmax2T!/D# 6 l, where r(T), development rate at temperature T and r, T max, D and l, Þtted parameters. Temperature thresholds and maximal development rate were estimated from that regression. Lower temperature threshold was also estimated from a linear regression. Based on the lower temperature threshold obtained, the thermal constant was calculated by use of the following equation (Varley et al. 1974): K 5 ( @ yi ~ti 2 x!#/n,

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URBANEJA ET AL.: DEVELOPMENT AND SURVIVAL OF C. SP. NEAR lyncus

Table 1. Mean development time (days) of male and female C. sp. near lyncus and stage specific survival percentages at 20°C when reared on P. citrella on citrus seedlings (mean 6 SE) Egg

Larva

Pupa

Total

Male 1.29 6 0.27a 6.14 6 0.46a 9.59 6 0.43a 17.02 6 0.50a (n 5 11)a Female 1.21 6 0.26a 6.18 6 0.24a 9.64 6 0.48a 17.03 6 0.45a (n 5 11)a Survival 96.0 95.8 95.7 88.0 (%) Within a column, means followed by the same letter are not signiÞcantly different (P . 0.05; LSD test). a Initial number was 25 eggs.

where K, thermal constant; yi, development time; ti, temperature; x, lower temperature threshold; and n, replicates. Adult Survival. Recently emerged C. sp. near lyncus adults were individually kept in glass vials under the following experimental temperatures: 15, 20, and 258C. Half of them were fed a mixture of honey and pollen (30:1 wt:wt), in small droplets directly deposited inside the vial. The other half received no food at all. Twelve males and 12 females were considered in each case. Statistical Analyses. Where appropriate, results were subjected to either one- or two-way analysis of variance (ANOVA) and the t-test was used for mean separation at P , 0.05 (STSC 1987). If necessary, data were transformed [ln(x)] before the analysis. Linear regression was calculated using the same software package mentioned above. Regression curve was Þtted by iterative nonlinear regresssion (SAS Institute 1989). Results Development at 20&C: Seedlings Versus Detached Leaves. Duration of the different stages of C. sp. near lyncus when reared on citrus seedlings are shown in Table 1. No signiÞcant differences were detected between sexes. When development time obtained on citrus seedlings at 208C was compared with that obtained using detached citrus leaves under the same temperature (Table 2), it appeared that there were no differences between sexes (F 5 0.048; df 5 1, 50; P 5

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Table 3. Stage specific survival percentage of C. sp. near lyncus at different temperatures (°C) when reared on P. citrella on detached citrus leaves Temp, 8C

EggÐLarva

LarvaÐPupa

PupaÐAdult

Total

1 6 8 10 13 15 20 25 30 35 38 40

0 0 0 91.7 83.9 96.9 97.1 93.9 97.0 89.7 0 0

Ñ Ñ Ñ 54.5 69.2 93.1 91.2 93.6 80.7 62.9 Ñ Ñ

Ñ Ñ Ñ 41.7 88.9 100 100 100 100 86.4 Ñ Ñ

0 0 0 20.8 51.6 90.2 88.6 87.9 78.1 48.7 0 0

0.8303), between methods (F 5 3.56; df 5 1, 50; P 5 0.0650), and there was no interaction between these factors (F 5 0.064; df 5 1, 50; P 5 0.8044). Therefore, the detached leaf method was chosen for further assays. Development at 1–40&C: Detached Leaves. Development only occurred between 10 and 358C (Table 2). Eggs exposed to temperatures below 108C did not hatch, but 100% egg mortality was not reached even at 18C. When eggs exposed to that temperature for 2 wk were transferred to a cabinet at 258C, 44.4% egg eclosion occurred. In contrast to these results, 100% egg mortality occurred both at 38 and 408C. Within the limits mentioned, development time shortened as the temperature increased. However, at 358C development time increased, and adult wasps obtained under this temperature presented a light yellow color in contrast to the intense pigmentation usually exhibited by this species. Furthermore, when 3 females obtained at that temperature were offered P. citrella 3rd instars for parasitization daily during 20 d, no oviposition occurred. Development times obtained were statistically different for sexes (F 5 14.03; df 5 1, 140; P 5 0.0003). The effect of lower temperatures appeared to be stronger on females than on males. Survival of the different stages is shown in Table 3. The larva was the stage most sensitive to temperature, whereas both eggs and pupae were more robust. Survival decreased when approaching the extreme temperatures tested. Although neither eggs nor larvae

Table 2. Mean development times (days) of male and female C. sp. near lyncus at different temperatures (°C) when reared on P. citrella on detached citrus leaves (mean 6 SE) Sex

Stage

108C

138C

158C

208C

258C

308C

358C

Male

Egg Larva Pupa Total

Female

Egg Larva Pupa Total

9.10 6 0.98 34.5 6 2.94 50.50 6 16.66 94.50 6 12.74 (n 5 2) 9.00 6 3.92 33.50 6 6.86 68.00 6 1.96 110.5 6 0.98 (n 5 2)

3.81 6 0.71 20.25 6 1.87 21.00 6 3.27 45.06 6 2.79 (n 5 8) 4.38 6 0.73 20.44 6 2.07 24.44 6 2.73 49.13 6 3.65 (n 5 8)

2.68 6 0.14 9.46 6 0.58 15.00 6 0.38 27.13 6 0.47 (n 5 23) 2.35 6 0.42 9.10 6 0.52 16.39 6 0.84 29.51 6 2.17 (n 5 6)

1.42 6 0.07 5.56 6 0.20 9.56 6 0.33 16.54 6 0.38 (n 5 26) 1.48 6 0.14 5.67 6 0.41 9.25 6 1.00 16.40 6 0.38 (n 5 6)

1.46 6 0.03 4.52 6 0.31 5.60 6 0.21 11.58 6 0.37 (n 5 25) 1.46 6 0.08 4.13 6 0.24 6.25 6 0.49 11.83 6 0.56 (n 5 4)

0.98 6 0.06 2.90 6 0.08 4.54 6 0.15 8.42 6 0.17 (n 5 23) 0.93 6 0.04 3.00 6 0.00 4.25 6 0.49 8.18 6 0.53 (n 5 2)

0.78 6 0.11 3.06 6 0.18 5.93 6 0.23 9.75 6 0.30 (n 5 15) 0.86 6 0.23 2.88 6 0.24 7.00 6 0.00 10.77 6 0.42 (n 5 4)

Initial numbers were 40 eggs for each temperature.

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Fig. 1. Development rate (days21) of C. sp. near lyncus when reared on P. citrella on detached citrus leaves plotted against temperature (8C). Fitted curve: Logan model and modiÞcation 2.

could complete their development at 388C, when healthy pupae obtained at 258C were exposed to this temperature, 37.5% of them (n 5 16) reached the adult stage. Temperature Thresholds and Thermal Constant. Development rates were Þt with a nonlinear regression (Lactin et al. 1995, Logan et al. 1976) (Fig. 1) [r(T) 5 erT 2 e[rTmax 2 (Tmax 2 T)/D] 6 l; r 5 0.0964 6 0.0013; D 5 4.0290 6 0.1305; l 5 20.7246 6 0.1092, estimate 6 asymptotic standard error]. The upper and lower development thresholds estimated from this equation were 37.8 and 23.38C, respectively, and the maximal development rate occurred at 31.88C. Because the lower development threshold was considered unrealistic (no development was observed for temperatures below 88C, Table 3), development rates between 10 and 308C were further Þt with a linear regression (y 5 0.5492x 2 4.7986; r2 5 0.9785; F 5 6,005.28; df 5 1, 132; P 5 0.0016). The lower development threshold estimated from this equation was 8.88C. Using this estimate of the lower development threshold, a thermal constant of 182.0 6 3.1 DD (n 5 135) was calculated. Adult Survival. Survival of adult C. sp. near lyncus is presented in Table 4. Unfed specimens had a very short life, ranging from 2 to 3 d, whereas adults provided food survived between 27 and 100 d, depending on the temperature. These differences were signiÞcative (F 5 18.010, df 5 2, 65; P , 0.0001). However, no signiÞcant differences were found between sexes (F 5 0.347; df 5 1, 65; P 5 0.5645).

Discussion As expected, the duration of the life cycle and adult survival decreased as temperature increased from 10 to 308C. Nevertheless immature stages suffered higher mortality rates both at high (.308C) and low (,158C) temperatures. In general, female development appeared to be longer than male, and females lived longer than males. However, these differences were not signiÞcative in all cases. These traits are quite common to most insect species, and similar results have been obtained on different eulophid leafminer parasitoids, like Cirrospilus pictus on Phyllocnistis labyrinthiella Bjerkander (Sundby 1957), Quadrastichus sp. (Lla´cer et al. 1998), and Pnigalio minio (Walker) (Duncan, R., and Pen˜ a, J., TREC-University of Florida, personal communication) on P. citrella, Diglyphus isaea on Liriomyza spp. (Diptera: Agromyizidae) (Minkenberg 1989), or different wasps (Sympiesis Table 4. Sex specific survival (days, mean 6 SE) of adult C. sp. near lyncus when either fed a mixture of pollen and honey or unfed at different temperatures (°C) Sex

Fed/Unfed

158C

208C

258C

Female

Unfed

3.8 6 0.4 (n 5 10) 100.7 6 25.5 (n 5 9) 3.5 6 0.9 (n 5 12) 72.2 6 26.0 (n 5 9)

3.3 6 0.1 (n 5 12) 59.7 6 8.2 (n 5 12) 3.9 6 0.6 (n 5 12) 46.3 6 25.0 (n 5 7)

2.2 6 1.1 (n 5 12) 27.2 6 10.3 (n 5 12) 2.0 6 0.2 (n 5 12) 45.0 6 19.8 (n 5 12)

Fed Male

Unfed Fed

Initial numbers were 12 adults for each treatment.

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URBANEJA ET AL.: DEVELOPMENT AND SURVIVAL OF C. SP. NEAR lyncus

marylandensis Girault, S. sericeicornis Nees and P. minio [5P. flavipes (Ashmead)]) on Phyllonorycter blancardella F. (Lepidoptera: Gracillariidae) (Ridgway and Mahr 1990, Hagley and Barber 1991). Temperatures registered along the western shore of the Mediterranean Basin usually remain within the same limits obtained for C. sp. near lyncus. In Valencia, for example, the mean of maximum temperatures of the hottest month (August) is 29.08C and the mean of minimum temperatures of the coldest month (January) is 7.18C. However, even in January the mean temperature is 11.58C. That means that both upper and lower lethal temperatures are very rarely attained. Minkenberg (1989) and Minkenberg and Helderman (1990) demonstrated both for L. bryoniae (Diptera: Agromyizidae) and its eulophid ectoparasitoid D. isaea that the effect of an alternating temperature regime on development parameters could be predicted from regression equations obtained at constant thermoregimes. If we take for granted that this is also true for C. sp. near lyncus, we can infer that even during the coldest months parasitoid development should not necessarily stop because January mean temperature does not reach the lower temperature threshold (8.88C). Furthermore, for poikilothermic animals, lower temperature thresholds estimated from linear regressions are usually higher than actual values, because of the sigmoid type of response found when plotting development rates against temperatures (Laudien 1973). Hence, it is presumed that the life cycle of C. sp. near lyncus could continue even during the coldest months under Mediterranean weather conditions. According to mean temperatures registered in Valencia and to the estimated thermal constant, C. sp. near lyncus could complete its cycle in '12 d in summer and up to 90 d in winter. The insect did not exhibit arrested development because of winter daylight shortage (our winter rearings are kept in greenhouse under natural light). Thus, it appears that C. sp. near lyncus would be able to develop continuously throughout the year provided that suitable hosts are available. P. citrella populations usually crash at the beginning of winter because of the combined effect of natural enemies (Urbaneja et al. 1998a), lower temperatures (Minsheng et al. 1995) and, especially, the lack of citrus ßushing (Jacas et al. 1997). In Spain, new ßush is rarely found until the beginning of spring, and P. citrella usually does not become conspicuous until the end of that season (June). That means that, even if C. sp. near lyncus could endure winter temperatures, the lack of P. citrella larval stages would presumably force it to look for its original hosts. Cirrospilus sp. near lyncus was Þrst noticed in Spain as a parasitoid of P. citrella, therefore its original host range remains unknown. Nevertheless, the host range of the genus Cirrospilus sp. in the Palearctic region consists mainly of lepidopteran leafminers (Boucek and Askew 1968). Leafminer parasitoid species are usually shared on the basis of plant host type (Hespenheide 1991): in this case, trees and shrubs versus herbs, and, indeed, a study on parasitoids of leafminers attacking horticultural crops in Valencia (Verdu´ 1991)

343

did not mention any of the most abundant species recorded later on P. citrella. If we keep in mind that at a regional level, citrus can be considered a monoculture in most Spanish citrus growing areas, it will become clear that when looking for alternative hosts, opportunistic parasitoids, such as C. sp. near lyncus, would probably be forced to abandon the area, and this could result in a delayed response of parasitoids to P. citrella early increasing populations, as it is actually observed in the Þeld (Urbaneja et al. 1998a). Therefore, as a 1st step in implementing conservation strategies aimed at favoring the winter permanence of C. sp. near lyncus in citrus orchards, its host range should be determined. In summary, our results have shown that C. sp. near lyncus is very well adapted to climatic conditions prevailing in the western part of the Mediterranean Basin. Nevertheless, its important role in regulating P. citrella populations has a serious handicap because this exotic host becomes extraordinarily rare during the winter months. Acknowledgments L. Navarro (IVIA, Montcada, Spain) and J. E. Pen˜ a (University of Florida, Homestead, USA) provided useful comments on an early draft of the manuscript. The authors thank R. Hinarejos for taking care of the rearings, M. J. Verdu´ (IVIA, Montcada, Spain) for taxonomical assistance, and E. Carbonell (IVIA, Montcada, Spain) for statistical advice. This work was partially funded by the Instituto Nacional de Investigacio´ n y Tecnologõ´a Agraria y Alimentaria, INIA, and the Conselleria dÕAgricultura, Pesca i Alimentacio´ de la Generalitat Valenciana. AU. and E.L. were recipents of a grant from IVIA.

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