Exploitation of the serpentine leafminer Liriomyza trifolii and tomato leafminer L. bryoniae (Diptera: Agromyzidae) by the parasitoid Gronotoma micromorpha (Hymenoptera: Eucoilidae)

The developmental time and size of a solitary koinobiont parasitoid, Gronotoma micromorpha (Perkins) (Hymenoptera: Eucoilidae), were measured in two host species: the serpentine leafminer, Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) and tomato leafminer, L. bryoniae (Kaltenbach). There was no significant difference in the developmental time of G. micromorpha in these two hosts. However, significantly larger G. micromorpha adults emerged from L. bryoniae than from L. trifolii puparia. Dissection of larvae revealed that when offered a choice G. micromorpha accepted larvae of L. bryoniae more often than those of L. trifolii. The number of wasps emerging from parasitized hosts did not differ significantly between host species. These results indicate that L. trifolii and L. bryoniae are both acceptable and suitable hosts for G. micromorpha. Gronotoma micromorpha may be a useful biological control agent of both L. trifolii and L. bryoniae.

Liriomyza trifolii is indigenous to the New World (Spencer, 1973), but has extended its geographical range to Asia, Africa and Europe (Saito, 1997).In contrast, L. bryoniae is of Palaearctic origin (Sasakawa, 1961;Spencer, 1973).Liriomyza trifolii and L. bryoniae are similar in morphology, so it is not easy for growers to distinguish between them.Both leafminers are polyphagous attacking vegetables and ornamentals (Minkenberg & van Lenteren, 1986).The total developmental times (oviposition to adult emergence) of L. trifolii and L. bryoniae on kidney bean, Phaseolus vulgaris L., are 16.5 and 19.3 days, respectively, at 25°C (Tokumaru & Abe, 2003).
The braconid Dacnusa sibirica Telenga and the eulophid Diglyphus isaea (Walker), originally used for the biological control of L. trifolii and L. bryoniae in Europe, have been imported and released in Japanese greenhouses to control Liriomyza leafminers (Ozawa et al., 2001).Japanese greenhouses are usually small and kept at high temperatures and humidities (Yano, 1993).The effectiveness of D. sibirica may be restricted in Japan, because this wasp is less effective at controlling leafminers on tomato at high temperatures (Minkenberg, 1990).
In contrast to D. sibirica, D. isaea is a more effective parasitoid at high temperatures (Minkenberg, 1989).
Diglyphus isaea occurs in Japan, where it is the dominant parasitoid attacking the garden pea leafminer, Chromatomyia horticola (Gourea) (Takada & Kamijo, 1979).The potential risks of introducing exotic natural enemies has recently received attention and the potential of hybridization needs to be included in risk analysis (van Lenteren et al., 2003).Many naturalists believe strongly that native genotypes should be preserved and that introduction of foreign genes constitutes genetic pollution (Williamson, 1996).Greenhouses in Japan are not completely insect proof (Yano, 1993) so hybridization between European and Japanese populations of D. isaea may have occurred.
Male-biased sex ratios may be common in Diglyphus (Heinz & Parrella, 1990).Such male-biased sex ratios need to be carefully monitored and manipulated in the mass production of these parasitoids (Rathman et al., 1991).Moreover, in mass rearing thelytokous wasps do not use expensive hosts for the production of males (Stouthamer, 1993).Thus, thelytokous parasitoids are more suited to mass production than arrhenotokous ones (Abe & Tahara, 2003).
The solitary koinobiont parasitoid, Gronotoma micromorpha (Perkins), occurs in subtropical regions, i.e., Okinawa, Florida, Hawaii, Guam and Tahiti (Yoshimoto, 1963;Beardsley, 1988;Abe & Konishi, 2004), and appears to be the dominant parasitoid of L. trifolii on Okinawa, Japan (Konishi, 1998), and Guam, USA (Johnson, 1993).Under laboratory conditions, this parasitoid has a high net reproductive rate when it parasitizes the host L. trifolii (Abe & Tahara, 2003).Wolbachia infection induces thelytoky in this wasp (Arakaki et al., 2001).This thelytokous wasp is an egg-pupal and larval-pupal parasitoid (Abe, 2001).Gronotoma micromorpha can develop at most of the temperatures at which L. trifolii damages crops, and the combination of high temperature and short photoperiod found in Japanese greenhouses in winter does not adversely affect its development (Abe & Tahara, 2003).The developmental rate of G. micromorpha increases linearly with increasing temperature between 18 and 30°C.The lower thermal thresholds for complete development and oviposition are 11.7°C and approximately 18°C, respectively (Abe & Tahara, 2003).These results indicate that should G. micromorpha escape from greenhouses, where they were released for controlling leafminers, they cannot overwinter in Japan, except in the subtropical regions.To clarify the potential of G. micromorpha as a biological control agent of L. trifolii and L. bryoniae, the acceptability and suitability of these two Liriomyza species for this parasitoid were determined.

Insects and plants
Laboratory cultures of G. micromorpha and L. trifolii were established from parasitized and non-parasitized L. trifolii larvae collected in Itoman City, Okinawa Prefecture, Japan, in April 1998 (Abe, 2001).The maintenance of stock colonies, and preparation of hosts and parasitoids for experiments are described in more detail in a previous paper (Abe, 2001).A laboratory culture of L. bryoniae was established from individuals collected from leaves of tomato plants in Kamigamo, Kyoto City, Japan, in June 1998.Phaseolus vulgaris was used as a host plant for colony maintenance and in all the experiments.Colony maintenance and all experiments were conducted at 25°C under a 15L : 9D photoperiod.
The effects of host (L.trifolii) age at oviposition (0 to 4 days = egg to mature larva) on percentage emergence and developmental time of G. micromorpha were previously determined in the laboratory (Abe, 2001).There was no significant difference in percentage emergence among host ages.However, the developmental time was significantly shorter in 3-or 4-day-old larvae, suggesting that mature larvae are a more suitable host stage for G. micromorpha.The mature larvae of L. bryoniae are similarly more suitable than earlier stages as a host for G. micromorpha (Abe, Y., unpubl.).Larvae of L. trifolii developing on P. vulgaris leaves pupate after 5-6 days at 25°C (Abe, 2001), and those of L. bryoniae after 6-7 days (Abe, Y., unpubl.).Therefore, 4-day-old L. trifolii and 5-day-old L. bryoniae larvae were used in the experiments.

Host pupal size
To estimate the size of the pupa of the two Liriomyza species, the head width of 15 pupae within their puparia was measured to the nearest 6.7 µm under a binocular stereomicroscope.

Host suitability
The base of the stem of a P. vulgaris plant with two true leaves infested with approximately 20-30 4-day-old larvae of L. trifolii or 5-day-old larvae of L. bryoniae was immersed in water in a 10-ml glass vial attached to the inside bottom of a cylindrical glass tube (6.4 cm diameter, 22 cm high).The top of the tube was covered with organdy and the bottom with Kim-wipe®.Undiluted honey was streaked on the inside of the tube as a food source for the wasps.Immediately thereafter, one naive female G. micromorpha (0-24 h old) was released into the tube and allowed to parasitize the Liriomyza larvae for 24 h before removal from the tube.Fifteen wasps were used for each Liriomyza species.After removal of the wasp, the two leaves were detached from each plant by cutting the petioles and then placed in a 250-ml plastic cup.The immatures of Liriomyza were reared and the emergence of G. micromorpha wasps recorded.The hind tibial lengths of 20 randomly selected wasps emerging from each host species were measured to the nearest 6.7 µm under a binocular microscope.

Host acceptance
Phaseolus vulgaris plants with two true leaves were infested with mature larvae of L. trifolii or L. bryoniae as in the previous experiment.Just before the experiment, one of the two true leaves was cut from each plant.The stems of one plant with mature larvae of L. trifolii and one with mature larvae of L. bryoniae were immersed in water in a 10-ml glass vial attached to the bottom of a glass tube (as above).Immediately after transfer of the plants, one 0-24 h-old naive female G. micromorpha was introduced into the tube and allowed to oviposit into L. trifolii and L. bryoniae larvae for 24 h before removal.After removal of the wasp, all host larvae were transferred from the leaves into Ringer's solution and dissected with minute pins under a binocular microscope to see whether they contained parasitoid eggs or not.Twenty replicates were performed.The ratio of the number of the two host species (L.trifolii / L. bryoniae) per replicate ranged from 0.54 to 2.12.

Host acceptance and larval survival
The experimental design was the same as in the host acceptance experiment, except that the host larvae were reared after exposure to G. micromorpha and the emergence of adult parasitoids was recorded.After the end of adult emergence, host puparia with no exit holes were dissected under a binocular microscope to record mortality of L. trifolii and L. bryoniae pupae, and larval, pupal, or adult mortality of G. micromorpha.Twenty replicates were performed.The ratio of the two host species (L.trifolii / L. bryoniae) per replicate ranged from 0.72 to 3.33.

Data analysis
One-way ANOVA was used to detect differences in pupal head width between L. trifolii and L. bryoniae.Effects of host species on developmental time and body size of G. micromorpha were analyzed using the same statistical tests.Host acceptance and combined effects of host acceptance and larval survival of G. micromorpha were analyzed in paired T-test after weighting data for proportional availability of hosts.The significance level for all statistical tests was set at P = 0.05.

Host acceptance and larval survival
There was no significant difference in rate of emergence of G. micromorpha between L. trifolii and L. bryoniae (Table 3, paired T-test, t = -0.394,d.f.= 19, P > 0.05).No dead larvae, pupae, or adults of G. micromorpha were found in the dissected host puparia.These puparia contained only dead host pupae.

DISCUSSION
Gronotoma micromorpha completed its development successfully in both L. trifolii and L. bryoniae.No signifi-cant difference was found in the developmental time of this wasp in the two host species.However, larger G. micromorpha adults emerged from L. bryoniae than from L. trifolii puparia.Developmental time (or developmental rate) and body size are both important traits for evaluating fitness of parasitoids (Roitberg et al., 2001).In solitary koinobiont parasitoids, the size of the offspring is often influenced by the host species from which it emerged (Visser, 1994).Pupae of L. trifolii and L. bryoniae can be regarded as fixed host resources available for offspring development of G. micromorpha, because the parasitoid is in the 1st instar when the host pupates (Abe, Y., unpubl.).Pupae of L. bryoniae were significantly larger than those of L. trifolii.Large hosts presumably contain more resources and, therefore, should be of relatively higher quality than small hosts (Nicol & Mackauer, 1999).Consequently, the difference in body size of G. micromorpha is probably due to the difference in the size of pupae of the two host species.Similarly, no significant difference was found in female developmental time of D. sibirica in L. trifolii and L. bryoniae, and larger parasitoid females emerged from L. bryoniae than from L. trifolii puparia (Abe et al., 2005).This difference in female size might be also explained by the higher quantity of food available in L. bryoniae.

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Means with the same letter were not significantly different in one-way ANOVA (P > 0.05).A female parasitoid can influence the fitness of her offspring through her choice of a host, since the quality of the host can directly affect offspring fitness (Luck & Nunney, 1999).Thus, it is predicted that foraging female parasitoids that encounter hosts of different species should select the most suitable.Female G. micromorpha exhibited a significant preference for L. bryoniae larvae.However, no significant difference was found in the number of wasps emerging between L. trifolii and L. bryoniae.These results indicate that G. micromorpha immatures parasitizing L. bryoniae had a higher mortality rate than those parasitizing L. trifolii.Why do female G. micromorpha prefer L. bryoniae larvae, which are of a lower quality than L. trifolii larvae in terms of their offspring's survival?It is possible that L. bryoniae larvae are a better quality host than L. trifolii larvae in terms of the fecundity of wasp's offspring.In general, large parasitoids are more fecund than small ones (King, 1987;Visser, 1994).Provided the size and fecundity of female G. micromorpha are positively correlated, as in female D. sibirica (Croft & Copland, 1993), female G. micromorpha emerging from L. bryoniae puparia would have a higher fecundity than those from L. trifolii puparia.Further study is needed to clarify the relationship between the size and fecundity in female G. micromorpha.
The geographical distributions of L. bryoniae and G. micromorpha do not overlap.However, the present study reveals that L. bryoniae is as good as L. trifolii as a host for G. micromorpha in terms of larval development and survival and is the preferred host for oviposition.These three factors are important for biological control because they influence a parasitoid's parasitism rate and its numerical response.In addition to the wide range of host stages (egg to mature larvae) suitable for oviposition (Abe, 2001), the reproductive capacity and no adverse effect of high temperature or short photoperiod on development (Abe & Tahara, 2003), the present results indicate that G. micromorpha could be an effective biological control agent of L. trifolii and L. bryoniae in greenhouses.

TABLE 2 .
Acceptability of L. trifolii and L. bryoniae as hosts for G. micromorpha.