International Symposium on Tropical Agriculture & Agro-biotechnology, Dec.

7-9, 2004

Parasitism of Halticoptera circulus (Walker) (Hymenoptera:Pteromalidae): A Parasitoid of the Liriomyza trifolii (Burgess) (Diptera:Agromyzidae) at Different Densities
Edwin D. F. Lopez1, Lekhnath Kafle2, Po-Yung Lai2 and Yin-Fu Chang2
1 2

Pan American School of Agriculture, Zamorano, Honduras.

Department of Tropical Agriculture and International Cooperation National Pingtung University of Science and Technology, 1 Hsuen Fu Road Neipu Hsiang, Pingtung 91201, Taiwan Abstract
This study was conducted on larval-pupal endoparasitoid Halticoptera circulus (Walker) to determine its parasitism when Liriomyza trifolii (Burguess) was a host. The parasitism and biological characters of parasitoid was tested at 4 different (1, 3, 5 and 7 pairs) densities when 50 L. trifolii larvae were provided daily. The longevity and fecundity of H. circulus females decreased with the increase in density of H. circulus adults. The sex ratio of the offspring of H. circulus was also affected as its density increased. The host feeding of female parasitoid and superparasitism was also observed. When more than 3 pairs of H. circulus adults were provided with 50 L. trifolii larvae for parasitization, inverse relationships were found on the adult longevity, number of L. trifolii larvae killed by host feeding, number of offspring production of a H. circulus female and the ability to find and attack suitable L. trifolii larvae of H. circulus female. The male progenies produced were higher than female progenies in all H. circulus densities. Three pairs of H. circulus adults was optimal for reducing the number of L. trifolii larvae and pupae from reaching adulthood as well as for maximizing the reproduction rate of H. circulus when 50 leafminer larvae were provided daily for parasitization. Key words: Parasitoid, Host, Instar, Halticoptera circulus, Liriomyza trifolii, Parasitization

Introduction
Liriomyza sativae (Blanchard), L. trifolii (Burgess), L. huidobrensis (Blanchard), L. bryoniae (Kaltenbach) and L. strigata (Meigen) were economically important leafminers in the world (Spencer, 1973). Chen and Chang (2002) reported that L. huidobrensis, L. trifolii, L. sativae and L. bryoniae had caused serious damage to crops between 1982 and 1999 in Taiwan. The wide geographical distribution of the leafminers could be attributed to a number of factors; including insecticide resistance, increase in monoculture of horticultural crops, increase in horticultural trade among countries and lack of inter-national quarantine (Murphy and LaSalle, 1999). Halticoptera circulus is a larva-pupal endoparasitoid. It became successfully established throughout the North America, Hawaii and Florida (Johnson and Hara 1987; Schuster et al, 1991).
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International Symposium on Tropical Agriculture & Agro-biotechnology, Dec. 7-9, 2004

The parasitoid used in this study was obtained from Agricultural Research Institute, Taichung that was introduced from Hawaii in 2000. The objective of this study was to determine biological characters as adult longevity, fecundity, sex ratio, developmental time and mortality of H. circulus at four different densities in laboratory conditions with L. trifolii as host.

Materials and Methods
Using L. trifolii larvae as test insects Through out this study the same procedures were followed to obtain the late 2nd or early 3rd instar L. trifolii larvae as test insects. Six pairs of Phaseolus ‘‘Henderson’’ lima bean plants (two leaves each) were placed for 6 hr in a screen cage containing 50-60 L. trifolii adults. After 6 hr, bean plants were removed from the cage and held for 5 days to allow L. trifolii eggs to hatch and develop until they reached the late second or early third instar. Rearing of H. circulus H. circulus parasitoids used in this study were obtained from a laboratory culture using L. trifolii as the host insect. L. trifolii colony was maintained in the laboratory using the methods described by Rathman et al. (1991). Stems of L. trifolii infested bean plants (two leaves each) were cut immediately above the roots and placed in a 200-ml flask filled with water. A honey-water solution (25%) was sprayed on lima bean leaves to provide food source for adult parasitoids. Plants were exposed to H. circulus for 24 hr and then removed. The leaves were cut at the base and kept in closed plastic containers to allow L. trifolii larvae to pupate. Leafminer puparia were held in the same container until H. circulus adults emerged. Parasitoid adults were returned to oviposition cages for either culture maintenance or used in subsequent studies. Longevity of H. circulus For this study, two leaves containing about 50 late 2nd or early 3rd instar leafminer larvae were used. Leaves were placed in a cage (0.008m3) and newly emerged 1, 3, 5 and 7 pairs of parasitoids were released into the different cages. After 24 hours, exposed leaves were removed and new 50 late 2nd or early 3rd instar leafminer larvae were added. This was done daily until the female parasitoids died. Honey-water solution (25%) was provided to males until they died. The number of days that a male and female lived was recorded. Fecundity and sex ratio of H. circulus Leaves exposed during the experiment of the longevity of parasitoids were used to determine the fecundity and sex ratio. The exposed leaves were held in a petri dish (9 cm diameter) until leafminer larvae pupated. The puparia were collected and held until adult parasitoids or leafminers emerged. The sex and numbers of H. circulus emerged were recorded daily. The equation 1 was used to calculate sex ratio. Sex ratio = m/(m+f) (1) Where “m” and “f” are the number of males and females produced.
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International Symposium on Tropical Agriculture & Agro-biotechnology, Dec. 7-9, 2004

Mortality of L. trifolii larvae Leaves exposed during the experiment of the longevity of parasitoids were used to determine the mortality factors of L. trifolii larvae. Host feeding and parasitic action of H. circulus were considered as mortality factors of L. trifolii larvae. The sum of all the mortality factors considered in the analysis is the total mortality coefficient, which was defined as the generation mortality (K) (Krebs, 2001). The number of death larvae due to host feeding or due to parasitic action and the number of H. circulus emerged or not emerged were recorded daily at each density. The equation 2 was used to calculate k-values. k = log10 (Ns)-log10 (Ne) (2) Where “Ns” is the number of individuals starting the stage, and “Ne” represents the number of individuals ending the stage. Additionally, 10 pairs of plants, each pair infested with 50 leafminer larvae, were placed separately in acrylic cylinders without parasitoid adults to serve as a check. The number of L. trifolii adults that emerged and the number of larvae or pupae died due to natural causes were recorded. All of the studies were replicated 4 times. All the experiments were conducted in a laboratory under the environmental conditions of 26±20C, 50±10% RH and a photoperiod of 14L: 10D. Mean were compared by using t-test and SNK test (SAS, 1999).

Results
Adult longevity The analysis of variance showed that there was significantly difference between the lifespan of males and females of H. circulus. The mean duration of the adult stage for females was almost 20 days longer (34.25±4.92 days) than that for males (14.25±2.36 days) when the parasitoids were observed in one pair. In addition, significantly differences were found in the longevity of females at the four different densities; however, longevity of females were not significantly different between 1 and 3 pairs and at 5 and 7 pairs of H. circulus densities per cage. Differences in the longevity of males at different densities were not significant (Table 1). The mean longevity of females decreased almost 10 days at the density of 7 pairs of H. circulus per cage as compared with the longevity of a female when only 1 pair of H. circulus were placed in a cage. Table 1. Longevity of males and females of H. circulus at four different densities. Longevity ( Days)a H. circulus density (Pairs per cage) 1 3 5 7
a

Males n 4 11 20 26 Mean ±SD 14.25±2.36 A 19.27± 8.71 A 21.15± 6.23 A 16.77±8.63 A n 4 12 18 23

Females Mean ± SD 34.25±4.92 A 33.17±4.97A 27.53±2.56 B 24.50±3.40 B

Means followed by different letters in the same column are significantly different by the SNK test (P<0.05).
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International Symposium on Tropical Agriculture & Agro-biotechnology, Dec. 7-9, 2004

Fecundity and sex ratio The analysis of variance revealed that the mean offspring produced by 1 pair of H. circulus per cage was significantly difference in comparison with 5 and 7 pairs parasitoids per cage. However, no significant difference was found between 1 and 3 pairs and among 3, 5 and 7 pairs of H. circulus per cage. It was not possible to explain the oscillating trend of the offspring sex ratios as the density of H. circulus increased. The values of the overall sex ratio first declined from 0.67 at 1 pair of H. circulus per cage to 0.57 at 3 pairs, then increased to 0.65 at 5 pairs and again declined to 0.54 at 7 pairs of H. circulus per cage (Table 2). Table 2. Mean number of offspring produced throughout the lifespan of individual females of H. circulus at four densities provided daily with 50 L. trifolii larvae for parasitization. H. circulus Density (Pairs per cage) 1 3 5 7
a

Mean no. of offspring produced per femalea Male progeny 97.25 A 64.05 AB 45.50 B 23.58 C Female progeny 48. 00 A 47.97 A 24.60 A 19.93 A Total offspring 145.25 A 112.02 AB 70.10 B 43.51 B

Sex ratio 0.67 0.57 0.65 0.54

Means followed by different letters in the same column are significantly different by the SNK test (P<0.05). Developmental time The analysis of variance showed significant differences in developmental time between males (17.87±4.68days) and females (18.58±4.30days), being shorter this time for males than for females at the density of 1 pair of H. circulus per cage. Developmental time was also significantly affected by the increases of H. circulus density. The developmental time was increased with the increases of the density (Fig. 1). The mean developmental time increased from 18.57±4.49 days when the progeny was produced at a density of 1 pair per cage to 18.64±5.86, 19.46±5.54 and 20.15±6.55 days at the densities of 3, 5 and 7 pairs, respectively. 20.5 20 Days to reach adulthood 19.5 19 18.5 18 17.5 1 3 5 7 Numbers of pairs of H. circulus per cage c c b a

Fig. 1. Days required for H. circulus offspring to emerge at four densities. Bars with different letters are significantly different by the SNK test (P<0.05).
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International Symposium on Tropical Agriculture & Agro-biotechnology, Dec. 7-9, 2004

Superparasitism Superparasitism was found. Often more than one H. circulus egg, 1st or 2nd instar larvae were found in parasitized host, but never was more than one 3rd instar larva found in a host. As the H. circulus adult density increased, the probability of encounter already parasitized host may be increased, resulting in the increase in the probability of superparasitism. Mortality of L. trifolii larvae Host feeding was found and the way in which the female fed upon the host was by constructing a feeding tube. The L. trifolii larvae killed by the action of host feeding turned into necrotic appearance identifiable by naked eyes. The action of host feeding resulted in the death of L. trifolii larvae. According to the analysis of variance, the mean number of L. trifolii larvae killed daily by the host feeding action increased in a decreasing rate with the increase of the density of H. circulus (Table 3). With the increase of the density of the H. circulus, the mean number of L. trifolii killed daily by a female decreased from 2.60 at the density of 1 pair of H. circulus adults per cage to 1.68, 1.07 and 0.93 larvae per female at 3, 5 and 7 pairs of H. circulus adults per cage, respectively (Table 3). In all of the densities, the host feeding activity began on the day of adult emergence and continued until the death of the adult. The mortality rates, expressed as k-values, increased as the number of H. circulus adults per cage increased (Table 3). Table 3. Mean number of L. trifolii larvae killed daily by host feeding action of H. circulus at four different densities. H. circulus density (Pairs per cage) 1 3 5
a

Total no. of tests 114 106 100

Mean no. of host provided/test 49.14 49.58 49.86

Mean no. of host killed/testa 2.60 C 5.05 B 5.37 B

k-value 0.024 0.047 0.049

7 76 49.34 6.54 A 0.062 Means followed by different letters are significantly different by the SNK test (P<0.05).

The analysis of variance revealed that the mean number of L. trifolii adults emerged daily from pupae at five different densities of H. circulus were significantly different. However, no significant difference was found between 3 and 5 pairs of H. circulus per cage. The emergence of L. trifolii declined as the number of H. circulus adults per cage increased (Table 4). It resulted in a direct relationship between the k-value and the H. circulus density. The k-values for the parasitic action increased in a similar magnitude with respect to the k values for the host feeding action for all of the densities, this increase approximately is 10 times the k-value for host feeding (Table 5). The generation mortality showed a direct relationship with the increase of the H. circulus density and that was higher than the mortality coefficient 0.094 obtained when L. trifolii larvae were not exposed to H. circulus for parasitization (Table 5).
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International Symposium on Tropical Agriculture & Agro-biotechnology, Dec. 7-9, 2004

Table 4. Mean number of L. trifolii adults emerged daily from pupae collected at five different densities of H. circulus. H. circulus density (Pairs per cage) 0 1 3 5
a

Total no. of tests 10 114 106 100

Mean no. of host pupa collected/test 49.86 42.84 41.76 40.47

Mean no. of host adults emerged/testa 40.14A 22.62B 13.72C 12.07C

k-value 0.094 0.277 0.483 0.525 0.655

7 76 38.03 8.41D Means followed by different letters are significantly different by the SNK test (P<0.O5).

Table. 5. Generation mortality of L. trifolii larvae and pupae with and without action of H. circulus at five densities. H. circulus k-value Density Host feeding Parasitization Generation mortality (K) (Pairs per cage) 0 --0.094 1 0.024 0.277 0.301 3 0.047 0.483 0.530 5 0.049 0.525 0.575 7 0.062 0.655 0.717

Discussions
It was feasible to successfully maintain and multiply colonies of H. circulus under laboratory conditions. The longevity of a H. circulus female was around one month; it laid eggs throughout its lifespan. An increase in the number of H. circulus adults in a cage affects the longevity of the female adults. Competition for food sources and suitable hosts for egg laying resulted in a decrease in the parasitization efficiency, a reduction of their lifespan and an increase in the probability of superparasitism of the H. circulus. Three pairs of H. circulus adults were the optimum number for reducing the number of L. trifolii larvae and pupae from reaching adulthood, as well as for maximizing the reproduction rate of H. circulus when a host density provided was at 50 L. trifolii larvae per 0.008 m3. The number of L. trifolii larvae killed were proportionally smaller as compared with the number of L. trifolii adults emerged when one H. circulus female searching for hosts. Based on the biological characteristics and parasitization performance of H. circulus found, it is suggested that when the environmental conditions are suitable for the H. circulus activity, it can be considered as a potential effective biological control agent of L. trifolii.

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References
Chen, C. C. and Chang, S. C. 2002. Occurrence and control strategies of Liriomyza leafminers in Taiwan. Information on plants pest, disease and control strategies. pp 31-44. (Department of Applied Zoology, Taiwan Agricultural Research Institute, Wufeng, Taichung, Taiwan, R.O.C) Johnson, M. W. and Hara, A. H. 1987. Influence of host crop on parasitoid (Hymenoptera) of Liriomyza spp. (Diptera: Agromyzidae). Environ. Entomol. 16:339-344. Johnson, M. W. and Mau, R. 1986. Effects of intercropping beans and onion on populations of Liriomyza spp and associated parasitic Hymenoptera. Proc. Hawaii. Entomol. Soc. 27: 95-103. Krebs, C. J. 2001. Ecology: the experimental analysis of distribution and abundance. Benjamin Cummings, San Francisco. pp 206-304. Murphy, S. T. and LaSalle, J. 1999. Balancing biological control strategies in the IPM of new world invasive Liriomyza leafminers in field vegetable crops. Bio. Control. 20: 91-104. Rathman, R. J., Johnson, M. W. and Tabashnik, B. E. 1991. Production of Ganaspidium utilis (Hymenoptera: Eucoilidae) for biological control of Liriomyza spp. (Diptera: Agromyzidae). Biol.Control 1: 256–260. SAS Institute. 1999. SAS/STAT user’s guide: Statistics. SAS Institute, Inc., Cary, NC. Schuster, D. J., Gireath, J. P., Wharton, R.A. and Seymour, P. R. 1991. Agromyzidae (Diptera) leafminer and their parasitoids in weed associated with tomato in Florida. Environ. Entomol. 20:720-723. Spencer, K. A. 1973. Agromyzidae (Diptera) of economic importance. The Hague, Netherlands. pp. 418.

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