Spatial distribution of parasitism on Phyllocnistis citrella Stainton, 1856 (Lepidoptera: Gracillariidae) in citrus orchards

Many species of microhymenopterous parasitoids have been registered on Phyllocnistis citrella, the citrus leafminer. The present study aimed to identify the spatial distribution pattern of the native and introduced parasitoids of P. citrella in two citrus orchards in Montenegro, RS. The new shoots from 24 randomly selected trees in each orchard were inspected at the bottom (0-1.5 m) and top (1.5-2.5 m) stratum and had their position relative to the quadrants (North, South, East and West) registered at every 15 days from July/2002 to June/2003. The leaves with pupae were collected and kept isolated until the emergence of parasitoids or of the leaf miner; so, the sampling was biased towards parasitoids that emerge in the host pupal phase. The horizontal spatial distribution was evaluated testing the fitness of data to the Poisson and negative binomial distributions. In Montenegrina, there was no significant difference in the number of parasitoids and in the mean number of pupae found in the top and bottom strata (χ = 0.66; df = 1; P > 0.05) (χ = 0.27; df =1; P > 0.05), respectively. In relation to the quadrants, the highest average numbers of the leafminer pupae and of parasitoids were registered at the East quadrant (χ = 11.81; df = 3; P < 0.05), (χ = 10.36; df = 3; P < 0.05). In the Murcott orchard, a higher number of parasitoids was found at the top stratum (63.5%) (χ = 7.24; df =1 P < 0.05), the same occurring with the average number of P. citrella pupae (62.9%) (χ = 6.66; df = 1; P < 0.05). The highest number of parasitoids and of miners was registered at the North quadrant (χ = 19. 29; df = 3; P < 0.05), (χ = 4.39; df = 3; P < 0.05). In both orchards, there was no difference between the numbers of shoots either relative to the strata as well as to the quadrants. As the number of shoots did not varied much relative to the quadrants, it is possible that the higher number of miners and parasitoids in the East and West quadrants would be influenced by the higher solar exposure of these quadrants. The data of the horizontal spatial distribution of the parasitism fit to the negative binomial distribution in all sampling occasions, indicating an aggregated pattern.


Material and Methods
Samplings were carried out in two adjacent organically grown orchards (approximately 2 ha each), one with Citrus deliciosa Tenore cv. Montenegrina and the other with a hybrid of Citrus sinensis L.Osbeck x C. reticulata Blanco Murcott, in Montenegro (29° 68' S and 51° 46' W), Rio Grande do Sul state. Each orchard, sampling was done in an area with approximately 0.6 ha, containing 315 plants.
Twenty-four randomly selected trees were inspected every 15 days from July/2002 to June/2003 and all new shoots of the bottom (0.5-1.5 m above the soil) and top (1.6-2.5 m) layers were examined and had their location relative to the quadrants (North, South, East and West), registered. All leaves containing pupae of P. citrella were collected and placed in identified plastic bags and transported to the laboratory, where they were individualized in sealed Petri dishes and kept until the emergence of either parasitoids or P. citrella.
The vertical distribution of the collected organisms in the plant was analyzed considering the bottom and top strata. The homogeneity of distribution among such strata was tested by the chi-square test of heterogeneity (χ 2 = Σ(O-E) 2 /E). The position of the collected leaves relative to the quadrants was also recorded and tested by Kruskal-Wallis.
The analysis of the horizontal distribution considered the total parasitism (without parasitoid species distinction). The sampling data was fitted to a Poisson and to a negative binomial series, respectively, by the dispersion index (I) and the dispersion parameter k, following Elliott (1983). Chi-square tests were utilized to test the goodness of fit of the observed distributions.

Introduction
Investigating patterns of spatial distribution of organisms in the environment is a central issue in population dynamics, considering that differences in the impact of several agents biotic and abiotic may occur in function of spatial variations in the proximity of individuals within a population (Heads and Lawton, 1983;Hassel, 1986).
The relationship between the spatial distribution of hosts and that of its natural enemies has a great influence on the dynamics of both populations. Many models describing this interaction assume that natural enemies have a random distribution relative to the host spatial distribution. This premise simplifies mathematical models, but is unrealistic because most natural enemies react to the spatial distribution of their prey (Pedigo, 1996).
The larvae of P. citrella, the citrus leafminer, dig into the subepithelial leaf tissue when feeding, affecting young leaves, shoots and, sometimes even small fruits. The miner causes reduction in the photosynthetic area and injuries that facilitate the infection by the bacteria Xanthomonas citri pv. citri, the citrus canker causal agent, considered one of the most important citrus pest (FUNDECITRUS, 2003).
The spatial distribution of the different stages of the leaf miner has been studied at different scales and in diverse species and cultivars of citrus in Brazil and abroad. In these studies was found a higher number of eggs in the terminal leaves of the shoots, a caterpillar preference by shoots in the north quadrant and an aggregated dispersion pattern of caterpillars (Peña and Schaffer, 1997;Paleari et al., 2001;Dantas, 2002).
According to Askew (1980), most miner insects are heavily attacked by eulophids. P. citrella has a number of natural enemies which belongs to this parasitoid family, and many are promising biological control agents of this pest. The effect of environmental spatial heterogeneity upon populations has been the target of several studies that seek to evaluate its role in the reduction of pest insect populations as well as in keeping their interaction with its natural enemies (Hassel, 1986). The aggregation of parasitoids in some areas of the environment seems to heavily influence the persistence of the interaction in non-cultivated areas as much as in agroecosystems (Hassel and May, 1974).
The differences found in the two orchards among quadrants, relative to the numbers of parasitoids and of pupae, may be explained by the plant arrangement in rows, and to the higher solar exposition of certain quadrants. In Montenegrina, rows were arranged in the northsouth direction and, due to a larger distance between trees in different rows than to the distance between plants within a row, the east and west orientations probably receive more solar radiation. In the Murcott area, rows are oriented east-to-west and thus the north orientation is the one receiving more solar radiation.
The analysis of the horizontal distribution of the parasitism revealed aggregated patterns ( Table 2). The data fit the Negative Binomial distribution in all tested occasions. This is reinforced by the high values of I obtained. The aggregated pattern of the citrus leaf miner distribution has been already registered in different cultivars (Vivas and Lopez, 1995;Peña and Schaeffer, 1997;Peña, 1998). In sweet orange orchards in São Paulo state, although Knapp et al. (1995) had considered the pest distribution as uniform, Paleari et al. (2001), when developing a sampling method to monitor P. citrella and its natu-All calculations were done with Microsoft Excel, Bioestat 2.0 (Ayres et al., 2000) and Ecological Methodology (Krebs, 2000).

Results and Discussion
A total of 498 individuals of microhymenopterous parasitoids were collected in the Montenegrina orchard and 212 in the Murcott, all belonging to Eulophidae and Encyrtidae.
The average percentage of parasitism was 30.2% in Murcott area and 37.6% in Montenegrina. In a simultaneous study carried out in these same areas it was found that the larval-pupal parasitism contributed 10.7% in average to the mortality of immature stages of the citrus leafminer (Jesus, 2005).
The observed parasitism rates among strata and quadrants of the tree canopy showed differences in the two cultivars (Tables 1). The high standard deviations registered on both cultivars could be explained considering flushing occurs only in certain periods of the year. In Montenegrina, there was no significant difference both in the number of parasitoids and of pupae found in the strata (χ 2 = 0.66; df =1; P > 0.05) and (χ 2 = 0.27; df = 1; P > 0.05), respectively. Also, the number of new shoots recorded in the two layers did not show a significant difference (H = 2.3472; P = 0.1255). In the Murcott orchard, a higher parasitoid number (63.5%; χ 2 = 7.24; df =1 P < 0.05), and a higher number of P. citrella pu- values occurs, indicating a lower aggregation of parasitism in these occasions. The scarcity of foliar resources to the miner also leads to an increase in aggregation, because the parasitism becomes restricted to young shoots where larvae and host pupae occur.
Two peaks in parasitoid populations were registered for both orchards, although not simultaneously (Figure 1). In Montenegrina, the total number of parasitoids registered was larger probably due to the higher vegetative growth of this cultivar (Koller, 1994) and also to the larger leaf miner total population registered in this same area in a study carried out simultaneously to the present one (Cristiane Ramos de Jesus, personal communication), which may be related to the preference/ suitability of this variety as a host.
In Montenegrina, a small populational peak was registered in February 17 th and a larger one in March 17 th , when 368 parasitoid individuals were collected; in ral enemies, verified that the number of damaged new shoots varied among the quadrants; in another words, it was found not homogeneous Thus, the parasitism distribution will occur associated to the host, even though different patterns may arise The processes observed in the field that generate aggregated patterns of parasitism, besides the host presence, may be diverse: the continuous exploitation by parasitoids of previously colonized regions; the greater concentration of kairomones resulting either in a shorter searching time for new hosts (Waage, 1983) or increased residence time, which leads to higher parasitism rates; as well as the limited flight ability of these eulophids (Godfray, 1994).
The data for all sampling occasions analyzed fit to the Negative Binomial distribution; nevertheless, k values tended to be lower at smaller densities, indicating a higher aggregation in these periods ( Figure 1). As the host and parasitoid populations increase, an increase in k   these occasions the largest k values were also obtained ( Table 2). In Murcott, the first population peak occurred in February 3 rd , concomitantly to those observed in Montenegrina. In these occasions, the calculated k, from the Negative Binomial, had larger values, also indicating in this orchard a weaker aggregation. It is important to emphasize that in spite of the continuing sampling until June/2003, after March 31 no pupae of the leaf miner were found in either orchard. The observed aggregate pattern of parasitoids of P. citrella as well as the influence of this pattern in the dynamics of the system highlights the importance of this information to the development of a sequential sampling plan. Furthermore, it could provide elements to the elaboration of mathematical models to describe the interactions that occurs in the citrus system under organic management.