Department of Entomology, National Taiwan University, Taipei 106, Taiwan. J. Med. Entomol. 45(4): 785Ð799 (2008)

Transcription

1 DIRECT INJURY, MYISIS, FORENSICS Larval Competition of Chrysomya megacephala and Chrysomya rufifacies (Diptera: Calliphoridae): ehavior and Ecological Studies of Two low Fly Species of Forensic Significance SHIUH-FENG SHIO 1 ND T-CHUN YEH Department of Entomology, National Taiwan University, Taipei 1, Taiwan J. Med. Entomol. 5(): 75Ð799 () STRCT Chrysomya megacephala and Chrysomya rufifacies are two predominant necrophagous species in Taiwan. Larvae of the latter can prey on other maggots, including that of their own species as facultative food. This facultative characteristic of C. rufifacies may enhance its competitive advantage over other maggots and could also change the situation of other coexisting colonies. In this study, these two species were colonized in the laboratory, and the main objective was to try to understand the effect of competition on larval development. ccording to our results, intraspeciþc competition mostly occurred as competition for food; when the rearing density was increased, larvae pupated earlier, resulting in a lighter adult dry weight. The tendencies were similar in both species, but C. megacephala developed smaller viable adults and had higher survivorship at high densities. lthough C. rufifacies could use the food resource of cannibalism, its survivorship was still low. Our results also showed there were signiþcant interactions between intraspeciþc competition and the density factor. However, with interspeciþc competition, the Þrst-instar larvae of C. rufifacies invaded maggot masses of C. megacephala to feed together. The third instars of C. rufifacies were able to expel C. megacephala larvae from food by using a ßeshy protrusion on their body surface; C. megacephala was usually forced to pupate earlier by shortening its larval stages. The results indicated that a temporary competitive advantage could only be obtained by C. rufifacies under a proper larval density. In addition, the effects on different larval stages, the responses to different competition intensities, and the temperaturedependent effects on interspeciþc competition are also discussed. In general, under mixed-species rearing at different temperatures and densities, larval duration, adult dry weight, and survivorship of both species decreased. However, our results did not completely agree with previous studies, and we suspect that the difference was partially caused by different experimental designs and different biological characters of different blow ßy colonies. Our results also suggest that both the predation ability and defense or escape activity should be taken into account when evaluating larval competitive advantages. The durations of larval stages of these two species could be decreased by 5 h when a single species was reared alone and food was limited; the largest reduction in larval duration, 5 h in C. megacephala and 3 h in C. rufifacies, caused by interspeciþc competition was under a high larval density. In conclusion, competition decreased the larval duration of these two species by up to d; this also draws attention to justifying the postmortem interval estimation of using larval developmental data when larval competition exists. KEY WORDS forensic entomology, Calliphoridae, larval competition, postmortem interval Developmental data of blowßies can be used to estimate the short-term postmortem interval (PMI), normally a few hours to a few days (Catts and Goff 199). However, there are many biotic and abiotic factors known to inßuence insect growth and activity, and determining these factors and their effects has been the most active area of research in forensic entomology (Wells and Lamotte 1). iological factors, especially the behavioral factors of competition and predation, have nevertheless seldom been thoroughly discussed in previous studies. Until now, only a few 1 Corresponding author, sfshiao@ntu.edu.tw. studies related to interactions between different species of blow ßy larvae have been published; including interactions between native and introduced blowßies (Wells and Greenberg 199a, b), larval predation (Faria et al. a), dispersal and predation behavior (Reigada and Godoy 5), competition for patchy resources (Zuben et al. 1), larval resistance to predation (Wells and Kurahashi 1997), cannibalistic behavior (Faria et al. b), and larval population density (Goodbrod and Goff 199). The habitat or ecological islands of animal carrion are small and distinct, and communities developing within them have at their disposal a limited amount of -55//75Ð799$./ Entomological Society of merica

2 7 JOURNL OF MEDICL ENTOMOLOGY Vol. 5, no. energy, which is gradually used up by the activities of the community members (eaver 1977). The substrate of carrion is ephemeral, and ßies that feed on it rarely complete more than one generation on a single carrion item (eaver 1977); so the number of eggs or larvae in carrion frequently exceeds its carrying capacity (Kneidel 19). So and Dudgeon (199) have also shown that competition is an important factor inßuencing the structure of necrophagous dipteran communities. Chrysomya megacephala (Fabricius) (Oriental latrine ßy) and Chrysomya rufifacies (Macquart) (hairy maggot blow ßy), two predominant necrophagous species in Taiwan, are indigenous to the ustralasian regions of the Old World tropics. These Old World blowßies of Chrysomya were also introduced to the mericas in the mid-197s and quickly became widespread and abundant in North and South merica (aumgartner and Greenberg 19). ccording to our general survey in the Taipei area (Chen et al. ), these two blow ßy species always Þrst arrive and are present simultaneously on corpses, and the same situation was also observed by Goff () in the Hawaiian Islands. This means that interactions caused by these two coexisting primary ßy species, such as competition and predation, may frequently play important roles in the successional dynamics of this early carrion faunal assemblage. The objectives of this study were to provide more detailed larval developmental and behavior data under intra- and interspeciþc competition and different temperature effects and to try to determine factors affecting competition when using these developmental data to estimate the PMI. Materials and Methods Insects and Rearing Conditions. Laboratory colonies of larvae of C. megacephala and C. rufifacies used in this study were originally collected from the Taipei area and initiated in. Maggots were reared on an artiþcial diet consisting of a mixture of Þsh meal:yeast: agar:water of g: g:. g:3 ml, in a (L) by 17.5 (W) by.5 (H)-cm plastic plate; the rearing methods basically followed those of Hung (1995). Mature third instars were transferred onto sawdust for pupation. dult males were randomly sampled and identiþed 5 d after emergence for species reconþrmation, and some males and females of the same species were kept in a 3-cm 3 rearing cage for mating. Sugar water and a mixture of milk powder and sugar were provided as food for the adults. Pork liver was used to induce females to lay eggs. Rearing of all stock colonies was performed in a growth chamber at C, % RH, and a 1:1 (L:D) photoperiod. For all experimental purposes, larvae were moved out from the stock colony and kept in a 5-ml plastic cup covered with cotton netting and containing g of the artiþcial diet. ecause postfeeding larvae of C. rufifacies are inactive and usually postpone pupation because of being stuck in the media, some openings were cut in the plastic cups so that mature larvae could move outside to facilitate pupation. The cup was inside a container (1 cm in diameter and 1.5 cm in height) with a 1-cm-deep layer of sawdust. The treatments were the same for C. megacephala. Effects of Density on Larval Development in Single- and -species Cultures. Larvae of both species from those single-species cultures were reared at the respective densities of 1,,,, 1, 3,, 1,, and 1, per g artiþcial diet in plastic cups ( -h-old newly hatched larvae were used, manually counted, and placed onto the artiþcial diet; Þve replications were performed for each density treatment). Experiments were performed in a growth chamber at C, 7% RH, and a 1:1 (L:D) photoperiod. Developmental duration, survivorship, and adult dry weight were recorded for each treatment. Larval developmental duration is the time since egg hatching to pupation (pupal stage not included), larval survivorship is the success rate for pupation, and because the food and sawdust could be easily attached onto the surface of larvae or pupae to further cause measuring errors, adult dry weight was used instead as a parameter in this study. dults were CO -knockdowned in h after emergence, kept in 95% alcohol for 3 d, and then kept in an oven at 5 C for3dtomeasure their dry weights. DuncanÕs new multiple-range test was performed to determine the density effects on larval development. For the mixed-species cultures, an equal ratio of larval numbers of the two species was placed in the same cup, and the experiments were performed at the same density and rearing condition as in the single-species culture. Two-way analysis of variance (NOV) was used to determine the combined effects of competition and larval density. To further compare the effects of density on the development of these two blow ßy species under inter- and intraspeciþc competition, larval survivorship (%) was multiplied by the adult dry weight (mg) as the production to represent the overall Þtness of these two species. Effects of the Intensity of Interspecific Competition on Development. To evaluate the effect of the intensity of interspeciþc competition, larvae consisting of the two different species were kept in a 5-ml plastic cup with g of artiþcial diet and different species ratios of 1: 3, :, and 3: 1 were used; eight replications for this experiment were performed. Larval developmental time and adult dry weight were recorded for each treatment. Linear regression was used to determine the effects of different competition intensities on larval development. Interspecific Competition Under Different Temperatures. Temperature is one of the key factors affecting larval development. To further understand the cross-effects of competition and temperature on larval development, Þve different temperature treatments (1, 3,, 33, and 3 C; the actual measured temperature in the growth chamber during experiments were 1.., 3..1, 7.., 33.1., and 3..1 C, respectively) were carried out under an equal species ratio (:) and Þxed rearing densities ( larvae/ g of medium). ecause the rise in temperature caused by the feeding maggot mass might be

3 July SHIO ND YEH: LRVL COMPETITION OF LOW FLY LRVE 77 Mean duration of larval development (hours) Survivorship (%) C C. megacephala C. rufifacies Density (larvae/ g medium) C. megacephala C. rufifacies Density (larvae/ g medium) obvious, we decided to select a condition of larvae/ g medium as our experimental condition, which provided sufþcient food resources for larvae, and the feeding maggot mass caused only about a 1 C rise above the ambient temperature in our experiments. t-test analysis was performed to determine the effects of competition, DuncanÕs new multiple-range test was used to analyze the different parameters at different temperatures, and two-way NOV was used to test the interactions of the two factors of temperature and competition. Predatory ehavior and Movement Orientation of Larvae. To understand the role that predation plays in larval competition, a simple experiment was designed to observe the predatory behavior of C. rufifacies on C. megacephala. Following the experimental design of Faria et al. (1999) and Faria and Godoy (1), 5- (second instar) and 5-h-old (third instar) larvae from mixed-species cultures were selected. Five larvae of the same instar stage from each species were placed into a 5-ml plastic cup without food. Predatory behavior was observed in a walk-in growth chamber under the conditions of C, % RH, and a 1:1 (L:D) photoperiod. Larval secretions and excretions were left on the food after the larvae had fed, and this may have altered Mean adult dry weight (mg) 1 1 C. megacephala C. rufifacies Density (larvae/ g medium) Fig. 1. Effects of different breeding densities on the duration of larval development (), adult dry weight (), and larval survivorship (C) under single-species rearing of C. megacephala and C. rufifacies at C. the nearby microenvironment. The mixture of the artiþcial diet and larval products (fecal material) was collected to test its effects on the movement orientation of larvae of both species. Fecal material was respectively obtained by collecting the mixture of arti- Þcial diet after each instar larvae have fed (rearing conditions: same as in stock colonies; rearing density: larvae/ g of medium). The fecal material (1 g) from C. megacephala and C. rufifacies was respectively placed on two ends of a 5-ml plastic cup Þlled with artiþcial diet on the bottom to 1 cm deep. Ten conspeciþc larvae of same stage (1 [Þrst instar], 3 [second instar[rsqb, and 7 h old [third instar]) were washed with distilled water and placed in the middle of the plastic cup. The bath was to try to remove any odor of the larvae in advance. The two-choice experiments were performed in a growth chamber of C, 7% RH, and a dark environment (: [L:D] photoperiod). Movements of the three instars were recorded every 3 and min. If more than six larvae were found to have aggregated at one end of the mixed diet, the orientation tendency was judged to be sustained and was recorded. The results were analyzed by test with YateÕs correction for continuity.

4 7 JOURNL OF MEDICL ENTOMOLOGY Vol. 5, no. Mean duration of larval development (hours) Survivorship (%) C Density (larvae/ g medium) Density (larvae/ g medium) Density (larvae/ g medium) Fig.. Effects of different breeding densities on the duration of larval development (), adult dry weight (), and larval survivorship (C) of C. megacephala under single- and mixed-species rearing at C., single-species culture; mixed, mixed-species culture. Results Effects of Density on Larval Development in the Single-species Culture. The larval developmental time of C. megacephala did not signiþcantly differ under the densities of 1Ð1 larvae/ g artiþcial diet (F.5; df,; P.73) and was l3.5 h at C on average, but it was signiþcantly reduced when the density exceeded 1/ g (F 37.7; P.1). The maximum reduction was 5.1 h, and the average developmental time was only.% of that at the lower-density conditions (Fig. 1). The developmental time of C. rufifacies also did not signiþcantly differ when the density was larvae/ g (F.7; df,5; P.9) and was 1.9 h on average, but it was signiþcantly reduced when the density exceed larvae/ g (F.19; P.3). The maximum reduction time was 53.9 h, and the developmental time was 33.5% of that at the lower-density conditions (Fig. 1). lthough maximum reductions in the time of larval development under density effects were similar in the two species, C. megacephala had a shorter life stage and thus the density effects were greater than those in C. rufifacies. Mean adult dry weight (mg) 1 1 For adult dry weight, both species had the lightest weight at a density of 3 larvae/ g and signiþcantly differed from those at other density conditions (P.5). The adult dry weights of C. megacephala were higher than those of C. rufifacies at low rearing densities, but were nevertheless lower when the density exceeded 3 larvae/ g (Fig. 1). s for larval survivorship, the survivorship of C. megacephala did not signiþcantly differ between densities of 1 and larvae/ g (.%; F.79; df,1; P.7) or between and 3 larvae/ g (.%; F.3; df,1; P.15) but signiþcantly dropped when the density exceeded 3 larvae/ g. The survivorships of C. rufifacies did not signiþcantly differ when density was 1 larvae/ g (1.%; F.59; df,; P.7). The survivorship of C. megacephala turned out to be higher than C. rufifacies when the density exceeded larvae/ g (Fig. 1C). Effects of Density on Larval Development in the -species Culture: C. megacephala. Compared with the results in single-species cultures, interspeciþc competition signiþcantly reduced the larval developmental time at densities of 1 larvae/ g but did

5 July SHIO ND YEH: LRVL COMPETITION OF LOW FLY LRVE 79 Mean duration of immature development (hours) Survivorship (%) Density (larvae/ g medium) C Density (larvae/ g medium) Mean adult dry weight (mg) Density (larvae/ g medium) Fig. 3. Effects of different breeding densities on the duration of larval development (), adult dry weight (), and larval survivorship (C) of C. rufifacies under single- and mixed-species rearing at C., single-species culture; mixed, mixed-species culture. not signiþcantly differ when density was at 3 or larvae/ g (P.5; Fig. ). Similar results were also found for adult dry weight (Fig. ). Except at a density of larvae/ g (t.7; df ; P.), survivorship did not differ at low rearing densities ( 1 larvae/ g medium) between single- and mixed-species cultures. However, when the density was 1 larvae/ g, competition strongly reduced larval survivorship. For example, differences in survivorship between single- and mixed-species cultures at densities of 3 and larvae/ g medium were up to 9.5 (t 9.; df ; P.1) and 3.% (t 15.; df 3; P.1), respectively. In general, except at low breeding densities ( 1 larvae/ g medium) and extremely high densities ( 1, larvae/ g medium), interspeciþc competition reduced larval survivorship as the density increased. Effects of Density on Larval Development in the -species Culture: C. rufifacies. Competition generally reduced the larval developmental time and adult dry weight of C. rufifacies except at extremely high densities ( 1 larvae/ g medium; Fig. 3 and ). Larval survivorship increased as the density increased at densities of 1 larvae/ g under interspeciþc competition but strongly decreased as density exceeded 1 larvae/ g (Fig. 3C). We used the product of the mean adult dry weight and the mean larval survivorship as indicators for the overall Þtness of these ßies; the results showed that the Þtness levels of both species were lower under interspeciþc competition than under intraspeciþc competition and gradually decreased as larval densities increased (Fig. and ). In summary, interspeciþc competition reduced both speciesõ developmental time, adult dry weight, and survivorship, and the results of the two-way NOV showed signiþcant interactions between the competition and density factors for both C. megacephala (F 1.7; df 5; P.1) and C. rufifacies (F 1.19; df 5; P.1). Effects of the Intensity of Interspecific Competition. Linear regressions of the adult dry weights of C. megacephala and C. rufifacies against the intensities of interspeciþc competition are presented in Fig. 5 (in which the competition intensities are represented by the numbers of the opposite species, and 1,, and 3 were the numbers of the opposite species in the treat-

6 79 JOURNL OF MEDICL ENTOMOLOGY Vol. 5, no. 1 Production of C. megacephala (mg) Production of C. rufifacies (mg) 1 ments with larvae in total). s the competition intensity increased, the adult dry weight of C. megacephala signiþcantly decreased (r.73, P.1), but the adult dry weight of C. rufifacies was not signiþcantly affected (r.1, P.7). However, a linear regression of developmental rate versus competition intensities showed opposite results. Figure shows that the larval developmental duration of C. megacephala did not change with different competition intensities (r.7, P.19) but was signiþcantly reduced in C. rufifacies when the intensity increased (r.5999, P.1). Effects of Interspecific Competition on Different Larval Stages. InterspeciÞc competition affected larval development differently at different larval stages. For C. rufifacies, only the second-instar larvae showed signiþcant differences of shortening their larval duration ( 9.7 h; t 3.95; df 1; P.3), with no signiþcant differences at the other three larval stages Density (larvae/ g medium) Density (larvae/ g medium) Fig.. Effects of the single- or mixed-species rearing on the production of C. megacephala () and C. rufifacies () in g of medium at different density treatments at C. The production is the value of the mean adult dry weight multiplied by the mean larval survivorship. (Fig. 7). This shortened the overall larval developmental time by. h for C. rufifacies. However, interspeciþc competition strongly changed the developmental duration of both the feeding and postfeeding stages of third instars of C. magacephala, (Fig. 7), and it was interesting to note that the developmental time was shortened in the feeding stages but prolonged in the postfeeding stages. The overall larval developmental time was shortened by.9 h for C. megacephala. In addition, interspeciþc competition did not change the body length of most larval stages of either species (Fig. ), except for the third instars of C. megacephala (Fig. ), the body length of which was signiþcantly reduced under competition stress. Interspecific Competition Under Different Temperatures: C. megacephala (Fig. 9). Except at 1 C, interspeciþc competition caused signiþcant reductions in both the larval developmental duration and adult dry weight at different temperatures (Table 1). Compared with the single-species cultures, the larval

7 July SHIO ND YEH: LRVL COMPETITION OF LOW FLY LRVE Mean adult dry weight (mg) Mean adult dry weight (mg) duration of mixed-species cultures were shortened by h, and these time reductions were..5% of those in single-species cultures. The adult dry weights stably dropped (by 3%) with interspeciþc competition at different temperatures. Interspecific Competition Under Different Temperatures: C. rufifacies (Fig. 9). InterspeciÞc competition also signiþcantly affected larval duration in most of the different temperature treatments except for that at 33 C (Table ). However, the adult dry weight did not seem to change very much under moderate temperature conditions (3 and C) and was only signiþcantly reduced ( %) at extremely high temperatures (33 and 3 C). Results of two-way NOV showed the interactions between the two factors of interspeciþc competition and temperature were statistically signiþcant in both species (for C. megacephala, F 3.759, df, P.9; for C. rufifacies, F 5., df 3, P.) y = x competition intensity y = -.13x competition intensity Fig. 5. Linear regression of the adult dry weights of C. megacephala () and C. rufifacies () to the intensities of interspeciþc competition. s the competition intensity increased, the adult dry weight of C. megacephala signiþcantly decreased (r.73, P.1); but the adult dry weight of C. rufifacies was not signiþcantly affected (r.1, P.7). Competition intensities are represented by the number of individuals of the different species; 1,, and 3 are the respective numbers of individuals of the different species in each treatment of larvae total. Predation by C. rufifacies. The third instars of C. rufifacies usually tightly truss their prey with their curved body and the heavily sclerotized spines on the body surface to keep the prey from moving; they use their mouthhooks to penetrate the preyõs body to extract ßuids. Figure 1 shows the predation actions of a third-instar larva of C. rufifacies against a third instar of C. megacephala. Observations also showed that a single C. rufifacies larva could solely and successfully complete predation of a third-instar larva of C. megacephala; but more frequently, several larvae worked together until all the body ßuid of the prey had been sucked out (Fig. 1). However, predation of second instars of C. rufifacies did not seem to be as easy as that of their larger late instars; our results showed that two or more second-instar larvae usually took 1 h to catch a C. magacephala larva. Movement Orientation of Larvae. The experiment on movement orientation showed that blow ßy larvae

8 79 JOURNL OF MEDICL ENTOMOLOGY Vol. 5, no. Mean duration of immature development (hours) Mean duration of immature development (hours) exhibit signiþcant preferences in their movement tendencies toward fecal material. C. megacephala larvae tended to move to their conspeciþc fecal material in all three larval stages (Tables 3 and ); nevertheless, the Þrst-instar larvae of C. rufifacies tended to move to fecal material of C. megacephala (Table 5). This test was performed for 3 and min for both species; as the time increased to min, C. rufifacies larvae did not show a special preference for moving toward the fecal material in any instar or for moving toward the fecal material of any one species in second instars (Table ). The results also imply the invasion of maggot masses of C. megacephala by C. rufifacies only occurred in their early larval stage. Discussion Intraspecific Competition. So and Dudgeon (199) proposed two different responses of dipteran larvae to y = x competition intensity y = -.57x competition intensity Fig.. Linear regression of the durations of larval development of C. megacephala () and C. rufifacies () in response to different competition intensities. The developmental time was not affected by competition intensities in C. megacephala (r.7, P.19); but as the intensity increased, the development time signiþcantly decreased in C. rufifacies (r.719, P.15). Competition intensities are represented as in Fig. 5. intraspeciþc competition: an instantaneous increase in larval mortality as the intensity of competition increases and the temporary maintenance of the number of individuals surviving, although each of them has reduced Þtness. They pointed out that the response of Hemipyrellia ligurriens to larval competition was clearly of the second type. Emergent adults were undersized (the adult dry weight was only 1.% of the potential maximum value) and had reduced fecundity and longevity. In our studies, both species of C. megacephala and C. rufifacies also exhibited similar trends under intraspeciþc competition of reduced larval developmental duration, larval survivorship, and adult dry weight at higher rearing densities. Nonetheless, it is still quite difþcult to tell into which model these two species should be classiþed. ccording to the results, under higher-density conditions, C. megacephala was more sensitive to density effects, especially in its responses to changing larval durations, and its body sizes

9 July SHIO ND YEH: LRVL COMPETITION OF LOW FLY LRVE 793 The duration of larval stages (hours) The duration of larval stages (hours) were more ßexible (dry weight was 19.5% of the potential maximum value). One possible explanation is that C. megacephala has the potential to produce smaller viable adults to maintain higher survivorship; in other words, it produces smaller adults as a trade-off for maintaining survivorship. However, C. rufifacies tended to maintain larger adult body sizes (dry weight was.% of the potential maximum value) and thus reduced its larval survivorship. Obviously, the different strategies above may more or less reßect individual species characteristics and also strongly indicate the important role that density plays in intraspeciþc competition. Traditionally, we use a maggotõs age to approach the PMI, and a maggotõs age is usually determined by its species, instar, length, and thermal history (Greenberg and Kunich ). This procedure also involves comparisons to previously existing experimental data for reference (Goff 199). Therefore, the results presented here remind us when using a singlespecies model to generate reference data in the laboratory, intraspeciþc competition and larval density 1 st instar nd instar feeding postfeeding Larval stages ** ** ** 1 st instar nd instar feeding postfeeding Larval stages Fig. 7. Effects of single- and mixed-species rearing on the developmental times of Þrst-, second-, and third-instar larvae (including the feeding and postfeeding stages) of C. megacephala () and C. rufifacies () at C (**P.1). factors should also be taken into account to prevent a bias of age determination, especially when using body size or length as an indicator. Interspecific Competition. Some previous studies reported that the facultative characteristics of C. rufifacies can help it survive under critical conditions of food shortage and probably have higher Þtness under competitive stress (Goodbrod and Goff 199, Wells and Greenberg 199c, aumgartner 1993). However, in our studies, interspeciþc competition generally caused reduced larval duration, adult dry weight, and survivorship in both species, and thereby reductions in the overall Þtness of both species (see Fig. ). If we evaluate the relative magnitude of the reduction of production in Fig., we may Þnd the effect of interspeciþc competition to those two blow ßy species could be shown by the difference between the two lines in Fig. and. Results showed the effect of C. megacephala on C. rufifacies and the effect of C. rufifacies on C. megacephala have no signiþcant difference (t.319; P.3); it implies that the competitive

10 79 JOURNL OF MEDICL ENTOMOLOGY Vol. 5, no. 1 ** ody length of larvae (mm) ody length of larvae (mm) Time (hours) Time (hours) Fig.. Effects of single- or mixed-species rearing on the body lengths of Þrst-, second-, and third-instar larvae of C. megacephala () and C. rufifacies () at C. The larval stages are represented by 19-, 37-, and -h-old larvae, respectively (**P.1). superiority of one species over another could not be well established. It is interesting that the maximum reduction of larval duration occurred in the low-density condition for C. megacephala but in at high densities for C. rufifacies. This suggests the larval density is an important and species-dependent factor in interspeciþc competition. In addition, Fig. 3C shows the temporary rise in survivorship for C. rufifacies in the density interval between and 1 larvae/ g medium, and survivorship quickly dropped when the density increased. This result further indicates that a temporary competitive advantage can only be obtained by C. rufifacies under a proper larval density, and there is no competitive advantage for C. rufifacies under a high larval density. In conclusion, C. megacephala had a more-sensitive response to the coexistence of other competitors, especially by expressing a shorter larval duration and lighter body weight. Nevertheless, C. megacephala had relatively stable survivorship under interspeciþc competition. s Ullyett (195) mentioned, a ßy of smaller growth weight required a smaller quantity of food for full development; its more rapid and earlier development on carrion has a distinct advantage over other species in the acquisition of food. However, we believe that C. rufifacies did not actually beneþt from its facultative characteristics in the mixed-species condition, and in contrast, it suffered from the early departure of C. megacephala under higher larval densities. Further explanations are given in the following sections discussing ecological and behavioral aspects. Ecological Role and Feeding ehavior of C. rufifacies. The ecological role of C. rufifacies was reviewed and discussed by aumgartner (1993); in terms of carrion insect succession, C. rufifacies is generally regarded as a secondary carrion ßy, but in southern Queensland, ustralia (OÕFlynn and Moorhouse 1979), and Hawaii (Goff et al. 19), it is believed to be a primary species. Whether the ecological role of C. rufifacies can be altered by different climates or geographic environments is still unknown; but in Taiwan (Chen et al. ) and other areas in the region,

11 July SHIO ND YEH: LRVL COMPETITION OF LOW FLY LRVE 795 Production of C. megacephala (mg) Production of C. rufifacies (mg) such as China (Zhu et al. ) and Thailand (Sukontason et al. 1), C. rufifacies is undoubtedly an active primary carrion species. ccording to our observations, as a primary ßy, predation and cannibalism are rare in C. rufifacies. lthough the Þrst-instar larvae of C. rufifacies was attracted toward feeding maggot Temperature ( ) Temperature ( ) Fig. 9. Production index of single- and mixed-species rearing at different temperatures. Forty larvae per g of medium were reared under Þve temperature treatments., C. megacephala;, C. rufifacies. masses of C. megacephala, we believe that predation is not the main intention of C. rufifacies. Kitching (197) and Goodbrod and Goff (199) observed that when Þrst placed onto the food medium, maggots tend to aggregate in a single mass and burrow in, feeding continuously. They also believed that the movement Table 1. Larval developmental time and adult dry weight of C. megacephala in single- (pure) and mixed-species (mixed) cultures at different temperatures (all at a density of larvae/ g medium) a Temperature ( C) Larval developmental time (h) dult dry weight (mg) P P a a a.3.33 a b b b.3.15 b c c c 7.. c d d.1.7. d d e e e.7. bc.1 Values in a column followed by the same letters do not signiþcantly differ (P.5); P values indicate the statistical differences between single- and mixed-species colonies at the same temperature. a Equal numbers of C. megacephala and C. rufifacies (:) larvae were reared in the mixed-species culture.

12 79 JOURNL OF MEDICL ENTOMOLOGY Vol. 5, no. Table. Larval developmental time and adult dry weight of C. rufifacies in single- (pure) and mixed-species (mixed) cultures at different temperatures (all at a density of larvae/ g medium) a Temperature ( C) Larval developmental time (h) dult dry weight (mg) P P 1 b Ñ Ñ Ñ Ñ Ñ Ñ a a. 7.. a 7..3 ab b b a..5 a c c a 7.. bc d d.3.3. a.39.3 c.37 Values are mean SE. Values in a column followed by the same letters do not signiþcantly differ (P.5); P values indicate statistical differences between the single- and mixed-species cultures at the same temperature. a Equal numbers of C. rufifacies and C. megacephala (:) larvae were reared in the mixed-species culture. b Larvae of C. rufifacies failed to pupate at 1 C. of numerous mouthhooks combined with the secretions of larval salivary and proteolytic enzymes increase the efþciency of the feeding process and the rate of larval development. ccording to our observations and results of the experiment on larval movement orientation, the maggot mass of C. megacephala always formed quickly after the Þrst instars hatched; however, the situation in C. rufifacies differed. The newly hatched larvae of C. rufifacies always climbed around on the medium for a while, usually 1 dor longer, and then gathered to form their own maggot Fig. 1. Larval predation of third instars of C. rufifacies on a third instar of C. megacephala. () Single third instar of C. rufifacies trusses the prey with its curved body and sclerotized spines, and uses its mouthhook to penetrate the prey to extract ßuid. () Several larvae usually work together until all of the body ßuid of the prey has been sucked out. mass. Furthermore, if other species, such as C. megacephala, are present, C. rufifacies larvae tend to invade the other speciesõ preexisting maggot mass. ecause predation only occurred with second- and third-instar larvae of C. rufifacies and also because a higher efþciency of food digestion is known to exist in maggot masses of C. megacephala (Goodbrod and Goff 199), we believe that the early invasion by C. rufifacies into maggot masses of C. megacephala is probably only to facilitate their feeding efþciency and larval development, instead of for predation or cannibalism. ased on our observation in this experiment, through invading the maggot masses of other species, C. rufifacies can also force other competitors to leave food earlier and thus it acquires more resources. Finally, the aggregation and coexistence is long been considered an important factor in carrion ßy competition. Ives (1991) has indicated the intraspeciþc aggregation would increases intraspeciþc competition and reduces interspeciþc competition, and although interspeciþc aggregation is rare, it was also believed could signiþcantly reduces interspeciþc competition. Defense Response of C. megacephala. Goodbrod and Goff (199) pointed out that in mixed-species culture of C. megacephala and C. rufifacies, the larval mortality of C. rufifacies remained relatively stable, but the pupal weight increased. However, we found different results in this study. Under our mixed-speciþc rearing, the larval developmental rate, adult dry weight, and larval survivorship of C. rufifacies signif- Table 3. ttraction rates to fecal material of C. megacephala and C. rufifacies by different instars of C. megacephala Stage ttraction to fecal material of C. megacephala ttraction to fecal material of C. rufifacies No attraction 3-min a attraction rate (%) First larval instar Second larval instar Third larval instar min a attraction rate (%) First larval instar Second larval instar Third larval instar a ttraction rates were recorded after 3 and min.

13 July SHIO ND YEH: LRVL COMPETITION OF LOW FLY LRVE 797 Table. analysis of the three larval stages of C. megacephala attracted to fecal material Stage ttracted to fecal material only (df 1) ttracted to fecal material and no attraction (df ) 3 min a First instar 1. b. b Second instar 1.77 b 37.1 b Third instar b 3. b min a First instar 19. b. b Second instar 15.3 b 15.3 b Third instar. b 1.5 b Values are. a ttraction rates were recorded after 3 and min. b Values signiþcantly differ (P.5);.5 (1) 3.15;.5 () icantly changed (Fig. 3). When comparing our experimental results with those of Goodbrod and Goff (199), we found that different experimental designs may have caused the different results. Goodbrod and Goff (199) used a 5-liter container as their experimental arena, but we used a 5-ml plastic cup with some openings cut in the sides and an outside container that allowed the C. megacephala larvae to escape predation. When considering body size and the mobility and defense abilities of C. megacephala, we believe that predation is actually not easy for C. rufifacies. Reigada and Godoy (5) performed an experiment to understand the dispersal and predation behaviors of blow ßy larvae in mixed-species culture; their results showed that C. megacephala signiþcantly changes its dispersal pattern when coexisting with a predator, Chrysomya albiceps; and they thought this change might be attributable to the predation and/or escape ability of the prey. In addition, C. megacephala usually has a larger body size and better moving and climbing abilities. ll of the above reasons led us to conclude that C. megacephala is quite resistant to predation by C. rufifacies, and this also agrees with another study. Wells and Kurahashi (1997) proposed an interesting hypothesis; they believed that C. megacephala, historically sympatric with C. rufifacies, is relatively resistant to predation by C. rufifacies, which can provide it with Table 5. ttraction rates to fecal material of C. megacephala and C. rufifacies by different instars of C. rufifacies Stage ttraction to fecal material of C. megacephala ttraction to fecal material of C. rufifacies No attraction 3-min a attraction rate (%) First larval instar Second larval instar Third larval instar min a attraction rate (%) First larval instar Second larval instar Third larval instar a ttraction rates were recorded after 3 and min. Table. analysis of the three larval stages of C. rufifacies attracted to fecal material Stage ttracted to fecal material only (df 1) ttracted to fecal material and no attraction (df ) 3 min a First instar. b.5 b Second instar 19.1 b.9 b Third instar 1.5 b 3.7 b min a First instar b Second instar. 1. Third instar b Values are. a ttraction rates were recorded after 3 and min. b Values signiþcantly differ (P.5);.5 (1) 3.15;.5 () a competitive advantage over the more vulnerable Cochliomyia macellaria when larvae of all three occur together. Finally, we would like to suggest that both the predation ability and the defense or escape activity should be taken into account when evaluating larval competitive advantages or estimating the PMI. Temperature, Larval Stage, and Competition Intensity. Temperature, larval stage, and competition intensity are three newly proposed factors that have not been discussed in previous studies on interspeciþc competition. Our results strongly suggest that the effect of interspeciþc competition is temperature dependent, especially the larval developmental time. Higher temperatures generally enhance the effects of interspeciþc competition, because high temperatures speed up the developmental rates of both species and also change their responses to competition. However, the cross-effect of temperature and competition is still unclear; our preliminary conclusion was that the interaction is probably caused by different degrees of temperature durability in the two species. The results also showed that larvae of C. megacephala are less temperature sensitive under interspeciþc competition than are those of C. rufifacies (Tables 1 and ). For the interspeciþc competition effect on different larval stages proposed in this article, we clearly found that the results exactly reßected the behavioral characteristics of these two species: the change in larval duration of second instars of C. rufifacies and in late instars of C. megacephala, respectively, represent predation/invasion and disturbance/escape situations. However, body sizes were relatively stable regardless of whether for single- or mixed-species rearing except for that of the late instars of C. megacephala. This particularly reminds us that careful veriþcation is needed when using body size of late instars C. megacephala as a PMI indicator. Competition intensity is another interesting issue that needs to be addressed. Competition intensity here was deþned as the degree of stress caused by a different species: the greater the number of individuals of the different species that is present, the higher the competition intensity it causes. Our results indicated that the factor of competition intensity works

14 79 JOURNL OF MEDICL ENTOMOLOGY Vol. 5, no. differently on these two species. The body size of C. megacephala was mostly affected by different competition intensities, whereas developmental time was otherwise signiþcantly affected in C. rufifacies. lthough the mechanism of how the competition intensity really works on these two species is still unknown, we found that the effect was actually profound and should not be overlooked in evaluating larval developmental condition. However, in actual applications, data on the initial population or species composition and proportion on a carcass are difþcult to acquire, thus making this correction hard to apply in most real cases. Concluding Remarks. Temperature, density (including inter- or intraspeciþc population densities), behaviors (including predation, cannibalism, defense, and dispersal behaviors), and interactions between and among species obviously and signiþcantly affect the outcomes of larval development and thereby directly inßuence PMI estimations. We are not able to completely clarify the complexity of this small ecosystem currently, but we believe the following issues should be further studied or reevaluated: Whether C. rufifacies is a primary or secondary ßy (Early and Goff 19)? The higher Þtness of C. rufifacies under competition? Using C. rufifacies as a more-reliable indicator for estimating PMI (Goodbrod and Goff 199)? Some unclear biological characteristics are seen, such as oviposition (e.g., egg-laying delay of C. rufifacies adults in Goff ), feeding inhabitant (e.g., different species inhabiting different parts of the carrion; Tullis and Goff 197, Goff et al. 19), predation, and dispersal behaviors (e.g., maggot migration in yrd and utler 1997). Our advice of using developmental data in estimating PMI could be further summarized as followed: Use a mixed-species rearing to generate the reference data if possible. Carefully apply the body length or body weight of maggots as the indicator of larval development, especially when two or more species coexisted on the carrion. Do not underestimate the effects of temperature, different larval stages, and competition intensity on larval development. cknowledgments This work was supported by the National Science Council, Republic of China (NSC95--H-- and NSC H--15) and the Institute of Forensic Medicine, Ministry of Justice, Republic of China (IFM9-M and IFM91-M). References Cited aumgartner, D. L Review of Chrysomya rufifacies (Diptera: Calliphoridae). J. Med. Entomol. 3: 33Ð35. aumgartner, D., and. Greenberg. 19. The genus Chrysomya (Diptera: Calliphoridae) in the new world. J. Med. Entomol. 1: 15Ð113. eaver, R Non-equilibrium ÔislandÕ communities: Diptera breeding in dead snails. J. nim. Ecol. : 73Ð 79. yrd, J. H., and J. F. utler Effect of temperature on Chrysomya rufifacies (Diptera: Calliphoridae) development. J. Med. Entomol. 3: 353Ð35. Catts, E. P., and M. L. Goff Forensic entomology in criminal investigations. nnu. Rev. Entomol. 37: 53Ð7. Chen, W. Y., T. H. Hung, and S. F. Shiao.. 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