Biology and morphometry of Lycoriella ingenua (Diptera: Sciaridae)

BIOLOGY AND MORPHOMETRY OF LYCORIELLA INGENUA 41 BIOL. LETT. 2004, 41(1): 41 50 Available online at http://www.biollett.amu.edu.pl Biology and morphometry of Lycoriella ingenua (Diptera: Sciaridae) MARIUSZ LEWANDOWSKI 1, AGNIESZKA SZNYK 2 and ANDRZEJ BEDNAREK 2 1 Department of Applied Entomology, 2 Department of Animals and Environment, Warsaw Agriculture University, Nowoursynowska 166, 02-787 Warsaw, Poland (e-mail: lewandowski@alpha.sggw.waw.pl) (Received on 6th February 2004; Accepted on 16th July 2004) Abstract: The morphometry of Lycoriella ingenua (Dufour, 1839) (Diptera: Sciaridae) was studied with the use of insects reared in the laboratory. The length and weight of eggs, as well as of the body of each larval instar, pupae, and adults were measured. The head capsule of larvae was also measured. Larval body length ranged from 0.36 to 7.9 mm, and weight from 0.0015 to 2.126 mg, so during development these parameters increased 19 and 119 times, respectively. Measurements of head capsule width showed that between the successive instars it increased about 1.45 times. This parameter proved to be the most reliable feature for identifying particular instars. The present study also includes lengths and widths of other developmental stages as well as their time requirements for complete development. At 24 o C the development of one generation lasted from 18 to 21 days. Key words: Diptera, Sciaridae, Lycoriella ingenua, mushroom fly, morphometry INTRODUCTION Flies of the family Sciaridae occur almost all over the world. Their larvae are known to feed on decaying organic matter, plants, and the spawn of fungi. Due to such a wide food spectrum, several species of this family are recognized as pests in horticultural production. Some species of the genus Lycoriella are acknowledged to be the most threatening pests, causing serious damage to the world mushroom production (MENZEL & MOHRIG 1997). Great economic losses caused by various sciarid species in the mushroom industry were reported from Australia (CLIFT 1979), USA (SNETSINGER 1972, CANTELO 1979, WHITE 1985), Russia (GERBATCHEVSKAYA 1963), United Kingdom (AUSTIN & JARY 1933, HUSSEY et al. 1969, BINNS 1973, WHITE 1985), and other West European countries (HUSSEY et al. 1969, WHITE 1986, GEELS & RUTJENS 1992, SCHEEPMAKER et al. 1995). However, according to the recent reclassification of Sciaridae (MENZEL & MOHRIG 2000), there are only two species of Lycoriella that attack mushroom cultures in Europe (MENZEL & MOHRIG 2000, WHITE

42 M. Lewandowski, A. Sznyk and A. Bednarek et al. 2000): (1) L. ingenua (Dufour), syn. L. mali (Fitch), L. solani (Winn.) and (2) L. castanescens (Lengersdorf), syn. L. auripila (Winn.). Elaboration of efficient methods for controlling pest populations requires a competence in identification of species and their developmental stages. With this aim, in studies on many insect groups, morphometric methods are widely used, but their application to the Sciaridae has been limited to very few species. PITCHER (1936) gave some head capsule parameters for Sciara fenestralis (Zett.) without any details of the used methods. WILKINSON & DAUGHTERY (1970a,b) published a biological and morphometric description of Bradysia impatiens (Johannsen), including measurements of egg width, body length and head capsule width for each larval stage. HUSSEY & GURNEY (1968) studied the morphometry of the head capsule in L. auripila (= L. castanescens) larvae. STEFFAN (1966) described immature stages of L. mali (= L. ingenua). All those studies determined the morphological features of larvae, which can be useful in identification of particular larval instars. Body weight of larvae of B. paupera (Tuomikoski) was given in one paper only (BERG 2000). The aim of this study was to determine morphological parameters for particular developmental stages of the sciarid fly L. ingenua. They could be useful for species identification, especially for quick identification of different larval instars in vivo. No detailed measurements and description of morphological features were made for this species before. MATERIALS AND METHODS Collecting the material and mass rearing Adult flies of Lycoriella ingenua (Dufour, 1839) were caught, by the use of aspirators, in commercial mushroom-growing cellars in the Warsaw region. They were then put into test-glasses (dimensions 10 2.7 cm) and transported to a laboratory in a portable refrigerator. These adults (about 100 individuals) were placed in glass isolators covered on both sides with fine gauze (about 0.1 mm). The isolators were then placed on Petri dishes (diameter about 10 cm each) filled with some peat, prepared according to BINNS s (1973) method. Sifted through a sieve (8 mesh), and then moistened, the peat was blended with chalk in quantities providing neutral ph and with a 2% addition of ground soybeans serving as food supply for larvae. The Petri dishes with isolators were put into climate chambers for cultivation. Flies were left for 24 hours at 24±0.3 C to lay eggs. The isolators with sciarids were removed afterwards and all the dishes were covered with parafilm, which made it possible to maintain humidity in the dishes at a level similar to that of mushroom-growing cellars. It also prevented the insects from escaping from the dishes. Development and morphological parameters Six Petri dishes, prepared according to the description above, were used in the experiment. Every day, until the end of development, 30 individuals from each dish were randomly selected and measured. Each instar was put into an aluminium cup (diameter about 5 mm and height 4 mm) and weighed on a Sartorius Supermicromicro scale (±0.0001 mg). Adult flies

BIOLOGY AND MORPHOMETRY OF LYCORIELLA INGENUA 43 just before weighing were caught with an aspirator and killed with ethyl acetate. Each emerging adult individual was subjected to the measurements. Eggs, larvae and pupae were weighed alive just after being taken from dishes. After weighing, larvae were placed in separate test-glasses with 70% ethyl acetate to kill and preserve them. They were measured after extraction from the liquid. The other stages were measured immediately after weighing. Morphometric measurements were taken under an Olympus stereoscope microscope equipped with a graduated eyepiece (10/100 ). The following measurements were taken: egg length and width; larval body length (from the tip of the head to the end of the abdomen) and head capsule width (in the widest place) in each instar; pupal and adult body length and width of the widest abdominal segment. The results of measurements were grouped according to developmental stage. In determining larval instars, features like head capsule width and type and arrangement of spiracles on the larval body were taken into account. The percentage share of each instar was calculated every day. Duration of development of each instar was estimated basing on the percentage share of individuals on the subsequent days of development. Statistical analysis of the data was carried out by using Statgraphics Plus v. 4.1. Mean values of body weight, length and head capsule width in subsequent instars were compared with Student s t-test for α=0.05. Body weight and length were transformed to receive a linear function. Linear regression between body length and weight was calculated. Correlation coefficients and regression were assessed. On the basis of mean head capsule width in each instar, the coefficient of head capsule enlargement at the moment of transition into the subsequent larval stage (Brook s ratio) was calculated. RESULTS Duration of development of the flies The time required for completing the development of one generation and duration of development of particular stages of L. ingenua are presented in Fig. 1. The time needed to complete the development of the first generation at 24 o C ranged from 18 to 21 days. The first-instar larvae hatched from eggs after 3 days. The developmental time of larvae was 12 13 days, out of which the fourth larval stage was the longest. The pupal period lasted 3 to 5 days, which subsequently resulted in the emergence of the first adult individuals after 18 days. Males appeared one day earlier than females. The percentage shares of developmental stages on successive days of the life cycle of L. ingenua are presented in Fig. 1. Body length and weight of particular developmental stages Body length at particular developmental stages revealed significant differences between successive developmental stages (Table 1). After egg hatching, the body length of larvae increased 19 times throughout their 4-instar development, while pupae were significantly shorter than the last larval stage. In the case of adult forms, length of the adult body increased after the emergence from pupa. Differences in body length

44 M. Lewandowski, A. Sznyk and A. Bednarek 100% Percentage share of individuals 80% 60% 40% 20% 0% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Day of development egg 1-st 1st instar larval st. 2-nd 2st instar larval st. 3-rd 3st instar larval st. 4-th 4st instar larval st. pupa female male Fig. 1. Percentage shares of individuals in various developmental stages of Lycoriella ingenua on successive days Table 1. Body length (mm) for developmental stages of Lycoriella ingenua Stage n Range Mean SD Student s t test Egg 48 0.243 0.329 0.290 0.024 1st instar 57 0.360 1.400 0.782 0.228 14.89* 2nd instar 61 0.183 2.632 1.550 0.446 11.65* 3rd instar 57 1.560 4.650 2.802 0.744 11.18* 4th instar 139 2.900 7.900 5.519 1.216 15.69* Pupa 68 1.800 3.200 2.539 0.353 19.78* Adult 70 2.500 3.950 3.098 0.369 9.08* Comparison of body length for female and male pupae Female 33 2.400 3.200 2.816 0.191 Male 35 1.800 3.000 2.277 2.258 9.74* Comparison of body length for female and male adults Female 33 3.080 3.950 3.421 0.208 Male 37 2.500 3.560 2.810 0.207 12.3* * Means significantly different at p < 0.01

BIOLOGY AND MORPHOMETRY OF LYCORIELLA INGENUA 45 among male and female pupae were significant. A similar relationship was observed for adults. The mean width of eggs was 0.164 mm (0.129-0.186, SD = 0.0002, n = 48). Significant differences in body weight between successive developmental stages are shown in Table 2. During larval development, body weight increased 119 times. The weight of the pupa was significantly lower than the weight of the last larval stage. Also body weight of males was significantly lower than that of females. Table 2. Body weight (mg) for developmental stages of Lycoriella ingenua Stage n Range Mean SD Student s t-test Egg 48 0.0015 0.0053 0.0031 0.0009 1st instar 57 0.0015 0.0244 0.0079 0.0056 5.9* 2nd instar 61 0.0108 0.7550 0.0545 0.0956 3.67* 3rd instar 57 0.0506 3.3999 0.1497 0.0820 5.79* 4th instar 139 0.1704 2.1260 0.9420 0.5165 11.5* Pupa 68 0.3197 1.2445 0.7574 0.3013 3.23* Adult 70 0.1352 1.0315 0.4585 0.2206 6.66* Comparison of body weight for female and male pupae Female 33 0.8040 1.2445 1.0466 0.1360 Male 35 0.3197 0.5851 0.4848 0.0619 22.13* Comparison of body weight for female and male adults Female 33 0.2627 1.0315 0.6149 0.2109 Male 37 0.1352 0.4030 0.2986 0.0592 8.59* * Means significantly different at p < 0.01 Daily increase in weight and length of larvae An increasing tendency in body weight and length of larvae was observed (Fig. 2). On days 6 or 7, the weight gain of larval bodies was visibly impeded, whereas on days 5 and 6 an increase in their length was noted. During the last two days of larval development, body weight no longer increased and on the last day a significant decrease in body length in relation to day 11 was observed. The correlation coefficient for these two parameters was r = 0.96 (r 2 = 91.55%), so p < 0.01. The relation between the parameters is linear and can be expressed by the following regression equation: y = -4.231 + 2.317x, where y = log(weight), and x = log(length). Comparison of the body width of pupae and adults and larval head capsule width Females had wider abdomens; similarly, pupae of females were considerably wider than those of males (Table 3). Brook s ratio showed a decreasing trend in the course of larval development (Table 4). Comparing larvae of the same head capsule width enabled the determination of the width range of head capsule for a particular larval stage. A decreasing number of larvae within an adequate width range could

46 M. Lewandowski, A. Sznyk and A. Bednarek Body weight [mg] 1.4 1.2 1.0 7 6 5 Body length [mm] 0.8 4 0.6 3 0.4 2 0.2 1 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 0 Weight Length Day of development Fig. 2. Changes in body weight and length of larvae of Lycoriella ingenua during development Table. 3. Comparison of abdomen width (mm) between female and male pupae and adults of Lycoriella ingenua Female Male Student s Stage n Mean SD n Mean SD t-test Pupa 33 0.998 0.029 35 0.752 0.0417 15.67* Adult 33 0.747 0.114 37 0.485 0.0523 12.61* * Means significantly different at p < 0.01 Table. 4. Head capsule width (mm) and Brook s ratio for larvae of Lycoriella ingenua Larval stage n Range Mean SD Student s t-test Brook s ratio 1st instar 59 0.0710 0.1200 0.0976 0.0109 2nd instar 59 0.1286 0.1557 0.1540 0.0143 3rd instar 63 0.2000 0.2429 0.2214 0.0145 4th instar 133 0.2571 0.3429 0.2912 0.0176 * Means significantly different at p < 0.01 24.14* 25.91* 27.40* 1.58 1.44 1.32

BIOLOGY AND MORPHOMETRY OF LYCORIELLA INGENUA 47 be considered the moment of proceeding from one larval instar to the subsequent one. Fig. 3 shows the numerical comparison of L. ingenua, where four peaks correspond to the amount of larvae in each instar. Number of individuals 70 60 50 1st instar 2nd instar 3rd instar 4th instar 40 30 20 10 0 0.07 0.09 0.09 0.10 0.11 1.12 0.13 0.14 0.16 0.17 0.19 0.20 0.21 0.23 0.24 0.29 0.30 0.31 0.34 Fig. 3. Head capsule width for particular larval stages of Lycoriella ingenua Width [mm] DISCUSSION Our experiments with L. ingenua contribute to the knowledge of the life cycle of this species. The developmental time of the first generation, from oviposition to the emergence of adults, was found to last 18 21 days at 24 o C. Similarly, WETZEL et al. (1982) estimated that the life cycle of L. mali (=L. ingenua) at 23.9 o C lasted about 19 days. In the case of L. auripila (=L. castanescens), a species common in Western Europe, it lasts 21 days (HUSSEY & GURNEY 1968), whereas for Bradysia impatiens it takes on average 21.4 days (WILKINSON & DAUGHERTY 1970b). The developmental time for the larvae was similar to that reported by WETZEL et al. (1982) for L. mali (about 10 days), and shorter than for another sciarid, S. fenestrialis, studied by PITCHER (1936). The latter author applied a temperature of 22 23 o C, which prolonged the development to 23 24 days. Development of particular larval stages was also proportionally longer. Similarly, larval development of L. auripila, according to HUSSEY & GURNEY (1968), was evidently longer. The time of development of the sciarid L. ingenua discovered in the course of the research was similar to that of L. mali reported by WETZEL et al. (1982), which is confirmed by FREEMAN s (1987) and MENZEL & MOHRIG s (2000) assurance that

48 M. Lewandowski, A. Sznyk and A. Bednarek this is the same species. Similarly, results obtained by PITCHER (1936) for S. fenestrialis showed that developmental time was proportionally longer in successive larval stages, which is evidently indicative of a similarity in the biology of the abovementioned species. The width of the larval head capsule of L. ingenua presented here is significantly different from that of S. fenestrialis given by PITCHER (1936). However, Brook s ratio calculated for all the stages is similar (Table 5). According to Brook s rule, this coefficient is constant for each species (CROSBY 1973). Table 5. Mean head capsule width (mm) and Brook s ratio (BR) for larvae of four sciarid species Larval stage L. ingenua (original data) S. fenestrialis (PITCHER 1936) L. castanescens (HUSSEY & GURNEY 1968) B. impatiens (WILKINSON & DAUGHERTY 1970a) width BR width BR width BR width BR 1st instar 0.098 0.087 0.07 0.072 1.58 1.33 1.57 1.45 2nd instar 0.154 0.116 0.11 0.105 1.44 1.34 1.55 1.85 3rd instar 0.221 0.156 0.17 0.195 1.32 1.60 1.41 1.43 4th instar 0.291 0.250 0.24 0.280 Mean 1.45 1.42 1.51 1.58 Mean egg weight for L. ingenua was 0.003 mg, whereas egg width (0.17 mm) and length (0.29 mm) were definitely lower than those reported for L. auripila (0.3 0.7 mm) by HUSSEY & GURNEY (1968). They are, however, similar to the results for L. mali eggs, (0.15 0.25 mm) presented by KIELBASA & SNETSINGER (1980). The mean body length of L. ingenua larvae ranged in this study from 0.78 to 5.52 mm, increasing 7.1 times during development. The maximum body length of L. mali larvae, according to KIELBASA & SNETSINGER (1980), reached 6 to 7 mm. These values are smaller than the maximum length recorded in our study (7.9 mm). Mean body weight of particular larval instars ranged from 0.008 mg of larvae in the first instar to 0.942 mg for the fourth instar, which means that this value increased 119 times during development. According to BERG (2000), the mean body weight of another sciarid species, B. paupera, ranged from 0.011 to 2.238 mg. Taking these extreme results into consideration, the upper limit of body weight for larvae was found to be similar to that obtained during this study, whereas the lower limit was ten times smaller in L. ingenua (0.0015). The mean body weight of pupae for L. mali presented by TUNG & SNETSINGER (1973) was 1.18 mg and 0.46 mg for female and male pupae, respectively. Pupae of L. ingenua in our study weighed on average 1.05 mg and 0.48 mg, for females and males, respectively. The measurements of body length and weight for larvae were compared by the use of linear regression. Both correlation and regression coefficients revealed a close relationship between these parameters, which is to say that body weight increased proportionally to body length. During the last three days the body weight of larvae remained at the same level whereas the length of larvae decreased significantly. It

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