Introduction
Taxonomy of the Chalcidoidea (Hymenoptera) is almost exclusively based on the comparison of morphological traits, but these can be related to fitness and thus are a possible target of natural selection. As a consequence, morphological differences are shaped by both speciation divergence and ecological adaptations. In particular, expression of numerous morphological traits may be strongly influenced by the larval environment and, especially, by developmental temperature (Gibert et al., Reference Gibert, Capy, Imasheva, Moreteau, Morin, Pétavy and David2004). Thus, errors are possible when species are differentiated based on variable morphological traits whose real variance is unknown. Morphological and biological diversity is well known in the Chalcidoidea, and their classification is complicated by variation in size and morphology as a function of the host, seasonal dimorphism and dichroism (Askew, Reference Askew1971).
In the Chalcidoidea, Pnigalio Schrank (Eulophidae) is a taxonomically problematic genus (Barrett et al., Reference Barrett, Brunner and Turner1988). Taxonomists dealing with this genus have often used differences in colour, propodeal morphology and position of the costulae to distinguish species, but have only rarely assessed intraspecific variation. Moreover, numerous species have been described from only a few individuals from a restricted geographic area. High levels of phenotypic plasticity in Pnigalio have already been reported for several traits of P. minio (Walker) (as P. flavipes (Ashmead)) (Barrett et al., Reference Barrett, Brunner and Turner1988). It is therefore important to determine the potential magnitude of intraspecific variation in these traits before they are used for species recognition.
The understanding of intraspecific variation in Pnigalio can be facilitated by investigating a specific system of plant–phytophage–parasitoid. Hitherto, these data have not been available, partly because of the difficulties in rearing species of this genus. For the present study we employed Parietaria diffusa M. & K. (Urticales: Urticaceae), Cosmopterix pulchrimella Chambers (Lepidoptera: Cosmopterigidae), and Pnigalio soemius (Walker) (Hymenoptera: Eulophidae). Pnigalio soemius is a polyphagous ectoparasitoid of late instar leafminer larvae belonging to different orders of insects, and of gall-forming sawflies.
In this study we evaluated the influence of rearing temperature on several quantitative traits using developmental reaction norms (DRN). A DRN is the response curve of a phenotype as a function of an environmental gradient, using a character state approach (Via et al., Reference Via, Gomulkiewicz, De Jong, Scheiner, Schlichting and Van Tienderen1995).
Materials and methods
Rearing
A culture of C. pulchrimella was maintained in an environmental chamber at 25±1°C, 60%±10% r.h. and 12L:12D on P. diffusa. Plants with a height of about 15 cm were collected in the field, and each placed in a pot of 10 cm diameter that was then covered by a plastic isolator. After a quarantine period in a greenhouse, the plants were exposed to about 20 adults of the leafminer by introducing them through the isolator. The isolator was cylindrical, with a diameter of 10.5 cm and a height of 19 cm, and was closed by a net on the top. After 24 h, the adults were collected to infest other plants. After reaching maturity in 14 days, the leafminer larvae were exposed to the parasitoids.
A culture of P. soemius was established using adults that emerged from parasitized larvae of C. pulchrimella collected in Parco Gussone, Portici (Na, Italy), in April, 2001. Identification of this parasitoid population as P. soemius was made by comparison with specimens from The Natural History Museum, London, that had been identified as P. soemius by Dr Z. Bouček. However, the specific limits of P. soemius have to be better defined because high variability in colour and other morphological traits, correlated with temperature and host species differences, have been demonstrated in this genus (Viggiani, Reference Viggiani1963; Askew, Reference Askew1971; Barrett et al., Reference Barrett, Brunner and Turner1988; Ateyyat, Reference Ateyyat2002; Bernardo et al., Reference Bernardo, Pedata and Viggiani2005).
Three pairs of parasitoids were offered a plant of P. diffusa with full grown leafminer larvae for 24 h. Parasitoid females were at least 72 h-old and were allowed to feed on honey and to host-feed (Bernardo et al., Reference Bernardo, Pedata and Viggiani2006).
Tests
Influence of temperature on gastral tergite pigmentation of P. soemius females was assessed at five temperatures (10°, 15°, 20°, 25°, 30°±1°C), whereas influence on size and other traits was evaluated at four temperatures (15°, 20°, 25°, 30°±1°C). Parasitoid pairs (10 replicas of 3 pairs) were offered for five consecutive days a single infested plant at each temperature, thus testing each insect at all the tested temperatures.
Six days after rearing at 20–30°C, and 15 days at 10–15°C, leaves with leafminer larvae were collected and put in aerated boxes (65 mm diameter and 85 mm high). Newly emerged parasitoid adults were counted, isolated and sexed. Colour comparison was performed on a reduced data set of 15 females for each temperature, except for 10°C because only 11 females were obtained at this temperature. For morphological comparison, 50 newly emerged females were isolated in glass vials (5.5×1.2 cm). After death, specimens were mounted on card rectangles and used to measure the following features and ratios: length of metatibia, a widely used comparative measure of body size (Bezemer et al., Reference Bezemer, Harvey and Mills2005; Chow & Heinz, Reference Chow and Heinz2005), length of clava/first segment of funicle, length/width of fourth segment of funicle, length of fore wing, length of marginal and stigmal veins, length of marginal vein/stigmal vein, length/width of propodeum, width of median carina near anterior margin of propodeum, insertion of costulae on median carina/length of propodeum, and number of costulae. The number of costulae was established by giving an integer value to each costula that reached the lateral margin of the propodeum (plicae), and a decimal value (0.25; 0.5; 0.75) given for each costula that did not, based on the distance it was separated from the plicae or median carina; width of the median carina at the anterior margin was ascribed to one of four qualitative classes we defined for our sample (fig. 1). The length/width ratio of the gaster is sometimes considered an important taxonomic character, but we did not take these measurements because the gaster quite often is collapsed in dead specimens and its use is therefore impractical. Gastral pigmentation was evaluated within 24 h after death. The percentage of black on the gastral tergites was calculated using the PDSview program.
Fig. 1. Propodeum of female Pnigalio soemius, illustrating width range of median carina at the anterior margin of propodeum. A, straight base (class 1); B, slightly widened base with small triangular inner cell (class 2); C, intermediate widened base with medium triangular inner cell (class 3); D, widely enlarged base with large triangular inner cell (class 4). c, costula; mc, median carina; p, plica; w1, w2, w3, w4, anterior base of median carina.
Voucher specimens are deposited in the insect collection of the Dipartimento di Entomologia e Zoologia Agraria ‘Filippo Silvestri’, Università degli Studi di Napoli ‘Federico II’.
Statistical analysis
Data satisfying conditions of normality and homoscedasticity, both untransformed or after appropriate transformation, were analysed by ANOVA and the means were separated at the 0.05 level of significance by a multiple range test (Tukey HSD). In all other cases a non-parametric test (Kruskal-Wallis) was used and medians were graphically separated by a Box-and-Whisker plot analysis (Statgraphics plus, 1997).
All data are presented non-transformed with their standard error within brackets.
Results
Pigmentation of gastral tergites
For gastral tergite pigmentation, a convex reaction norm was observed. The percentage of black pigmentation ranged between 5 and 100%, with all females reared at 10°C having the tergites completely black, and with a minimum of pigmentation around 25°C (Kruskal-Wallis test, P<0.01; df=4) (fig. 2). At 10 and 15°C, the legs also showed a larger proportion of dark colour, although this trait was not evaluated quantitatively.
Fig. 2. Reaction norm of percentage of black on the gastral tergites of female Pnigalio soemius according to its rearing temperature (mean±SE).
Size
Metatibia length ranged between 0.4 and 0.8 mm and was influenced significantly by temperature (ANOVA test, P<0.01; F=2.9; df=3) (fig. 3). The DRN showed a concave curve, with the metatibia being longest at an intermediate temperature (20°C). Fore wing length ranged between 1.18 and 2.26 mm and showed a decreasing DRN, with specimens reared at 30°C having the shortest wing length (ANOVA test, P<0.01; F=9.15; df=3) (fig. 4).
Fig. 3. Reaction norm of length of metatibia of female Pnigalio soemius according to its rearing temperature (mean±SE).
Fig. 4. Reaction norm of length of fore wing of female Pnigalio soemius according to its rearing temperature (mean±SE).
Antenna
The DRN of the ratio between the length of the clava and of the first segment of the funicle showed a convex curve, with the highest ratio at 15°C (ANOVA test, P<0.01; F=8.01; df=3) (fig. 5). The clava varied from being shorter than to longer than the first funicular segment, with their ratio ranging between 0.77 and 1.37. The DRN of the ratio between the length and the width of the fourth funicular segment showed a shape resembling a sigmoid function, with specimens reared at 15° and 20°C having the higher values (Kruskal-Wallis test, P<0.05; df=3) (fig. 6). The fourth funicular segment varied from sub-quadrate to rectangular, with the length:width range varying between 1.12 and 2.33.
Fig. 5. Reaction norm of length of clava/first funicular segment of female Pnigalio soemius according to its rearing temperature (mean±SE).
Fig. 6. Reaction norm of length/width of fourth funicular segment of female Pnigalio soemius according to its rearing temperature (mean±SE).
Fore wing venation
The marginal and stigmal veins showed different DRN trends. The marginal vein had a concave curve, with its greatest length at intermediate temperatures (ANOVA test, P<0.01; F=5.42; df=3) (fig. 7), whereas length of the stigmal vein was inversely proportional to the rearing temperature (ANOVA test, P<0.01; F=19.53; df=3) (fig. 8). The ratio between the two measures increased with temperature (ANOVA test, P<0.01; F=24.1; df=3) (fig. 9). Minimum and maximum values of this ratio were 3.05 and 5.58; marginal and stigmal vein length ranged between 0.40 to 0.77 mm and 0.10 to 0.20 mm, respectively.
Fig. 7. Reaction norm of length of marginal vein of female Pnigalio soemius according to its rearing temperature (mean±SE).
Fig. 8. Reaction norm of length of stigmal vein of female Pnigalio soemius according to its rearing temperature (mean±SE).
Fig. 9. Reaction norm of length of marginal vein/stigmal vein of female Pnigalio soemius according to its rearing temperature (mean±SE).
Propodeum, median carina and costulae
Females reared at 15°C had the highest average value of the length:width ratio of the propodeum (Kruskal-Wallis test, P<0.01; df=3) (fig. 10), with the range of variation being between 0.77 and 1.67. The mean number of costulae increased when rearing temperature increased (Kruskal-Wallis test, P<0.01; df=3) (fig. 11). Even though correlation between the size of a female and the number of costulae was weak (Y=0.611+0.022 X, F=6.61, P<0.05, R2=0.032), about 77% of females with more than three costulae were larger than the average size of a female at that rearing temperature. An influence of rearing temperature on the position of the costulae relative to the median carina was also observed. Females reared at 30°C had the costulae situated more proximally on the propodeum than when reared at lower temperatures (ANOVA test, P<0.01; F=12.44; df=3) (fig. 12). The number of costulae ranged between 2 and 4, and the position of their junction with the median carina ranged between its middle and proximal 1/6 length. The shape of the costulae was very variable because they were often incomplete (fig. 1).
Fig. 10. Reaction norm of length/width of propodeum of female Pnigalio soemius according to its rearing temperature (mean±SE).
Fig. 11. Reaction norm of the number of costulae of female Pnigalio soemius according to its rearing temperature (mean±SE).
Fig. 12. Reaction norm of the intersection of the costulae with the median carina/length of propodeum of female Pnigalio soemius according to its rearing temperature (mean±SE).
The median carina varied in structure close to the anterior margin of the propodeum, being straight and narrow (fig. 1a) or widened anteriorly and then forming a small (fig. 1b), medium (fig. 1c) or large triangular cell (fig. 1d). The number of individuals with a larger triangular cell increased with the rearing temperature (ANOVA test, P<0.01; F=20.57; df=3), and specimens assigned to class 4 (n=6) were reared only at 30°C (fig. 13).
Fig. 13. Reaction norm of the anterior width of the median carina of female Pnigalio soemius according to its rearing temperature (mean±SE).
Discussion
Pigmentation of gastral tergites
Temperature showed a strong influence on pigmentation of the gastral tergites and other body parts, with both the tergites and tarsi tending to be darker at lower temperatures. Results for the gastral tergites gave a convex DRN with a minimum around 25°C, which is similar in pattern to responses for body pigmentation that have been recorded for other insects (Capy et al., Reference Capy, David and Robertson1988; Laudonia & Viggiani, Reference Laudonia and Viggiani1993; Pétavy et al., Reference Pétavy, Moreteau, Gibert and David2002; Mound, Reference Mound, Ananthakrishnan and Whitman2005).
The classical interpretation of the adaptive function of such variation is the thermal budget hypothesis, i.e. a darker colour absorbs more solar radiation, visible or infrared, at low temperatures (David et al., Reference David, Capy, Payant and Tsakas1985, Reference David, Capy and Gautier1990; Kingsolver & Wiersnaz, Reference Kingsolver and Wiernasz1991; Goulson, Reference Goulson1994; Solensky & Larkin, Reference Solensky and Larkin2003; Gibert et al., Reference Gibert, Capy, Imasheva, Moreteau, Morin, Pétavy and David2004). In species that are polymorphic for dorsal colour pattern, individuals that belong to dark morphs generally warm up more rapidly and attain higher body temperatures than paler individuals (De Jong et al., Reference De Jong, Gussekloo and Brakefield1996; Forsman, Reference Forsman1997, Reference Forsman2000). For example, black individuals of the pygmy grasshopper, Tetrix subulata (Linnaeus) (Orthoptera: Tetrigidae), attain a mean temperature exceeding that of grey individuals by 49% (Forsman, Reference Forsman1997). Furthermore, darker forms may have the thermal optimum at a lower level than light-coloured ones; for example, dark coloured (nigra) specimens of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) have the thermal optimum 3.7°C lower than light coloured (aulica) specimens (Soares et al., Reference Soares, Coderre and Schanderl2003). It also seems that geographical distribution affects the colour of specimens, with darker individuals at higher latitudes probably due to lower average temperatures (de Oliveira et al., Reference de Oliveira, Lopes, Dias and Nault2004). Variation in thermal capacity is likely to have pervasive implications for individual fitness, affecting activity period, energy budget, escape capability, dispersal, mating success, and fecundity (De Jong et al., Reference De Jong, Gussekloo and Brakefield1996; Gilchrist, Reference Gilchrist1996; Kingsolver, Reference Kingsolver1996; Forsman & Appelqvist, Reference Forsman and Appelqvist1999; Forsman, Reference Forsman2000).
Temperature had less influence on P. soemius males for colour of the gaster because at 10°C males did not have completely black gastral tergites (data not shown). A lesser effect of temperature on males has been observed also for other insects (David et al., Reference David, Gibert, Gravot, Pétavy, Morin, Karan and Moreteau1997; Marriott & Holloway, Reference Marriott and Holloway1998; Gibert et al., Reference Gibert, Capy, Imasheva, Moreteau, Morin, Pétavy and David2004). Forsman (Reference Forsman2000) found a lower preferred temperature for males than for females of pygmy grasshoppers, which partially reflected the sexual difference in body size and variation in colour. Males are smaller than females and because of the proportionally larger surface area could be more susceptible to desiccation, but compensate behaviourally for this disadvantage by maintaining lower body temperatures, thereby reducing evaporative water loss. The preference for a higher body temperature in females in pygmy grasshoppers could reflect the strong effect of temperature on reproductive performance (Forsman, Reference Forsman2000). The latter author pointed out the strong association between thermal preferences and the ability to attain high temperatures across colour morphs of females of pygmy grasshoppers. However, even though colour may have a profound effect on the capacity for thermoregulation, this may not necessarily translate into morphospecific differences in performance and fitness (Gilchrist, Reference Gilchrist1996; Forsman, Reference Forsman2000).
Seasonal dichroism in Eulophidae is well documented (Askew, Reference Askew1971; Barrett et al., Reference Barrett, Brunner and Turner1988) and chromatic variation has been recorded in several other families of Hymenoptera (Viggiani, Reference Viggiani1963, Reference Viggiani1999; Laudonia & Viggiani, Reference Laudonia and Viggiani1993; Zaviezo & Mills, Reference Zaviezo and Mills1999). Thus, the present results suggest that less importance should be placed on colour in the construction of identification keys for species of Pnigalio.
The present results showed that extent of gastral tergite pigmentation did not have a linear correlation with decreased temperature because females reared at 30°C were darker than those reared at 20 and 25°C (fig. 2). This effect has also been recorded for other insects (Laudonia & Viggiani, Reference Laudonia and Viggiani1993; Pétavy et al., Reference Pétavy, Moreteau, Gibert and David2002). It is possible that stress may be responsible for the development of pigmentation and it is the amplitude of the stress rather than the quality (cold or heat) that determines the magnitude of the effect (Pétavy et al., Reference Pétavy, Moreteau, Gibert, Morin and David2001, Reference Pétavy, Moreteau, Gibert and David2002).
Size
The size of insects is often measured as total body length, but in some cases this measurement is not easily taken because it is influenced by several variables (head position, inclination of junctures, segments of the gaster telescoping to varying degrees in different individuals) (Gauld & Fitton, Reference Gauld and Fitton1987). The choice of a trait that is simple to measure objectively is not easy; however, fore wing or tibia lengths are often used to estimate body size variation (Gauld & Fitton, Reference Gauld and Fitton1987; Bezemer et al., Reference Bezemer, Harvey and Mills2005; Chow & Heinz, Reference Chow and Heinz2005; Lalonde, Reference Lalonde2005). The present results showed that the choice of a representative size trait is critical because different traits showed very different DRNs.
Pnigalio soemius is an idiobiont and, for this kind of parasitoid, host resources either are static or slowly diminish in quality over time because the host does not feed and grow. Thus, idiobiont parasitoid offspring size is usually correlated with the size of the host when it is parasitized (Harvey, Reference Harvey2005). Temperature may have influenced the final body size of P. soemius adults by two different and somewhat opposing ways: at higher rearing temperatures larval hosts could have attained a larger body size before being parasitized, but host degrading processes may be faster at higher temperatures during the growth of the parasitoid larva.
Antenna
Calculated ratios between the length of the clava and first segment of the funicle and between the length and width of the fourth funicular segment were both highly variable. The clava varied from being shorter to longer than the first segment of the funicle, and the fourth funicular segment ranged from sub-quadrate to rectangular. Because the first ratio and the shape of fourth funicular segment are both used in taxonomic keys (Graham, Reference Graham and de1959; Askew, Reference Askew1968), their variability could result in the ranges of different species overlapping.
Fore wing
Fore wing length decreased with increased rearing temperature, which is consistent with the pattern typical of ectothermic animals, and as has been recorded for two different species of Drosophila (Diptera: Drosophilidae) (Atkinson, Reference Atkinson1994; David et al., Reference David, Gibert, Gravot, Pétavy, Morin, Karan and Moreteau1997; Morin et al., Reference Morin, Moreteau, Pétavy and David1999; Gibert & De Jong, Reference Gibert and De Jong2001; Pétavy et al., Reference Pétavy, Moreteau, Gibert, Morin and David2001). A possible adaptive explanation for longer wings at lower temperatures is for a lower wing loading when flying in colder temperatures, as was suggested for Drosophila by Pétavy et al. (Reference Pétavy, Morin, Moreteau and David1997).
Taxonomists often use relative measurements, and especially ratios between the length of wing veins, because these are often considered a means of discriminating species, but the present data showed that wing veins are strongly influenced by overall size variations, and that different lengths and ratios exhibited very different DRNs. The only linear relationship we found was between stigmal vein and fore wing lengths. Moretau et al. (Reference Moretau, Imasheva, Morin and David1998) and Gibert et al. (Reference Gibert, Capy, Imasheva, Moreteau, Morin, Pétavy and David2004) demonstrated similar results for Drosophila melanogaster Meigen and D. simulans Sturtevant, with several parts of the wings of these species exhibiting different DRNs. Hoffmann et al. (Reference Hoffmann, Woods, Collins, Wallis, White and McKenzie2005) also showed differences in size and shape of the wings for different insects reared under conditions of different stresses that involved low food quality, addition of ethanol, and cold shock.
Wing morphology is subject to sexual dimorphism (Gauld & Fitton, Reference Gauld and Fitton1987; Pretorius, Reference Pretorius2005; Teder & Tammaru, Reference Teder and Tammaru2005); therefore, analogous studies should be performed for males before extending the present results to this sex.
Propodeum, median carina and costulae
The number of costulae and where these intersect the median carina are considered important characters in all keys of Pnigalio species (Bouček, Reference Bouček1958; Graham, Reference Graham and de1963; Askew, Reference Askew1968; Miller, Reference Miller1970; Yoshimoto, Reference Yoshimoto1983). The intraspecific variation of these traits has been poorly investigated (Barrett et al., Reference Barrett, Brunner and Turner1988), but the high variability shown by the present results suggests that they should be used carefully to discriminate among species. Differences in propodeal sculpturing of reared males were less pronounced, but smaller specimens showed a reduced number of costulae in accordance to what has been reported by other authors (Miller, Reference Miller1970; Barrett et al., Reference Barrett, Brunner and Turner1998).
Conclusions
Several studies have suggested previously that insects are characterized by physiological and behavioural traits that are temperature dependent, and that the optimal temperature for these traits may vary (Forsman, Reference Forsman2000). When plotted as a function of temperature, several traits we measured show a curvilinear relationship with a peak in value at some intermediate rearing temperature. The phenotype of almost all the traits we tested was affected by temperature, and the variation in these was larger than previously known. As a consequence, a high number of our specimens could have not been correctly keyed on the available keys for the European Pnigalio species (Bouček, Reference Bouček1958; Graham, Reference Graham and de1963; Askew, Reference Askew1968). Consequently, any future revision of Pnigalio should more carefully assess variation in these traits for species recognition.
It is important to emphasize that we analysed only one potential source of morphological variability because we used a single host species and the same larval instar of that species as host. Several authors have shown for other insects that very dissimilar morphs can be correlated with size or physiological condition of the host (Pinto et al., Reference Pinto, Velten, Platner and Oatman1989; Salvo & Valladares, Reference Salvo and Valladares1995). Polyphagous parasitoids, which utilize a wide range of host species and sizes, could be most markedly affected, even though Salvo & Valladares (Reference Salvo and Valladares1995) did not show a definite trend between parasitoid size and host size of polyphagous parasitoids of leafminers.
Furthermore, the present study evaluated the influence of only a single environmental factor (temperature) using parasitoids cultured from a single population from one locality. Generally, there is a broad parallelism between geographic genetic variation and morphological plasticity, and this parallelism is often considered as adaptive (Atkinson, Reference Atkinson1994; Gibert et al., Reference Gibert, Capy, Imasheva, Moreteau, Morin, Pétavy and David2004). Moreover, with respect to size, it is likely that insects developing under natural conditions are much more variable than laboratory-reared insects, as has been reported for Drosophila (Moretau et al., Reference Moretau, Capy, Alonso-Moraga, Munoz-Serrano, Stockel and David1995; David et al., Reference David, Gibert, Gravot, Pétavy, Morin, Karan and Moreteau1997; Gibert et al., Reference Gibert, Moreteau, David and Scheiner1998; Pétavy et al., Reference Pétavy, Moreteau, Gibert, Morin and David2001). For example, Pétavy et al. (Reference Pétavy, Moreteau, Gibert, Morin and David2001) showed that the adults of D. melanogaster and D. simulans reared under alternating conditions were smaller than adults grown under a constant temperature, and that the magnitude of this effect was proportional to the amplitude of the thermoperiod. Pétavy et al. (Reference Pétavy, Moreteau, Gibert and David2002) also showed that variability in body size of wild flies sometimes can be 10 times that of laboratory-reared flies. Therefore, the variance of the different features we measured for P. soemius could be even larger if a greater number of host species and other environmental factors (i.e. humidity) had been included.
This study demonstrates the risk of using any single feature when distinguishing species, and that ratios, although certainly more reliable also can be problematic because reaction norms based on ratio calculations can have different shapes. Consequently, not only is phenotypic plasticity of great importance to the ecologist and evolutionary biologist, it is of basic importance to the taxonomist (Ananthakrishnan, Reference Ananthakrishnan, Ananthakrishnan and Whitman2005).
Acknowledgements
The authors are grateful to an anonymous referee for extensive revision of the manuscript, Dr Andrew Polaszek (The Natural History Museum, London, UK) for his valuable comments and Lucio Bernardo for his technical help in evaluating gastral pigmentation on specimens used in this study.
