Wing Morphometry As a Tool for Correct Identification of Primary And

Bulletin of Entomological Research, Page 1 of 8 doi:10.1017/S0007485309006762 Ó 2009 Cambridge University Press

Wing morphometry as a tool for correct identiﬁcation of primary and secondary New World screwworm ﬂy

M.L. Lyra1,2 *, L.M. Hatadani2, A.M.L. de Azeredo-Espin1,2 and L.B. Klaczko2 1Laborato´rio de Gene´tica Animal, Centro de Biologia Molecular e Engenharia Gene´tica e Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil: 2Departamento de Gene´tica e Evoluc¸a˜o, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil

Abstract

Cochliomyia hominivorax and Cochliomyia macellaria are endemic Neotropical Calliphoridae species. The former causes severe myiasis in hosts while the latter is Sarcosaprophagous, but commonly found as a second invader in wounds. Due to the morphological similarity between them and the potential losses that C. hominivorax represents for cattle breeders, the rapid and correct identification of these two species is very important. In addition to a correct identification of these species, a good knowledge of C. hominivorax biology can be helpful for designing control programs. We applied geometric morphometric methods to assess wing differences between C. hominivorax and C. macellaria and conduct a preliminary analysis of wing morphological variation in C. hominivorax populations. Canonical variate analysis, using wing shape data, correctly classified 100% of the individuals analyzed according to sex and species. This result demonstrates that wing morphometry is a simple and reliable method for identifying C. hominivorax and C. macellaria samples and can be used to monitor C. hominivorax. Both species show sexual dimorphism, but in C. hominivorax it is magnified. We suggest that this may reflect different histories of selection pressures operating on males and females. Significant differences in wing size and shape were obtained among C. hominivorax populations, with little correlation with latitude. This result suggests that wing variation is also a good morphological marker for studying population variation in C. hominivorax.

Keywords: Cochliomyia sp., species identiﬁcation, wing morphometry, sexual dimorphism, populations

(Accepted 18 December 2008)

Introduction The genus Cochliomyia Townsend is now considered to consist of four endemic new world Calliphoridae species, *Author for correspondence C. hominivorax (Coquerel, 1858), C. macellaria (Fabrı´cius, Fax: 55-19-3521 10 89 1775), C. aldrichi (Del Ponte, 1938) and C. minima (Shannon, E-mail: [email protected] 1926) (FAO, 1993). 2 M.L. Lyra et al.

Cochliomyia hominivorax, commonly called the New are no well-characterized morphological markers; and World screwworm fly or primary screwworm, is the only little is known about morphological variation in natural species of the genus known to be of great economic populations of C. hominivorax (Gagne´ & Peterson, 1982; importance. The larvae of this species are obligatory wound Richardson et al., 1982; Azeredo-Espin, 1987). parasites of mammals and are only able to develop in nature The purpose of the present study is: (i) to provide a on living tissues, causing severe myiasis in hosts (Guimara˜es morphological analysis of wing size and shape variation & Papavero, 1999). The historical range of this insect of C. hominivorax and C. macellaria; (ii) to assess how extended from southern United States to Argentina, with suitable geometric morphometric methods are for identifica- seasonal fluctuations and with the greatest abundance in the tion of these species and for studies of population Neotropical region; but it has been successfully eradicated morphological variation; and (iii) to conduct a preliminary from North and most of Central America by the sterile insect analysis of population variation in C. hominivorax. technique-SIT (IAEA/FAO, 2000; Wyss, 2000). Cochliomyia macellaria, the secondary screwworm, is morphologically similar to C. hominivorax; and it is widely Materials and methods distributed and abundant in the new world, ranging from Data acquisition southern Canada to Argentina (Baumgartner & Greenberg, 1985; Dear, 1985). Cochliomyia macellaria is a sarcosapropha- Cochliomyia hominivorax were obtained as third instar gous species that develops primarily on carrion but are larvae found in wounded sheep, dogs or cattle from 12 commonly confused with C. hominivorax in cases of myiasis different localities, six in Brazil and six in Uruguay, totalling because it tends to be a secondary invader. Old records of 476 individuals. A total of 119 individuals of C. macellaria myiasis attributed to this species most certainly should be were obtained as third instar larvae in carcasses of dead transferred to C. hominivorax (Guimara˜es & Papavero, 1999), animals (horse and cattle) from three different localities, two but few cases of myiasis in humans by this species have been from Brazil and one from Uruguay. Geographic locations of recorded (e.g. Smith & Clevenger, 1986; Josephson & the sampled areas and the number of individuals analyzed Krajden, 1993). This species has also been reported to be a are shown in table 1. All collections were carried out in the mechanical vector of human and animal diseases due to its summer months between January 2003 and March 2005. synanthropic behaviour (Greenberg et al., 1963). Collected larvae were brought into the laboratory for Little is known about the habits or relative abundance of species identification and allowed to pupate in sawdust. the other two species of the genus, C. aldrich and C. minima. The pupae were maintained at constant temperature (25 C) They both have a restricted geographical distribution to and humidity (70%), and the adults were frozen 24 h after some Caribbean islands and to Florida (Dear, 1985). The emergence. Sex identification was carried out after emer- immature stage of both species is undescribed and the flies gence, considering that in this genus, like some other are rarely considered, if at all, in identification guides to Calliphoridae, males are holoptic or subholoptic, and myiasis species (FAO, 1993). Adults are apparently attracted females are dichoptic (Dear, 1985). to fresh horse manure and carrion but a single case of The right wing of each fly was removed and mounted in C. minima myiasis has been reported in a dog in Puerto Rico water on a slide under a coverslip (Bitner-Mathe´ & Klaczko, (De Leoń & Fox, 1980). 1999). Wing images were captured with a video camera The taxonomic status of the species in the genus has been attached to a microcomputer and slide preparations were very controversial, mainly due to misidentification of and retained. TpsDig version 2.11 (Rohlf, 2006) was used to misapplications of names to C. hominivorax and C. macellaria. obtain Cartesian coordinate data for 16 landmarks (fig. 1). This, allied to the morphological similarity between the two species, their geographical overlap and the potential losses Data analyses that C. hominivorax represents for cattle breeders, give great importance to the rapid and correct identification of these Centroid size (CS) was used as an overall measure of two species. wing size. This was calculated as the square root of the sum Different methods based on molecular markers for the of the squared distances between the centroid and its characterization of the two species have been successfully landmarks (Bookstein, 1991) using tpsRegr version 1.31 investigated (Pomonis, 1989; Taylor et al., 1996; Litjens et al., (Rohlf, 2005a). Analysis of variance (ANOVA) was con- 2001). But the current methods for species identification ducted to ascertain the effects of species, sex or locality on based on morphology are relatively restricted to specialists size. since, in adults, identification characters are mainly based on Partial warps and uniform component scores were male genitalia, female basicostal coloration and pollinosity calculated with the program tpsRelw version 1.42 (Rohlf, differences in the fifth tergite (FAO, 1993). 2005b) (no weights assigned to any of the landmarks), and In addition to a correct identification of these species, a the matrix obtained was then interpreted as the set of shape good knowledge of C. hominivorax biology can contribute variables (Zelditch et al., 2004). Effects of species, sex or to the design of control programs (IAEA/FAO, 2000; locality on shape were evaluated by multivariate analysis of Krafsur, 2005). In the New World screwworm fly, molecular variance (MANOVA) on partial warps and uniform compo- markers have also been used and have provided important nents scores. In order to account for allometric effects, we information about population structure and genetic varia- performed a multivariate regression of shape variables on bility (e.g. Roehrdanz, 1989; Infante-Vargas & Azeredo- size (CS). Espin, 1995; Taylor et al., 1996; Lyra et al., 2005, Torres et al., The matrix of shape variables was used for canonical 2007, Lyra et al., in press). However, although phenotypic variate analysis (CVA) to examine the pattern of among- traits might provide interesting results that could not be species/-sexes variation in total shape space and to obtain a obtained with neutral markers at the molecular level, there classification matrix. Regression analysis of shape variable Wing geometry in Screwworm 3

Table 1. Geographic locations of the sampled areas and number of analyzed individuals of Cochliomyia hominivorax and Cochliomyia macellaria (F, females; M, males).

Species Locality Latitude (S) Longitude (W) F M Total C. hominivorax Brazil Caiapoˆnia – GO 16570 51480 19 23 42 Saõ Seb. do Paraı´so – MG 20550 46590 17 15 32 Caraguatatuba – SP 23380 45280 22 19 41 Carambeı´ –PR 24570 50060 11 11 22 Fagundes Varela – RS 28310 55140 23 25 48 Pinheiro Machado – RS 31340 53230 22 15 37 Uruguay Banãdos de Medina – C.L. 32230 54210 20 21 41 Cerro Colorado – Flo. 33520 55330 22 23 45 Colonia – Col. 34200 57440 25 21 46 Juaquıń Suarez – Can. 34440 56050 15 20 35 San Antonio – Sal. 31230 57510 19 23 42 Paso Munõz – Sal. 31270 56230 17 28 45 Subtotal 232 244 476 C. macellaria Brazil Goiaˆnia – GO 16430 49150 20 20 40 Mogi-Guaçu – SP 22220 46560 19 20 39 Uruguay Colonia – Col. 34200 57440 20 20 40 Subtotal 59 60 119 Total 293 305 595

1 2 95%: 686.46–695.23) and CSfemales = 704.57+2.75 (CI 95%: 3 699.10–710.04). Wing size alone was not sufficient to reliably identify the two species, but the overlap between the groups 15 4 10 5 was small. 11 12 A significant variation was found between species and a 16 13 7 striking sexual dimorphism for wing shape. Table 2 shows 14 the results of MANOVA on wing shape for C. hominivorax 8 6 and C. macellaria. In general, male wings were narrower than female wings in both species (fig. 2). Variation in wing shape was significantly correlated 9 to size in both females and in C. hominivorax males (C. hominivorax females: R2 = 0.028, P < 0.001; C. macellaria Fig. 1. Wing of Cochliomyia sp. showing landmarks. females: R2 = 0.042, P < 0.001; C. hominivorax males: R2 = 0.031; P < 0.001) but was not significant in C. macellaria males (C. macellaria males: R2 = 0.023, P = 0.071). However only a on CVA roots (CVA1 and CVA2) was used to illustrate small proportion (< 5%) of shape variation was due to size, shape changes by constructing the thin plate spline distor- indicating a negligible allometric effect. tion graphics. Canonical variate analysis (CVA) conducted on the shape Multivariate or linear regressions were used as an variables showed that all individuals of C. hominivorax and exploratory analysis to examine the association between C. macellaria (100% of samples, according classification C. hominivorax population traits and a geographic variable, matrix) were correctly classified according to sex and latitude. species. The first canonical root (CVA1) accounted for 77% Linear regression of size in latitude, ANOVA and of total variation and completely separated the sexes in MANOVA were conducted in Minitab14 (Ó 2004 by Minitab C. hominivorax and most males and females in C. macellaria. Inc.). Multivariate regression of shape variables on size, The CVA2 accounted for 21% of total variation and could latitude and CVA roots was performed in the tpsRegr 1.31 roughly separate the two species (fig. 2). The CVA3 (Rohlf, 2005a). Canonical variate analysis was conducted in accounted for the last 2% of total variation. Statistica for Windows (version 6.0).

Cochliomyia hominivorax population variation Results C. hominivorax populations were significantly differen- Interspecific variation tiated for both wing size and shape. ANOVA and MANOVA The ANOVA on the centroid size of the wings showed a results for effects of localities on size and shape are shown highly significant difference, according to sex and species in table 2. Since sexual dimorphism was present in (table 2). In C. hominivorax, male wings were approximately C. hominivorax, a linear regression was performed separately 12% larger than in females: CSmales = 753.06+3.56 (CI 95%: for each sex. We were not able to find any obvious 746.02–760.09) and CSfemales = 675.77+2.99 (CI 95%: 670.14– geographic pattern for the variation observed. Figure 3 681.40). In contrast, in C. macellaria males were approxi- represents observed variation of size for males and females mately 2% smaller than females: CSmales = 690.85+2.32 (CI in populations. The regression of size on latitude was 4 M.L. Lyra et al.

Table 2. Results of ANOVA on centroid size and MANOVA on non-uniform components of partial warps for Cochliomyia hominivorax (Ch) and C. macellaria (Cm) and for C. hominivorax populations.

Ch X Cm Centroid size (ANOVA) Partial warps (MANOVA) df F P df F P Species 1 15.78 < 0.0001 28 99.26 < 0.0001 Sex 1 48.49 < 0.0001 28 104.59 < 0.0001 Sex X species 1 86.97 < 0.0001 28 27.46 < 0.0001 Ch populations df F P df F P Sex 1 293.04 < 0.0001 28 281.36 < 0.0001 Local 11 9.78 < 0.0001 308 4.82 < 0.0001 Sex X Local 11 1.13 0.332 308 1.56 < 0.0001

5.0

2.5

0.0

CVA2 (21%) –2.5

–5.0

–7.5 –5 0 510 CVA1 (77%)

Fig. 2. Scatterplot of individual scores from canonical variate analysis. The amount of variation expressed for each CVA is in parentheses. Grids represent the consensus and negative and positive deformations over the CVA1 and CVA2. Groups: ChF ( ), C. hominivorax females; ChM (&), C. hominivorax males; CmF ( ), C. macellaria females; CmM (K); C. macellaria males.

significant for both females and males, but the percentage Andersson, 1994; Fairbairn, 1997). Sexual dimorphism has of explained variation was very small (females: R2 = 0.043, been studied in relation to physiology, ecology and behavior. P = 0.01; males: R2 = 0.023, P = 0.01). The results from multi- The two major explanations for its evolution are intra- variate regression of shape on latitude were very similar to specific niche divergence and natural (and/or sexual) those obtained for size (females: R2 = 0.016, P < 0.001; males selection (Lande, 1980; Shine, 1989; David et al., 2003). R2 = 0.009, P < 0.001). In insects, females are usually larger than males (e.g. Huey et al., 2006), probably due to the fecundity advantage for larger females (but see David et al., 2003). A larger male Discussion size is rare and is believed to be favored in cases of competition among males, such as fight for females or Interspecific variation and sexual dimorphism territory, or when they carry females during nuptial flight Differences between males and females are common (Andersson, 1994; David et al., 2006a). in animals. Since Darwin’s (1871) first investigations, these In C. macellaria, we found that females are larger than differences have been the subject of discussion by numerous males and that this size difference may be interpreted as a evolutionary biologists (e.g. Maynard Smith, 1978; fecundity advantage, as in some Drosophila species. On the Wing geometry in Screwworm 5

850

800

750

700 Centroid size (CS)

650

600 16 20 24 28 32 36 Latitude

Fig. 3. Graphic of centroid size (mean CS+SE) vs. latitude for Cochliomyia hominivorax (Ch) populations. L, the mean CS for females; &, the mean CS for males. other hand, C. hominivorax males are significantly larger than meaning of the variation found and to confirm and/or females, suggesting the existence of selective pressures generalize these tentative conclusions. probably related to male behaviour. Even though adult flies of C. hominivorax are often difficult to observe in nature, Wing morphometry as a tool for species identification Guillot et al. (1978) and Krafsur (1978) were able to observe an aggressive behaviour in field C. hominivorax males, show- Geometric morphometrics methods applied to wing ing that they are territorial and have a striking behaviour venation have been successfully used in previous studies towards screwworms, and even towards other species of to discriminate between different insect species (Dujardin small insects. et al., 2003; Houle et al., 2003; Villemant et al., 2007; Hatadani The observation of larger males than females would agree & Klaczko, 2008; Ludoski et al., 2008). The results described with adaptive interpretations mentioned previously herein demonstrate that wing morphometrics is a simple although other factors are probably related to the dimorph- and reliable method for differentiating C. hominivorax and ism. For example, different sexual behavior of females in the C. macellaria samples, since canonical variate analysis of two species may be considered, since C. hominivorax females wing shape variables presented a 100% good match for sexes mate only once (Crystal, 1967), a fact that could reinforce and species. Our results suggest that the partial warp scores sexual selection and increase competition among the males could be used as the basis of a test to distinguish of this species. Furthermore, to our knowledge, there is no C. hominivorax and C. macellaria to monitor the expansion record of monandry for C. macellaria in the literature. and control of C. hominivorax throughout infested and non- Results reported here also found a significant sexual infested countries. dimorphism for wing shape in C. hominivorax and A major advantage of a wing shape test is the ease and C. macellaria (males have narrower wings when compared speed of collecting test material. Data collection for wing to females in both species). Sexual shape dimorphism is shape analysis is both relatively cheap (requiring only a poorly understood, and it is believed to arise from ecological digitized image) and faster than DNA-based markers; causes or natural selection, that is, the adaption of each sex to several hundred wings can be digitized and analyzed on different ecological niches (Shine, 1989; Andersson, 1994; the same day (e.g. Houle et al., 2003). It can also be easily Bonduriansky, 2006). Although there is no direct evidence collected from both live individuals and long-dead speci- for the functional significance of shape variation in the mens. However, since the landmarks used in this study were species analyzed, it seems reasonable to suppose that the distributed over the entire wing area, the major disadvan- differences in foraging habitats and behavior, reproductive tage of a wing shape test is that it requires non-damaged activities and flight agility or resistance may influence the wings. shape dimorphism observed, as has been proposed for other species (e.g. Sivinski & Dodson, 1992; Bonduriansky, 2006). Cochliomyia hominivorax population variation The observed sex dimorphism in C. hominivorax is among localities consistent with a hypothesis that males engage in combat, either for territory or for females, when larger males would Most of the literature on dipteran wing morphology be favoured. However, more detailed studies of social variation concentrates on the genus Drosophila, in which behaviour and life history are needed to understand the latitudinal clines have been observed for body size in several 6 M.L. Lyra et al. species (e.g. Imasheva et al., 1994; Bitner-Mathe´ et al., 1995; the International Atomic Energy Agency (IAEA, grant Gilchrist et al, 2004; Collinge et al., 2006; David et al., 2006b). 11822/RO). Although alternative explanations have been proposed (Santos et al., 2006), it is generally believed that the selective References pressures acting on the formation of these clines are due to, or related to, temperature variation along latitude transects Andersson, M.B. (1994) Sexual Selection. 624 pp. Princeton, N, (Imasheva et al., 1994). Phenotypic plasticity occurs in the Princeton University Press. same direction of temperature and is expected to produce Azeredo-Espin, A.M.L. (1987) Ana´lise cariotı´pica, morfome´trica larger sizes in flies developing under lower temperatures e de compatibilidade sexual em linhagens brasileiras than in those growing under higher temperatures (Partridge de Cochliomyia hominivorax (Diptera: Calliphoridae). PhD et al., 1994; Imasheva et al., 2000; Pe´tavy et al., 2001; David dissertation, University of Campinas (UNICAMP), Campi- et al. 2006b). In fact, in various papers, about 50% of the total nas, SP, Brazil. variation can be explained by a regression on latitude (James Baumgartner, D.L. & Greenberg, B. (1985) Distribution and et al., 1995; Gilchrist et al., 2004). medical ecology of the blow flies (Diptera: Calliphoridae) of Cochliomyia hominivorax is an obligate parasite of Peru. Annals of the Entomological Society of America 78, 565– endothermic animals, and all larval stages develop in an 587. almost constant environment inside the host. Hence, if Bitner-Mathe´, B.C. & Klaczko, L.B. (1999) Plasticity of Droso- temperature variation in developmental time of flies is one phila melanogaster wing morphology: effects of sex, hypothetical explanation for size variation, it should not be temperature and density. 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