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 Leo´n & 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 Sa˜o 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 Ban˜ados de Medina – C.L. 32230 54210 20 21 41 Cerro Colorado – Flo. 33520 55330 22 23 45 Colonia – Col. 34200 57440 25 21 46 Juaquı´n Suarez – Can. 34440 56050 15 20 35 San Antonio – Sal. 31230 57510 19 23 42 Paso Mun˜oz – Sal. 31270 56230 17 28 45 Subtotal 232 244 476 C. macellaria Brazil Goiaˆnia – GO 16430 49150 20 20 40 Mogi-Guac¸u – SP 22220 46560 19 20 39 Uruguay Colonia – Col.