Advances in Environmental Biology

Advances in Environmental Biology, 9(19) Special 2015, Pages: 64-70

AENSI Journals

Advances in Environmental Biology ISSN-1995-0756

EISSN-1998-1066

Journal home page: http://www.aensiweb.com/AEB/

Describing Dimorphism in Wing Shapes in the Blowfly Lucilia Sericata Meigen (Diptera: Calliphoridae) using Geometric Morphometrics 1Aileen

S. Espra, 2Sharon Rose M. Tabugo, 2Mark Anthony J. Torres, 3Jessie G. Gorospe, Michael E. Manting and 2Cesar G. Demayo 1 2 3

2

Muhmin

University of Mindanao, Matina Campus, Davao City, Philippines Department of Biological Sciences, College of Science and Mathematics, MSU-Iligan Institute of Technology, Iligan City, Philippines School of Graduate Studies, MSU-Naawan Campus, Naawan, Misamis Oriental, Philippines

ARTICLE INFO Article history: Received 23 June 2015 Accepted 25 July 2015 Available online 30 August 2015 Keywords: Dimorphism, wing shapes, L. sericata, geometric morphometrics

ABSTRACT The study aims to describe dimorphism in the shape of the wings in Lucilia sericata using geometric morphometric methods. Images of the wings which were detached from the body of males and females of the insects were digitized and the coordinates taken were subjected to procrustes and relative Warp analysis using the TPS program software version 1.45. Multivariate methods of statistical analysis of the relative warp scores and euclidean distances show dimorphism in wing shapes. The variations observed between sexes could be genetic or could be mere reflections of the existence of high phenotypic plasticity brought about by varied environmental conditions or changes during growth and development of the larvae. Since the differences among the individual configurations of each sex were captured using mathematical functions varying according to the position of each landmark in the wing image, the geometric variation detected showed that these landmarks can be considered key characters to distinguish differences in wing morphotypes among the two sexes of L. sericata. These characters maybe important for insect dispersion, migration and sexual selection.

© 2015 AENSI Publisher All rights reserved. To Cite This Article: Aileen S. Espra, Sharon Rose M. Tabugo, Mark Anthony J. Torres, Jessie G. Gorospe, Muhmin Michael E. Manting and Cesar G. Demayo., Describing Dimorphism in Wing Shapes in the Blowfly Lucilia Sericata Meigen (Diptera: Calliphoridae) using Geometric Morphometrics. Adv. Environ. Biol., 9(19), 64-70, 2015

INTRODUCTION One of the flies that was observed to be large in numbers in one of the piggery in Iligan City was the Lucilia sericata (Meigen). This blow fly is a member of the family Calliphoridae. The common green bottle fly, Lucilia sericata, formerly Phaenicia sericata, is a common visitor to carrion, feces, and garbage. In addition, it has forensic, medical and veterinary importance [1-10]. Despite its importance in forensic and veterinary this blow fly once they are in significant numbers they become a nuisance in the community and even threaten animal production because they are transmitters of disease.In fact in other countries, it has been reported that over 80 per cent of UK sheep flocks are affected by blowfly strike annually. The welfare of between 500,000 and 1 million animals is compromised each year as a result of these infestations. For the farmers, this brings economic costs both in the loss of animals as well as in the treatment and control of the disease [11]. In this study, dimorphism in morphological shapes of the wings of this species was investigated since this morphological character is important in insect movement and epidemiology. Male and female differences have been studied extensively in several aspects such as physiological [12-13], biochemical, movement [14], morphological and other traits. Sexual dimorphism in size (SSD) has attracted the attentions of numerous biologists after Darwin [15] and have been attempted to be explained in evolutionary biology [16]. Gender differences in lifespan and mortality rates have been recorded in two seed beetle species [17]. Color, development of some parts of the body such as horns in beetles[18], presence of sting in bees, the size of eyes[19-20] and morphologic differences in some instars[21] are some other sex specific differences. This study used landmark-based geometric morphometry to describe wing variation among and between sexes of L. sericata which account sexual dimorphism. The study used geometric morphometry as a tool to analyze wing shape of Lucilia sericata population. Geometric morphometrics is a relatively new technique that has generated valuable results in many fields of classic morphometry. In addition, it was used for the Corresponding Author: Aileen S. Espra, Faculty University of Mindanao, Matina Campus, Davao City, Philippines E-mail: [email protected]

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Aileen S. Espra et al, 2015 Advances in Environmental Biology, 9(19) Special 2015, Pages: 64-70

discrimination of species, identification of the group variations, display of the evolutionary relations, discrimination of sexes and determination of the relationships between growth and figure [22]. Furthermore, a major advantage of the geometric framework is a comprehensive use of information about shape, available from a set of landmarks [23]. Variations in body shape (and wings as part of the body) have important fitness consequences because they can affect the ability to occupy habitats successfully [24-25] to prevail in predatorprey interactions [26-27] to reproduce successfully and provides a link between the genotype and the environment [28]. Thus we can expect selection to act upon wing phenotype [29]. In this study, the wing morphology of the L. sericata is an ideal model or an excellent object for qualitative or quantitative morphological studies. Furthermore, the wing is flat and has many morphological landmarks at the points where the wing veins intersect or meet the wing margin and is fairly distinctive and an important character for classification and identification of species which could help in the proper management and control of pest infestation [30]. MATERIALS AND METHODS Collection, Identification, and Preparation of Flies: Adult flies were collected from the piggery located at Abuno, Iligan City, Philippines (Figure 1). The flies were collected using aerial nets. Sampling was done once only. In the laboratory, the collected flies were sorted and identified using taxonomic keys for the proper confirmation of the species. Male and female L. sericata (Figure 2) were utilized in this study. After which, the pair of wings of the male and female was detached from their body through the use of small scissors or tweezers. Male and female wings were mounted separately. The wings were captured using digital CANON camera (Power shot SX210 IS 14.1 Megapixels) under STL Leica microscope. Twenty one (21) landmarks were identified and tagged for geometric morphometric analysis following the method of Rohlf [31]. The landmarks identified are the intersections of wing veins with the wing margin, intersection of cross vein with major vein branch point. The description and locations of the landmarks are presented in Table 1 and Figure 3.

Fig. 1: The map of Iligan City showing the sampling area. Legend:

Fig. 2: Lucilia sericata (a) unmounted (b) female (c) male.

- Abuno

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Table 1: The description of assigned landmarks on Lucilia sericata wings. Landmark Description of the Landmark 1 Humeral cross vein 2 Subcostal vein 3 Anterior branch of radius R1 vein 4 R2+3 vein, the distal end of Radius 5 R4+5 vein, the distal end of Radius 6 Branching endpoint of Media vein 7 Curve point of Media vein 8 Intersection between Media vein and bm-cu vein 9 Intersection between bm-cu vein and CuA1 vein 10 Branching point of CuA1 vein 11 Branching point of A1+CuA2 vein 12 Intersection between A1+CuA2 vein and CuA2 vein 13 Intersection between CuA2 vein and bm-cu vein 14 Point of origin of A1+CuA2 vein and Median vein 15 Intersection between bm-cu and Median vein 16 Intersection between Median vein and r-m vein 17 Intersection between r-m vein and R4+5 vein 18 Branching point of vein Rs vein 19 Intersection of Rs vein and R1 vein 20 Intersection between humeral cross vein and R1 vein 21 Subcosta vein Legend: Anal veins (A); Cubitus Anterior (CuA) vein; Radius (R) vein

Fig. 3: Landmarks on Lucilia sericata wings used for geometric morphometric analysis. Geometric Morphometric Analysis and Statistical Analysis: For geometric morphometric analysis, TPS Program of Rohlf [31] was used. TPS file was built for the captured wings and landmarks were placed using tpsDig. The landmark data were superimposed using General Procrustes Analysis for translation and rotation parameters [22]. After superimposition, the deformation or “warping” in shape of each individual from a consensus form is given by partial warp scores [22, 32]. The partial warp scores were analyzed using EXCEL 2007 and PAST 2.17c [33] for the multivariate analysis. Canonical Variate Analysis (CVA) was used to compare variation in shape within pooled left and right wings of female and male flies. On the other hand, Discriminant Function Analysis (DFA) was used to compare wing shape variation between sexes. Finally, the relative warps correspond to the principal components and define a shape space in which individuals are replaced [32]. Histograms were generated using PAST version 2.17c [33] from the relative warps of the left and right wings of both female and male samples. RESULTS AND DISCUSSION The superimposed images showing the distribution of the landmark points for all the left and right wings of both sexes of L. sericata is shown in Fig. 4. It can be seen from the figure that variations in shapes between the left and right wings can be qualitatively seen mostly in landmark 11 although other landmark points also show differences between sexes. The mean shapes of the left and right wings of L. sericata based on thin-plate splines in Figure 5 show the wings show differences in shapes especially in the coastal region. The descriptions of the variations observed is summarized in Table 2. To further quantitatively described the nature of the variations observed, Euclidean distance matrix analysis (EDMA) was done and results show a total of 31 interlandmark distances were found significant to explain quantitatively the variations in shapes within and between the left and right wings of male and female L. sericata (Table 3, Fig. 6). Canonical variate analysis of the relative warp scores of the wings was also performed to further determine if the existence of dimorphism described in L. sericata was significant. Results show significant dimorphism in shapes for the left and right wings of the insect (Tables 4, 5, 6, Fig. 7,). Wings were also observed to be correctly classified (>70%) (Table 3) confirming the wings in L. sericata are sexually dimorphic.

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Fig. 4: Procrustes-fitted landmark coordinates of the wings of L. sericata. (Legend: red- left female wing; blueright female wing; Green – left male wing, violet- right male wing)).

Fig. 5: Morphological deformation grids showing the shape of the wings of the (a) left and (b) right wings of female and (c) left and (d) right wings of male L. sericata. Table 2: Descriptions of variations in the left and right wings in male and female wings of L. sericata. Variance Variance Male Left Wings Female Left Wings % % Variations in the costal part and Variations in the costal, median part, distal RW 1 37.32 25.17 median of the wing and posterior part of the wings Variations in the costal part of RW 2 12.31 15.30 Variations in the median part of the wing the wing Variations in the costal part of RW 3 8.72 12.61 Variations in the median part of the wing the wing Variations in the posterior part RW 4 .88 8.60 Variations in the proximal part of the wing of the wing Variations in the posterior part RW 5 5.96 6.45 Variations in the proximal part of the wing of the wing Variations in the median part of RW 6 5.18 5.43 Variations in the proximal part of the wing the wing RW 7 5.01 Variations in the distal part of the wing Male Right Wings Female Right Wings Variations in the costal part of Variations in the costal and median part of the RW 1 27.80 28.70 the wing wing Variations in the costal part of RW 2 18.51 14.57 Variations in the median part of the wing the wing Variations in the costal part of RW 3 11.22 9.42 Variations in the median part of the wing the wing Variations in the costal part of RW 4 8.68 8.36 Variations in the proximal part of the wing the wing Variations in the costal part of RW 5 6.30 6.12 Variations in the proximal part of the wing the wing Variations in the costal part of RW 6 5.08% 5.55 Variations in the median part of the wing the wing

The variations observed in the wing morphology of L. sericata indicate that this population has a diverse gene pool. It can be assumed that the variations in wing shapes between sexes could be genetic but some authors argue these are mere reflections of the existence of high phenotypic plasticity brought about by varied environmental conditions [35] or changes during growth and development of the larvae [36-38]. Since the differences among the individual configurations of each sex were captured using mathematical functions varying according to the position of each landmark (landmarks 4-8, 11-15 and 18-21) in the wing image, the geometric variation detected showed that these landmarks can be considered key characters to distinguish

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differences in wing morphotypes among the two sexes of L. sericata. These characters maybe important for insect dispersion, migration and sexual selection [39-43]. Table 3: Interlandmark distances which show the sources of variations between the wings of the male and female L. sericata. INTERLANDMARK DISTANCES VALUES INTERLANDMARK DISTANCES VALUES 1-4 0.1188 5-18 0.1131 1-5 0.1338 5-19 0.1301 1-6 0.1381 5-20 0.136 1-7 0.1131 6-11 0.1183 4-11 0.1061 6-12 0.1297 4-12 0.1133 6-13 0.1232 4-13 0.1063 6-14 0.1404 4-14 0.1227 6-15 0.1209 4-15 0.1031 6-18 0.1172 4-19 0.1156 6-19 0.1343 4-20 0.1213 6-20 0.1404 5-11 0.1158 6-21 0.102 5-12 0.1261 7-19 0.1075 5-13 0.1195 7-20 0.1145 5-14 0.1365 8-20 0.1004 5-15 0.1169

Fig. 6: Interlandmark distances which showed the sources of variations in the wings of L. sericata.

Fig. 7: Scatterplot showing the distribution of individuals along the the CVA axis. Table 4: CVA of RWA scores of procrustes-fitted landmark coordinates of the wings of L. sericata. Wilk’s Lambda 0.1097 Pillai trace df1 108 df1 df2 243.5 df2 F 2461 F P(same) 4.094E-09 P(same)

1.479 108 249 2.241 1.125E-07

Table 5: Pairwise comparison. Female left wing Female right wing Male left wing

Female right wing 0.65207

Male left wing 0.094461 0.110734

Table 6: Confusion Matrix showing the percentage reclassification of the wing. Female left wing Female right wing Male left wing Female left wing 23 (77%) 3 (10%) 4 (13%) Female right wing 3 (10%) 26 (87%) 1 (3%) Male left wing 2 (6.67%) 0 24 (80%) Male right wing 0 1 (3%) 5 (16.67%)

Male right wing 0.01680 0.03108 0.71907

Male right wing 0 0 4 (13.33%) 24 (80%)

Total 30 (100%) 30 (100%) 30 (100%) 30 (100%)

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Conclusion and Recommendation: The use of other geometric morphometric tools to determine wing variation of L. sericata population is also highly recommended to further support its result particularly wing variability for proper identification and control of L. sericata population. ACKNOWLEDGMENT The Researcher would like to thank the Commission on Higher Education – Faculty Development Program (CHED-FDP) for the scholarship grant, the University of Mindanao (SHEI) for the faculty privileges and support, to Prof. Muhmin Michael E. Manting for the technical assistance and the Department of Biological Sciences - College of Science and Mathematics, MSU-IIT for the use of laboratory equipment. REFERENCES [1] Heath, A.C.G., 1982. Beneficial aspects of blowflies (Diptera: Calliphoridae). New Zealand Entomologist, 7-3. [2] Mackerras, M.J., 1933. Observations on the lifehistories, nutritional requirements and fecundity of blowflies. Bull. Entomol. Res., 24: 353–362 [3] Strikewise, 2007. Blowfly strike. http://www.strikewise.com/blowfly.html . [4] Whitworth, T., 2006. Keys to the genera and species of blow flies (Diptera: Calliphoridae) of America north of Mexico. Proceedings of the Entomological Society of Washington, 108: 689-725. [5] Whitworth, T., 2010. Keys to the genera and species of blow flies (Diptera: Calliphoridae) of the West Indies and description of a new species of Lucilia Robineau-Desvoidy. Zootaxa, 2663: 1–35. [6] Nandi, B.C., 2004. Checklist of Calliphoridae (Diptera) of India. Rec. zool. Surv. India, Occ., 231: 1-47. [7] Singh, D. and I.S. Sidhu, 2007. Two New species of Melinda Robineau-Desvoidy (Diptera:Calliphoridae) from India, with a key to the Indian species of this genus. [8] Smith, D.R. and R.R. Clevenger, 1986. Nosocomial nasal myiasis. Arch Pathol Lab Med 110: 439–440. [9] Rueda, L.C., L.G. Ortega N.A. Segura, V.M. Acero, F. Bello, 2010. Lucilia sericata strain from Colombia: Experimental colonization, life tables and evaluation of two artificial diets of the blowfly Lucilia sericata(Meigen) (Diptera: Calliphoridae), Bogota, Colombia strain. Biological Research, 43: 197-203. [10] Ronges, K., 2009. Revision of Oriental species of Bengalia peuhi species group (Diptera, Calliphoridae) Zootaxa, 2251: 1-76. [11] Wall, R., 2012. Early Season of Sheep could reduce the incidence of blowfly strike as climate warms. http://www.bristol.ac.uk/research/im. [12] Yurkiewicz, W.J., 1969. Sexual dimorphism in neutral lipid metabolism in the Indian-meal moth, Plodia interpunctella (Hubner). Ohio Journal of Science, 69(2): 70. [13] Porco, D., L. Deharveng and Gers, Ch., 2004. Sexual discrimination with cuticular lipids in Schoettella ununguiculata (Tullberg, 1869) (Collembola: Hypogastruridae). Pedobiologia, 48: 581-583. [14] Krasnov, B.R., S.A. Burdelov, I.S. Khokhlova, N.V. Burdelova, 2003. Sexual size dimorphism and morphological traits and jump performance in seven species of desert fleas (Siphonaptera). Journal of Zoology of London, 261: 181-189. [15] Darwin, C., 1871. The descent of man and selection in relation to sex. 528 pp. John Murray, London. [16] Webster, M.S., 1997. Extreme sexual size dimorphism, sexual selection and the foraging ecology of Montezuma oropendolas. The Auk, 114(4): 570-580. [17] Fox, C.W., L. Dublin, S.J. Pollitt, 2003. Gender differences in lifespan and mortality rates in two seed beetle species. Functional Ecology, 17: 619-626. [18] Emlen, D.G., J. Hunt, L.W. Simmons, 2005. Evolution of sexual dimorphism and male dimorphism in the expression of beetle horns: phylogenetic evidence for modularity, evolutionary lability, and constraint. American Naturalist 166, S42-S68. [19] Land, M.F., 1989. Variations in the structure and design of compound eyes. pp. 90-111 in Stavenga, D. G. & Hardie, R. C. (Eds) Facets of Vision, 454. Springer-Verlag. [20] Land, M.F., 1990. The design of compound eyes, 55-64 in Blakemore, C. (Ed.) Vision: coding and efficiency, 448. Cambridge University Press. [21] Cook, L.G., P.J. Gullan, A.C. Stewart, 2000. First-instar morphology and sexual dimorphism in the gallinducing scale insect Apiomorpha Rübsaamen (Hemiptera: Coccoidea: Eriococcidae). Journal of National History, 34: 879-894. [22] Adams, D.C., F.J. Rohlf, D.E. Slice, 2004. Geometric morphometrics: ten years of progress following the “revolution”. Ital. J. Zool, 71: 5–16.

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