Describing Compartmentalization in the Forewing of Populations of the Rice Grasshoppers , Oxya sp . ( Acrididae : Orthoptera )

The fore-wings of Rice grasshoppers, Oxya sp. are divided into compartments by a complex network of veins. There has been a long term hypothesis suggesting that wing compartments bounded by veins may correspond to distinct cell lineages and domains of gene expression. Hence, this study was conducted to determine the patterns of modularity and integration in the fore-wings of the Rice grasshoppers, Oxya sp. A total of 180 points were used to outline the margins of the fore-wings. Results showed that the fore-wing of Oxya sp. is partitioned into 6 developmental modules. The consistency in the number and pattern of developmental modules in the fore-wings suggests that the wings are highly conserved indicating genetic conservatism in the morphological spaces. Results also suggest that compartments are considered an autonomous unit of gene regulation and major veins served as boundaries and as active center of integration.


INTRODUCTION
Wing is an essential part in the daily life of an insect and is considered to be an important organ.Wing traits evolve rapidly to respond to various environmental conditions (Cui and Liu, 2001) and therefore, have large contribution to the unparallel success and wide diversity of insects.Since wing venation is species-specific it is also used taxonomically (Francoy et al., 2009).Several studies on compartmentalization in insect wings suggest that one or set of genes control wing development (Garcia-Bellido and de Celis, 1992;Sturtevant and Bier, 1995;Gilbert et al., 1996;Biehs et al., 1998;Torres et al., 2010).Wing compartments may respond to distinct cell lineages and domains of gene expression (Lawrence, 1992;Klingenberg et al., 2002;Demayo et al., 2011;Tabugo et al., 2011), thus are promising candidates for being separate developmental modules.
Modularity has recently become an active area for investigation in evolutionary developmental biology and other related discipline (Klingenberg, 2008).This describes the general idea that biological systems at many hierarchal levels are about the differences in the degree of integration of part within and between sets of traits (Klingenberg, 2008).It is related to the concept of "morphogenetic field" (Gilbert et al., 1996;Klingenberg et al., 2001;Wilkins, 2002) as they are constituted by localized developmental processes that takes place within them; herewith morphological modules have concrete spatial dimensions (Klingenberg et al., 2002).Since modules are structural units that are internally integrated by developmental interactions (Klingenberg et al., 2002;Demayo et al., 2011) and modularity and integration are 60 concerned with the degree of covariation between parts of a structure, these can be studied by means of morphometric methods (Klingenberg, 2009).The question however is whether the entire wing is a single module or whether the compartments, even the smaller parts of the wings, could be considered as autonomous units of morphological variation.The issue that may correspond to domains of gene expression, still remained ambiguous, thus this study was conducted.The investigation focused on Oxya sp.
Various studies have been conducted looking into the wings of other insects such as fruit fly, bees and dragonfly (Klingenberg et al., 2001;Klingenberg, 2009;Tabugo et al., 2011).However, there are no studies yet looking into the wings of grasshoppers.Grasshopper wings provide many morphological landmarks bounded by veins thus are potential candidates of developmental modules.
In this study, Oxya sp.grasshoppers were collected at three provinces of Mindanao: Lanao del Norte, Misamis Occidental and Zamboanga del Sur.The morphometric data in the fore-wings were used for studying modularity and integration.Modularity and Integration (MINT) analysis tool (Marquez, 2008a) was used to determine the autonomous unit of morphological variation that could be considered as developmental modules.Morphological approach in identifying developmental modules is not different from the approach based on genetic interaction, thus the objectives of this study are to determine the possible number and pattern of developmental modules defining the fore-wings of Oxya sp. using the Modularity and Integration Analysis Tool (MINT ver 1.5, Marquez, 2008b).This study aims to delimit the spatial domain of developmental modules in the fore-wings of Rice grasshoppers, Oxya sp. and to determine whether such pattern of developmental modules between populations of Oxya sp.holds true even for different geographical areas.This study also aims to determine whether the compartments, even smaller parts of the wings, could be considered as "autonomous unit of morphological variation".
Understanding modules in the wings of the grasshoppers can be informative about the underlying biological process of compartmentalization in the wings.Likewise, understanding covariation between modules can have substantial implications for understanding genetic variation and the potential of the species for evolutionary change (Klingenberg, 2008).

MATERIAL AND METHODS
Samples were collected at selected provinces in Mindanao namely: Lanao del Norte, Misamis Occidental and Zamboanga del Sur by using sweep nets and were placed in a properly labelled container filled with 70% ethanol.Through visual inspection of genitalia the sex of samples were identified.
The fore-wings were detached and were mounted neatly on clean and clear slides and were properly labelled.Mounted slides were scanned using a Hewlett-Packard Jacket 2400 Scanner with 1200 dots per inch (dpi).Images were cropped, labelled and saved one by one.Through TPSdig2 (ver 2.12, Rohlf, 2008), a total of 180 points were used for outlining the shape, as well as the wing venation pattern of the fore-wings of Oxya sp. (Fig. 1).After outlining, the TPS curve was then converted into landmarks points (XY) using TPSutil (Rohlf, 2009) which served as the raw data for the analysis.Modularity and Integration Tool (MINT) for Morphometric Data version 1.5 (Marquez, 2008b) was used to study modularity and integration in the fore-wings of the selected population of Oxya sp.The software calculates the matrix correlations between expected and observed covariance matrices.The data sets were loaded, and then a set of models were built and loaded.A total of 11 a priori models for the forewings (Table 1, Fig. 2) Oxya sp. were constructed with the help of the model building tool option of the software.MINT assumes that the data themselves have modular structure, and that by partitioning the entire data space into orthogonal subspaces, covariance matrices were then computed based on the modified data structures (Marquez, 2008a).The patterns of variational modularity were tested using Gamma (γ*) test for the Goodness of fit (GoF) on the alternative a priori models to evaluate whether a proposed model or hypothesis is good enough to explain variation in the data set.The lower γ* value imply high degree of similarity between the observed data and the proposed model.Meanwhile, a low P-value (<0.05) corresponds to large values of γ* value, which implies a large difference between data and the model and thus a poor fit model (Marquez, 2008a).

RESULTS AND DISCUSSION
Table (2) shows the gamma (γ*) and p-values computed for each model in the fore-wing of Oxya sp.Results show that Model 2 yielded a P-value of 1 and a lowest gamma (γ*) value, indicating that the proposed model and observed data are not significantly different.Model 2 therefore qualify as the best fit model and was fairly consistent best-fit model for the fore-wings for both male and female Oxya sp. (Fig. 3).In this model, the fore-wing is partitioned into six (6) developmental modules: (1) Module 1 is bounded by anterior margin and Costa (C), ( 2   The consistency of Model 2 as the best fit model of the fore-wings of Oxya sp. in all geographical location is evident.This indicates that the modularity in the forewings is highly conserved and there is genetic conservatism in the morphological spaces in the fore-wing.Consistency of the best fit model suggests that there is not much variation in the spatial arrangement of developmental modules in the fore-wings of Oxya sp.Each fore-wing appears to be internally coherent developmental processes that produce shape variation thus the wings can be considered as single developmental module (Klingenberg et al., 2001) occupying a specific morphological domain and corresponds to a single morphogenetic field (Gilbert et al., 1996).The different modules can evolve independently, at least to some extent, without disrupting functions at the level of whole organism (Klingenberg, 2005;Klingenberg, 2008;Torres et al., 2010;Tabugo et al., 2011).
However, the results of this study confirmed a number of studies suggesting that insect wings are being partitioned into compartments, and that these modules serve as autonomous units of morphological variation and each compartment is a separate developmental module (Cowley and Atchley, 1990;Cavicchi et al., 1991;Zimmerman et al., 2000;Klingenberg et al., 2001;Torres et al, 2010;Tabugo et al., 2011).Results show that main veins serve as boundaries for each developmental module.Compartment boundaries (veins) not only serve as a delimiter between autonomous unit of developmental domains or modules but also serves as active center of integration from which crucial patterning signal originate (Lawrence and Struhl, 1996;Milan and Cohen, 2000;Klingenberg and Zaklan, 2000;Tabugo et al., 2011).These signals initiate regulatory interactions that subdivide the wings into series of sectors with discrete boundaries (Sturtevant and Bier, 1995;Lawrence and Struhl, 1996;Sturtevant et al., 1997;Biehs et al., 1998;Milan and Cohen, 2000).

CONCLUSION
Results showed that in all provinces, the fore-wings of the Rice grasshoppers, Oxya sp for both male and female and between left and right, are partitioned into 6 developmental modules: (1) Module 1 is bounded by anterior margin and Costa (C), (2) second module is bounded by Costa (C) and Subcosta (Sc), (3) Third module is bounded by Subcosta (Sc) and Media posterior (MP), (4) fourth module bounded by Media posterior (MP) and Cubitus (Cu), (5) fifth module is bounded by Cubitus (Cu) and Anal vein (A), and (6) sixth module is bounded by anal vein (A) and posterior margin of the fore-wing.
Fair consistency in the number and patterns of the hypothesized developmental modules in the fore-wings imply that the wings of Oxya sp. are highly conserved indicating genetic conservatism.

Fig. 2 :
Fig. 2: Models used in this study for the fore-wings of Rice grasshoppers, Oxya sp.
) second module is bounded by Costa (C) and Subcosta (Sc), (3) Third module is bounded by Subcosta (Sc) and Media posterior (MP), (4) fourth module bounded by Media posterior (MP) and Describing Compartmentalization in the Fore-wing of Populations of the Rice Grasshoppers, Oxya sp.63 Cubitus (Cu), (5) fifth module is bounded by Cubitus (cu) and Anal vein (A), and (6) sixth module is bounded by anal vein and posterior margin of the fore-wing.

Fig. 3 :
Fig.3: Best fit model for grasshopper for both male and female of Rice grasshoppers, Oxya sp. in all sites which defines the shape of the fore-wing.

Table 1 .
A priori developmental modules of modularity tested in this study.Modules correspond to regions of the fore-wing of Oxya sp. as hypothesized.First module is bounded by anterior margin and Costa Second module is bounded by Costa (C) and Subcosta (Sc) Third module is bounded by Subcosta (Sc) and Media posterior (MP) Fourth module is bounded by Media posterior (MP) and Cubitus (Cu) Fifth module is Bounded by Cubitus (Cu) and Anal Vein (A) Sixth module is bounded by Anal vein (A) and Posterior margin of the wings.First module is bounded by the anterior margin of the wings and Radius (R3) Second module is bounded by Radius (R3) and Media posterior (MP) Third module is bounded by Media posterior (MP) and Posterior margin of the wings.
Fifth module is bounded by Media anterior (MA) and Media posterior (MP) Sixth module is bounded by Media posterior (MP) and Cubitus (Cu) Seventh Module is bounded by Cubitus (Cu) and Anal vein (A) Eighth module is bounded by Anal vein (A) and Posterior margin of the wings.M8:H 7 5 First module is bounded by the anterior margin of the wings and Radius (R1) Second module is bounded by Radius (R1 and R3) Third module is bounded by Radius (R3) and Media posterior (MP) Fourth module is bounded by Media posterior (MP) and Anal Vein (A) Fifth module is bounded by Anal vein (A) and Posterior margin of the wings.

Table 2 .
Computed γ-value and P-value for the left and right fore-wings of both male and female of Rice grasshoppers, Oxya sp.Only the top three (3) best fit models are tabulated.