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Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela



Carlos Vasquez, Gerardo Castillo, Martha Davila and Alexander Hernandez
 
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ABSTRACT

In this study, variations in idiosomal setae length and genetic similarity using RAPD-PCR technique were evaluated in Oligonychus punicae (Hirst) or Oligonychus biharensis (Hirst) (Acari, Tetranychidae) females collected on grapevines or mango trees growing in Tarabana and El Tocuyo counties, Lara state, Venezuela. Idiosomal seta analysis (v2, sc1, sc2, c1, c2, c3, d1, d2, e1, e2, f1, f2, h3) showed significant differences in sc1 and sc2 in O. punicae from both localities, meanwhile in O. biharensis setae v2, sc1, c1, d1, e1 and f1 were found to be different in same localities. The Component Principal Analysis (ACP) on idiosomal setae showed that O. punicae population from Tarabana (TARVID) is similar to those from El Tocuyo (TOCVID). Meanwhile, O. biharensis populations exhibit higher variability in idiosomal setae length than O. punicae populations. Genetic analysis, DNA amplification by RAPD yielded 218 bands, being 175 (80%) polymorphic and 43 (20%) monomorphic. Higher number of bands was obtained with primer OPB10, suggesting it would be able to detect higher polymorphism in individuals studied.

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Carlos Vasquez, Gerardo Castillo, Martha Davila and Alexander Hernandez, 2011. Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela. Journal of Entomology, 8: 341-352.

DOI: 10.3923/je.2011.341.352

URL: https://scialert.net/abstract/?doi=je.2011.341.352
 
Received: December 03, 2010; Accepted: February 28, 2011; Published: March 25, 2011



INTRODUCTION

Tetranychidae includes about 1,200 described species and some of them have been economic importance pests (Bolland et al., 1998), mainly species belonging to genus Tetranychus and Oligonychus (Zhang, 2003). In Oligonychus, 213 species have been reported feeding mainly on trees, shrubs and perennial grasses (Bolland et al., 1998).

The Avocado Brown Mite (ABM), Oligonychus punicae (Hirst), is a main pest in several avocado cultivars (Persea americana L.) in most of the producer areas in California, where high population levels have been observed causing defoliation (Cerna et al., 2009). O. punicae has been reported on more than 20 host plant species, including Mangifera indica L., Musa sapientum L. and Punica granatum L. (Ochoa et al., 1994; Bolland et al., 1998) and it is widely distributed in Neotropical countries like Colombia, Costa Rica, Cuba, El Salvador, Honduras, Mexico, Nicaragua, Panama and Venezuela (Bolland et al., 1998). In Venezuela, the ABM has been reported on Musa spp. and causing considerable damage on leaves from various Vitis vinifera L. cultivars (Vasquez et al., 2008).

On the other hand, the Cassava Red Mite (CRM), Oligonychus biharensis (Hirst) has been observed on guava in Malaysia (Gould and Raga, 2002) and more recently CRM has been reported occurring on Clitoria sp. in Venezuela (Vasquez et al., 2009). In China, it is a major pest on litchi, primarily during summer and autumn seasons (Chen et al., 2005). According to Bolland et al. (1998), O. biharensis occurs largely in Asian countries but it has been registered in Brazil and Mexico, however, so far it has not been recorded in Venezuela, so that this work constitutes the first record in the country.

Frequently, as response to environmental variations some morphological variations are verified in Tetranychid species as response to environmental variations ranging from phenotypic plasticity to speciation (Meyers and Bull, 2002) with genetic polymorphism and biotypes and semi-isolated races formation in intermediate position (Magalhaes et al., 2007). Furthermore, population density and geographical distances are considered as main factors affecting genetic flux in natural populations of Tetranychus urticae Koch (Tsagkarakou et al., 1997; Carbonelle et al., 2007). Additionally, changes of host plant could originate new species reproductively isolated from sympatric progenitors (Tsagkarakou et al., 1999; Magalhaes et al., 2007) which reproductive incompatibilities are considered as genetic divergences evidence (Navajas et al., 1994; Tsagkarakou et al., 1997).

Several statistical tools have been used to distinguish between species and intraespecific variations in different arthropod taxa. Moder et al. (2007) based on morphometric data sets from the ant genera Cardiocondyla, Lasius and Tetramorium used a discriminant analysis procedure in species distinction for finding the optimal character combination. Gettinger and Owen (2000) suggested three distinct host-associated Androlaelaps rotundus populations in Paraguay, by using a multivariate analysis of morphometric data. Furthermore, although the value of each band may measured by RAPD not have the same weight in an evolutionary context (Hance et al., 1998), technique has shown to be an valuable tool to better understand genome relationship of related plant species (Patra et al., 2011; Lakshmi et al., 2008) but also it has been widely used for microorganisms (Cumagun et al., 2007) and invertebrates (Hlaoua et al., 2008), including predatory and phytophagous mite species (Rodrigues et al., 2004; Yli-Mattila et al., 2000).

Despite the importance of both species of Tetranychidae worldwide, information about chaetotaxic length and genetic variation is not available in Venezuela, thus we intended to evaluate them in O. punicae and O. biharensis as effect of host plant and geographic distribution in state of Lara, Venezuela.

MATERIALS AND METHODS

Collection and identification of mite populations: O. punicae from V. vinifera and O. biharensis from M. indica were collected in Tarabana County (Palavecino municipality, 522 masl, 10°01'10" N and 69°16'55" W) and El Tocuyo County (Moran Municipality, 604 m, 9°47'55" N and 69°49'38" W), respectively in state of Lara, Venezuela from May 2007-December, 2008. Leaves showing damage symptoms produced by Tetranychidae were randomly collected from each host plant species and location. Damage was evidenced by yellowish spots on adaxial leaf surface (Jeppson et al., 1975). Subsequently, samples were placed in plastic bags and taken to the laboratorio de Zoología Agrícola, Decanato de Agronomia, Universidad Centroccidental Lisandro Alvarado, Venezuela. Each sample was labeled with data of location, host plant and collection date.

At the laboratory, females or males from both localities and host plants were mounted using Hoyer’s medium (Krantz, 1978). Each slide was labeled, oven-dried at 44°C during 3-5 days and sealed. Species were identified using taxonomical key provided by Pritchard and Baker (1955) and by comparison with the aedeagus morphology (Ochoa et al., 1994). Once species were confirmed, females and males were reared in rearing units according to Helle and Overmeer (1985).

Seta analysis: Dorsal setae (v2 = external ventrals; sc1 = 1st scapulars; sc2 = 2nd scapulars; c1 = 1st dorsocentrals; c2 = 1st dorsolaterals; d1 = 2nd dorsocentrals; d2 = 2nd dorsolaterals; e1 = 3rd dorsocentrals; e2 = 3rd dorsolaterals; f1 = inner sacrals; f2 = outer sacrals; h3 = anterior para-anals) of O. punicae and O. biharensis females were measured as illustrated by Zhang (2003). Forty females were examined for each location and host plant species under a contrast-phase microscope magnification (Zeiss DM 1000). Data were subjected to a Principal Component Analysis (PCA) using the program NTSYS-PC version 1.7 (Sneath and Sokal, 1973). After PCA, variables resulting in significant differences were subjected to variance analysis using SAS JMP 5.0.1 (SASJMP, 2003) to determine morphological variations in populations.

Molecular studies: DNA extraction: Twenty females of each species/location were used for DNA extraction at Laboratorio de Virologia, Postgrado de Fitopatologia, Decanato de Agronomia, Universidad Centroccidental Lisandro Alvarado. DNA was extracted following Cenis (1993).

DNA amplification: Each single DNA was estimated in 10 ng by comparison methods and then amplified by Polymerase Chain Reaction (PCR) technique following Osakabe et al. (2000). Primers used were OPA01, OPA03, OPB10 and OPB13 (2ng) (Operon Technologies Co, from series A and B) in 1.5 μL Buffer (25 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3), desoxinucleotids (dNTPs) (10 mM), Taq polymerase (5 μL-1) and genomic mites DNA.

PCR was performed in a thermocycler (Perkin Elmer Gene Amp PCR System 2400) under following conditions: Pre-denaturing at 93°C for 1 min, followed by 45 denaturing cycles at 92°C for 1 min, annealing at 36°C for 1 min and extension at 72°C for 1 min, post-extension at 72°C for 5 min and preservation at 4°C.

Electrophoresis: Nine microliter of DNA from each sampled female was separated by electrophoresis on 1.5% agarose gel and run for 1.25 h at 80 V in a 1X TAE buffer (Tris base 40 mM; Acetato de Sodio 5 mM; EDTA 7, 7 mM, pH 8). Gels were stained with 1% ethidium bromide (BrEth) for 10 min. A 100 pb-pair of base titer was used. Finally, gels were photographed on an UV transilluminator using a Polaroid camera.

Statistical analysis: Data set were subjected to Principal Component Analysis (PCA) using NTSYS-PC version 2.1 (Rohlf, 1992). Afterward, variance analysis was performed to those variables resulting significant using SAS JMP 5.0.1 (SASJMP, 2003).

RESULTS

Seta analysis in O. punicae and O. biharensis populations: Both O. biharensis populations exhibited greater variability in the idiosomal setae length when compared to O. punicae populations. O. biharensis exhibited higher number of setae (v2, sc1, c1, d1, e1 and f1) showed to be different (p<0.05), whereas in O. punicae females only differences in sc1 and sc2 length of mites collected in Tarabana (TAR) and El Tocuyo (TOC) were found (Table 1). Thus, O. punicae populations collected from Tarabana (TARVID) and El Tocuyo (TOCVID) showed to be similar, since there is a tendency to remain in a single group (Fig. 1).

Image for - Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela
Fig. 1: Clustering of setae data from O. punicae (V = vid) and O. biharensis (M = mango) populations collected in Tarabana (Ta) and El Tocuyo (To) according to PCA

Table 1: Idiosomal dorsal setae mean length (μm) of O. punicae and O. biharensis from grape and mango trees collected in two localities (Tarabana (TAR) and El Tocuyo (TOC)) from Lara state, Venezuela
Image for - Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela
Values are as Mean±SD, Values in a column followed by same letter did not show significant differences, according to t-student test (p>0.05)

Table 2: Principal Components Analysis (PCA) for idiosomal seta characterization
Image for - Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela

Table 3: Component weights of used characters in setae characterization
Image for - Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela
v2 = External ventrals; sc1 = 1st scapulars; c1 = 1st dorsocentrals; d1 = 2nd dorsocentrals; e1 = 3rd dorsocentrals; f1 = inner sacrals; sc2 = 2nd scapulars; c2 = 1st dorsolaterals; d2 = 2nd dorsolaterals; e2 = 3rd dorsolaterals; f2 = outer sacrals; h3 = anterior para-anals

According to the Principal Component Analysis (PCA), about 50% of variability in populations was accounted for the first two components (Table 2) and c2, d2, e2 and f2 setae showed more weight in component 1, whereas sc1, c1 and d1 obtained more weight in component 2 (Table 3).

RAPD analysis: DNA amplification by RAPD yielded 218 bands, being 175 (80%) polymorphic and 43 (20%) monomorphic. Greater number of bands was obtained with primer OPB10, suggesting it would be able to detect higher polymorphism in individuals studied (Table 4).

Similarly, out of 121 total bands obtained in locality TAR, higher number of bands was obtained with primer OPB10 (37), being 31 bands polymorphic and 6 bands monomorphic. When primer OPB13 was used, 33 total bands were produced, all of them being polymorphic. Lower numbers of bands were observed by using primers OPA01 and OPA03 (Table 2). Primer OPB10 separated larger number of bands in populations from locality TOC, followed by OPA01 and OPA03 with 24 bands each and OPB13 with 19 bands (Table 5). No difference in number of polymorphic bands was observed in both locations (TAR = 90 bands and TOC = 85).

When number of DNA bands for mite species were analyzed, larger number was observed in O. biharensis (from Mango), showing 113 bands when primer OPB10 (37 total bands) was used, followed by OPB13 (27), OPA03 (25) and OPA01 (24).

Table 4: Number of bands of DNA in O. punicae and O. biharensis using different primers
Image for - Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela
NMB = Number of monophormic bands; NPB = Number of polymorphic bands; NTB = Number of total bands. OPA01 = 5-CAGGCCCTTC-3`; OPA03 = 5-AGTCAGCCAC-3`; OPB10 = 5`-CTGCTGGGAC-3`; OPB13 = 5`-TTCCCCCGCT-3

Table 5: Number of bands of DNA in O. punicae or O. biharensis according to localities
Image for - Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela
NMB = Number of monophormic bands, NPB = Number of polymorphic bands, NTB = Number of total bands, TAR = Tarabana County; TOC = El Tocuyo County

Table 6: Number of bands of DNA in O. punicae or O. biharensis according to host plant
Image for - Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela
NMB = Number of monophormic bands, NPB = Number of polymorphic bands; NTB = Number of total bands, grape (O. punicae) and mango (O. biharensis)

Similarly, the highest number of polymorphic bands was obtained with OPB10, followed by OPB13 in samples collected from grapevine, being 105 bands similar to those in mango (Table 6).

On the other hand, when number of bands by crop was analyzed in TAR, higher number was obtained with primers OPA03 and OPB10 in grapevine, while in mango samples, higher number was obtained with OPB10 and OPB13 (Table 7). Furthermore, higher number of polymorphic bands was observed in samples collected on grape (55 polymorphic bands) compared to 35 bands in samples from mango.

Conversely, in samples from TOC, higher number of total and polymorphic bands was observed in samples from mango with primers OPB10 and OPB13 (18 and 11, respectively) (Table 7).

Genetic variation in O. punicae and O. biharensis: Higher variability was observed in O. punicae populations collected in TOC (33%) and TAR (28%), meanwhile in O. biharensis, variation was 21 and 12% in TOC and TAR, respectively. Similarity percentage between populations was 43, 37, 32 and 73% for TOCVID, TARVID, TOCMAN and TARMAN, respectively (Fig. 2).

Table 7: Number of bands of DNA from O. punicae or O. biharensis by host plant from localities Tarabana (TAR) and El Tocuyo (TOC)
Image for - Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela
NMB = Number of monophormic bands; NPB = Number of polymorphic bands; NTB = Number of total bands; grape (O. punicae) and mango (O. biharensis)

Image for - Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela
Fig. 2: Mean test for idiosomal setae sc1 (top) and d2 (bottom) in O. punicae and O. biharensis females collected in El Tocuyo and Tarabana. Mean values followed by the same letter are not significantly different according to least square mean test

O. biharensis populations showed tendency to be more separated than O. punicae populations (Fig. 3). In addition, based on RAPD analysis which accounted for 36% of variability, similarity coefficients for TOCVID, TOCMAN, TARVID and TARMAN were 0.56, 0.40, 0.47 and 0.78, respectively (Table 6). Thus, O. biharensis population from El Tocuyo showed higher similarity to O. punicae population from Tarabana.

Image for - Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela
Fig. 3: Dendrogram obtained by UPGMA algoritm of Jaccard’s index between O. punicae (VID) and O. biharensis (MAN) collected in Tarabana (TAR) and El Tocuyo (TOC)

DISCUSSION

The Principal Component Analysis (PCA) of the idiosomal seta data showed that subset of characters could be useful for species characterization. On the other hand, setae in O. biharensis populations from Tarabana (TARMAN) and El Tocuyo (TOCMAN) seem to be more discriminating, thus indicating that populations are separated by location possibly due to environmental conditions in each area (Carbonelle et al., 2007).

Although studied species are morphologically related, seta analysis by PCA could be useful in species discrimination due to, on one hand, analysis separated both species, being O. punicae in the upper left and O. biharensis in the lower right and on the other hand, disaggregated O. biharensis individuals and grouped them by locality (Fig. 1).

Moreover, the combined analysis of variance revealed that sc1 and d2 showed greater variability proving to be useful for species discrimination by location and/or host plant, thus corroborating those setae length could be more discriminating for O. punicae and O. biharensis characterization (Fig. 4). Despite of the value of setae length for characterization in Tetranychidae species, little information is available. As mentioned by Sandoval (2005), O. peruvianus females collected from avocado and cassava trees were discriminated by Principal Components Analysis and differences in v2, sc1, c1, c2, c3, d1, d2, e1, e2, f1, f2 and h3 were observed in O. peruvianus populations from both host plant species collected from same location. Similar to our results, setae analysis showed to be a valuable basis for Oligonychus perseae female characterization, a species commonly found in avocado trees and often misidentified as O. peruvianus.

Other idiosomal morphological characters have been used also for characterization of Tetranychidae species. According to Boudreaux and Dosse (1963), cuticle lobe shape can be used for species characterization. Hence, Hance et al. (1998) obtained valuable information to differentiate races of Tetranychus urticae Koch and Tetranychus cinnabarinus (Boisduval).

Image for - Idiosomal Setae and Genetic Analysis in Oligonychus punicae and Oligonychus biharensis (Acari, Tetranychidae) Populations from State of Lara, Venezuela
Fig. 4: Principal Coordinate Analysis (PCA) graphic based on RAPD data from O. punicae (VID) and O. biharensis (MAN) from localities Tarabana (TAR) and El Tocuyo (TOC)

RAPD analysis: Based on present results, primer OPB10 showed to be more liable to amplify DNA polymorphism sectors in O. punicae and O. biharensis populations from TAR and TOC, thus this primer could be used for discriminating these two Oligonychus species. Primer OPB13 showed more restricted potential for species-specific discrimination. Similarly, Osakabe et al. (2000) found that out of 40 primers used to evaluate genetic variability in Panonychus citri (McGregor), only OPA01, OPB10 and OPB12 yielded diagnostic bands, however, clonation was exclusively achieved in polymorphic bands by using primer OPB10. Garg et al. (2009) showed usefulness of RAPD technique to elucidate the complementariness approaches to make diversity analysis more explanatory and powerful for optimum genetic amelioration and effective conservation of genotypic variability.

Although initiator OPB10 yielded the higher polymorphism in O. punicae and O. biharensis populations; we recommend more detailed studies of these two species including a greater number of individuals, locations and/or host plants to improve the technique.

Although previous studies have demonstrated usefulness of primer OPB10 as molecular markers detector in mites, we recommend the use of more specific primers to detect genetic differences between O. punicae and O. biharensis. Furthermore, due to RAPD-PCR technique measures DNA polymorphism in a random pattern; value of each band may not have the same weight in an evolutionary context (Hance et al., 1998) as in other more advanced molecular techniques which consuming more time and resources (Ndjiondjop et al., 2006). Thus, variability obtained by the RAPD technique is considered a preliminary result for the two populations of Oligonychus studied.

Genetic variation in O. punicae and O. biharensis: Although the dendrogram constructed for all individuals using cluster analysis (UPGMA) was able to separate mite populations per location, it showed no separation by species. O. punicae and O. biharensis populations from locality TAR showed higher similarity between them than with their respective homologous population. Accordingly, Paulauskas et al. (2006) found that Ixodes ricinus L. populations from different localities in Norway and Lithuania were not clearly separated in the dendrogram constructed from genetic distances based on RAPD.

Lower variability observed in O. biharensis collected in Tarabana (TAR) could be related to limitative gen flux between individuals due to low population levels were observed in this population. Previous studies have demonstrated that genetic differentiation in T. urticae populations laid on density and geographical distance between populations but not on colonized host plant species (Tsagkarakou et al., 1998, 1999; Hinomoto and Takafuji, 1995). On the other hand, Tsagkarakou et al. (1999) found that genetic differentiation was positive and significantly correlated with geographic distance between populations in field and greenhouses and genetic exchange increased as population density get higher in a microgeographical habitat (<100 m2).

CONCLUSION

Present results confirm usefulness of morphological characters for species characterization purposes, mainly in case of morphologically similar Tetranychid species, however molecular studies to determine genetic variations prove to be an auxiliary tool to determine molecular variations in tetranychid species of economic importance. It is therefore recommended that more detailed studies involving other molecular techniques and more populations to better understand variability of these mite populations.

ACKNOWLEDGMENTS

To Instituto de la Uva (IUVA) and Estación Experimental Miguel Luna Lugo (Universidad Centroccidental Lisandro Alvarado) for helping to this research.

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