Morphometric Analysis and Host Range of the Genus Pentalonia Coquerel ( Hemiptera : Aphididae ) Infesting Banana in Java

Pentalonia nigronervosa is the most important pest on banana and known as vector of Banana bunchy top virus (BBTV). This species plays an important role on the spread of banana bunchy top disease (Dale, 1987; Hu et al., 1996; Watanabe et al., 2013). Infection of BBTV has been reported on various banana genotypes in the world and resulted in 100% yield losses (Dale, 1987). Incidence of BBTV reached 100% and 96% on banana cv. Cavendish Williams and plantain hybrid variety PITA 23, respectively in Southern region of Cameroon, Africa (Ngatat et al., 2017). In contrast, incidence of BBTV in Indonesia is low, i.e. ranged from 0 % to 38.6% (Nurhayati, 2003). Since Indonesia and other countries in Southeast Asia are considered having the highest genetic diversity of wild banana, it is important to be prepared for an appropriate management strategy of BBTV and its vector. In genus Pentalonia, P. nigronervosa and P. caladii are known as “cryptic species”. Previously, Hardy (1931) placed P. caladii in synonymy with P. nigronervosa but recently Foottit et al. (2010) reestablished the identity of P. nigronervosa and P. caladii each as full species based on morphometric and molecular analysis. Furthermore, the host range of the two species is slightly different. P. nigronervosa mostly feeds on banana and occasionally feeds on Heliconia species (Nordam, 2004; Bhadra & Agarwala, 2010; Foottit et al., 2010; Suparman et al., 2011; Miller et al., 2014), whereas P. caladii feeds on Alocasia sp., Caladium sp., Elettaria sp., Hedychium sp., Xanthosoma sp., and Zingiber sp. (Nordam, 2004; Miller et al., 2014). Identification of aphid infesting banana in Java has been done a long time ago by Nordam (2004). He reported P. nigronervosa as the single species of aphid infesting banana in Java based on the specimens collected by van der Goot in 1916 and D. Nordam in 1976–1977. Due to the most recent taxonomy of banana aphids, it is necessary to update the status of banana aphids in Indonesia, including in Java. The two cryptic species, P. nigronervosa and P. caladii, are only distinguished by the ultimate ABSTRACT


INTRODUCTION
Pentalonia nigronervosa is the most important pest on banana and known as vector of Banana bunchy top virus (BBTV). This species plays an important role on the spread of banana bunchy top disease (Dale, 1987;Hu et al., 1996;Watanabe et al., 2013). Infection of BBTV has been reported on various banana genotypes in the world and resulted in 100% yield losses (Dale, 1987). Incidence of BBTV reached 100% and 96% on banana cv. Cavendish Williams and plantain hybrid variety PITA 23, respectively in Southern region of Cameroon, Africa (Ngatat et al., 2017). In contrast, incidence of BBTV in Indonesia is low, i.e. ranged from 0 % to 38.6% (Nurhayati, 2003). Since Indonesia and other countries in Southeast Asia are considered having the highest genetic diversity of wild banana, it is important to be prepared for an appropriate management strategy of BBTV and its vector.
Identification of aphid infesting banana in Java has been done a long time ago by Nordam (2004). He reported P. nigronervosa as the single species of aphid infesting banana in Java based on the specimens collected by van der Goot in 1916and D. Nordam in 1976-1977 Due to the most recent taxonomy of banana aphids, it is necessary to update the status of banana aphids in Indonesia, including in Java. The two cryptic species, P. nigronervosa and P. caladii, are only distinguished by the ultimate rostrum segment (URS) in which the URS of P. caladii is shorter than P. nigronervosa (Nordam, 2004;Foottit et al., 2010;Miller et al., 2014). Multivariate analysis approach on morphometric of aphids has been used by some authors to investigate morphological variation within and between populations of aphids (Blackman, 1987;Foottit et al., 2010).
In this study, there were some morphometric characters of aphids infesting bananas were studied using multivariate analysis and t-test to get accurate identification to confirm morphological identification keys by Blackman & Eastop (2006) and Miller et al. (2014). In addition, we also conducted the host plants survey of aphids infesting banana to collect appropriate data for the purpose of developing a strategy of controlling banana aphids and BBTV.

Collection of Aphids
Aphids were collected by purposive sampling method from different plants belong to the Family Musaceae, Araceae, Zingiberaceae, Heliconiaceae, Costaceae, and Strelitziaceae. Aphid collection was conducted in several locations in West Java, Central Java and East Java in August 2017 to January 2018 (Table 1). Aphids were collected from the plants using a soft brush and then stored in eppendorf tube containing 70% alcohol. Information of location, altitude, collection date, and host species were recorded.

Preparation of Aphid Slide Specimens
Aphids were mounted with modified procedure of Blackman & Eastop (2000)  The aphids were then transferred to the syracuse watch glasses and the ventral side of the abdomens was punctured using a small needle. The aphids were transferred to test tube containing 10% KOH solution and heated until the aphid body became transparent, then the aphids were rinsed with aquadest for 2 times. In order to remove the water content from the aphid body, subsequent immersion in alcohol solution with concentration of 50%, 70%, 80%, 95% and 100% was proceeded for 5-10 minutes on each concentration. Finally, the aphids were immersed in clove oil for 10 minutes to remove the alcohol from the aphid body and then aphids were mounted using Canada balsam for permanent slide purpose.
The specimens were then dried on warmer slider at 35 to 40°C for 2 to 3 weeks.

Identification and Morphometric Measurements
Identification of aphid was conducted morphologically following key identification in "Aphids on the World's Herbaceous Plants and Shrubs" by Blackman & Eastop (2006) and "Review and Key to Aphid (Hemiptera: Aphididae) in Micronesia" by Miller et al. (2014). Eleven morphometric measurements were performed according to Foottit et al. (2010), i.e. length of body (BL), head width (HW), antennal length (AL), length of antennal segment I-II (A1-2), length of antennal segment III-V (A3−5), length of antennal segment VI (A6), length of ultimate rostrum segment (URS), length of siphunculus (Sph), length of hind femur (Fem), length of hind tibia (Tib) and length of caudal (Cau). The morphometric measurement was only conducted on apterous samples. Morphometric characters measurement was derived from image-measurement using Stereo Microscope Leica M 205C and Leica Application Suit Software Version 4.4.0.

Data Analysis
Data of morphometric characters of aphids were analyzed using principle component analysis (PCA) followed by analysis of significant difference of morphometric characters using t-test. Data analysis was performed using PAST version 3.18 (Hummer et al., 2001) and no data transformation was done before analysis.

Morphometry of Aphid Species Infesting Banana
A total of 151 specimens were examined for morphological characters. Two species of aphids were identified, i.e. P. nigronervosa and P. caladii (Table 1). The two species have closely similar characters: length of the antennae is longer than the body, abdomen I and VII without marginal tubercles, apterous frequently having rhinaria at the third antennal segment, parallel or divergent frontal antennal tubercles, siphunculus elongate or swollen with 0-2 rows apical reticulation, femur pale basally and dark distally. The two species were only distinguished by the length of ultimate rostrum segment (URS), in which URS of P. caladii is shorter than those of P. nigronervosa (Figure 1). Foottit et al. (2010) and Miller et al. (2014) recorded that the URS length of P. caladii is generally less than 0.13 mm whereas those of P. nigronervosa is more than 0.13 mm; the mean length of URS length of P. nigronervosa and P. caladii was 0.152 ± 0.005 mm and 0.123 ± 0.003 mm, respectively.
Principle component analysis (PCA) was performed to observe morphological variations between P. nigronervosa and P. caladii. Of the eleven principle components, only the first two principle components were suitable for further analysis due to its eigenvalue, i.e. more than 1 (Figure 2). The first two principle components explained 75.1% of morphology variation in both species (Table 2). The first and second principle component (PC1 and PC2) contributed 66.4% and 9.7% of variance data, respectively. Component loading of the eleven morphological traits showed evenly contributed to PC1, but loading of URS length showed strongly contributed to PC2 with score 0.797.
Principle component analysis (PCA) showed morphometric differences of P. nigronervosa and P. caladii. The morphometric scatter plot of P. nigronervosa and P. caladii exhibited separation projection (Figure 3). The character of URS length was the most contributed in the differentiation of both species. This result confirmed previous study by Foottit et al. (2010) that examined P. nigronervosa collected from banana and P. caladii collected from non-banana host. We also demonstrated that P. caladii collected from banana and non-banana host had no differences on URS length.
The aphid infesting different host plants frequently exhibits variation in morphometric and fitness characters. Aphids reared on poor nutrient quality of host plants are smaller than those reared on rich nutrient quality (Dixon & Kindlmann, 1994). The nutrient quality of a host plant and aphid nutritional biology affects the fitness of aphid (Awmack & Leather, 2002;Powel et al. 2006). The previous study showed that P. nigronervosa reared on banana plant had the longest longevity than taro and red ginger flower (Robson et al., 2007). Badhra & Agarwala (2010) also reported that two congeneric species of banana aphid, P. nigronervosa and P. caladii showed differences in biological character when host plant transfer was performed.

Host Range of P. nigronervosa and P. caladii
Two banana aphid species, P. nigronervosa and P. caladii, were found on 3 and 6 host plants, respectively (Table 4). P. nigronervosa was mostly found on banana (Musa spp.), and frequently found on Heliconia sp. and Ravenala madagascariensis. Laboratory experiment conducted previously indicated that banana aphid P. nigronervosa can feed and live on some species of zingiberaceous and araceous (Suparman et al., 2017). However, P. nigronervosa    Table 3 was rarely found on those alternative plants in the present survey. On the other hand, P. caladii was observed more on Colocasia esculenta (Araceae), Dieffenbachia sp. (Araceae), Curcuma longa (Zingiberaceae), Costus sp. (Costaceae), and rarely on banana and R. madagascariensis (Figure 4). Furthermore, the highest population of P. caladii was observed on C. esculenta and Dieffenbachia sp. Other host plants of P. caladii that was reported previously including Alocasia sp., Caladium sp., Elettaria sp., Hedychium sp., Xanthosoma sp., and Zingiber sp. (Nordam, 2004;Duay et al., 2014;Miller et al. , 2014). This finding suggested that the host range of P. caladii is wider than those of P. nigronervosa.
Survey conducted across Java confirmed that banana was the main host plant of P. nigronervosa and alternative host plant of P. caladii. Out of 151 specimens collected from banana plants, 142 specimens (94% of samples) were P. nigronervosa and 9 specimens (6% of samples) were P. caladii. The specimens of P. caladii were collected from cv. Raja in Pati, Central Java (seven specimens), cv. Barangan in Sukabumi, West Java and Musa veluntina in Bogor, West Java (each one specimen). Although the occurrence of P. caladii is low in banana, its ability to transmit viruses should be considered. P. caladii was reported to transmit Cardamom bushy dwarf virus (CBDV), a new Babuvirus within the family Nanoviridae (Venugopal, 1995;Mandal et al., 2004).
Understanding the host range of banana aphids is important to determine the potency of plant species, especially those adjacent to banana plants, as the reservoir of insect vector of BBTV. Banana aphids were typically found in large number only on suckers, rather than on mature plant parts (Young & Wright, 2005). Aphids prefer feeding on phloem and less commonly on xylem of herbaceous shrubs, trees,  1 mm 1 mm 1 mm 1 mm weeds and cultivated plants (Blackman & Eastop, 2000). On the large colonies, the aphids dispersed to the upper leave, and winged adults (alatae) can also be found although in small number. The alatae play more important role in transmission and spread of BBTV due to its mobility.

CONCLUSION
Two species of aphids infesting banana in Java were identified, i.e. P. nigronervosa and P. caladii. The two species could be differentiated using morphometric characters and its host range. The host plants of P. nigronervosa were mostly Musaceae and rarely on Heliconiaceae and Strelitziaceae, while P. caladii were found on Araceae, Zingiberaceae, Costaceae, and rarely on Musaceae and Strelitziaceae. The alternative host plants will provide sustainable food for the aphids, therefore the existence of this alternative host plants must be considered seriously in the controlling strategy of the banana aphids and BBTV.