Pacific Science, vol. 68, no. 3 February, 10, 2014 (Early view) 

Pentalonia nigronervosa Coquerel and Pentalonia caladii van der Goot (Hemiptera: Aphididae) and their relationship to banana

bunchy top virus in Micronesia

By Julie Anne M. Duay, Ross H. Miller*, George C. Wall, Keith S. Pike, and Robert G. Foottit

Abstract The geographical distribution of Pentalonia nigronervosa and P. caladii, and the incidence of banana bunchy top virus (BBTV) were determined in Micronesia. Aphids and plant tissues were collected from banana and non-banana hosts of Pentalonia aphids in Palau, Yap, Guam, Rota, Tinian, Saipan, Pohnpei, Kosrae, and Majuro. Aphids were identified based on the host species they were collected from, and on morphometric analysis of the length of the ultimate rostral segment. All plant samples were tested for the presence or absence of BBTV using a triple antibody sandwich enzyme linked immunosorbent assay (TAS-ELISA). TAS-ELISA analysis confirmed BBTV to be present on Guam, Saipan, and Rota, but BBTV was not detected elsewhere in Micronesia. *Corresponding Author E-mail: rmiller@uguam.uog.edu

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Introduction

Pentalonia nigronervosa Coquerel, the banana aphid, occurs on banana (Musa

sp.) throughout the tropical and subtropical regions of the world (Dale 1987; Blackman and

Eastop 2000; Magee 1967). Coquerel first described P. nigronervosa from specimens collected

on Réunion Island, although the aphid likely originated in Southeast Asia as did the genus Musa

(Waterhouse 1987; Robinson 1996). P. nigronervosa was likely distributed throughout the

Pacific on bananas carried by ancient Polynesians beginning about 1000 A.D. (Waterhouse

1987).

A closely related aphid species, Pentalonia caladii van der Goot, infests plants in the

genera Heliconia (Heliconiaceae), Alpinia and Zingiber (Zingerberaceae), and Colocasia

(Araceae) (Waterhouse 1987). Initially described as a separate species by van der Goot in 1917

from specimens collected on Caladium in Java, P. caladii subsequently lost its species status and

was considered a form of P. nigronervosa (Waterhouse 1987). Foottit et al. (2010) restored it to

full species status based on significant genetic and morphological differences observed on

Pentalonia aphids occurring on different hosts. They noted that P. nigronervosa appeared to be

restricted to the genus Musa in the field in Micronesia, while P. caladii was commonly found on

Heliconia, Alpinia, and Colocasia, and rarely on Musa. Morphologically, P. caladii possessed an

ultimate rostral segment length (URSL) less than 0.13 mm in length, while that of P.

nigronervosa was always longer.

P. nigronervosa is economically important because it vectors banana bunchy top virus

(BBTV) (Hu et al. 1996), a single-stranded, circular DNA virus first identified in Fiji in 1889

(Magnaye and Valmayor 1995). BBTV currently threatens banana production in one-fourth of

the world’s banana growing areas (Dale 1987; Magee 1967), causing significant banana

mortality in Australia, the Bonin Islands, China, Congo, Egypt, Tuvalu, Fiji, Gabon, Guam,

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Hawaii, India, Kiribati, the Philippines, Samoa, Sri Lanka, Taiwan, Tonga, Vietnam, Wallis

Island, and other Pacific island groups (Stover 1972; Dale 1987; Ferreira 1991). The purpose of

the present study was to determine the geographic and host distribution of P. nigronervosa and

P. caladii and to determine the incidence of BBTV in Micronesia.

Materials and Methods

Site Description

Aphids and disease surveys were conducted throughout Micronesia from 2010 to 2012.

Aphid and plant tissue samples were collected from Guam, Saipan, Tinian, and Rota in the

Mariana Archipelago, from the Caroline Islands of Kosrae, Pohnpei, and Yap in the Federated

States of Micronesia, from Babeldaop in the Republic of Palau, and from Majuro in the Republic

of the Marshall Islands.

Survey of Pentalonia and BBTV in Micronesia

Eight hundred fifty-seven plant tissue samples and 440 samples of adult aphids were

collected from banana and non-banana host plants in Micronesia. Aphids collected from plants

were stored in 95% ethanol in 2 ml cryogenic vials (Corning Inc., Corning, NY). The leaf blade

and midrib of all plant samples collected with or without aphids were cut into small pieces and

wrapped in sterile gauze fabric within hours of being collected. They were then placed in clear

styrene tubes containing CaCl2 as a desiccant, and stored at approximately 24°C until processed

in the laboratory.

Aphids destined for morphometric analysis were cleared and mounted in Canada balsam

on micro-slides using methods adapted from Foottit and Maw (2000), and cured for two weeks at

50°C.

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Adult apterous aphids were first identified to species using conventional taxonomic keys

(Blackman and Eastop 2000), and confirmed by measuring the URSL as described by Foottit et

al. (2010).

All plant samples were tested for BBTV using a triple antibody sandwich enzyme linked

immunosorbent assay (TAS-ELISA; Agdia Inc., Elkhart, IN). A humidity box was prepared by

lining an airtight container with wet paper towels to prevent testwell samples from evaporating.

A capture antibody solution was prepared by diluting 100 µl of concentrated capture antibody

with 10 ml of carbonate coating buffer. A 96-well microtiter plate was then coated with 100 µl of

diluted capture antibody solution. The microtiter plate was placed in the humidity box at ~24°C

for 4 h or at 4°C overnight. After incubation, the plate was washed three times with phosphate

buffered saline Tween 20 (PBS-T). Approximately 0.3 g of leaf tissue was homogenized with a 3

ml general extraction buffer and the supernatant containing the antigen was collected. 100 µl of

supernatant containing each sample antigen were loaded into each of two testwells. Positive and

negative controls consisting of samples previously testing positive or negative for BBTV were

added to other testwells. 100 µl of general extraction buffer were added to two assigned testwells

as a blank sample control. The microtiter plate was incubated at either room temperature for 2 h

or at 4°C overnight. After incubation the plate was washed eight times with PBS-T. An enzyme

conjugate solution was prepared using ECI buffer containing 0.2% bovine serum albumin and

2% polyvinylpyrrolidone in PBS-T buffer held at pH 7.4. The ECI buffer contained a

concentrated detection antibody and an alkaline phosphatase enzyme conjugate, which were

pipetted into each testwell and incubated for 2h at 24°C. After incubation, the plate was washed

eight times with PBS-T. A p-nitrophenyl phosphate (PNP) substrate was added to the testwells

and incubated in the dark for about 60 min. Testwells were visually examined for color change

and then measured on a Synergy HT micro-plate reader at 405 nm for confirmation. Testwells

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that emitted any shade of yellow color were considered positive for BBTV while testwells that

remained completely colorless were considered negative.

Results

Survey of Pentalonia and BBTV in Micronesia

P. nigronervosa was identified on Musa sp. on Guam, Saipan, Tinian, Rota, Kosrae,

Pohnpei, Palau, Yap, and Majuro. P. caladii was identified on plant species in the families

Zingerberaceae, Araceae, and Heliconiaceae on all the islands sampled except for Tinian. No

Pentalonia aphids were collected on any non-banana host plants on Tinian (Table 1).

<<Table 1 near here>>

Of the 858 plant samples collected from Palau to Majuro, 56 tested positive for BBTV

using TAS-ELISA. This included 17 samples from Guam, 3 samples from Saipan, and 36

samples from Rota (Table 1; Figure 1). Plants testing positive for BBTV were collected from

five locations on Guam, one location on Saipan, and three locations on Rota. Plant samples

collected from Tinian, Kosrae, Pohnpei, Palau, Yap, and Majuro all tested negative for BBTV.

<<Figure 1 near here>>

Discussion

P. nigronervosa and P. caladii were present on Guam, Saipan, Rota, Palau, Yap, Majuro,

Kosrae, and Pohnpei. Although aphids on Zingerberaceae, Araceae, and Heliconiaceae were not

collected on Tinian, previous studies found P. caladii on Alpinia purpurata, Heliconia sp., and

Strelitzia reginae Banks (Miller, CNAS-WPTRC, Univ. of Guam unpubl. data). Although no

samples were collected on Chuuk in the present study, Nafus (1997) had previously identified P.

nigronervosa collected from Musa sp. on Chuuk, although BBTV was not found there or on any

other island in Micronesia.

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P. nigronervosa has previously been found in tropical Africa, on Atlantic islands, and in

California, Central America, China, Egypt, Florida, India, Indonesia, Malaysia, Madagascar,

Mauritius, Mexico, the Middle East, Mozambique, Philippines, Réunion, northern South

America, Taiwan, and the West Indies (Waterhouse 1987). In addition, Waterhouse (1987)

indicated that P. nigronervosa also occurred in eastern Australia, the Cook Islands, Fiji, Hawaii,

Kiribati, the Marshall Islands, Micronesia, Papua New Guinea, Samoa, Tokelau, Tonga, Tuvalu,

Vanuatu, and Wallis Island. However, because P. caladii has only recently been elevated to

species status, there were no records of its geographic distribution in Micronesia. The present

study showed that P. nigronervosa and P. caladii are distributed throughout Micronesia

dependent on the presence of the appropriate host for each aphid on each island. P. nigronervosa

was found on Musa sp., while P. caladii was found on non-banana host plants such as Alpinia

spp., Heliconiaceae, Colocasia spp., and Strelitziaceae.

Within Micronesia, BBTV was found only on Guam, Rota, and Saipan. Most BBTV

positive samples on these islands were collected from farms and backyard banana plants in

residential areas. On Saipan, BBTV did not occur as frequently as on Guam and Rota, with

samples from a single farm testing positive. On Tinian, where no BBTV was found, local

farmers reported past banana mortality due to BBTV, suggesting that BBTV may be under

current control on Tinian due to the use of a variety of surveillance and sanitation measures.

Many farmers on Saipan and Tinian control BBTV by killing and removing infected

banana plants when early symptoms first become visible. Banana aphids are first killed with

insecticides applied to the infected banana plant and mat, and then the entire infected banana mat

is killed with herbicide. Once the entire mat is dead it may be removed and disposed of (Wall

2010).

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Acknowledgements

We thank Christina Nguyen, Eric Maw, and Glenn Meno for assistance. Funds were

provided by the USDA Tropical and Subtropical Agriculture Research (TSTAR) Special Grant

Program, number 2010-34135-21267.

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