Taxonomy, hosts, and distribution of an emerging invasive mealybug, Phenacoccus miruku (Hemiptera: Coccomorpha: Pseudococcidae), in Florida

2.1 Morphology

We examined 90 adult female specimens of P. miruku from 13 counties in Florida, USA, from 15 host plants, which are deposited in the Florida State Collection of Arthropods (FSCA). These specimens were previously reported as P. sisymbriifolium in Ahmed and Miller (2019), the Florida Department of Agriculture and Consumer Services, Division of Plant Industry (FDACS-DPI) database (FDACS-DPI 2024), and FDACS-DPI’s Tri-ology reports through 2023.

The following Florida P. miruku material was examined: USA: FLORIDA: Alachua County: Gainesville, 17 July 2023, on Coreopsis sp. (Asteraceae), L. Line (2023-07275) (two adult ♀♀ on two slides); Gainesville, 2 April 2022, on Geranium carolinianum L. (Geraniaceae), J. Brambila (2022-02838) (three adult ♀♀ on three slides); Gainesville, 3 September 2020, on unknown host, G. Ouwinga, J. Allen, L. Deeter, M. Ahmed (2020-3411) (eight adult ♀♀ on eight slides); Brevard County: Melbourne, 29 September 2023, on Solanum quitoense Lamarck (Solanaceae), A. Tasi, V. Benjamin (2023-10088) (two adult ♀♀ on two slides); Melbourne, 5 October 2023, on I. batatas (L.) Lam. (Convolvulaceae), A. Tasi, V. Benjamin (2023-10383) (one adult ♀ on one slide); Melbourne, 5 October 2023, on S. quitoense, A. Tasi, V. Benjamin (2023-10393) (one adult ♀ on one slide); Melbourne, 23 October 2023, on Solanum melongena L. (Solanaceae), A. Tasi, V. Benjamin (2023-11004) (two adult ♀♀ on two slides); Melbourne, 12 April 2024, on Solanum lycopersicum L. (Solanaceae), A. Tasi, V. Benjamin (2024-03643) (one adult ♀ on one slide); Broward County: Hollywood, 13 March 2023, on unknown Compositae, M. Zenoble, R. Tordi (2023-02544) (one adult ♀ on one slide); Dixie County: Old Town, 20 March 2023, on Physalis heterophylla Nees (Solanaceae), A. Barrios (2023-02586) (two adult ♀♀ on two slides); Hernando County: Brooksville, 8 January 2020, on Gomphrena serrata L. (Amaranthaceae), N. Marquez (2020-79) (one adult ♀ on one slide); Brooksville, 29 January 2020, on G. serrata, N. Marquez (2020-417) (one adult ♀ on one slide); Lake County: Eustis, 8 January 2019, on B. alba (L.) DC. (Astaraceae), N. Marquez (2019-93) (two adult ♀♀ on two slides); Eustis, 29 January 2019, on B. alba, N. Marquez (2019-322) (three adult ♀♀ on three slides); Eustis, 12 March 2019, on B. alba, N. Marquez (2019-1068) (one adult ♀ on one slide); Eustis, 12 March 2019, on B. alba, N. Marquez (2019-1069) (one adult ♀ on one slide); Clermont, 26 March 2019, on B. alba, N. Marquez (2019-1410) (two adult ♀♀ on two slides); Eustis, 26 March 2019, on B. alba, N. Marquez (2019-1411) (six adult ♀♀ on six slides); Eustis, 2 April 2019, on B. alba, N. Marquez (2019-1596) (two adult ♀♀ on two slides); Fruitland Park, 3 April 2019, on B. alba, N. Marquez (2019-1625) (three adult ♀♀ on three slides); Tavares, 3 April 2019, on B. alba, N. Marquez (2019-1622) (three adult ♀ on three slides); Tavares, 3 April 2019, on B. alba, N. Marquez (2019-1616) (three adult ♀♀ on three slides); Mount Dora, 8 April 2019, on B. alba, N. Marquez (2019-1748) (three adult ♀♀ on three slides); Grand Island, 11 April 2019, on B. alba, N. Marquez (2019-1841) (two adult ♀♀ on two slides); Clermont, 17 April 2019, on Gamochaeta antillana (Urb.) Anderb. (Asteraceae), N. Marquez (2019-2068) (one adult ♀ on one slide); Tavares, 18 April 2019, on B. alba, N. Marquez (2019-2066) (two adult ♀♀ on two slides); Astatula, 19 April 2019, on A. artemisiifolia L. (Asteraceae), N. Marquez (2019-2142) (one adult ♀ on one slide); Eustis, 22 April 2019, on G. antillana, Marquez (2019-2174) (three adult ♀♀ on three slides); Tavares, 24 April 2019, on B. alba, N. Marquez (2019-2207) (one adult ♀ on one slide); Mount Dora, 6 May 2019, on G. antillana, N. Marquez (2019-2494) (two adult ♀♀ on two slides); Fruitland Park, 17 June 2020, on B. alba, N. Marquez (2020-2369) (three adult ♀♀ on three slides); Clermont, 18 April 2022, on B. alba, N. Marquez (2022-03335) (two adult ♀♀ on two slides); Clermont, 8 March 2022, on Baccharis halimifolia L. (Asteraceae), B. Danner (2022-01954) (two adult ♀♀ on two slides); Eustis, 17 May 2022, on Achillea sp. (Asteraceae), M. Sellers (2022-05621) (two adult ♀♀ on two slides); Manatee County: Parrish, 13 May 2023, on B. alba, C. Poock (2023-05078) (five adult ♀♀ on five slides); Marion County: Dunnellon, 26 August 2022, on unknown host, T. Deuel (2022-07883) (one adult ♀ on one slide); Orange County: Apopka, 3 October 2020, on B. alba, G. Ouwinga, L. Deeter, M. Ahmed (2020-3889) (two adult ♀♀ on two slides); Palm Beach County: Lake Worth, 17 March 2023, on Bidens sp. (Asteraceae), D. Miller (2023-02641) (one adult ♀ on one slide); St. Johns County: St. Augustine, 9 May 2019, on Gnaphalium sp. (Asteraceae), L. Deeter (2019-3077) (two adult ♀♀ on two slides); Sumter County: Sumterville, 5 April 2019, on B. alba, N. Marquez (2019-1744) (one adult ♀ on one slide); Sumterville, 5 April 2019, on A. artemisiifolia, N. Marquez (2019-1747) (two adult ♀♀ on two slides); Volusia County: Tomoka Springs, 17 March 2022, on Ruellia blechum L. (Acanthaceae), N. Marquez (2022-02236) (one adult ♀ on one slide).

We also examined a series of seven adult female paratype specimens of P. miruku borrowed from Ehime University Museum, Matsuyama, Japan with the following verbatim label data: JAPAN,/ Okinawa prefecture,/ Okinawa Is. Ogimi-son,/ on B. pilosa/ var. radiata,/ 26. vi.2021,/ coll. J. Choi (seven adult ♀♀ on seven slides). ‘/’ indicates the positions of the line breaks on the label. Furthermore, we have contacted Dr. Cristina Granara de Willink (pers. comm., November 2023) and she has provided information based on her examination of the type series of P. sisymbriifolium with the following label data: URUGUAY: Montevideo, I-1943, on Solanum sisymbriifolium, A. Silveira (eight adult ♀).

2.2 Molecular methods

Whole specimens of P. miruku (Florida: Alachua: Gainesville, 3 April 2022, on B. alba, D. Miller, 2022-02798; Florida: Lake County: Clermont, 18 April 2022, on B. alba, N. Marquez, 2022-03335) were non-destructively DNA extracted with a Qiagen DNeasy Blood and Tissue Kit (Hilden, Germany). Voucher specimens were slide-mounted and deposited in the FSCA. The polymerase chain reactions (PCRs) were conducted as 25 µl reactions with the Kapa HiFi HotStart ReadyMix Kit (Basel, Switzerland). Primer sets and thermocycler conditions for 18S, 28S D10, 28S D2, and COI-5′ followed the methods in Ahmed et al. (2020), except for an additional COI-3′ fragment. This PCR was conducted with reverse complement of HCO2198 (Folmer et al. 1994) and TL2-N-3014 (Simon et al. 1994). The thermocycler conditions for this primer pair was as follows: 1) 95 °C for 2 min, [32× steps 2–4] 2) 98 °C for 30 s, 3) 50 °C for 30 s, 4) 72 °C for 75 s, 5) 72 °C for 7 min, 6) 4 °C indefinite hold. PCR products were visualized by gel electrophoresis and purified for bidirectional sequencing. All PCRs targeting the COI-5′ barcode region failed in these specimens. Sanger sequencing was conducted on the ABI SeqStudio platform with BigDye Terminator v3.1 chemistry. Sequence chromatograms were trimmed and assembled in Geneious Prime 2023.2.1. New sequences were queried to the GenBank nucleotide database by MegaBLAST searches (Altschul et al. 1990). Newly generated sequences were deposited in GenBank (COI: PP503189, 18S: PP503320, 28S D2: PP532812, 28S D10: PP532813). Highly similar sequences returned from MegaBLAST searches were downloaded for further comparison. Sequences were aligned using the default settings of MUSCLE (Edgar 2004) as implemented in MEGA7 (Kumar et al. 2016) and trimmed to 100 % data coverage. Sequence similarity was then calculated using p-distance for each target in MEGA7 (Kumar et al. 2016).

3.1 Morphology

P. miruku diagnosis based on seven adult female paratype specimens from Japan: with 18 pairs of cerarii; nine-segmented antennae; 1–6 dorsal multilocular pores on margins of abdomen; ventral multilocular pores extending to margin of abdomen, on abdominal segments III–VIII; 0–3 quinquelocular pores from widest part of clypeus up to anterior margin of head; quinquelocular pores abundant on thorax, on abdominal segments II–V, present in anterior row on abdominal segment V only; two sizes of ventral oral collar tubular ducts; dorsal oral collar tubular ducts present marginally from abdominal segment VIII to prothorax or head, a few also present medially on thorax; small translucent pores on each hind tibia; and circulus present or absent, highly variable in shape and size, sometimes split into two circular pieces.

P. miruku diagnosis based on 90 adult females from Florida: with 18 pairs of cerarii; nine-segmented antennae; 0–10 dorsal multilocular pores on margins of abdomen; ventral multilocular pores extending to margin of abdomen, on abdominal segments III–VIII; 0–5 quinquelocular pores from widest part of clypeus up to anterior margin of head; quinquelocular pores abundant on thorax, on abdominal segments II–V, present in anterior row on abdominal segment V only; two sizes of ventral oral collar tubular ducts; dorsal oral collar tubular ducts present marginally from abdominal segment VIII to prothorax or head, a few present medially on thorax; small translucent pores on each hind tibia; and circulus present or absent, highly variable in shape and size, sometimes split into two circular pieces.

P. sisymbriifolium diagnosis (translated and adapted from Granara de Willink and Szumik 2007): with 18 pairs of cerarii; nine-segmented antennae; 0–2 dorsal multilocular pores on margins of abdomen; ventral multilocular pores extending to margin of abdomen, on abdominal segments III–VIII; about 32 quinquelocular pores ahead of mouthparts, 0–16 quinquelocular pores from widest part of clypeus up to anterior margin of head (pers. comm. Cristina Granara de Willink, November 2023); quinquelocular pores abundant on thorax, on abdominal segments II–VII; two sizes of ventral oral collar tubular ducts; dorsal oral collar tubular ducts present marginally and submarginally from abdominal segment VIII to prothorax or head, absent medially; small translucent pores on each hind tibia; and with large, anvil-shaped circulus.

`When we examined the paratype specimens of P. miruku, we observed quinquelocular pores on the abdomen as far posteriorly as the anterior area of abdominal segment V; small, inconspicuous translucent pores on the hind tibiae typical of Phenacoccus; a few dorsal oral collar tubular ducts in the medial area of the thorax, and two sizes of ventral oral collar tubular ducts. That is, we found that specimens of P. miruku were morphologically more similar to the description of P. sisymbriifolium in Granara de Willink and Szumik (2007). However, differences remain between P. miruku and P. sisymbriifolium as described in Granara de Willink and Szumik (2007), including in the form of the circulus (variable vs. large anvil-shaped), the distribution of the quinquelocular pores (to the anterior row of abdominal segment V vs. to abdominal segment VII), the number of quinquelocular pores anterior to the widest part of the clypeus (consistently 0–5 in P. miruku and more variable in P. sisymbriifolium (0–16)), and the dorsal oral collar tubular ducts (present on medial area of thorax vs. absent from this area). We found that the shape of the circulus in Florida specimens is highly variable, ranging from small to large oval, or oxbow shaped, or split into two separate circular pieces, or even absent in some specimens, fitting the description of P. miruku. Quinquelocular pores extended from the head as far posteriorly as the anterior area of abdominal segment V in P. miruku paratypes and Florida specimens. Finally, a few dorsal oral collar tubular ducts in the medial area of the thorax were found to be present on the paratypes of P. miruku and on our Florida material.

P. miruku is also similar morphologically and molecularly to P. parvus, a species that was first detected in Florida in 1983 (Williams and Hamon 1994). P. miruku differs by having (character states of P. parvus are presented in parentheses): zero to two marginal oral collar tubular ducts laterad of the antennae (a cluster of four or more oral collar tubular ducts laterad of the antennae); zero to two marginal oral collar tubular ducts laterad of the anterior spiracle (a cluster of four or more marginal oral collar tubular ducts laterad of the anterior spiracle); quinquelocular pores as far posteriorly as abdominal segment IV or the anterior of V (quinquelocular pores as far posteriorly abdominal segment VI); 0–5 quinquelocular pores on the head (numerous quinquelocular pores on the head); ventral multilocular pores not reaching the body margins (ventral multilocular pores reaching the body margin); and dorsal multilocular pores absent (dorsal multilocular pores present marginally).

P. miruku is also similar morphologically and molecularly to P. montevidensis Pacheco da Silva & Kaydan, a species recently described from Uruguay (Pacheco da Silva et al. 2020). P. miruku differs by having (character states of P. montevidensis are presented in parentheses): dorsal multilocular pores restricted to the margin of the abdomen (dorsal multilocular pores in the medial areas of the posterior abdominal segment VI and VII) and dorsal lanceolate setae with typical acute apices (some dorsal lanceolate setae with a protrusion on the apex).

All of the type specimens of P. sisymbriifolium are currently still in Argentina and could not be examined by the authors. The species description states that P. sisymbriifolium has about 32 quinquelocular pores anterior to the mouthparts (Granara de Willink and Szumik 2007), however it was unclear whether this meant starting at the posterior part of the labrum and everything above it, or only from the anterior of the clypeus to the anterior margin of the head. Direct communication with the author of P. sisymbriifolium shed light on this character and the variability of the number of quinquelocular pores around the mouthparts in this species. From the widest part of the clypeus and anterior to the mouthparts, the holotype has 14 quinquelocular pores, one paratype has 16 quinquelocular pores, and the other six paratypes have 0–3 quinquelocular pores in this area (pers. comm. Cristina Granara de Willink, November 2023).

3.2 Molecular analysis

BLAST searches of COI-3′ of the Florida specimens recovered the highest sequence similarity of 99.68 % to Japanese specimens of P. miruku (ON533756) and P. parvus (GU134696 and KJ530609). These COI sequences differed by a single nucleotide polymorphism. The Florida sample was unique among all available Phenacoccus COI sequences in GenBank. BLAST searches of 18S of the Florida specimens recovered 100 % matches to Phenacoccus manihoti Matile-Ferrero and P. parvus, with P. miruku inexplicably absent from the returned results. The 18S sequence from Japanese P. miruku (ON527953) contains two gap regions that were trimmed out during assembly (pers. comm. Jinyeong Choi, August 2023). Comparison between the untrimmed Japanese 18S sequence demonstrated a 100 % match with the Florida population (pers. comm. Jinyeong Choi, August 2023). BLAST searches of 28S D2 of the Florida specimens recovered 100 % matches to several specimens of P. manihoti, P. miruku, and a Phenacoccus sp. (MW251837). BLAST searches of 28S D10 recovered only one high match to a specimen of P. parvus, from which it differed by a single nucleotide polymorphism.

3.3 Ecological associates and distribution

P. miruku is frequently found in association with the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae). On B. alba, P. miruku has been found in mixed infestations with solanum mealybug, P. solani (05152023-05078; 04252024-04239) and in mixed infestations with trochanter mealybug, Pseudococcus sorghiellus (Forbes) (10032022-08824). P. miruku also has been found in mixed infestations with citrus mealybug, Planococcus citri (Risso), on three plant hosts: A. artemisiifolia (2019-2142), S. quitoense (10052023-10393), and S. lycopersicum (04152024-03643). Evidence of parasitism has been noted but the specific wasps attacking this species have not yet been identified. In two instances, predatory flies in the family Chamaemyiidae, which specialize on mealybugs, scales, or aphids, were found with infestations of P. miruku (10262021-5969). Five specimens of chamaemyiid flies that emerged from a mixed infestation of P. miruku and P. citri on S. quitoense were identified as Leucopina bella (Loew) (10052023-10393) (Figure 3). The L. bella specimens are deposited in the FSCA.

Figure 3:

Leucopina bella (Loew) (Diptera: Chamaemyiidae) reared from a mixed infestation of Phenacoccus miruku and Planococcus citri on Solanum quitoense Lam. from Brevard County, Florida. Photograph by Erin Powell.

P. miruku has been recorded in the landscape in the following 20 Florida counties: Alachua, Brevard, Broward, Collier, Dixie, Hernando, Hillsborough, Indian River, Lake, Marion, Okaloosa, Orange, Palm Beach, Pasco, Polk, Putnam, St. Johns, St. Lucie, Sumter, and Volusia (Figure 4). P. miruku has been found feeding on 25 host plants belonging to 10 families in Florida (Table 1). To date, the most common host is B. alba.

Figure 4:

Map of Florida with recorded Phenacoccus miruku distribution (counties shaded in grey) from 2019 to 2024. Map created by K. Burnette.

3.4 A key to the slide-mounted adult females of Phenacoccus species in Florida

*Note that P. solani and P. solenopsis can be difficult to distinguish morphologically and a combination of character states using multiple specimens may be required. We have arranged character states in the couplet in order of diagnostic reliability based on our analysis of Florida specimens and that of Williams (2004), Hodgson et al. (2008) and Zhao et al. (2014).

1. (0) Dorsum with multilocular pores … 2

   1. Dorsum without multilocular pores … 4

2. (1) Dorsal multilocular pores forming rows across some abdominal segments … 3

   1. Dorsal multilocular pores restricted to body margins … P. miruku Tanaka & Choi (in part)

3. (2) Ventral quinquelocular pores absent … P henacoccus  multicerarii Granara de Willink

   1. Ventral quinquelocular pores present … Phenacoccus madeirensis Green

4. (1) Quinquelocular pores present … 5

   1. Quinquelocular pores absent … 6

5. (4) Ventral multilocular pores absent from body margin; with conspicuous cluster of four or more marginal oral collar tubular ducts laterad of anterior spiracle … Phenacoccus parvus Morrison

   1. Ventral multilocular pores present near body margin; with 0–2 marginal oral collar tubular ducts laterad of anterior spiracle … P. miruku Tanaka & Choi (in part)

6. (4) Ventral multilocular pores on abdominal segment VII present in row along posterior edge only; circulus usually small, rounded; antennae usually 8-segmented … Phenacoccus solani Ferris

   1. Ventral multilocular pores on segment VII present between anterior and posterior margins; circulus usually large, more flaccid; antennae usually 9-segmented … Phenacoccus solenopsis Tinsley