Sentinel egg masses of Hal. halys and N. viridula were deployed at three agricultural sites in Lake County, Florida, between March and August of 2021. Deployments were conducted weekly from March to June with a shift to bi-weekly deployments starting in July due to a shortage of eggs. For all but one week where egg mass availability was low, 120 egg masses were deployed per week. Of the 120 egg masses, approximately half were Hal. halys eggs and half N. viridula eggs.

The first site, located in Okahumpka, FL (28.744666N, – 81.895032E), includes 8 acres where uncertified organic practices are utilized. The farm is surrounded by woodlands, and over 120 varieties of crops are rotated within the farm seasonally. The second site, located in Clermont, FL (28.629214N, – 81.76725E), includes 69.4 acres of muscadine grapes, but the study was conducted in a 25.4-acre plot of the vineyard. The vineyard used integrated pest management practices that include conventional pesticides. The vineyard is bordered by a thin strip of forest and is adjacent to a lake. The third site, located in Lady Lake, FL (28.927383N, – 81.904568E), is a 2.5-acre orchard of peach trees within a residential community. It has a border of weedy plants and hardwood trees. The past management style was conventional with some organic practices. However, management practices did not occur during this study.

The N. viridula egg masses were sourced from a colony at the University of Florida’s Entomology and Nematology Department. The stink bugs were reared in mesh cages (39.9 cm × 39.9 cm × 61.0 cm). They were fed a diet of tomatoes, peanuts, baby carrots, and sweet corn on a Monday, Wednesday, Friday schedule. The photoperiod in the rearing rooms was 16L:8D, and the average temperature and relative humidity were 24.5 °C and 42%, respectively.

The Hal. halys egg masses were sourced from a quarantine colony at FDACS-DPI and the New Jersey Department of Agriculture (NJDA) Phillip Alampi Beneficial Insect Lab. The FDACS-DPI colony of H. halys was reared in round, mesh-ventilated, polystyrene containers of various sizes. The temperature and relative humidity were ±1 °C and ±5% of the conditions in Medal et al. (2012), respectively. The colony was fed a diet of whole sweet corn, snap peas, peanuts, and organic baby carrots.

The NJDA colony of Hal. halys was reared in quarantine according to protocols by Dorsey and Lovero (2014) and was fed a diet designed by Cohen (2000). Eggs were shipped to FDACS-DPI and were either directly frozen or reared to adulthood according to the protocols described above.

All egg masses were frozen at -80 °C prior to their deployment and were glued using Elmer’s glue to 1.5 × 1.5 cm squares of waterproof paper (Rite in the Rain, Tacoma, WA). The sentinel egg mass cards were then pinned to the undersides of the leaves of plants within the crop field and at its periphery. If the egg masses could not be pinned directly to plants, they were clipped to orange and yellow flags which were staked approximately 12 cm above the ground. These measures were taken to reduce damage to plant products that would eventually be sold.

The egg masses were left in the field for 72 hours, then returned to the lab where they were recounted, and predation damage was documented. The egg masses were then placed into small petri dishes sealed with parafilm and stored in a quarantined room. The temperature and relative humidity in this room were 26.5 °C and 48%, respectively. The photoperiod in this room was 16L:8D. The egg masses were checked daily for parasitoid emergence, which often occurred between 20–25 days of storage and were stored for a total of 30 days before being discarded. Reared parasitoids were stored in 95% ethanol prior to identification.

A single, wild stink bug egg mass, unidentified at the time, was collected on a squash plant in Lake County, on May 25^th, 2021. The egg mass was brought back to the lab for assessment of parasitism and identification of the stink bug species via COI barcoding.

Eggs, nymphs and adults of the redbanded stink bug Piezodorus guildinii (Westwood, 1837) were collected from an unmanaged planting of hairy indigo (Indigofera hirsuta L.) located at the University of Florida Plant Science Research and Education Unit in Marion County, Florida (29.4079530N, 82.1459287W). Eggs were hand collected and placed into 10-dram plastic vials until eclosion, after which the emerged Pi. guildinii nymphs were transferred to a larger cage for rearing. This was done to prevent accidental introduction of egg parasitoids into the main Pi. guildinii colony as the dark color of Pi. guildinii eggs makes it difficult to identify parasitism. Collections were made throughout the spring and summer of 2018.

Yellow, 3D-printed cylinder traps (Figure 1) were deployed in Florida by the Cooperative Agricultural Pest Survey (CAPS Program) throughout the year to monitor for exotic pests, especially psyllids in a variety of agricultural ecosystems (Snyder et al. 2019). These traps also serve well for collecting other insects that are attracted to yellow, and residues were screened for parasitoid wasps. The preservative in the collection tube (Figure 1B) was propylene glycol (Prestone LoTox) with salt.

Figure 1. 

Yellow cylinder trap employed by CAPS Program in Florida A exterior view of trap; the inside of the funnel is coated with Insect-a-Slip B interior of trap where insects are preserved in a vial containing propylene glycol and salt.

The collection data and images associated with the specimens examined were deposited in the database of the Museum of Biological Diversity, The Ohio State University (mbd-db.osu.edu), and a DarwinCore archive file is provided in Suppl. material 1. Collecting unit identifiers associated with specimens are listed by county below.

Psix striaticeps: 54 females, 3 males, deposited in Florida State Collection of Arthropods (FSCA). Florida: Alachua Co. (FSCA 00095801, 00095797); Brevard Co. (FSCA 00095791–00095792, 00095830); Hardee Co. (FSCA 00034061, 00034693, 00034696); Hillsborough Co. (FSCA 00095795–00095796, 00095798, 00095800); Lake Co. (FSCA 00094219, 00094237); Lee Co. (FSCA 00095788–000-5789); Marion Co. (FSCA 00095892–00095894); Miami-Dade Co. (FSCA 00095831); Orange Co. (FSCA 00034688–00034689, 00095793–00095794, 00095813–00095823); Pinellas Co. (FSCA 00095826–00095827, 00095790); St. Johns Co. (FSCA 00095799, 00095803–00095809, 00095811–00095812, 00095828–00095829, 00095891, 00095896–00095899); St. Lucie Co. (FSCA 00095824–00095825).

Specimens of Psix striaticeps were identified using the key to Psix species by Johnson and Masner (1985).

Genomic DNA was nondestructively isolated from the whole adult specimens using the Qiagen DNeasy kit (Hilden, Germany) as described by Giantsis et al. (2016). The host eggs were pulverized prior to analysis and genomic DNA was isolated using the Qiagen DNeasy kit (Hilden, Germany) as described by Giantsis et al. (2016). Polymerase chain reaction (PCR) was carried out to amplify the DNA barcode region of the Cytochrome Oxidase Subunit I (COI) using the LCO/HCO primers of Folmer et al. (1994). For the host eggs, the PENT_F2/HCO primer was used as described by Gariepy et al. (2014). The PCR was performed in a 25 µl reaction volume using the KAPA HiFi Hotstart Ready Mix (Roche) per the manufacturer’s standard protocol. PCR conditions were as follows: 95°C for 2 min, followed by 32 cycles of 95 °C for 30 s, 50 °C for 40 seconds, 72 °C for 1 min with a final extension at 72 °C for 7 min. All PCR products were electrophoresed through an agarose gel (1.5%) and were purified and prepared for sequencing using BigDYE Xterminator (Applied Biosystems) per the manufacturer’s protocol. Products were then sequenced in both directions at FDACS-DPI, Gainesville, Florida. Chromatograms were then trimmed in Sequencher 5.4.6. and assembled into contigs. Amino acids were translated using MEGA7 (Kumar et al. 2016). Data was deposited in GenBank under accession numbers OK157284–OK157305, OK327010–OK327011 (Table 1).

Both GenBank (Altschul et al. 1990) and BOLD (Ratnasingham and Hebert 2007) were queried, and sequences for the Psix group of genera (Psix Kozlov & Lê, Paratelenomus Dodd, Nirupama Nixon) barcodes were downloaded from BOLD. Based on the phylogenetic topology of Chen et al. (2021), a species of Telenomus was selected as an outgroup for the neighbor-joining analysis. DNA barcodes were aligned using MUSCLE with default settings as implemented in MEGA7 (Tamura et al. 2013). Neighbor-joining of DNA barcodes was performed in MEGA7 using K2P distance (Kimura 1980) with 10,000 bootstrap replicates. The resulting Newick tree file (Suppl. material 2.) was altered in the Interactive Tree of Life (Letunic and Bork 2021) portal to collapse terminal clusters and apply bootstrap support values to branches.

We identified specimens as Ps. striaticeps based on the following characters in the key to species by Johnson and Masner (1985): radicle black; frons without submedian carina; width of frons greater than height of compound eyes; metascutellum (dorsellum) protruding weakly; ventral lip of metascutellum punctulate; posterior margin of T2 punctulate; lateral sulci on S2 widely separated; area between sulci on S2 covered with large seta-bearing punctures (Figures 4–8).

Figures 4–8. 

Psix striaticeps, female (FSCA 00095790) 4 head, anterolateral view 5 head and mesosoma, lateral view 6 mesosoma, posterior view 7 metasoma, posterodorsal view 8 metasoma, ventral view. msct: metascutellum; r: radicle; S2: second metasomal sternite; T2: second metasomal tergite.

Figures 9–10. 

Wild, parasitized egg masses 9 Chinavia marginata (FSCA 00095810) 10 Piezodorus guildinii (FSCA 00095895).

Our analysis of COI sequences retrieved two clades of Ps. striaticeps that differ from each other by approximately 2.5% K2P distance (Suppl. material 3.). One of the specimens in BIN:BOLD:ADV1998 was identified by Taekul et al. (2014), and the specimens that match the Floridian population were previously unidentified (BIN:BOLD:ADX9028) (Figure 11). The Floridian specimens are identical to each other and to one of the specimens from South Africa (KMPUD3236-19).

The wild egg mass of C. marginata (Figure 9) was identified by a BOLD query that matched its COI sequence to specimens from Costa Rica (99.8%; BIN BOLD:AEE5175). Chinavia marginata occurs in Florida, the West Indies, and the southwestern US south to northern South America (Rolston 1983).

Figure 11. 

K2P Neighbor-joining tree of COI for Psix group of genera. Bootstrap support values over 80 are labelled on the branches.

The two species of Psix that are now found in the Nearctic can be separated by sculptural differences on the frons: lateral to the central keel, the frons in Ps. striaticeps is rugulose (Figure 12) and in Ps. tunetanus it is smooth (Figure 13).

Figures 12–13. 

12 Psix striaticeps, female (FSCA 00095790), head, anterior view 13 Psix tunetanus, female (FSCA 00095802), head, anterior view. ck: central keel.

The twelve counties where we identified Ps. striaticeps span peninsular Florida from north to south and include localities on the Atlantic and Gulf coasts (Figure 14). The earliest record is from 2018 in Marion County, followed by a single specimen collected in a hemp plantation in Miami-Dade County in 2019. The broader distribution that we detected in 2021 demonstrates the importance of continuous monitoring programs, such as CAPS, and their value for detecting a range of insects. Given that Ps. striaticeps is widespread in Florida and has been in the state for at least three years, we consider this species to be well-established.

Johnson and Masner (1985) reported that Ps. striaticeps was collected year-round in Zimbabwe, suggesting that it is multivoltine and possibly polyphagous. Our data on Ps. striaticeps in Florida supports these ideas. Specimens were collected in yellow cylinder traps continuously from December 2020 to August 2021. The females of Ps. striaticeps in Figures 2–3 are presented at identical scale size, and the specimen in Figure 2 is 1.5 times as long as the specimen in Figure 3. This size variation is consistent with Ps. striaticeps parasitizing eggs of various stink bug species, which vary in the size of their eggs, directly affecting the size of the parasitoid (Medal and Smith 2015). In Florida, Ps. striaticeps has been reared from the eggs of Pi. guildinii, N. viridula, and C. marginata, indicating that it is at least oligophagous.

Figure 14. 

Distribution map indicating the counties (red) in Florida where Ps. striaticeps has been collected.

The establishment of Ps. striaticeps in Florida has implications for stink bug management and ecology. It offers the prospect of biological control of N. viridula, from which it has been recorded previously (Johnson and Masner 1985), but the nontarget effects of Ps. striaticeps are yet unknown. Johnson and Masner (1985) also reported Acrosternum acutum (Dallas, 1851) (Pentatomidae) and Clavigralla scutellaris (Westwood, 1842) (as Acanthomia brevirostris Stål, 1873) (Coreidae) as hosts, indicating that its host range includes at least two superfamilies of true bugs. It has yet to be determined if Ps. striaticeps can parasitize Hal. halys eggs, and if it does, there is the possibility of its use for biological control of this species, particularly because there may be a suitable match in the phenologies of these species. Halyomorpha halys typically has two generations per year in mild climates, but in warmer climates, such as Florida, the species can give rise to several more generations per year (Hoffman 1933; Kistner 2017). Part of this effect may be that warmer climates tend to be at lower latitudes and have less drastic changes in daylight hours, and Hal. halys nymphs have been found to develop faster with a 12L:12D photoperiod than 15L:9D (Musolin et al. 2019; McDougall et al. 2021). Another factor is the year-round availability of host plants. Sweet corn, grown throughout the winter, is one of the top grossing crops in Florida and is a known host of Hal. halys (Poplin 2013). Tomatoes also have two cropping seasons in Florida, providing a second, economically important host for H. halys (Sargent et al 2014; Zoebel et al. 2016). Because the data from this study and from Johnson and Masner (1985) suggest that Ps. striaticeps is multivoltine as well, it may be able to provide control throughout the year. The logical next step is to determine the host range of Ps. striaticeps. This includes determining if it can parasitize Hal. halys eggs, if it targets beneficial stink bugs such as Euthyrhynchus floridanus (L., 1767) and Podisus maculiventris (Say, 1831), and how it might impact other native species of true bugs.