Abstract
In 2025, a swarm of giant honey bees, Apis dorsata dorsata, was detected on board a cargo vessel prior to arrival at the port of Elizabeth, New Jersey, USA. Apis dorsata is a quarantine species not native to the United States, and further inspection of the bees uncovered 28 mites that were identified as Tropilaelaps mercedesae by morphological and DNA barcoding analysis. Tropilaelaps mercedesae is an ectoparasite of honey bees that is not yet present in the USA. An additional mite species, Kuzinia morsei, was also observed. Molecular screening of common honey bee pathogens in 42 intercepted bees showed a 5% prevalence of trypanosomatid infections and a 2% prevalence of American foulbrood. Additionally, Black queen cell virus (BQCV) and Deformed wing virus B (DWV-B) were detected in 38% and 7% of the samples, respectively. To our knowledge, this is the first detection of T. mercedesae and K. morsei on a vessel bound for the USA, as well as the first interception of a whole swarm of A. dorsata dorsata. Phylogenetic analysis of the CO1 sequence data indicated that the intercepted bee swarm originated from a southern India lineage. Our findings indicate that exotic bee swarms can harbor parasitic mites and multiple bee pathogens, both critical to the apiculture industry. Additionally, our findings reveal that Tropilaelaps mites can survive extended periods associated with adult bees and in the absence of brood, suggesting greater potential for long-distance movement and introduction risk. This study provides new insights into Tropilaelaps dispersal and swarm-mediated pathogen movement, thereby improving our ability to predict establishment and outbreak risk crucial for countering invasive agricultural threats. The findings also emphasize that early detection, through coordinated port and shipboard surveillance, and strong interagency collaboration, is vital for reducing the risk of agricultural pest and pathogen introductions into the USA.
Introduction
The expansion of international apicultural trade, and global trade of commodities, have facilitated the intercontinental movement of honey bee pests and pathogens worldwide (). Among these, the ectoparasitic mites in the genus Tropilaelaps Delfinado & Baker 1961 are increasingly recognized as one of the most significant threats to honey bees in North America. Tropilaelaps spp. are native to Asia, initially found on dead honeybees and on field rats nesting near apiaries at Mataas Na Kahoy, Lipa, Philippines (). Laigo and Morse 1968 () stated that the giant honey bee Apis dorsata Fabricius is the primary host of Tropilaelaps clarae Delfinado & Baker, but these mites have also successfully infested managed Apis mellifera Linnaeus colonies, possibly due to interactions between A. dorsata and A. mellifera ().
Of the four described Tropilaelaps species, T. mercedesae Anderson & Morgan 2007, is considered the most widespread and destructive species to Apis mellifera, where it has only been found to feed on the larvae and pupae (, ). Infestation by this mite species results in brood mortality, malformations in the adult stage, and colony collapse. The impact on the colony is often more severe than that of the parasitic mite Varroa destructor Anderson & Trueman 2000, due to the shorter phoretic/quiescent phase on adult bees and faster reproductive rate of T. mercedesae (, ). Tropilaelaps mites not only inflict direct damage through feeding on bee larvae but can also vector honey bee viruses. Tropilaelaps mercedesae has been found to carry several devastating honey bee viruses, including deformed wing virus (DWV), black queen cell virus (BQCV), acute paralysis virus (ABPV) and sacbrood virus (SBV), with viral loads increasing significantly in infested colonies () (, ). Replication of deformed wing virus (DWV) has been demonstrated in T. mercedesae () and further studies have shown that mite feeding facilitates viral amplification in developing brood, with higher DWV and BQCV loads in A. mellifera larvae and pupae infested with T. mercedesae compared to mite-free controls (). Viral transmission by T. mercedesae is compounded by multiple feeding wounds compromising bee larval immunity and providing favorable conditions for other opportunistic pathogens like American foulbrood (Paenibacillus larvae) and Nosema species () to overwhelm the infested colonies.
The dispersal mechanisms of T. mercedesae remain poorly understood, especially for inter-colony transmission and survival during host migration periods. Recent research by Uzunov et al. () demonstrated that T. mercedesae can successfully transfer between A. mellifera colonies via natural swarming events. However, their study revealed limited survival capacity, with mites surviving only 4–6 days in the absence of brood when associated with A. mellifera, a non-native host species (). This short survival duration contrasts with the extended broodless periods experienced by T. mercedesae’ natural host, the giant honey bee (Apis dorsata), which spends several weeks in migratory cycles with no brood production (, ). This apparent mismatch between T. mercedesae survival capacity on non-native hosts and the bio-ecological requirements of the natural host migration patterns suggests that T. mercedesae may possess host-specific adaptations that allow its survival during extended broodless periods on native hosts. Understanding these survival mechanisms is critical for predicting T. mercedesae dispersal and developing effective management strategies.
While T. mercedesae is considered endemic to eastern Asia, it has been detected in South Korea, a region characterized by cold winters and a temperate climate (). More recently this species has expanded into new regions, with new populations reported in western Russia in 2021-2022, indicating the ability of this species to establish outside the known range and overwinter in temperate climates (). Additional detections of T. mercedesae were reported in Uzbekistan and Georgia in 2024 (, ).
Several other mite species have been observed associated with bees, among them Kuzinia spp (–). While little is known about Kuzinia morsei (), several Kuzinia species are known to be associated with bumble bees (Bombus spp.) and distributed across the Neartic, Paleartic, Neotropical and Oriental biogeographic realms (, ). These species are known to feed on pollen, honey, bumble bee cocoon material, nematodes, and fungi, but the effect on bees living with these mites is currently unknown (, ). The immature Kuzinia deutonymphs are known to disperse on adult bumble bees (). The co-occurrence of multiple mite species on A. dorsata dorsata raises important questions about potential mite-mite interactions, their impact on colony health, and whether different mites have evolved similar host-specific survival adaptations.
Herein, we report for the first time the interception of Apis dorsata dorsata and two mite species associated with the swarm, aboard a container vessel bound for the U.S. port of Newark-Elizabeth, New Jersey. The two mites were identified as adult T. mercedesae and phoretic hypopi of Kuzinia morsei (). The potential economic impact of introducing K. morsei to the USA is unknown. We also describe pathogen screening results from the intercepted bees, since the introduction of vector-borne diseases is also a key risk factor. These findings raise serious concerns about inadvertent introductions of bees through global trade and transport.
Materials and methods
Vessel route reconstruction and timeline analysis
The vessel route reconstruction was conducted using documented port arrival dates, vessel characteristics, standard commercial shipping practices and availability of the exact date, time and location of the swarm interception.
Swarm establishment scenarios were analyzed using vessel documentation that included port arrival dates and route reconstruction. Three possible swarm establishment periods were evaluated: Scenario A: Mundra, India (April 16 – June 25, 2025); Scenario B: Karachi, Pakistan (June 26 – July 4, 2025), and Scenario C: Salalah, Oman (July 6 – 8, 2025) (Figure 1). Duration of swarm establishment (and mite survival) was calculated from estimated establishment to swarm interception at the coordinates 06°04.3’N, 030°00.6’W on July 24, 2025.
Figure 1
Sample collection
A swarm bivouac of several hundred adult A. dorsata dorsata was detected by crew members of the container vessel in July 2025 (Figures 2A, B). Under the guidance of Customs and Border Protection and USDA-APHIS inspectors, the bees were contained by the crew in a plastic bag and frozen aboard the ship at -6 °C to prevent potential release at the port. The intercepted bees were all adults, and the swarm did not include brood or comb material. From this frozen swarm, a sample of about 70 bees was sent to the USDA-ARS’ Crop BioProtection Research Unit, at the National Center for Agricultural Utilization Research (Peoria, Illinois, USA), for initial processing and analysis. Bees were washed in 100% ethanol, and the resulting debris examined for mites using a Zeiss Stemi 508 stereoscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). This initial inspection identified a Tropilaelaps mite and 10 Kuzinia mites. The collected Tropilaelaps mites and a bee leg were sent to the USDA-APHIS’ Pest Identification Technology Laboratory (PITL) (Fort Collins, Colorado, USA), for molecular confirmation. Upon this initial discovery, the whole swarm containing hundreds of bees, was requested and inspected for further presence of Tropilaelaps and Kuzinia mites at the USDA-ARS’ Systematic Entomology Laboratory (SEL) and the Beltsville Bee Research Laboratory, Beltsville Agricultural Research Center (Beltsville, Maryland, USA). The SEL’s Electron and Confocal Microscopy Unit washed bees according to the methods described in Monfreda et al., 2007 () and prepared samples for variable pressure scanning electron microscopy. This further inspection of the frozen swarm samples yielded an additional 27 Tropilaelaps and 38 Kuzinia mites.
Figure 2
Morphological identification
Bee and mite species were confirmed morphologically using the species descriptions and taxonomic keys available in the literature (–), the online Lucid Keys () and following the Tropilaelaps genus description by Delfinado and Baker () and subsequent species-level descriptions by Anderson and Morgan (). Variable Pressure Scanning Electron Microscopy micrography was performed using an ultra-high-resolution Schottky Field-emission Hitachi SU-7000 microscope (Hitachi Ltd, Tokyo, Japan) at the USDA-ARS Systematic Entomology Laboratory and the Electron and Confocal Microscopy Unit to support species confirmation.
Molecular identification
From the specimens found in the swarm aboard the cargo vessel, one leg from one A. dorsata dorsata individual and a single whole Tropilaelaps specimen were sent to the USDA-APHIS Pest Identification Technology Laboratory (PITL) for molecular identification. DNA from both samples was extracted using a Lucigen MasterPure DNA extraction kit (LGC, Biosearch Technologies, Teddington, UK) following manufacturer’s instructions with modifications (). In the case of the A. dorsata leg, the sample was crushed with 2.3-mm zirconia/silica beads in a 1.5-ml microcentrifuge tube by agitation on high for 1 min in a mini-beadbeater (Biospec Products, Bartlesville, OK, USA). The Tropilaelaps specimen was sampled non-destructively. The whole mite was placed in a 1.5-ml microcentrifuge tube and allowed to incubate overnight in tissue and cell lysis solution, after which the mite was removed and preserved for further examination while the remaining solution was processed for DNA extraction. The CO1 DNA barcode region was amplified from both specimens using universal primers LCO1490 and HCO2198 (). The 50.0 µl PCR mixture utilized the TaKaRa Ex Taq HS polymerase kit (Takara Bio, Shiga, Japan) and included 37.75 µl water, 5.0 µl 10X Ex Taq buffer, 4.0 µl dNTPs, 200 nM of each forward and reverse primer, 0.25 µl Taq containing 1.25U, and 1.0 µl of template DNA (except for the negative control in which water was substituted) per reaction. The PCR thermocycling conditions were an initial denaturation step of 94 °C (3 min), 32 cycles of 94 °C (20 s)/50 °C (20 s)/72 °C (30 s), and a final extension step of 72 °C (5 min), a lid temperature of 105 °C was maintained through all steps. PCR was carried out on a Bio-Rad PTC Tempo Deepwell Thermal Cycler (Bior-Rad Laboratories Inc., Hercules, CA). PCR products were visualized on 1% agarose gels to ensure correct product length and successful amplification. The PCR products were Sanger sequenced using the same primers as those for PCR amplification on a SeqStudio instrument using the BigDye Terminator v3.1 kit (ThermoFisher, Waltham, MA, USA) following the manufacturer’s instructions. Forward and reverse reads were trimmed of low-quality calls at both ends and merged into single consensus barcode sequences using Geneious Prime 2025 (www.geneious.com). The resulting barcode sequences were searched against the GenBank database using default BLASTn settings to corroborate species ID (). Mitochondrial cytochrome c oxidase subunit 1 (CO1) sequences for each species from Genbank were downloaded separately and aligned using MAFFT (, ). Alignments were trimmed to the maximum overlap size to sample size ratio resulting in a 350bp aligned region for A. dorsata and a 286bp aligned region for T. mercedesae. Apis florea was used as the A. dorsata outgroup, and Varroa destructor was used for the T. mercedesae outgroup. Unique sequences from each species were aligned together with reference taxa. Neighbor-joining trees () were inferred from genetic distance matrices with 1,000 jackknife replicates, and trees were subsequently rooted using designated outgroup taxa to aid interpretation of the geographic origin of the intercepted samples.
To examine the origin of the Tropilaelaps mite, a haplotype network was constructed using the full CO1 sequence from the intercepted mite with the sequences used in Namin et al. () (indicated in Supplementary Table S1). The resulting 537bp alignment was used for a TCS statistical parsimony analysis () implemented in PopART v. 1.7 (Population Analysis with Reticulate Trees ();).
Additional whole genome sequencing was carried out at the USDA-APHIS Pest Identification Technology Laboratory for the single Tropilaelaps sample using Oxford Nanopore Technology long-read sequencing. Whole genome amplification was completed to increase the total mass of DNA available for sequencing using a Qiagen Repli-g Midi kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. Library preparation was completed using the ONT Ligation Sequencing Kit v14 (SQK-LSK114; Oxford Nanopore Technologies, Oxford, United Kingdom) with the NEB Next ONT Module enzymes (New England Biolabs Inc., Ipswich, MA) following manufacturer’s instructions with modifications to maximize DNA retention. The prepared library was sequenced on a GridION device for 72 hours using a MinION/GridION Flow Cell (FLO-MIN114; ONT). Sequencing data were basecalled live using the ONT Super Accuracy basecaller model. Post-sequencing, data were imported into Geneious Prime 2025. Mitochondrial and cytochrome P450 genes were pulled out using Minimap2 () using OR400173.1 and GCA_002081605.1 respectively as references. Successfully isolated reads were assembled using Flye v. 2.9.6 () set to assemble Oxford Nanopore Raw Reads with genome size varying by assembly target, and three polishing iterations.
Individual A. d. dorsata, Tropilaelaps and Kuzinia samples were also analyzed at the USDA-ARS Beltsville Agricultural Research Center Bee Research Laboratory, and at the APHIS-PPQ National Identification Services’ Molecular diagnostics Unit at the Smithsonian National Museum of Natural History (Washington DC, USA). Here, DNA was extracted from A. d. dorsata legs and whole Tropilaelaps and Kuzinia mites by placing each in 100µL of 5% Chelex-100 (Bio-Rad) in molecular-grade water. Samples were ground with a disposable pestle, and the resulting slurry was incubated at 60 °C for 10 min. The Chelex and cellular debris were pelleted by centrifugation, and the supernatant was subjected to Proteinase K digestion (0.6 mg/ml final concentration) for 60 minutes at 55°C followed by 15 minutes at 99 °C. The CO1 barcoding region was amplified from each specimen using the primers LCO1490 and HCO2198 () or Lep.F1 and Lep.R1 () in parallel. Two microliters of each extract along with 10-fold and 100-fold serial dilutions were used in separate 25 µl PCR reactions of 94°C x 2 min, followed by 35 cycles of 94°C x 30s, 45°C x 30s, 72°C x 2 min). were carried out for each template. Prominent bands from the LCO1490 and HCO2198 pairing, based on agarose gel electrophoresis, were sequenced using Sanger sequencing. Attempts at amplification for the Kuzinia mite failed for both primer pairs at all dilutions. Sequence-based identifications for Apis and Tropilaelaps were concordant with results obtained by the PITL laboratory.
All CO1 sequence data are available in Genbank under accessions (PX980322-PX980329) and long-read data is available as an SRA under BioProject (PRJNA1420927).
Pathogen screening
Molecular pathogen screening was conducted at the USDA-ARS’ Crop BioProtection Research Unit, National Center for Agricultural Utilization Research. DNA and RNA were extracted from 42 individual bees via the ALL-prep Qiagen kit (Qiagen, Hilden, Germany) with modifications. Bee samples were macerated individually in RLT+ buffer in a microcentrifuge tube containing two metal beads using a sterile plastic mortar and further macerated in Tissue-lyser II (ThermoFisher, Waltham, MA, USA) for four rounds of 30 seconds each. Quality and concentration of DNA and RNA were assessed via NanoDrop (ThermoFisher). RNA was normalized across samples and cDNA synthesis was generated using the QuantiTec Reverse Transcription Kit (Qiagen) using 0.35 µg of total RNA. qPCR assays were conducted using a SybrGreen reagent with 1 µl of template (DNA or cDNA) and primers targeting Vairimorpha spp. = Nosema spp. (N. ceranea and N. apis), trypanosomatids (C. mellificae, L. passim, C. expoeki, and C. bombi), the bacterial pathogen American foulbrood (Paenibacillus larvae), and eight viruses including: Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Chronic bee paralysis virus (CBPV), Deformed wing virus A (DWV-A), Deformed wing virus B (DWV-B), Israeli acute paralysis virus (IAPV), Lake Sinai virus (LSV), and Sacbrood virus (SBV). Further amplification was carried out by targeting bee reference genes (Actin and 28S rRNA) and microbial markers (16S rRNA and fungal ITS) to confirm nucleic acid presence and effective extraction. Primers used for pathogen screening were those from Milone and Tarpy 2021 (for targets: Vairimorpha spp., trypanosomatids, IAPV, DWV-A, DWV-B, ABPV, CBPV, LSV, Apis Actin, and Apis 28s rRNA) (), Martinez et al. (for Paenibacillus larvae) (), Cavigli et al. (for SBV) (), Iredale et al. (for BQCV) (), Nadkarni et al. (for bacterial load) (), and Bell et al. (for fungal load) ().
Results
Route reconstruction and timeline analysis
Route reconstruction revealed that the vessel arrived in Mundra, India on April 16, 2025, after which time it remained in port until June 25, 2025 at the latest. From there it transited through Karachi, Pakistan on June 26, 2025, and Salalah, Oman on July 6, 2025, and proceeded south southeast toward the Cape of Good Hope. After rounding the Cape a northwest route was plotted to cross the Atlantic Ocean toward Port Elizabeth, New Jersey. The swarm was discovered in the North Atlantic Ocean and intercepted while still 3,600 nautical miles (6,667 kilometers, at position 06°04.3’N, 030°00.6’W) from its final destination (see Figure 1), at which time it was reported to the US Customs officials. The cargo manifest indicated containerized shipments that included locomotive spare parts, clothing articles, hazardous materials, rice and wooden crates containing sandstone. No bulk cargo outside of a container was documented that would provide suitable habitat for the establishment or survival of a bee swarm during transit.
Swarm establishment scenario analysis indicated swarm establishment (and Tropilaelaps survival) of between 29 to 99 days (Scenario A: Mundra, India establishment), 20 to 28 days (Scenario B: Karachi, Pakistan establishment) and 16 to 18 days (Scenario C: Salalah, Oman establishment) (Figure 1A).
Honey bee host and mite identification
Bees were identified morphologically as Apis dorsata dorsata (Figure 2B) using published taxonomic keys and by comparison with museum specimens. The mite recovered from the initial bee washes was identified as Tropilaelaps mercedesae based on diagnostic morphology (Figures 3A, B), supported by electron microscopy (Figures 4A, B) and comparison to published descriptions (Delfinado & Baker 1961; Anderson & Morgan 2007). In addition to Tropilaelaps, in this initial examination, we also recovered 10 mites morphologically consistent with Kuzinia morsei deutonymphs based on morphological features (Figure 3C).
Figure 3
Figure 4
Through CO1 DNA barcoding, the intercepted bees were found to belong to a distinct clade of A. d. dorsata that is only known from southern India and may constitute a cryptic species or a distinct subspecies (). The CO1 sequence was a 100% match to a reference specimen from Karnataka, India (Figure 5). The T. mercedesae CO1 sequence showed 100% sequence identity to publicly available sequences reported from multiple countries in Asia, including South Korea, India, China, Thailand, Vietnam, and Nepal (Figure 6A). From the long-read sequence data, a full-length assembly of CO1 was generated as part of a full-length assembly of the mitochondrial genome. Read depth was too shallow to confidently resolve other genes of interest. The full length CO1 assembly was used to replicate the phylogeography analysis by Namin et al. () and more closely resolve the possible origin of the mite. In the resulting analysis, the intercepted mite shared the same haplotype, H20 (sensu Namin et al. ()), as a specimen collected from Bangalore, India (Figure 6B). This sequence is also highlighted in the Tropilaelaps neighbor-joining tree (Figure 6A).
Figure 5
Figure 6
Subsequent examinations of additional material at the USDA-ARS Systematic Entomology Laboratory recovered 27 additional Tropilaelaps and 38 Kuzinia mites from three sample sets. Thus, in total, the sample examinations yielded 28 T. mercedesae and 48 K. morsei mites. All Tropilaelaps samples were females, with no immature stages or males collected from the swarm. In the same manner, all Kuzinia samples were deutonymphs. These specimens were morphologically consistent with T. mercedesae and K. morsei (Figure 4A, B and 4C, D respectively). The CO1 barcoding confirmed the identities of Tropilaelaps and A. d. dorsata in the corresponding samples, but DNA barcodes from the Kuzinia immatures could not be amplified using the standard CO1 workflow. One additional collected mite was identified as an adult female spider mite Tetranychus sp. (Acariformes, Tetranychidae) with gut contents presenting a light green tone. Sample coloration and body shape across all samples (bees and mites) strongly indicate that these organisms were alive at the time of collection, with vivid body coloration and flexible, turgid bodies rather than the deflated or sunken appearance typical of post-mortem specimens.
Pathogen screening
Our pathogen screening of A. d. dorsata did not detect any Nosema spp. but detected two individuals with trypanosomatid infection. The bacterial pathogen Paenibacillus larvae (causative agent of American Foulbrood) was detected in 2% of the bees evaluated. BQCV was detected in 38% of individuals, while DWV-B was found in 7% of the samples. All other RNA viruses tested were negative (Figure 7A). From the samples examined, 52.4% were uninfected (n=23), 42.9% (n=18) had a single infection, while 4.8% (n=2) were coinfections (Figure 7B), with one sample presenting trypanosomatid and BQCV infections, and the other coinfected with BQCV and DWV-b viruses. Reference genes amplified consistently at qPCR cycle threshold (Ct) Ct=10 (Apis 28s rRNA) and Ct=27 (actin) (), while pathogens showed higher Ct values (>Ct=30). Given that the intercepted samples have not been maintained under optimal storage conditions for RNA preservation, and that suboptimal sample storage significantly affects viral detection sensitivity in bee samples (), the prevalence estimates should be considered as lower-bound estimates. The higher pathogen Ct values may reflect low viral loads, RNA degradation, or both, potentially leading to underestimation of true pathogen prevalence. All pathogens detected during our molecular screening are already present in the United States and are not considered quarantine pathogens; however, they are of significant concern for honey bee health, as several (i.e. Paenibacillus larvae (American foulbrood) and Deformed wing virus) are strongly associated with colony morbidity and collapse.
Figure 7
Discussion
To our knowledge, this interception represents the first detection of Apis d. dorsata as well as Tropilaelaps mercedesae and Kuzinia morsei on cargo inbound to the USA. Morphological and molecular evaluation from multiple laboratories corroborated that the Tropilaelaps specimen is consistent with descriptions and DNA barcodes for Tropilaelaps mercedesae (, ). The A. d. dorsata specimens were identified as such by comparing their CO1 sequence with published barcodes and were morphologically congruent with published descriptions and museum specimens of A. d. dorsata. The A. d. dorsata subspecies was resolved in a distinct clade (with 100% JK support) within A. d. dorsata; found only in southern India and morphologically indistinguishable from A. d. dorsata ( (); Figure 5). These results place the intercepted swarm within a southern India lineage, based on the resolution of these individuals in the NJ tree. This is relevant because southern India is a region where Tropilaelaps mercedesae has been reported, and it provides a plausible source for the mites recovered from the swarm. Our phylogenetic analysis conducted on the intercepted Tropilaelaps mites, places it within a well-supported clade, also, alongside sequences of Indian origin, including a reference from Bangalore. The haplotype network analysis (albeit limited by available sequences) corroborated this placement, with the intercepted sample (H20) falling within an India-associated haplotype, further supporting an Indian geographic origin for this interception. The finding is particularly significant given the history of invasions of Tropilaelaps into new regions, which have repeatedly followed host movements into new areas. The successful introduction of T. mercedesae is predicted to be a major threat to U. S. apiculture because it has become a serious pest of honey bees both where it is endemic and where it is introduced (, ).
The threat posed to honey bees by Tropilaelaps is substantial and derives from their distinctive biology and epidemiology. These mites reproduce faster than Varroa destructor due to their short lifecycle and ability to feed on both pre- and post-capped brood (, –). Experimental studies have shown that infestations of Tropilaelaps lead to multiple feeding wounds per larva or pupa, resulting from repeated penetration of the bee cuticle by the mite’s sharp chelicerae (, ). This extensive tissue damage leads to weight loss, deformities in the adult bee, and increased mortality (). In addition to the direct feeding damage that Tropilaelaps mites inflict on bees, they are also competent vectors of DWV, with active replication confirmed within the mites (, ). Dissemination of the virus through feeding facilitates viral proliferation in brood, with pupae infested with Tropilaelaps exhibiting higher DWV and BQCV loads compared to Tropilaelaps-free controls ().
Our pathogen screen of intercepted bees found no microsporidia but revealed BQCV infection in about a third of the bees tested. This aligns with the virus’ association with mite feeding wounds (). Our pathogen survey analysis must be interpreted within the context of sample storage limitations. The prevalence estimates we observed (38% BQCV, 7% DWV-B) likely reflect the suboptimal sample preservation following the interception and during transport to the lab (at -6°C rather than at -80°C). Recent studies have demonstrated that storage conditions can significantly impact viral RNA detection in bee samples (). Despite these limitations, our findings add to the limited research on pathogen associations with A. d. dorsata bees. The detection of multiple pathogens, even under suboptimal preservation conditions, demonstrates that multiple regulated bee pathogens circulate in wild giant honey bee populations.
Sample coloration, turgidity and body positioning strongly indicates that all collected organisms (bees and mites) were alive at the time of collection. The presence of Tropilaelaps mites in a broodless A. d. dorsata swarm that stayed aboard a ship for several weeks at sea is biologically and epidemiologically significant, demonstrating that adult Tropilaelaps mites can survive extended periods associated with adult bees and without access to brood. Although Tropilaelaps mites are considered to be highly dependent on brood, and do not exhibit prolonged phoretic behavior compared to Varroa destructor, previous studies have shown that they can remain alive on adult bees for up to six days in conditions devoid of brood (–).
Route reconstruction and vessel timeline analysis revealed three potential establishment scenarios where the A. d. dorsata swarm might have established on the vessel. Thus, the period of association of the broodless swarm (along with Tropilaelaps and Kuzinia mites) with the vessel varies significantly depending on the establishment timing and location, ranging from 29 to 99 days (Scenario A: Mundra, India), 20 to 28 days (Scenario B: Karachi, Pakistan) and 16 to 18 days (Scenario C: Salalah, Oman). The molecular analysis of both the A. d. dorsata bees and Tropilaelaps mites confirmed an Indian subcontinent geographic origin for the swarm, which combined with the documented extended vessel operations at Mundra Port (70 days), provides strong support for swarm establishment at Mundra (India). The scenario B (Karachi, Pakistan) swarm establishment is the most conservative and remains biologically plausible (based on geography and length of time on the vessel). However, this scenario lacks the molecular support for Pakistani geographic origin, and it has a shorter transit stop compared to the longer Mundra operations period; which provides an ample opportunity to establish aboard the vessel with possible access to land-based resources to forage and only adds, at a minimum, one day to the survival window over the Karachi scenario. The third scenario, establishment at the Salalah Port (Oman, Scenario C), is highly unlikely, given that there are no published records of A. d. dorsata in Oman (), and it could only occur if the swarm moved from a different vessel during harbor operations at this location.
Although a Mundra establishment appears to be extreme for a broodless swarm to survive, Robinson (, ) found that the time bivouacs (migrating swarms) of A. d. dorsata stayed at a particular location, ranged widely from two hours to more than 57 days on their migration route. The remarkable capabilities for migration of this species, which include seasonal migrations of up to 200 km and the ability to fly 1.9 km to forage, coupled with its adaptation to build open nests suspended from elevated structures and survive exposure to sun, wind and rain (, , , ), make this scenario entirely plausible.
The observation that Tropilaelaps mites from this interception were surviving at least 29 days without access to brood when associated with its natural host A. d. dorsata, is a significantly longer survival duration compared to the 6-day maximum reported for A. mellifera by Uzunov et al. (). This extended survival period aligns well with A. d. dorsata’s migratory ecology, in which seasonal migrations last several weeks during which colonies experience prolonged broodless phases (, , ). These data are important because it demonstrates the resilience of migrating A. d. dorsata swarms when there are no adequate conditions for a permanent nest (such as during oceanic crossings), and their potential capacity for foraging for resources when the vessel was stationed at ports (India, Pakistan and Oman), enabling survival for extended periods without stored resources in the absence of comb. This survival capacity also suggests the existence of host-specific adaptations by Tropilaelaps, that may have co-evolved with the natural migratory behavior of A. d. dorsata. These potential adaptations would likely include physiological tolerance to starvation and stress, modified host-seeking behavior, or exploitation of alternative nutritional sources available on the natural host but absent in A. mellifera (). Intact swarms might also provide dense adult-host contact that facilitates mite survival during broodless swarming periods, thus serving as a viable transport for Tropilaelaps mites, and increasing the potential for establishment if brood hosts were to become available soon after arrival (, ).
Thus, the extended survival capacity on native hosts has significant implications for understanding the invasion and dispersal potential of T. mercedesae. In contrast to the limited dispersal, due to short survival periods on A. mellifera (), the 3–4 week survival on its native host A. d. dorsata would facilitate long-distance dispersal during migration cycles. This could explain the historical distribution patterns of T. mercedesae across Southeast Asia (, ). These findings would also suggest that successful T. mercedesae establishment is dependent not only on suitable climatic conditions but also on associations with the specific host ().
Little is known about Kuzinia morsei, but they were encountered in larger numbers than Tropilaelaps, with more than 40 individuals from alcohol washes of the intercepted A. d. dorsata. A Kuzinia mite was found still attached near the rastellum and another mite located on the thorax of A. d. dorsata. Our records show that this is the first documented interception of K. morsei in a vessel bound for the United States. The original description of Kuzinia morsei is from samples collected by Roger Morsei in the Philippines in 1968, and its association with A. d. dorsata was not recognized until its description 22 years later in 1990 (). It took another 26 years, from the presently described interception, for this association to be corroborated. Given that the likely origin of the intercepted materials is India, we predict that these mites could occur in other parts of Asia. The economic impact of Kuzinia species is unknown, and they could not be identified with CO1 DNA barcodes as PCR failed to produce amplicons from these samples. It should be noted that Kuzinia morsei is currently under taxonomic revision and the resulting resolution may help to better understand the importance of this species. The presence of a Tetranychus spider mite among the samples is likely accidental but given that the material gut contents were still green, it is indicative of some recent contact with plant material by the intercepted bees. The extended survival we observed in T. mercedesae may not be unique among other A. d. dorsata ectoparasites, as Kuzinia morsei likely faces similar selective pressures during migration periods () but currently little is known about its biology or ecological role.
Apis dorsata dorsata is typically associated with forested habitats, where colonies nest openly on tall trees or cliffs in tropical and subtropical regions of South and Southeast Asia (, , ). Our observation and previous reports indicate that swarming or absconding by A. d. dorsata can include highly modified or urban-adjacent environments (60, 61). From a biosecurity perspective, the ability of A. d. dorsata to occupy modified landscapes and to move over long distances during foraging or migration cycles, may increase the likelihood of contact with human-mediated transport pathways, including vessels and associated port environs.
The results found here support previous remarks by Smith-Pardo et al. (62) about the importance of vigilance at ports of entry to prevent the introduction of non-native invasive species of honey bees (genus Apis) and their parasites and potential diseases into the USA. Social insects are particularly serious because they can survive for long periods of time in adverse conditions and because a single queen or swarm can found a colonial population in a short period of time. In the case of giant honey bees, this is particularly problematic as they are close relatives to the only species present in the USA, Apis mellifera (also introduced but naturalized and exploited commercially) and can compete with this species or serve as hosts of parasites that can easily jump from one species to another. In addition, recent reports of T. mercedesae establishment in Europe (), further highlight the species ability to adapt to non-native habitats.
The interception described here resulted in the immediate destruction of the foreign swarm, preventing the potential establishment of Tropilaelaps in the USA and demonstrating that invasive bee pests can arrive as hitchhikers with cargo. Collectively, this interception underscores how exotic species of honey bees can serve as vehicles for the transport of parasitic mites and associated pathogens that threaten domestic beekeeping and agriculture. These findings suggest that stowaway pests may be transported on inbound vessels regardless of declared cargo type, and that coordinated port and shipboard surveillance, together with communication with shipping companies, can strengthen early detection and risk awareness.
Conclusions
Molecular phylogenetic analysis of the intercepted bees and mites confirmed their placement within a distinct clade of A. d. dorsata and Tropilaelaps mite endemic to India, establishing the port in Mundra (India) as the most probable origin of the swarm. Combined with the vessel’s travel timeline and official communications of the swarm interception, we estimate that the broodless swarm and mites survived aboard the ship between 29 to 99 days from establishment to interception, with 16 of those days at sea with no possibility for the bees to forage for resources. The observations described in this study highlight the remarkable adaptations of A. d. dorsata and its co-evolved mites, and fundamentally challenges previous assumptions about the survival limitations of A. d. dorsata bees and Tropilaelaps mites. The interception of A. d. dorsata carrying T. mercedesae mites aboard a vessel inbound to a USA port highlights the importance of proactive surveillance and diagnostic readiness. Effective policies and mitigation measures prevented the entry of T. mercedesae and the mite K. morsei in the USA. However, the co-occurrence of BQCV, DWV-B and P. larvae pathogens further indicates the multifaceted risks posed by exotic bee introductions. Our findings underscore the value of early detection and interagency coordination for protecting USA apiculture and agriculture.
Statements
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.
Ethics statement
The manuscript presents research on animals that do not require ethical approval for their study.
Author contributions
JR: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Validation, Visualization, Supervision, Writing – original draft, Writing – review & editing. LT: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing. FZ: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. AF: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing. TG: Conceptualization, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing. YC: Funding acquisition, Investigation, Resources, Supervision, Writing – original draft, Writing – review & editing. JE: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Supervision, Writing – original draft, Writing – review & editing. JM: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Writing – original draft, Writing – review & editing. AS-P: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing. RO: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This research was supported in part by the U.S. Department of Agriculture, Agricultural Research Service (Project Numbers: 5010-30400-001-000-D to JR and 8042-22000-313-000D to RO) and Animal and Plant Health Inspection Service (Project Number: 8042-30500-002–0231 to YC and JE).
Acknowledgments
We would like to thank U.S. Customs and Border Protection (CBP) at Port of Newark, U.S. Coast Guard of New York, the New Jersey State Entomologists, and Tiffany Mauro (USDA-APHIS PPQ Port Director) for coordinating incident response at the port and the sample collection from the vessel. We also thank the vessel captain, Capt. O.C.L. Amarasinghe and crew for providing photographs and documenting the mitigation measures taken on July 24, 2025 and to Dr. Jesse Hardin (APHIS-PPQ-NIS) for insightful comments to the manuscript and for assisting in the reconstruction of the vessel route and timeline. We would like to acknowledge Jacob McDowell and “CBP Agriculture Programs and Trade Liaison Process Resolution and Risk Management team” for assistance in obtaining ship tracking information that was fundamental for the reconstruction of the vessel route and timeline. We extend a special thank you to Dr. Barry OConnor (University of Michigan) for providing reference materials and assistance with the identification of Kuzinia morsei, to Haley M. Gore (USDA-ARS NCAUR CBP) for her invaluable assistance with pathogen diagnostics and for contributing to the development of the pathogen ID methodology, to Dr. Robert Kula (USDA-ARS) and Dawn Boncristiani (USDA-ARS BRL) for their help coordinating additional specimen identification; and to Dr. Andrew Brower for his careful review of the manuscript and for the helpful comments and suggestions. We also acknowledge the Smithsonian National Museum of Natural History (NMNH), the National Agricultural Library (NAL), and the USDA SEL for support and assistance with specimens, references and equipment. The authors also wish to express their sincere gratitude to the two journal reviewers for their constructive feedback and insightful comments, which significantly enhanced the quality of this manuscript.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors JR, JE, and RO declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/finsc.2026.1829350/full#supplementary-material.
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Summary
Keywords
black queen cell virus, deformed wing virus B, giant honey bee, honey bee mites, honey bee pathogens, interception, Tropilaelaps
Citation
Ramirez JL, Tembrock LR, Zink FA, Fife A, Gilligan TM, Chen Y, Evans JD, Mottern J, Smith-Pardo AH and Ochoa R (2026) Interception of an Apis dorsata swarm with Tropilaelaps mercedesae and Kuzinia morsei mites on a cargo vessel inbound to the United States. Front. Insect Sci. 6:1829350. doi: 10.3389/finsc.2026.1829350
Received
12 March 2026
Revised
13 April 2026
Accepted
17 April 2026
Published
08 May 2026
Volume
6 - 2026
Edited by
Mariana Bulgarella, Victoria University of Wellington, New Zealand
Reviewed by
Antoine Felden, Victoria University of Wellington, New Zealand
Rogan Tokach, North Dakota State University, United States
Updates
Copyright
© 2026 Ramirez, Tembrock, Zink, Fife, Gilligan, Chen, Evans, Mottern, Smith-Pardo and Ochoa.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Jose Luis Ramirez, jose.ramirez2@usda.gov; Allan H. Smith-Pardo, allan.h.smith-pardo@usda.gov; Ron Ochoa, ron.ochoa@usda.gov
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.