First record of a phlebotomine sand fly (Diptera: Psychodidae) on the Dutch Caribbean island of Curaçao

23 INTRODUCTION Phlebotomine sand flies are small blood-feeding insects belonging to the family Psychodidae (subfamily Phlebotominae) and are widely distributed worldwide in tropical and subtropical regions, and to a lesser degree in temperate regions. Sand flies are opportunistic blood feeders that feed on various vertebrate hosts (Cotteaux-Lautard et al. 2016, Pérez-Cutillas et al. 2020). While the vast majority of species prefer to feed on mammalian hosts, other species, such as those from the Old World genus Sergentomyia, prefer reptiles (Maia and Depaquit 2016). A subset of sand fly species has been found to transmit human pathogens, such as the phlebovirus causing Toscana fever and the bacteria Bartonella baciliformis causing Carion’s disease, and zoonotic pathogens like Leishmania spp. (Costa and Souza 2018). Other sand fly species have been shown to ONLINE JOURNAL OF THE NATURAL HISTORY MUSEUM ROTTERDAM, WITH CONTRIBUTIONS ON ALL ASPECTS OF NATURAL HISTORY WWW.DEINSEA.NL First record of a phlebotomine sand fly (Diptera: Psychodidae) on the Dutch Caribbean island of Curaçao Jordy G. van der Beek1,2,3,4, Maarten J.J. Schrama1,3, Pasquale Ciliberti1, Pepijn J.J. Helleman1,3, Roel M. Wouters1,3,5, Francis Schaffner6 & Marieta A.H. Braks3,7 1 Naturalis Biodiversity Center, Leiden, The Netherlands 2 Pandemic and Disaster Preparedness Center, Delft, Rotterdam, The Netherlands 3 Institute for Environmental Sciences, Leiden University, Leiden, The Netherlands 4 Natural History Museum Rotterdam, Rotterdam, The Netherlands 5 Department of Ecology, Faculty of Science, Charles University, Prague, Czechia 6 Francis Schaffner Consultancy, Riehen, Switzerland 7 Centre for Zoonoses and Environmental Microbiology, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands ABSTRACT Phlebotomine sand flies are known vectors of pathogens important for public and veterinary health. They are well known to transmit protozoa parasites of the genus Leishmania. Despite their importance as vectors, sand fly distributions remain poorly understood, particularly in the Caribbean. We present the first observation of the sand fly Micropygomyia trinidadensis in Parke Hatun on Curaçao, an island of the Dutch Leeward Antilles. This is the first record of sand flies in the Dutch Caribbean. We discuss the ambiguities in biting behavior, as well as the potential role of this species in the transmission of Leishmania parasites. This finding expands the knowledge of sand fly occurrence in the Caribbean and offers insights for public and veterinary health risk assessments. Furthermore, this discovery underscores the need for continued sand fly monitoring to understand their distribution, ecology, and impact on local health. Keywords Phlebotomine sand flies, Dutch Caribbean, vector-borne diseases, public health, veterinary health Cite this article Van der Beek, J.G., Schrama, M.J.J., Ciliberti, P., Helleman, P.J.J., Wouters, R.M., Schaffner, F. & Braks, M.A.H. 2024 - First record of a phlebotomine sand fly (Diptera: Psychodidae) on the Dutch Caribbean island of Curaçao - Deinsea 22: 23 - 30 Submitted 9 October 2023 Revised 14 June 2024 Accepted 18 June 2024 Published 25 June 2024 Author for correspondence jordy[email protected] Editors of this paper Jelle W.F. Reumer Bram W. Langeveld Copyright 2024 Van der Beek, Schrama, Ciliberti, Helleman, Wouters, Schaffner & Braks Distributed under Creative Commons CC-BY 4.0 DEINSEA online ISSN 2468-8983 Van der Beek et al.: A phlebotomine sand fly from Curaçao DEINSEA 22: 23-30 [2024] 24 diseases have previously been documented on these islands (Schaffner & Braks 2016). In this article, we report the first observation of a single specimen of the sand fly species Micropygomyia (Sauromyia) trinidadensis (Newstead, 1922) on Curaçao, and discuss its medical and veterinary health relevance. This record is the first finding of phlebotomine sand flies in the Dutch Caribbean. METHODS From 13 November 2022 to 6 December 2022, a mosquito biodiversity survey was carried out on the Dutch Caribbean islands of Aruba, Curaçao, and Bonaire. The aim of the survey was to obtain an overview of disease vectors and design new identification tools. The survey was a joint enterprise of Dutch and French entomologists, public health officials, local public health agencies, vector control units, and local nature conservation agencies. The primary focus of the survey was the sampling of the local mosquito fauna (Diptera: Culicidae), but other disease vectors were also collected albeit with lower sampling effort, including biting midges (Diptera: Ceratopogonidae), kissing bugs (Hemiptera: Reduviidae), hard ticks (Arachnida: Ixodidae), and a single phlebotomine sand fly. The results of the sampling of mosquitoes and other disease vectors will be reported in separate articles. During the survey, CO2-baited BG-Pro® traps (Biogents, Regensburg, Germany) were used to trap mosquitoes at a total transmit a wide range of animal pathogens that are relevant to veterinary and wildlife health only (Jancarova et al. 2023). For example, some species biting reptiles can transmit specific pathogens, including Sauroleishmania, commonly known as “lizard Leishmania” (Sardaneta et al. 2018). The largest diversity of sand flies can be found in the Nearctic and Neotropics (Cecílio et al. 2022). While in the Caribbean region, hotspots of diversity have been reported from Trinidad, Tobago, and Cuba (Tikasingh 2011, Galati 2018), no sand flies have been reported from most other Caribbean islands (Rueda et al. 2015, Galati 2018). For these islands, it is difficult to assess whether the absence of records is the result of a gap in the distribution or simply the result of missing or insufficient research effort. This, and the gap in our knowledge of the natural history of this species group limits our understanding of the distribution of vectors of medical and (wildlife) veterinary importance. The Dutch Leeward Antilles, comprising the islands of Aruba, Bonaire, and Curaçao, is an example of a region with no previous reports on sand fly occurrence, despite their geographic position in the neotropical region near the above-mentioned sand fly diversity hotspots. The islands are located in the southern Caribbean Sea and are home to a diverse range of flora and fauna (Naturalis Biodiversity Centre 2017). Despite the omnipresence of vertebrate hosts, including humans, livestock, rodents and wild birds, as well as the presence of suitable habitats on these islands, no sand flies or sand fly-related Figure 1: Map depicting the trapping sites across the Dutch Leeward Antilles (Aruba, Curaçao, and Bonaire) during the mosquito biodiversity survey. Traps included CO2-baited BG-Pro traps (circles) and sticky traps (triangles). The location of the sand fly species Micropygomyia trinidadensis (Newstead, 1922) discovery site on Curaçao is marked by a red star. Van der Beek et al.: A phlebotomine sand fly from Curaçao DEINSEA 22: 23-30 [2024] 25 After habitus photographs were taken, we tried to mount the specimen in Euparal as described in Ciliberti (2019), the only difference being the clearing step. We cleared the specimen in a solution of cold KOH 10% for 24 hours at room temperature, instead of heating it in KOH 10% in a water bath for seven minutes. Unfortunately, the specimen was lost while transferring the specimen between the clearing and washing step, making morphological identification impossible. The identified sand fly was registered in the collection of Naturalis Biodiversity Center (Leiden, the Netherlands) under collection number RMNH.INS.1565650, and the sequence is stored in the Barcode of Life Data System (BOLD) under process ID CAMOZ007-23. RESULTS This expedition resulted in the collection of over 4,400 mosquitoes and over 1,000 biting midges. Within the 95 BG-Pro trap samples, only one sand fly was found (Fig. 2). The sticky traps did not result in the collection of additional specimens. Based on the GenBank BLAST results of the COI barcode, the specimen was identified as Mi. trinidadensis, with a similarity between 98.7% and 96.2% with the available fourteen Columbian sequences (Gutiérrez et al. 2014, Romero-Ricardo et al. 2016, Duque Granda et al. 2023, Posada-López et al. 2023) and one sequence from Panama (Azpurua et al. 2010). Our sequence fell within the cluster of available sequences (Supplementary Fig. S1). The closest available sequence of other species is Mi. (Sau.) peresi (Mangabeira, 1942) from Brazil, with 87.7% similarity (Pinto et al. 2015). Morphological confirmation was not possible due to the destruction of the specimen during slide mounting. The sand fly was retrieved (12.178083, -68.936944, 26 November 2022) from Parke Hatun (Fig. 3), a forest patch in the Seru Fortuna neighbourhood on the island of Curaçao. Parke Hatun is a relatively old forest patch with little understory and relatively tall trees. The patch is dominated by manchineel trees (‘manzaliña’ - Hippomane mancinella), along with only a few other trees in low numbers, such as Bahama strongbark (‘watakeli’ - Bourreria succulenta), seagrape (‘dreifi di laman’ - Coccoloba uvifera), and great morinda or Indian mulberry (‘noni’ - Morinda citrifolia). The trap was placed close to a 25m wide pond between a group of low manchineel trees next to a fallen tree, in an area where the forest floor was covered in a layer of dry litter consisting of leaves, twigs, and other organic matter (Figs. 3b,c). DISCUSSION AND CONCLUSION Our study provides the first record of sand flies in the Dutch Leeward Antilles, specifically the sand fly species Mi. trinidadensis. The relevance of this finding, particularly in the light of veterinary and public health, is further discussed below. The new location on Curaçao falls within the wide distribution range of this species, which extends from southern Mexico to southern Brazil (Young and Duran 1994), suggesting the of 95 locations (Aruba: 34, Curaçao: 33, and Bonaire: 28) (Fig. 1). The traps were set up for 24 h, with the exception of a few traps (n = 9) that remained in the field for 48 h. The traps were used as EVS-style traps and were hung on a tree branch within the vegetation, approximately 100 cm above the ground (Johnston et al. 2014). Carbon dioxide was produced by sugar fermentation using a mixture of 150 g beet sugar, 5 g active-dry yeast for fruit fermentation (Saccharomyces bayanus, SafSpirit™ FD-3), 0.5 g yeast nutrient salt, and 1200 ml tap water. The samples were stored in a freezer (-20°C) until identification. After identification, specimens were stored in 70% ethanol. In addition, sticky traps on A5 format white copy paper (148 × 210 mm) coated with castor oil were hung at two sites on Aruba and a single site on Bonaire (adapted from Cazan et al. 2021) (Fig. 1). This passive trapping method intercepts rather than attracts sand flies and is used to sample habitats showing high potential for sand fly breeding (i.e., rocks and stone walls with a good layer of old debris at the base and in cavities). Each site was equipped with 5-10 paper sheets that were hung close to the potential breeding sites and left in the field for two or three days equaling a sampling effort of 0.150.31 m2/night. Identification of the sand fly collection was performed by DNA barcoding of a 658 bp long fragment of the mitochondrial cytochrome oxidase I (COI) gene (Folmer et al. 1994). To extract DNA, three legs were removed from the sand fly, washed in ddH2O for 2x 5 min to remove any traces of ethanol, and then fully dried before being placed in 20 μl of QuickExtract DNA Extraction Solution for DNA extraction, following the manufacturer’s protocol. PCR amplification was performed in a 35 μl volume with 1x AceTaq® DNA buffer with Mg2+ (Vazyme Biotech Co. Ltd, Nanjing, China), 0.2 mM of each dNTP, 0.4 μM LCO1490/HCO2198 primer pair (Folmer et al. 1994), 1 U AceTaq® DNA polymerase, and 2 μl of DNA extract. The amplification protocol included an initial denaturation step for 4 min. at 95°C, followed by 35 cycles of each for 30 s denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, followed by a final extension for 7 min. at 72°C. The amplification products were checked on a 1% agarose gel and then sent for PCR purification and Sanger sequencing at Macrogen Europe BV (Amsterdam, the Netherlands). The generated forward and reverse reads were cleaned and aligned using Unipro UGENE software (version 41.0) (Okonechnikov et al. 2012). The resulting consensus sequence was blasted with a ‘blastn’ search against the GenBank database, employing default settings for identification (Altschul et al. 1990). The obtained results were then aligned with our newly acquired sequence using the MUSCLE algorithm (Edgar 2004), followed by the construction of an unrooted maximum likelihood phylogenetic tree utilizing PhyML (Guindon et al. 2010) with the default settings in Unipro UGENE. Visualization of the phylogenetic tree was performed using the online Iroki tool (Moore et al. 2020). Van der Beek et al.: A phlebotomine sand fly from Curaçao DEINSEA 22: 23-30 [2024] 26 species is native to Curaçao and potentially to the other Dutch Leeward Antilles. Although Parke Hatun is located close to an airport (approximately 1 km away), it is unlikely that the specimen was introduced by human-mediated transport. Sand flies are weak flyers, typically using hopping flights and dispersing only a few hundred meters (Service 2008). The short flight distance, combined with the specific habitat needs of many sand fly species, explains why the subfamily of phlebotomine sand flies lacks invasive species (ECDC 2017); these insects do not colonize distant areas through passive transport. The sampling effort during the expedition did not specifically target sand flies, except with the occasional setting of interception sticky papers. Due to the specific behaviour and specific trapping requirement, sand flies might therefore have been overlooked at certain trapping sites. More targeted research is needed to evaluate the distribution and abundance of sand flies on the island of Curaçao. The specimen collected in the trap was not blood fed. Literature from other areas within its range has been inconclusive regarding the blood-feeding preferences of Mi. trinidadensis. A preference for cold-blooded animals has been hypothesized based on the overlapping species of Leishmania flagellates between forest geckoes and Mi. trinidadensis in Panama (Christensen & Telford 1972). This was confirmed by bloodmeal analyses, in which at least 75% of the bloodmeals originated from reptiles (Tesh et al. 1971). In contrast, more recently blood-feeding of this species on mammals, including humans, was observed in urban environments in Venezuela (Scorza et al. 1979) and genetic bloodmeal analyses showed feeding on humans, dogs, and rodents in Brazil (Guimarães-e-Silva et al. 2017), anteaters in Columbia (Paternina et al. 2016), and chickens in Ecuador (Anaguano et al. 2015). Intraspecific preference for feeding on both reptiles as well as mammals is considered unusual for most hematophagous insects. In that context, Williams (1988) questioned whether the reptile feeding in Central America (Tesh et al. 1971, Christensen & Telford 1972) and the urban mammal feeding from Venezuela (Scorza et al. 1979) arise from the existence of two or more distinct species under the same name. This might have originated from the problematic taxonomic history of Mi. trinidadensis, resulting from misidentification of this species both in the present and the past, among others, with Mi. (Micropygomyia) cayennensis (Floch & Abonnenc, 1941), Lutzomyia (Tricholateralis) gomezi (Nitzulescu, 1931), and Lu. (Lutzomyia) longipalpis (Lutz & Neiva, 1912) (Williams 1988). It remains unclear whether the Mi. trinidadensis found on Curaçao is of the ‘mammalophilic type’ or the ‘herpetophilic type’. Although the vector competence of Mi. trinidadensis for Leishmania spp. is unclear, specimens, probably of the Figure 2: Photographs of the Micropygomyia trinidadensis (Newstead, 1922) sand fly specimen collected on Curaçao during the expedition: (A) Scales and hairs on dry specimen; (B) Close-up of thorax and head; (C) Habitus of wet specimen. Photograph courtesy Figure 2A: Jordy van der Beek and Figure 2B,C: Pasquale Ciliberti. Van der Beek et al.: A phlebotomine sand fly from Curaçao DEINSEA 22: 23-30 [2024] 27 mammalophilic type, have been found infected with unknown Leishmania promastigotes in Brazil (Gil et al. 2003, Lainson 2010, Guimarães-e-Silva et al. 2017) and Venezuela (Bonfante-Garrido et al. 1990). The Venezuelan sand fly promastigotes were indistinguishable from the promastigotes of Leishmania venezuelensis isolated from humans and produced a similar tumor-like inflammation in a hamster (Bonfante-Garrido et al. 1990). Given the ambiguities in the host preference of specimen called Mi. trinidadensis, the potential risk for the public and (wildlife) veterinary health caused by sand flies on Curaçao is unknown, and difficult to assess based on our findings. For a good indication, more information on the blood-feeding preference and its local distribution needs to be collected. So far, there have been no reports of human or animal leishmaniasis cases on the islands. For now, we assume that based on the absence of any reports of sand fly-borne diseases and the low number of sand flies that have been encountered, there is currently no reason to assume that there is a major risk for public or veterinary health. Overall, our findings indicate that further monitoring of sand flies is required to improve our knowledge on the geographical distribution of this species and its relevance to human, veterinary, and wildlife health. ACKNOWLEDGEMENTS First and foremost, we would like to express our gratitude to everyone who contributed to the mosquito trapping efforts on Aruba, Curaçao, and Bonaire. This includes the governmental vector control teams of the islands for their invaluable (logistical) support. In particular, we wish to extend our appreciation to Gisette Seferina for her crucial assistance on Curaçao. We would also like to acknowledge the significant contributions of Arjan Stroo and Jelle Davelez to our sampling efforts on the island of Bonaire. Our heartfelt appreciation to Carel de Haseth for his invaluable guidance in showing us around the island, in particular at the collection site Parke Hatun. His local expertise has greatly enriched our study. Finally, we also express our gratitude to Odette Doest from Dierenartsenpraktijk Doest, Curaçao, for sharing insights into sand fly related diseases in animals on the island. 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Van der Beek et al.: A phlebotomine sand fly from Curaçao DEINSEA 22: 23-30 [2024] 29 30 SUPPLEMENTARY MATERIAL Supplementary figure 1: Genetic maximum-likelihood tree illustrating the relationship of the collected Micropygomyia trinidadensis (Newstead, 1922) sand fly specimen COI barcode sequence with existing sequences from Genbank. The figure shows that the Curaçao specimen (in red) falls within a cluster of sequences identified as Mi. trinidadesis from multiple sources. The taxonomy has been updated according to Shimabukuro et al. (2017). (Jordy van der Beek) DEINSEA - the online open-access Journal of the Natural History Museum Rotterdam publishes contributions on all aspects of natural history editor-in-chief Jelle W.F. Reumer | editors Bram W. Langeveld & Cornelis W. 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