Trinidad and Tobago Tropical & Field Biology Study Abroad 2019 On the Toad Again: A Study of Cane Toad (Rhinella marina) Tick Infections in Northeastern Trinidad

Prepared for: Dr. Adrienne Brundage Dr. Kevin Conway Texas A&M University

Prepared by: Kaitlyn Skinner Texas A&M University, College Station, TX

Date: 11 June 2019

Abstract Hard ticks (Ixodidae) are significant vectors of human and animal pathogens throughout the Caribbean and are known to commonly infect the Cane Toad (Rhinella marina). The focus of this study is to analyze cane toad populations of northeastern Trinidad (Toco region) and determine tick species diversity, tick distribution on host body, and relationship between toad size (snout to vent length [mm]) and tick burden. Five genera and thirteen species of ticks were collected from thirty-nine specimen of Rhinella marina collected at 4 different locations. No significant correlation was found between toad size and tick burden. Ticks were only commonly found attached to the ventral surface of Rhinella marina, especially the throat region, and may exhibit preference in the site at which they attach to this host. This is thought to be due to the Cane toad’s preferred environment of forest edge as well as being commonly submersed in water.

Introduction The Cane Toad (Rhinella marina), has an extensive native range in Central and South America (Brown, 2006), including Trinidad and Tobago (Burgon, 2012). The Cane toad’s most prominent feature is a pair of large parotid glands behind their eyes containing bufotoxin, which is involved in defense against predators. This toxin can irritate domestic animals as well as humans, especially if bufotoxin comes into direct contact with the eyes or any mucus membranes (Brant, 2009). The highest density of Cane toads is recognized to be in close proximity to human habitation, and therefore these amphibians are recognized for their ability to thrive in urban environments (Brant, 2009). Roads are thought to facilitate rapid dispersal of the Cane Toad, allowing them to infiltrate more rapidly into previously unoccupied areas (Brown, 2006). This resilience in relation to human habitation is pertinent, as tick infestations are common on cane toads throughout their native range (Lampo, 1996). Hard ticks (Ixodidae) in particular, are important vectors of human and animal pathogens found in temperate climates, including Lyme borrelioses, the most prominent among tick-borne diseases (Heylen, 2019). Tick-borne pathogens are believed to be the culprit of more than 100,000 human illnesses throughout the world and are second to mosquitoes as vectors of human diseases (Fuente, 2008). The most important disease transmitting ticks in the Caribbean islands are Amblyomma variegatum (vector of cowdriosis and associated with acute dermatophilosis), Amblyomma cajennense (a potential vector of cowdriosis), and Riphicephalus (Boophilis) microplus (a vector of babesiosis and anaplasmosis) (Basu, 2017). A recent study by Burgon (2012) reported two species of ticks as ectoparasites of Rhinella marina in northern Trinidad: Amblyomma dissimile and Amblyomma rotundatum. Once ticks attach to their host, they become a more significantly harmful parasite than perceived by the naked eye. Ticks remain attached to their hosts for significant periods of time (i.e., weeks or months) to endure conventional transit periods from one area to another (Kelehear, 2017). This intense period of infestation causes detrimental effects to the host on top of potential disease transmission, including large wounds, potential infection, and discomfort. Three physical factors may influence infestation: the width of a host; the area of ventral surface, which occasionally touches the ground or vegetation; and the amount of body surface available for parasitism (Mohr, 1961). The current study analyzes such data concerning the parasite toll of Rhinella marina, including tick species diversity, tick distribution on host body, and relationship between toad size and tick burden, within northeasten Trinidad (Toco region).

Methods

Sample sites Four sample sites were used in this study: 1. Jammev Guest House: Well-lit area surrounded by maintained vegetation and bordering roadway (10.8264 ͦ N, -61.9328 ͦ W). 2. NE roadside: This site is northeast of Jammev. The roadway is enclosed on both sides by maintained private property with light vegetation (10.8314 ͦ N, -60.9291 ͦ W). 3. SE roadside: This site is southeast of Jammev. On one side of the road is a river surrounded by a mixture of maintained and dense vegetation, and on the other side is staggered maintained vegetation and private property (10.8233 ͦ N, -60.9350 ͦ W). 4. Beachside estuary: Stagnant stream ~100 m from the northeastern coastline of Trinidad. This area is still within a modified and maintained area surrounded by shops and near a main roadway (10.8346 ͦ N, -60.9219 ͦ W).

Toad collection and inspection for ticks During May 27 – June 3, 2019, I inspected Cane toads for tick infestations in northeastern Trinidad (Toco region). Cane Toads were collected randomly at night from four different locations and visually inspected for ticks. Thirty-nine toads were examined across the region collectively. Photographs of the dorsal and ventral surface of each individual toad were taken to maintain a record for later reference (Fig. 1). Each toad was assigned a number and snout to vent length (mm) was taken using a digital caliper. Ticks were removed with tweezers and immediately placed into vials of 75% alcohol for later identification. Tick scarring was also noted for later investigation (Fig. 1b). Ticks were identified to the lowest taxonomic level using information available from Basu (2017) and Brundage (2017).

Fig. 1 – (a) Ambylomma ovale (red circle) attached to inner thigh of Rhinella marina. (b) tick scarring on throat of Rhinella marina.

Tick distribution Using photographs taken during tick collection, a distribution body map was created as toads were captured across the study period. The final diagram is comprised of all 39 toads and collectively displays ticks harvested from all locations.

Results

Sample Size Thirty-nine toads were collected from all sites: Jammev Guest House – 19 toads; NE roadside – 10 toads; SE roadside – 4 toads; Beachside – 6 toads. 15 toads (38.462%) were infected with ticks and 24 toads (61.538%) were found clean of ticks. A total of 55 ticks were collected from the 15 infected toads representing 5 genera and 13 species: Amblyomma cajennense (4 individuals), Ambyomma calcaratum (3 individuals), Amblyomma dissimile (2 individuals), Amblyomma ovale (9 individuals), Haemaphysalis juxtakochi (9 individuals), Hyalomma spp. (4 individuals), Ixodes downsi (5 individuals), Ixodes luciae (5 individuals), Ixodes spp. (2 individuals), Rhiphicephalus annulatus (1 individuals) , Rhiphicephalus microplus (5 individuals), Rhiphicephalus sanguineus (1 individuals), Rhiphicephalus spp. (2 individuals). Two specimen were unable to be identified. The average tick burden was 1.41 ticks/toad. When one outlier (a juvenile individual with 28 ticks), the tick burden lowered to 0.69 ticks/toad.

Relationship between size and tick burden No significant difference was observed between tick burden of large toads and small toads (Fig. 2). The data shows a slight negative correlation in both graphs, but the R^2 value is weak (0.1168 with outlier and .0068 without). The toads with zero ticks were seen displayed across the collected population. The largest tick load (28 ticks) was carried by the smallest of all toads gathered (75.4 mm), but this outlier does not influence the remaining data.

Fig. 2 – Scatterplot displaying the relation between tick size (snout to vent length (mm)) and tick burden with and without the outlier (a juvenile individual with 28 ticks). There was found to be a slight negative trend in both graphs.

Tick Distribution Figure 3 summarizes the distribution of ticks across the surface of the bodies of the 15 infected toads, 41.8% of the ticks collected (23 individuals) were found on the dorsal surface of the body and 58.2% (32 individuals) were found on the ventral surface of the body. The throat/neck region was the most heavily infested area of the body (17 ticks, 30.9% of all ticks sampled), followed by the head region (12 ticks, 21.8%), the back (11 ticks, 20%), the auxiliary region (7 ticks 12.7%), the stomach (6 ticks, 10.9%) and leg/groin region (2 ticks, 3.6%).

- Tick - Tick Scarring

Fig. 3 – Tick distribution displayed on the dorsal and ventral side of Cane Toad (Rhinella marina). Image from https://commons.wikimedia.org/wiki/File:Litoria_aurea.jpg

Discussion

Effect of toad size and tick burden The relationship between toad size (snout to vent length) was found to be non-substantial and more based on random acquisition. Although the couple higher tick burden cases were found to be on the smaller length toads, overall the statistics do not support a dominant trend, as toads with no ticks were seen displayed evenly throughout the studied population. The graph including the outlier (a juvenile individual with 28 ticks) resulted in a stronger negative correlation then compared to the graph excluding it. Both graphs, with and without the outlier, showed a slight negative trend, but neither is statistically significant to prove a direct correlation. The outlier was an interesting find, for on top of being the smallest individual at 75.4 mm it was also the most significantly infected. Overall though, this individual does not follow the remaining specimens in average tick burden who appeared more sporadic. This result mirrors that of Burgon et al. (2012) who found no significant correlation between toad size and tick abundance based on toads from northern Trinidad. This data also refutes the study done by Mohr (1961), concerning the amount of body surface available has a direct relationship with parasite load. The tick burden in relation to the toad size overall had a slight negative correlation, but which future research may prove more distinct.

Tick Distribution on host body Tick distribution was discovered to be dependent on body region. Overall, the ventral surface was found to be more desirable for tick attachment (58.18%) vs. the dorsal side (41.82%). On top of that, the throat and head region was found to be highly desirable for attachment of ticks, occupying 17 ticks (30.9%). These results are similar to Burgon et al. (2012) in which the majority of tick were found on the throat and head region as well as the dorsum. This tick distribution can be related to the fact that toads bottom ventral surface comes in contact with the ground more often and therefore has more resistance and friction that can deter ticks from attaching. Therefore, ticks tend to prefer areas of least resistance and detrimental removal. Another reasoning for this distribution could be that of the environment in which Rhinella marina is found within northeastern Trinidad (Toco region). A study conducted by Tack et al. (2012) determined that tick abundance increased in the case of forest edge as well as shrub cover. An alternative explanation may be toad behavior. Cane toads tend to be found near water sources and therefore spend some time submerged. A study conducted by Oliver et al. (1993) found that toads who became submerged would see tick distribution more heavily in non- submerged areas. The ventral surface overall provides the least probability of host interaction, but with wet conditions surrounding the study locations, it would be plausible that the dorsal surface would also be a dominant choice for tick attachment. Although the ventral side was more favorable overall, the dorsal side was still considerably close behind in attachment sites with only a 16.36% difference. Both desire for little interaction as well as host behavior would justify the highly favorable throat region.

Sample Size A recent study of ticks in northeastern Trinidad by Burgon (2012) reported two species of ticks as ectoparasites of Rhinella marina: Amblyomma dissimile and Amblyomma rotundatum. This study found in total 5 genera and 13 species; a significantly higher species list than previously found in northeastern Trinidad. All species found were noted to be in Trinidad and Tobago by Basu (2017). On top of Amblyomma dissimile and Amblyomma rotundatum, species also found were Amblyomma cajennense, Ambyomma calcaratum, Amblyomma ovale, Haemaphysalis juxtakochi, Hyalomma spp., Ixodes downsi, Ixodes luciae, Ixodes spp., Rhiphicephalus annulatus, Rhiphicephalus microplus, Rhiphicephalus sanguineus, Rhiphicephalus spp. This important compilation of species found on Rhinella marina is significant in acquiring more reliable tick host information for future research. These tick species are vectors for a variety of diseases that affect both animals and humans and can have increased risks in more tropical areas as well as with increased global temperatures (Suss, 2008).

Conclusion Overall, tick distribution was noted more the ventral surface than the dorsal surface of Rhinella marina. The throat region especially saw the highest accumulation of ticks (30.9%). This result is thought to be the results of tick preference for little host disturbance combined with host behavior in relation to water submergence. No significant relationship was observed between toad size (snout to vent length) and tick burden. Only a slight trend was seen in analysis but was not statistically significant enough to prove a direct correlation. Future research is needed in larger quantity in order to investigate this correspondence. These results suggest that ticks, although thought to be found on larger hosts, are seen abundant throughout the Rhinella marina population of northeastern Trinidad (Toco region). These ticks and their known diseases should therefore be considered with human interaction within the surrounding area.

References

Basu, Asoke & Charles, Roxanne. Ticks in the Caribbean Region. 2017.

Brundage, Adrienne. ENTO 423 Medical Entomology Lab Manual. Texas A&M University. 2017.

Brown, Gregory P., et al. Toad on the Road: Use of Roads as Dispersal Corridors by Cane Toads (Bufo Marinus) at an Invasion Front in Tropical Australia. 1st ed., vol. 133, Biological Conservation, 2006, pp. 88–94.

Burgon, J. D., et al. An Investigation into the Amblyomma Tick (Acari: Ixodidae) Infections of the Cane Toad (Rhinella Marina) at Four Sites in Northern Trinidad. Living World, Journal of The Trinidad and Tobago Field Naturalists’ Club, 2012, pp. 60–66.

Fuente, Jose de la, et al. Overview: Ticks as Vectors of Pathogens That Cause Disease in Humans and Animals. Frontiers in Bioscience, 2008, pp. 6938–6946.

Heylen, D., et al. Ticks and Tick-Borne Diseases in the City: Role of Landscape Connectivity and Green Space Characteristics in a Metropolitan Area. vol. 670, Science of The Total Environment, 2019, pp. 941–949.

Kelehear, Crystal, et al. First Report of Exotic Ticks (Amblyomma Rotundatum) Parasitizing Invasive Cane Toads (Rhinella Marina) on the Island of Hawai‘i. 2nd ed., vol. 8, Ticks and Tick-Borne Diseases, 2017, pp. 201–334.

Lampo, M., and P. Bayliss. Density Estimates of Cane Toads from Native Populations Based on Mark-Recapture Data. 3rd ed., vol. 23, Wildlife Research, 1996.

Mohr, Carl O. Relation of Ectoparasite Load to Host Size and Standard Range . 6th ed., vol. 47, The Journal of Parasitology, 1961, pp. 978–984.

Oliver, James H., et al. Establishment of the Foreign Parthenogenetic Tick Amblyomma Rotundatum (Acari: Ixodidae) in Florida. 5th ed., vol. 79, The Journal of Parasitology, 1993.

Suss, Jochen, and Christine Klaus. What Makes Ticks Tick? Climate Change, Ticks, and Tick‐ Borne Diseases. 1st ed., vol. 15, Journal of Travel Medicine, 2008.

Tack, Wesley, et al. Local Habitat and Landscape Affect Ixodes Ricinus Tick Abundances in Forests on Poor, Sandy Soils. vol. 265, Forest Ecology and Management, 2012, pp. 30–36.