Ozolaimus megatyphlon and Ozolaimus cirratus parasitizing the Iguana iguana (Linnaeus, 1758) from Marajó Island, Pará, Brasil: new occurrence and morphological redescription

Abstract This study aimed to redescribe two species of Ozolaimus, parasites of free-living green iguanas native to Marajó Island. The gastrointestinal system of four iguana specimens was evaluated for the presence of helminths. Altogether, 12,028 nematodes were found, with a prevalence of 100%, an infection range of 780 to 7,736 nematodes, an infection intensity of 3.007, and a mean abundance of 3,007. Light microscopy and scanning electron microscopy were used to determine the species of nematodes found. The cecum was the site of infection that had the highest parasitic load. Morphologically, the nematodes were compatible with the genus Ozolaimus Dujardin, 1844, with the species Ozolaimus megatyphlon (Rudolphi, 1819) Dujardin, 1845, and Ozolaimus cirratus Linstow, 1906. Scanning electron microscopy showed the presence of small structures (serrated in Ozolaimus cirratus and rounded in Ozolaimus megatyphlon) located below the esophageal leaves. We also evidenced the phasmids in both species; this is the first record of these structures in nematodes of the genus Ozolaimus. In addition, this work expands the records on the geographic distribution of these parasites.


Introduction
Iguana iguana (Linnaeus, 1758), popularly known as the green iguana, belongs to the order Squamata (Lepidosauria: Reptilia), the most diversified among the group and one of the most important, which includes about 19 families and 4,500 species of lizards (Barten, 2006;Vitt & Caldwell, 2008). Iguana iguana is distributed throughout the Americas, including Brazil and occurs in the Amazon, Caatinga and Pantanal biomes (Campos & Desbiez, 2013).
Due to their low cost and ready availability, green iguanas have become one of the most popular unconventional pets in Brazil, making them economically important (Bauer & Bauer, 2014). Additionally, they are herbivorous, which many owners prefer, as they do not have to deal with providing insects or rodents for food (Barten, 1993).
These reptiles are hosts for a wide variety of parasites, which can be acquired by ingestion of contaminated plant material, coprophagy, geophagy, or active penetration by nematode larvae (Anderson et al., 2009). The authors Loukopoulos et al. (2007) and Breves et al. (2011) demonstrated that helminths of the Oxyurida order are commonly found parasitizing the gastrointestinal system of green iguanas.
Despite the growing number of studies related to host parasites of green iguanas in Brazil, most studies are concentrated in the Southeast, Midwest, and Northeast regions of the country, leaving gaps regarding helminth fauna in the northern region. The Amazon region still lacks information on helminths related to lizards. With that in mind, this study aimed to redescribe two species of the genus Ozolaimus of Iguana iguana from Marajó Island in the State of Pará.

Material and Methods
From 2019 to 2021, four free-living specimens of I. iguana were acquired dead from residents of the municipality of Soure (00º 43' 00" S; 48º 31' 24" W), Marajó Island, State of Pará. The research was carried out under authorization from Sisbio nº 68028. The organs of the digestive system, such as the esophagus, stomach, small intestine, and large intestine (colon, cecum, and rectum), were transported refrigerated to the laboratory. In the laboratory, each organ was isolated in plastic trays containing 0.9% NaCl saline and analyzed under a Leica ES2 stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany) to investigate the presence of helminths.
The collected nematodes were washed in 0.9% NaCl physiological solution, fixed in AFA solution (93 parts of 70% ethyl alcohol, 5 parts of formaldehyde, and 2 parts of glacial acetic acid), and stored in 70% alcohol. For light microscopy (LM), the nematodes were clarified in a 30% Lactophenol Aman solution and photographed in a Leica DM2500 microscope with a DFC310 FX digital capture system with Leica Application Suite V4.4 software (Leica Microsystems GmbH, Wetzlar, Germany). They were drawn and measured using a Leica DM2500 microscope with an imaging tube attached. The drawings were measured with the aid of a ruler, and the measurements were converted to micrometers or millimeters according to the Leica DM2500 (Leica Microsystems GmbH, Wetzlar, Germany) measurement conversion table. For morphometric analysis, 25 males and 20 females were used. After those procedures, the nematodes were stored in glycerin alcohol (70% ethanol with 5% glycerin). Measurements are given in millimeters, unless otherwise noted, and are presented as average values followed by minimum and maximum values in parentheses.
For scanning electron microscopy (SEM), the nematodes fixed in AFA solution were washed with distilled water, post-fixed in 1% osmium tetroxide for 2 hours, and then submitted to dehydration in an increasing series of ethanol from 70% ethanol until 100% for 1 hour in each battery of alcohol, subsequently subjected to the critical point of CO 2 model K850 Critical Point Dryer (Quorum Technologies Ltd., England), mounted on metallic aluminum supports (stubs), metallized with gold+palladium, and analyzed in a scanning electron microscope model VEGA 3 LMU (TESCAN, Brno, Czech Republic). Scientific articles and dichotomous keys were used to identify the species: Dosse (1942), Leussink (1958), Vicente et al. (1993), Anderson et al. (2009), andGibbons (2010).
Voucher specimens for the parasites were deposited in the Coleção Helmintológica do Instituto Oswaldo Cruz (CHIOC), Manguinhos, Rio de Janeiro, Brazil, as: CHIOC 39620 a-h for males and females of O. cirratus and CHIOC 39621 a-h for males and females of O. megatyphlon, correspondingly.

Results
A total of 12,028 nematodes were recovered from four specimens of I. iguana, with prevalence of 100% (n=4), mean intensity of infection of 3,007, mean abundance of 3,007, and range of infection of 780 to 7,736 nematodes. The parasites were found (mixed infection) in the small and large intestine (colon, cecum, and rectum).
The cecum was the site with the highest rate of infection. The specimens collected are morphologically compatible with the genus Ozolaimus Dujardin, 1844. In our study, we identified the species Ozolaimus megatyphlon (Rudolphi, 1819), Dujardin, 1845, and Ozolaimus cirratus, and their morphometries were compared to others already described in the literature for these two species (Tables 1 and 2). Family Pharyngodonidae Travassos, 1920 Genus Ozolaimus Dujardin, 1844 Specie Ozolaimus megatyphlon (Rudolphi, 1819) Dujardin, 1845 Ozolaimus cirratus Linstow, 1906  Medium-sized parasite, rounded body with cuticle completely striated transversely. Two lateral lips and a triangular oral opening, with small, rounded projections below the esophageal leaves. Esophagus long, thin, almost cylindrical, and divided into two portions: the first comprises the dilatation region, and the second goes to the bulb, the second portion being longer than the first. Bulb well developed; excretory pore immediately anterior or at the level of the bulb; nerve ring near the first portion of the esophagus. Males have a projection of six cuticular membranes from the esophageal segment. Spicule short and pointed. Genital cone present. Tail differentiated, short, and curved, containing a pair of precloacal papillae, a pair of postcloacal papillae, and a pair of caudal papillae. Females with vulva covered by a very prominent vulvar lip and a long uterus. Gravid females have ovoid, thin-shelled eggs and no embryonated.
Based on 25 male specimens: body 5.31 mm (4.71-6.28) length and 0.42 (0.33-0.61) wide at the bulb region. Distances from anterior end to nerve ring and excretory pore 0.26 (0.10-0.80) and 1.74 (1.08-2.14), respectively.     Ozolaimus cirratus (based on light microscopy and scanning electron microscopy. Figures 5 to 8; Table 2) Medium-sized parasite, rounded body with cuticle totally striated transversely. Dorsoventrally elongated buccal capsule with two lateral lips, and a triangular-shaped oral opening, and with serrated projections below the esophageal leaves. Esophagus long and divided into two portions: the first portion comprises the dilatation region, and the second portion is the one that goes to the bulb, the first portion being longer than the second. Bulb well developed; excretory pore immediately anterior or at the level of the bulb; nerve ring near the first portion of the esophagus. Males have a curved tail. Spicule long, distal end of the spicule is curved, and it is pointed. Genital cone present. Females with long uterus and vulva covered by a prominent vulvar lip. Gravid females thin-shelled eggs and no embryonated.    Based on 20 female specimens: body 6.47 mm (4.46-7.77) length and 0.68 (0.53-0.86) wide at bulb region. Distances from anterior end to nerve ring and excretory pore 0.32 (0.29-0.37) and 2.19 (1.66-3.14), respectively.

Discussion
The genus Ozolaimus Dujardin, 1845, comprises medium-sized nematodes with a dorsoventrally elongated mouth with two lateral lips and a long esophagus divided into a short anterior portion and a thinner posterior portion ending in a distinct bulb. Side wings absent. Excretory pore at the anterior end; long spicule; short, truncated tail; well developed, curved distally. Females have a vulva anterior to the anus and covered by the vulvar lip, with a long and sinuous uterus (Rudolphi, 1819;Dujardin, 1845;Vicente et al., 1993). In our study, the morphological characters were compatible with this genus. Five species of Ozolaimus parasites on lizards are currently described: Ozolaimus megatyphlon was initially described by Rudolphi (1819) found in the caeca of Iguana iguana in Berlin; Ozolaimus cirratus Linstow, 1906, in the large intestine of Iguana tuberculata Laurenti, 1768, in Germany; Ozolaimus monhystera (Linstow, 1902) in Cyclura cornuta (Bonnaterre, 1789) in Haiti; Ozolaimus ctenosauri Caballero, 1938, in the small intestine of Ctenosaura pectinata (Wiegmann, 1834) in Mexico; and Ozolaimus linstowi Malysheva, 2016 parasitizing the large intestine of I. iguana in Mexico. Of these authors, Linstow (1902Linstow ( , 1906 described the drawings referring to the two species O. cirratus and O. megatyphlon in more morphological detail. This was also the case with Ortlepp (1933), in his drawing of the esophagus and tail of these species. The images and descriptions contributed significantly to differentiating the species in our study. As observed in Table 1 of O. megatyphlon and Table 2 of O. cirratus, the morphometry of these species shows little difference between them, and in relation to other works, no significant differences were observed either.
In our research into the morphological analysis by LM, the species O. cirratus and O. megatyphlon differ from each other by the shape of the esophagus, position of the excretory pore, and shape of the spicule, as observed by Ortlepp (1933). And, for the first time through SEM, the presence of a small, serrated structure in the esophageal leaves of O. cirratus was described, while in O. megatyphlon, it presented small, spaced, and rounded structures in the esophageal leaves. In addition, phasmids were described for the first time in both species.
Parasites of this genus have been recorded in free-living iguanas in Midwest and Northeast Brazil (Breves et al., 2011;Teles et al., 2017;Otávio et al., 2018). The parasitological indices found in this work differ from those obtained by Teles et al. (2017), who obtained an average of approximately 1.600 Ozolaimus sp. per host. These parasites have also been recorded in iguanas in Peru by Arrojo (2002), in Panama by Bursey et al. (2007), and in Colombia and Suriname by Ávila & Silva (2010), demonstrating that these infections are very frequent. In the North region of Brazil, this was the first occurrence in the State of Pará, where 12.028 adult O. megathyphlon and O. cirratus were registered with a prevalence of 100% (n = 4), differing from the research by Otávio et al. (2018), which recorded 388 adult pinworms of O. megatyphlon and O. cirratus with a prevalence of 60% (n = 5), and Teles et al. (2017), which obtained a prevalence of 66.6% (n = 18), both in Brazil. There are still few records of Ozolaimus spp. in iguanids in Brazil.
The nematodes studied here, O. megathyphlon and O. cirratus, have also been recorded in Iguana rhinolopha, Iguana tuberculate, and I. iguana (Linstow, 1906;Dosse, 1942;Caballero, 1938;Loukopoulos et al., 2007;Breves et al., 2011;Otávio et al., 2018). Both Jacobson (2007) and Teles et al. (2017) report that the most common genera of Iguana parasites are Alaeuris, Ozolaimus, and Tachygonetria, and these pinworms have high host specificity, which is common in lizards, chelonians, and some snakes. The eggs, when ingested by the reptile, hatch in the upper digestive tract, which releases the larvae; when mature, adults migrate to the rectum (Greiner & Mader, 2006). They are usually elongated eggs with a flattened side, and most have a subpolar operculum (Anderson, 2000). The form of infection is mainly fecal-oral (Klingenberg, 2007). They are considered commensal organisms and can help in the digestion of foods of plant origin; however, in cases of massive infections, they can cause obstructions of the gastrointestinal tract, cloacal prolapse, and sometimes a slight local inflammatory reaction (Greiner & Mader, 2006;Klingenberg, 2007;Jacobson, 2007).
The iguanas in this research were free-living and had a high infection rate. According to van Marken Lichtenbelt (1993), free-living herbivorous lizards commonly present high loads of oxyurids, reaching up to 5.000 parasites per lizard. Kehoe et al. (2020) recorded an effective measure for controlling these helminths using oxfendazole to treat oxyurid nematodiasis. Lent & Freitas (1948) identified a single site of infection in the genus Ozolaimus sp., the large intestine. Breves et al. (2011) also recorded infection by this nematode in the large intestine, and in coproparasitological and morphological analysis of adult helminths, Carvalho (2018) recorded Ozolaimus in the colon of a green iguana. In the present study, these nematodes were present throughout the large intestine, which corroborates the findings of the researchers mentioned above.

Conclusion
In this work, we report for the first time the infection by O. megatyphlon and O. cirratus in green iguanas in the state of Pará, thus expanding the geographical occurrence of the genus. Under scanning electron microscopy, we added distinguishing morphological characters between the two species, such as the presence of a small, serrated structure below the esophageal leaves in O. cirratus and small, spaced, and rounded structures below the esophageal leaves in O. megatyphlon. In addition to showing for the first time the phasmids in both species.