University of Florida Thesis Or Dissertation Formatting

SURVEY AND MOLECULAR CHARACTERIZATION OF NEMATODES ASSOCIATED AS EXTERNAL EPIBIONTS ON THE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS

RAFAEL A. GONZALEZ

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2020

To my Mother and Father, thank you for believing in me

ACKNOWLEDGMENTS

I thank my parents Mario and Linda Gonzalez for their patience and support throughout my life. I also thank my committee members Robin Giblin-Davis, William H.

Kern Jr., and Natsumi Kanzaki for their support, guidance, and patience during this work. Thanks are owed to Cathy Beck, Bob Bonde, and Susan Butler for allowing me to participate in the health assessment manatee capture events and for their advice about how to keep both myself and the animals safe from harm. I would also like to thank

Seemanti Chakrabarti, Ulrich Stingl, and Brian Bahder for their valuable advice during this work. Lastly I thank my wife, Tina Fregeolle-Gonzalez for her love and support.

4 TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 6

LIST OF FIGURES ...... 7

LIST OF ABBREVIATIONS ...... 8

ABSTRACT ...... 9

CHAPTER

1 INTRODUCTION ...... 11

2 METHODS ...... 16

Manatee Sampling and Assessments ...... 18 Nematode Culturing Attempts ...... 22 Molecular Protocols ...... 24

3 RESULTS AND DISCUSSION ...... 32

4 CONCLUSION ...... 47

LIST OF REFERENCES ...... 49

BIOGRAPHICAL SKETCH ...... 53

5 LIST OF TABLES

Table page

2-1 Manatee skin sampling results, 2018 ...... 28

2-2 Manatee skin sampling results, 2019 ...... 30

3-1 Parameters for Bayesian inference from GTR+G+I model...... 44

3-2 Molecular sequences compared in the phylogenetic analysis...... 45

6 LIST OF FIGURES

Figure page

3-1 Left image is Cutidiplogaster manati laying an egg with thread attached from vulva. Very long tail is coiled and tangled between the egg and the nematode. Right side image is closeup view of head, showing simplified mouthparts compared to “ST” and “LT” morphospecies. November 17, 2014. Courtesy of Rafael Gonzalez...... 38

3-2 Head of diplogastrid morphospecies “LT”, showing robust mouth parts compared to C. manati. November 17, 2014. Courtesy of Robin M. Giblin- Davis...... 39

3-3 Tail of diplogastrid morphospecies “LT”. Head is out of focal plane to right side of image. Tail is visibly equal to or slightly less than length of body. November 17, 2014. Courtesy of Rafael Gonzalez...... 40

3-4 Two diplogastrid morphospecies “LT” individuals are seen with their long tails entangled. The head of the second worm on the right side of image is not visible. November 17, 2014. Courtesy of Rafael Gonzalez...... 41

3-5 Composite image of diplogastrid morphospecies “ST”. A, D and E show the robust head of “ST”. B and C both show the short tail, with this male specimen’s spicule and gubernaculum visible near the cloacal opening in image B and with the spicule emerging in image C. November 17, 2014. Courtesy of Robin M. Giblin-Davis...... 42

3-6 The combined Bayesian tree inferred from near full length of SSU and D2-D3 LSU. GTR+G+I model was applied for both loci. The parameters are listed in Table 3-1. Posterior probability values >50% are shown...... 43

7 LIST OF ABBREVIATIONS

AIC Akaike information criterion

CCR Capture, Crystal River (USGS yearly animal capture designation: denotes the last two digits of the year hyphenated by the capture number)

DIC Differential interference contrast optics used in microscopy

DNA Deoxyribonucleic acid

FLREC University of Florida Fort Lauderdale Research and Education Center

FWC Florida Fish and Wildlife Conservation Commission

IACUC Institutional Animal Care and Use Committees

LSU Large subunit ribosomal RNA (28S subunit)

NCBI National Center for Biotechnology Information

PCR Polymerase chain reaction

SSU Small subunit ribosomal RNA (18S subunit) rDNA Ribosomal DNA

RNA Ribonucleic acid tDNA Template DNA, synonym for sample DNA

TSB Tryptic soy broth (agar growth medium)

USGS United States Geological Survey

8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

SURVEY AND MOLECULAR CHARACTERIZATION OF NEMATODES ASSOCIATED AS EXTERNAL EPIBIONTS ON THE FLORIDA MANATEE, TRICHECHUS MANATUS LATIROSTRIS

Rafael A. Gonzalez

May 2020

Chair: Robin Giblin-Davis Major: Entomology and Nematology

Three morphospecies of diplogastrid nematode were identified from the skin of the Florida manatee, Trichechus manatus latirostris. One was morphologically confirmed to be a reisolation of Cutidiplogaster manati, previously found on a captive

West Indian Manatee Trichecus manatus manatus in an aquarium in Japan. Adequate molecular data for inclusion of C. manati in a modern phylogenetic framework for

Diplogastridae/Diplogastromorpha is provided for the first time. The three diplogastrid nematodes found on wild Florida manatees from several years of sampling (2013-2019) were molecularly characterized using LSU and SSU genetic sequences and placed in a modern phylogenetic tree revealing a putative new morphospecies in Cutidiplogaster sister to C. manati which was clearly monophyletic with several named Mononchoides species and another putative new morphospecies with shared ancestry with several undescribed nematodes morphologically similar to Mononchoides which are all in a highly supported clade including Tylopharyx, Eudiplogasterium, Paroigolaimella and

Sachsia.

9 The occurrence of diplogastrid nematodes in other biofouling-like environments, such as dung, or inside rotting palm trees, suggests that this group shares a tolerance for the waste metabolites of other organisms as well as an ability to tolerate changes in osmotic pressure. These adaptations may have allowed diplogastrids to exploit a niche

within the sloughing dead skin layer of the manatee epidermis while intermittently being

exposed to both saltwater and freshwater. The 100% association rate of the three

diplogastrid morphospecies with healthy wild adult Florida manatees from Crystal

Springs, Florida over two years of systematic sampling helps refute the assertion that C.

manati is causative of skin lesions observed in the original species description. This

also lends support to the hypothesis of a horizontally tansmitted epibiont association

between the worms and the manatees, when considered in the context of population

genetics.This association rate and ubiquitous occurrence on animals could be similar to

that shared by humans (Homo sapiens) and their associated skin mites (Demodex spp.).

CHAPTER 1 INTRODUCTION

The Diplogastridae/Diplogastromorpha are a particularly diverse and broadly adapted clade of nematodes which can be found in many habitats (Fürst von Lieven &

Sudhaus, 2000; Sudhaus & Fürst von Lieven, 2003; Kanzaki & Giblin-Davis, 2015).

While all nematodes are inherently aquatic metazoan animals, many Diplogastridae live in micro-aquatic biomes within terrestrial environments. Some are free-living predators in soil. This group has members that specialize in feeding on bacteria or fungi, and so

Diplogastridae commonly occur in decomposing substrates or in association with organic materials that are rich in bacteria and/or fungi and/or nematode prey (Fürst von

Lieven & Sudhaus, 2000; Sudhaus & Fürst von Lieven, 2003; Atighi et al., 2013;

Ragsdale et al., 2015; Ahlawat & Tahseen, 2016). Some Diplogastridae have evolved specialized parasitic/necromenic relationships with their insect hosts (Luong et al.,

2000; Giblin-Davis et al., 2006; Sommer, 2015) and many share phoretic associations with invertebrates (Sudhaus & Fürst von Lieven, 2003; Kanzaki & Giblin-Davis, 2015;

Kanzaki et al., 2017). Overall, Diplogastridae are rarely parasitic but are indeed mostly omnivorous and free-living as predators, or as grazers specialized on bacteria and/or fungi (Giblin-Davis et al., 2006; Ragsdale et al., 2015; Kanzaki et al., 2017). In the cases where they are free-living but phoretically associated with insects, this condition is considered to be an adaptation for the types of very intimate relationships as seen in the example of the fig wasps (Giblin-Davis et al., 2006; Kanzaki et al., 2009; Kanzaki et

al., 2014; Wöhr et al., 2014; Kanzaki et al., 2016; Susoy et al., 2016). In these cases,

the Diplogastridae involved have been long associated with their fig wasp/fig hosts and

adapted to a type of ‘island ecosystem biogeography’ where strange morphological and

life history outcomes have manifested from drastically differing selection pressures compared with those faced by free-living Diplogastridae.

The mouthparts of a nematode largely define what it will be able to feed upon, and so the evolution of mouthparts well suited to a particular niche should be expected as a consequence of natural selection. A nematode truly ‘is what it eats’ in the sense that it is distinguishable from other nematodes most easily by its mouthparts, and these mouthparts have evolved to best exploit the associated food source for the matching niche. Diplogastridae have shown an amazing capacity to be adaptive to shifting resource availability even without modification of their genotype. In seven different species of Pristionchus, a wide variation of feeding-type morphology was found to be produced by the same genotype (Susoy et al., 2016). This is thought to potentially

convey a significant adaptive advantage for exploiting available ecological niches

without the cost of maintaining genetic polymorphism (Susoy et al., 2016). In the case of

some fig wasp associated nematodes, this phenotypic plasticity has allowed precisely

evolved timing of mouth form with food-type availability as the fig lumen composition changes during maturation of the fruit (Susoy et al., 2016). This adaptation of

mouthform without genetic polymorphism can also be seen in necromenic/free-living

species of Pristionchus, and the development of a wide-mouth predatory form

(eurystomatous) versus a narrow-mouth grazing form (stenostomatous) seems to be

dependent on hormone signaling, possibly as a response to population density (Wilecki

et al., 2015; Ragsdale, 2015).

Although it is suggestive that behavioral necessity and morphological adaptation

through development of new forms are intrinsically linked, the direct relationship

between these is not well understood (Wilecki et al., 2015). Despite many available examples of the wide adaptability manifested by diplogastrid nematodes, there are few

examples of members of this group being associated with marine environments other than Cutidiplogaster manati described from the skin of manatees (Fürst von Lieven et

al., 2011) and Eudiplogaster pararmatus, now changed to Allodiplogaster pararmata, described from intertidal mudflats and feeding upon diatoms (Romeyn et al., 1983).

The striata group of the genus Allodiplogaster (to which A. pararmata belongs)

contains aquatic and semi-aquatic species but, like Cutidiplogaster, has never been

sequenced and placed into a modern molecular phylogenetic framework (Kanzaki &

Giblin-Davis, 2015). The position of the dorsal tooth and stegostomatal tube in C.

manati are typologically similar to that manifested in the fungal feeding genus

Tylopharynx (Fürst von Lieven et al., 2011), and may provide a clue to the feeding

preferences and phylogenetic placement of this nematode, but its actual form of

trophism is unknown. Also, it is not known if this morphological similarity is due to

convergence or because of shared ancestry. Thus, there is a major gap in our

knowledge concerning the phylogeny of marine associated diplogastrids such as C.

manati and A. pararmata that would benefit from the reisolation, culturing and

sequencing of either of these species. Nematodes are tiny (usually less than 1 mm long

and 10-20 microns wide) and can be difficult to re-isolate from a general locality after a

long period of time with any guarantees. However, reverse taxonomy (see Kanzaki et

al., 2012) could be attempted in the case of C. manati by re-isolating it from its putative

host, the West Indian/Antillean manatee Trichechus manatus manatus. This was

possible because of the opportunity to sample manatees in Florida from the same

species but from a different subspecies, the Florida manatee T. m. latirostris. It was

originally discovered in skin samples from a captive T. m. manatus isolated from the

east coast of Mexico and kept at the Okinawa Churaumi Aquarium in Japan (Fürst von

Lieven et al., 2011). In addition to its brackish water/marine habitat and association with

a vertebrate host, C. manati is unusual because it possesses and uses a radically long

tail (greater than its body length) to anchor itself to substrates. As stated in the original

species description for C. manati “the specimens were found anchored with their long

tails to the small, thick epidermal bumps within the thread algae (cyanobacteria?) that,

together with pennate diatoms, make up the major part of the epizoic community on the

manatee skin” (Fürst von Lieven et al., 2011, p. 51).

Reverse taxonomy for the phylogenetic elucidation of C. manati from the skin of

the manatee T. m. latirostris is a worthwhile endeavor because its putative host T. m.

manatus is a closely related subspecies of manatee for which sampling access was

available for this study. If the association between C. manati and manatees from the

Caribbean is ancient and host specific, then the hypothesis that populations of both

subspecies of manatees could have an association with C. manati or a closely related

species/cryptic species can be made. Bledsoe et al. (2006) identified nematodes as part

of the general community of microorganisms living in the biofouling community on both

captive and wild population members of the Florida manatee, T. m. latirostris. Ulcerative

skin lesions were observed in the captive population from Homosassa Springs, Florida

but not from wild populations sampled from Tampa and Naples, Florida. The proportion

of nematodes making up the overall invertebrate population in the biofouling skin layer

was about the same on the captive manatees (70%) as found on healthy wild animals

(63%) (Bledsoe et al., 2006). Nematodes were not identified below phylum level in

Bledsoe et al. (2006) but the high association rates in both healthy wild and captive animals (>60%) was seen as an encouraging sign for attempting reverse taxonomy with

C. manati from the Florida manatee in its native range in the current study.

CHAPTER 2 METHODS

The molecular data provided with the description of Cutidiplogaster manati was insufficient for placement in modern phylogenies. In the original species description, the researchers chose mitochondrial Cytochrome Oxidase I (mtCOI) and 12S ribosomal

DNA (rDNA) for barcoding (Fürst von Lieven et al., 2011). For the mtCOI protocol,

Folmer et al. (1994) is referenced. In that work, they developed a universal primer set for many different marine invertebrates. The primers were effective for eleven different invertebrate phyla including: Echinodermata, Mollusca, Annelida, Pogonophora,

Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, and

Coelenterata, as well as the putative phylum Vestimentifera (Folmer et al., 1994).

Nematoda is noticeably absent from this list and this is probably why no one prior to the

2011 work has published diplogastrid nematode sequences generated using these primers. Furthermore, Fürst von Lieven et al. (2011) state that they used the LCO forward x HCO reverse primer pair which is expected to generate a 700 base pair sequence (Folmer et al., 1994). Fürst von Lieven et al. (2011) state that they generated a mtCOI sequence 600 base pairs long, but the corresponding sequence uploaded to

The National Center for Biotechnology Information (NCBI), GenBank accession number

FN688897.1, is only 333 base pairs long and does not support alignment with other available diplogastrid mtCOI sequences. Attempting to amplify newly collected DNA during this study using the LCO and HCO primer pair was not successful. The other barcode region chosen for use in the original species description of C. manati, 12S rDNA, has not been used in other diplogastrid nematode studies, so it was not helpful for placement of C. manati in modern diplogastrid phylogenies. In order to be useful for

barcoding and molecular phylogenetic anlyses, the chosen region should also be used by other scientists investigating similar organisms for meaningful comparisons.

It was unknown whether the same nematode (C. manati) identified at the aquarium in Japan on a captive West Indian manatee (Trichechus manatus manatus) could also be found on the Florida manatee (T. m. latirostris). Prior to my work it was

also unknown if C. manati is harmfully associated with skin lesions on manatees,

potentially as a causative agent. The title of the species description states that the

worms were collected on West Indian manatee, specifically from skin lesions. The

authors do not conclusively state that C. manati could be a causative agent of the skin

lesions, but this potential causative link is implied because the nematodes were said to

have been sampled directly from the lesions (Fürst von Lieven et al., 2011).

To clarify these uncertainties, skin sampling of members of the wild Florida

manatee population near Crystal Springs, Florida (28°53’28”N, 82°35’50”W) was

conducted between 2013-2019. The skin scraping samples were taken during the

annual Florida Fish and Wildlife Conservation Commission (FWC) health assessment

capture of wild manatees (Bonde et al., 2012). These health assessment captures

represent a cooperative effort between FWC, the United States Geological Survey

(USGS) and the University of Florida College of Veterinary Medicine. Initial surveying

was conducted to determine which areas of the animals would provide a satisfactory

sampling location for nematodes, while minimally interfering with the FWC health

assessment protocols. Once the mid-tail region was chosen as suitable, two

subsequent years of sampling were conducted in order to evaluate the population

association rates, relative abundance and identity of nematodes present across a range

of individuals. The manatee capturing and research was conducted under the Federal

Fish and Wildlife permit number MA791721-5 as well as The Institutional Animal Care and Use Committees (IACUC) permit number USGS/WARC/GNV2019-01.

Manatee Sampling and Assessments

Skin samples from different individuals of the Florida manatee, T. m. latirostris were initially taken using a variety of scraping tools to determine what most efficiently separated the dead skin layer from the living tissue. Efficiency was qualitatively judged based on the volume of material which could be scraped and transferred using the same tool onto a 100 mm plastic Petri dish (Genesee Sci Inc. brand) containing sterile

1.5% (w/v) water agar media. Collected samples were then sealed inside the Petri dish with Parafilm M™ before being placed in a container maintained at or below ambient temperature and kept shaded for the duration of the sampling trip.

Tools that were evaluated were chosen to be blunt in order to eliminate the possibility of injuring the animal and to remove only the dead skin matrix without drawing blood or damaging the living epidermal layer. The amount of downwards pressure applied while dragging the sampling tool parallel across the sampling area was initially comparable to using a spoon to slice into a chilled gelatin dessert surface. When the initial amount of pressure resulted in very little dead-skin-matrix removal, it was increased slightly, repeating the scraping procedure on the same spot and for the same surface area. The maximum pressure applied was comparable to a ‘firm back scratch’ on human skin. The ideal tool for manatee skin sampling for this study was determined to be a Fisherbrand™ Spoonula™ Lab Spoon, utilizing the lateral rounded spoon side.

The concave surface of the Spoonula™ was oriented downwards while dragging it across the surface and the material was then sliced into the agar media. Other

implements tested included plastic cutlery, plastic credit card edge, stainless steel

Fisherbrand™ Scoopula™, and a metal teaspoon which were all tested using a similar

level of pressure in the range of a “firm human back scratch”. It was observed that the

thin stainless steel lateral edge of the Spoonula combined with the slightly concave

center made removal and transfer of the collected materials much easier when

compared with the other tested tools, and a visibly larger amount of material was

consistently collected and then concentrated for removal from a variety of manatee skin

areas. Once the Spoonula tool was identified as being optimal, more were procured for

future use to reduce overall sampling/cleaning times.

It is critically important to minimize the amount of time that the animals are

removed from the water and under stress. During any capture when an animal shows

signs of stress, as determined by the veterinarians, the health assessment is

immediately ended and the animal must be returned to the water as quickly as possible

(Bonde et al., 2012). Due to limitations created by the inherent danger of handling a

large animal, and the limited amount of time that was available for my sampling while

the rest of the team had to delay beginning their tasks of the health assessment, sample

areas that were relatively easy to access were compared: i.e., dorsal mid-back, dorsal

mid-tail, dorsal mid-flank area, and dorsal peduncle fold. The same morphospecies of

diplogastrid nematodes were recovered in similar frequencies and abundances from

most surfaces that were examined in these preliminary assessments. The manatees

sampled during the preliminary assessment in 2014 were identified by the USGS

sample numbers CCR14-14, CCR14-15, CCR14-16, CCR14-17, CCR14-18, CCR14-

19. No sampling was conducted towards the head of the manatee to avoid any

unnecessary stress during these important and necessary health assessments.

Sampling was also not attempted on the ventral surface of the animals due to safety and access issues.

For the last two years of this study (2018-2019), all skin samples were taken from five to nine arbitrarily chosen 5 cm x 5 cm square areas from the dorsal mid-tail region of each captured wild Florida manatee (Tables 2-1 and 2-2). The sample area was

scraped from right to left going from top to bottom, and collected material was

transferred to water agar plates using the sampling tool as stated above. Between new

samples, scraping tools were wiped with a Kimwipe and sterilized with 95% ethanol

before being reused.

For one of the 2018 animals (ID: CCR18-20) sequential sampling was performed

once (two total samples) on each of four sample areas, all near the mid-tail region, and

each involving a 5 cm x 5 cm area. The initial scrape was performed with nearly zero

downward pressure applied, and with the tool being dragged from right to left across the

sampling area. This low level of pressure resulted in removal of the biofilm/biofouling

layer only with no dead skin remnants present. Each of the four areas was then

sampled again with pressure approximating that necessary to slice a thin layer from the

top of a chilled gelatin dessert. This was done to compare nematode

presence/absence/relative abundance confined to the biofilm layer external to the

epidermis, versus presence/absence/relative abundance being recorded with dead skin

material being shed from the stratum corneum epidermal layer. The stratum corneum is

the outermost layer of dead skin from which the most superficial portion of dead cells is

sloughed and characterized by being entirely composed of dead cells that lack nuclei

and which are cornified (Graham, 2005). The estimated measurement of abundance

was recorded for each morphospecies observed on each sample plate and categorized

as 0 = absent, + = 1-15 nematodes visible, ++ = 15-99 nematodes visible, and +++ =

100 or more nematodes visible (Tables 2-1 and 2-2) . In all cases this analysis was

performed on sealed plates with no dissection performed to search for additional worms

which could have been potentially obscured by the dead skin matrix. This means that

relative abundance could have been understated. Some samples did not result in any

collection of dead skin material and this was also recorded (Table 2-1 and Table 2-2).

During 2019 all sampled animals, except manatee CCR-19-03, received four

sequential samplings of the same 5 cm X 5 cm area beginning with the biofilm layer and

gradually increasing pressure for each of the next three samples (Table 2-2). CCR-19-

03 was a juvenile animal, and the stratum corneum seemed to be removed very easily

with minimal pressure so sequential sampling was deemed inappropriate and the

animal was arbitrarily sampled in four 5 cm x 5 cm square areas from the dorsal mid-tail

region as done for most animals in the 2018 study (Table 2-1). Also in 2019, one animal

had three additional pseudoreplicate samples taken from three additional locations in

the midtail region. This was manatee CCR-19-02, samples E, F and G and occurred

because there was a delay in manatee captures providing an opportunity to collect

additional material (Table 2-2). 2019 samples were evaluated for nematode abundance

following the 2018 method (see above) and recorded in Table 2-2. Presence or absence

of dead skin in each sample was also recorded. Although dragging the tool across the

sampling areas with no downward pressure applied was intended to capture only the

biofilm layer without any dead skin remnants, this method was successful with only

three of the six total adult animals sampled during 2019 (Table 2-2). The reason for this is unknown, but may be due to different rates of sloughing skin across the entirety of individual manatees’ epidermal tissue. The adult animals sampled during 2019 had

USGS identifying numbers CCR19-01, CCR19-02, CCR19-04, CCR19-05, CCR19-06, and CCR19-07.

Nematode Culturing Attempts

Petri dishes containing samples were taken to the University of Florida, Fort

Lauderdale Research and Education Center (FLREC), Nematology lab and immediately checked for the presence of visible nematodes emerging from the sampled material via light microscopy on an Olympus JM stereo microscope. The travel time from the sampling area to the laboratory, by air-conditioned automobile, was approximately five hours. Sample material was also later dissected under magnification to search for the presence of additional worms. Special attention was placed on dissection of the epidermal bump remnants described by Fürst von Lieven (Fürst von Lieven et al.,

2011). It was found that C. manati individuals could sometimes be hidden inside the tent-like epidermal bumps when the bumps were dissected under magnification.

Multiple attempts were made to culture the three diplogastrid nematode morphospecies recovered from the manatees. Unsuccessful methods attempted primarily included water agar plates from which agar had been excised and water added. This was done both with and without the addition of tryptic soy broth (TSB) agar chunks. TSB (Difco brand via Fisher Scientific) was formulated as follows: 1.125 gm tryptic soybean casein digest broth (solidified) was combined with 11.25 gm granulated agar and 750 ml of deionized water. The flask containing the ingredients was loosely sealed with aluminum foil and autoclaved for 25 min on a liquid cycle before being poured into 100 mm plastic

Petri dishes to solidify under sterile conditions. TSB chunks were added to potentially

support the growth of microbes which could be possible food for the nematodes

because in attempts with full TSB plates they were quickly overgrown killing the

nematodes. Initially, deionized water was used for formulating the agar and adding

liquid to the culture attempt plates. Subsequently water collected from the Crystal River,

Florida sampling location was obtained and used to formulate the liquid media. This was

autoclaved as part of the usual media preparation procedure. Some plates were placed

on an benchtop orbital shaker at 20 rounds per min, and this was done continuously for

several weeks. The hypothesis was that by providing some active circulation of liquid

material in the culture plates similar to what might occur on the surface of manatee skin,

worm feeding-ability would be enhanced and lead to culturing success. None of these

methods resulted in success for any of the three diplogastrid morphospecies. However,

on two occasions an unidentified Plectus species was cultured from the scrapings using

1.5% (w/v) water agar media and two populations have been maintained in the

laboratory for five years (first isolation) and two years (second isolation). The water agar

used in the initial isolation and subsequent cultures of Plectus were not formulated

using the specially sourced Crystal Springs water. In order to culture Plectus

successfully, about 50% of the solidified water agar was sectioned and removed from a

fresh water agar plate using sterile technique. Agar material containing live worms was

then transferred from the original sample plate into the concavity of the new plate.

Sterile deionized water was then added to the new culture plate. Lastly, to enhance the

availability of whatever microbe the Plectus was utilizing as a food source, 100 μL of

liquid material from the older plate was aseptically transferred via pipette into the fresh

liquid of the new plate. Comparisons showed that the addition of TSB chunks resulted in

Plectus cultures that died-out more quickly. Subculturing of the plates consisting of water agar without TSB was required about every six months, whereas the plates with

TSB added required subculturing every six to eight weeks due to more luxuriant microbial growth. Because Plectus was only rarely observed and could have been arbitrarily associated with the manatee skin samples, no further work was done on this

species (except for pending amplification and sequencing attempts to see if it matches a described and sequenced species in GenBank).

Molecular Protocol

Photo- and video-documentation of hand-picked living worms was performed

using a BH-2 light microscope (Olympus, Tokyo) equipped with DIC (Differential

interference contrast) optics and a drawing tube. The photomicrographs were captured

with a digital camera system, model AM-7023 Dinoeye Eyepiece Camera (AnMo

Electronics Corporation, Taipei) attached to the microscope. Photomicrographic plates

were assembled using Adobe Photoshop Elements. From the photo and video

documented samples, the same individual worm from a specific diplogastrid

morphospecies was transferred into ISOHAIR enzyme digestion solution and processed

to yield template DNA (tDNA) as described by Tanaka et al. (2012). The ISOHAIR

enzyme digestion step was increased to two hours, followed by an additional heating at

80°C for three hours. This was done because the initial amplifications did not work as

expected.

The amplifications using these individual worm templates proved to be

problematic/sporadic and did not provide good sequencing results. Multiple gradients

and alternate primer pairs were attempted but the amplicons generated did not produce

satisfactory results. Dilutions of template were also attempted, as well as vacuum centrifugation and re-elution of DNA extraction into a smaller volume of buffer to concentrate available template. Despite the problems with the individual worm samples, sequence data for each of the three observed diplogastrid morphospecies was achieved

using pooled DNA samples as template. These pooled DNA samples each consisted of

between 3-7 worms which had been confirmed to all be one of the three observed

diplogastrid morphospecies. These morphological confirmations and sample collections

were checked and confirmed by Drs. Natsumi Kanzaki/Giblin-Davis from manatees

collected in 2018 or 2019.

The sequencing focused on the D2/D3 expansion segments of the large subunit

(LSU) ribosomal RNA gene sequences as well as near full-length small subunit (SSU)

gene sequences for each of the three different diplogastrid morphospecies that were

routinely observed from manatee skin samples. The LSU dataset is currently

considered to have the greatest value for distinguishing close relatives in the

Diplogastridae while still possessing reasonable deeper level phylogenetic signal

(Kanzaki & Giblin-Davis, 2015). LSU amplification was performed using the primer pair

forward D2a (5’-ACAAGTACCGTGAGGGAAAGTTG-3’) and reverse D3b (5’-

TGCGAAGGAACCAGCTACTA-3’) (Ye et al., 2007). Each 25 μL total volume

polymerase chain reaction (PCR) was formulated using Genessee Sci brand Apex™

Master Mix taq polymerase (Genesee Sci Inc., San Diego CA, USA) in accordance with

the manufacturer’s protocol, inclusive of 1 μL of tDNA per reaction. Thermal cycling was

conducted in the following steps using a Veriti VeriFlex™ Thermal Cycler (Applied

Biosystems by Thermo Fisher Scientific): an initial heating to 95°C for 5 min, then 35

cycles each consisting of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. A final extension step was performed at 72°C for 10 min. PCR products was purified using ExoSap-IT (Affymetrix Inc., Santa Clara CA,

USA) and sent to EUROFINS USA for final sequencing from the purified PCR products.

Small subunit (SSU) ribosomal RNA gene sequence products were also amplified for

corroboration of LSU results. SSU PCR was conducted using the same thermal cycling

parameters described above, and two sets of primer pairs. The ‘front’ primer pair for

SSU were forward SSU988F (5’-CTCAAAGATTAAGCCATGC-3’ and reverse

SSU1912R (5’-CTCAAAGATTAAGCCATGC-3’), while the ‘rear’ primer pair were forward SSU1813F (5’- CTGCGTGAGAGGTGAAAT-3’ and reverse SSU2646R (5’-

GCTACCTTGTTACGACTTTT-3’) (Holterman et al., 2006). Each pair of primers

amplifies about one half of the near full length SSU and then the two sequencing reads

must be assembled into the complete sequence using Geneious 8.1.3

(https://www.geneious.com) software. LSU and SSU sequences were aligned and

reconciled to consensus using Geneious 8.1.3 (https://www.geneious.com) software.

LSU and SSU sequence data for each of the three representative diplogastrid

morphospecies was uploaded to The National Center for Biotechnology Information

(NCBI) GenBank as accession numbers MT160762 (C. manati LSU), MT160758 (C.

manati SSU), MT160763 (“LT” LSU), MT160759 (“LT” SSU), MT160764 (“ST” LSU) and

MT160760 (“ST” SSU). The model of base substitution used for informing the final

phylogenetic analysis from the LSU data was as determined using MEGA X software

(Kumar et al., 2018) using the Akaike information criterion (AIC) model selection

criterion. Bayesian analysis was done using MrBayes 3.2 (Huelsenbeck & Ronquist,

2001; Ronquist et al., 2012) and according to Kanzaki et al. (2018). Finalized results

were used for tree construction as per Ye et al. (2007).

Table 2-1. Manatee skin sampling results from Crystal River, Fl, 2018 USGS Sample plate C. Manati “LT” “ST” Visible Manatee skin ID remnants CCR18-19 Sample A +++ +++ +++ P (Female) Sample B +++ +++ +++ P Sample C 0 +++ 0 P Sample D +++ +++ +++ P Sample E 0 +++ +++ P Sample F +++ +++ 0 P 67% 100% 67% CCR18-20 Sample A-1 0 +++ 0 A (Female) Sample B-1 +++ +++ +++ A Sample C-1 0 +++ 0 A Sample D-1 0 +++ +++ A 25% 100% 50% Resample A-2 0 +++ 0 P Resample B-2 0 +++ 0 P Resample C-2 0 +++ 0 P Resample D-2 +++ +++ 0 P 25% 100% 0% CCR18-21 Sample A 0 0 0 A (Male) Sample B 0 + 0 P Sample C 0 ++ ++ A Sample D ++ ++ ++ A Sample E ++ ++ ++ P Sample F ++ ++ ++ P 50% 84% 67% CCR18-22 Sample A 0 0 0 A (Male) Sample B + + 0 A Sample C 0 ++ ++ P Sample D 0 ++ ++ A Sample E ++ ++ ++ P Sample F ++ ++ ++ A 50% 84% 67% CCR18-23 Sample A +++ +++ +++ A (Female) Sample B +++ +++ +++ A Sample C 0 +++ +++ A Sample D 0 +++ +++ A Sample E +++ +++ +++ A 60% 100% 100%

Table 2-1. Continued USGS Sample plate C. Manati “LT” “ST” Visible Manatee skin ID remnants CCR18-24 Sample A +++ +++ +++ A Sample B +++ +++ +++ A (Male) Sample C +++ +++ +++ A Sample D +++ +++ +++ A Sample E +++ +++ +++ A Sample F +++ +++ +++ A 100% 100% 100% CCR18-25 Sample A +++ +++ +++ P (Male) Sample B +++ +++ +++ P Sample C +++ +++ +++ P Sample D +++ 0 +++ P Sample E +++ +++ +++ P Sample F +++ +++ +++ P Sample G +++ +++ +++ P Sample H +++ +++ +++ P Sample I +++ +++ +++ P 100% 89% 100% Estimated measurement of abundance was recorded for each morphospecies observed on each sample plate and categorized as 0 = absent, + = 1-15 nematodes visible, ++ = 15-99 nematodes visible, and +++ = 100 or more nematodes visible. Visible skin remnants were categorized as A = absent, or P = present. CCR18-20 was first sampled with minimal pressure (samples A-1 to D-1) and then resampled with increased pressure at each corresponding location (samples A-2 to D-2). For all other sampled manatees, each different sample plate represents a pseudoreplicate sample taken from a different arbitrary location near the mid-tail doral surface. Samples labelled under a specific manatee ID with a different letter represent a different 5 cm X 5 cm mid-dorsal tail sampling on the same animal. CCR refers to the USGS animal collection number. For example Capture #25 at Crysal River in 2018 = CCR 18-25

Table 2-2. Manatee skin sampling results, from Crystal River, Fl, 2019 USGS Manatee Sample plate C. Manati “LT” “ST” Visible ID skin remnants CCR19-01 Sample A +++ +++ +++ A (Female) 100% 100% 100% resample A-1 +++ 0 +++ P resample A-2 +++ +++ +++ P resample A-3 +++ +++ +++ P 100% 75% 100% CCR19-02 Sample A +++ +++ +++ P (Female) 100% 100% 100% resample A-1 +++ +++ +++ P resample A-2 +++ +++ +++ P resample A-3 ++ ++ ++ P (non-sequential) Sample B +++ +++ +++ P Sample C +++ +++ +++ P Sample D +++ +++ +++ P 100% 100% 100% CCR19-03 Calf not sequentially sampled Sample A 0 0 0 P (Female, calf) Sample B ++ ++ 0 P Sample C 0 + 0 P Sample D 0 0 0 P 25% 50% 0% CCR19-04 Sample A 0 0 + A (Male) 0% 0% 100% resample A-1 ++ ++ ++ P resample A-2 ++ ++ ++ P resample A-3 ++ ++ ++ P 75% 75% 100% CCR19-05 Sample A 0 ++ ++ P (Female) 0% 100% 100% resample A-1 0 ++ ++ P resample A-2 ++ ++ ++ P resample A-3 ++ ++ ++ P 50% 100% 100% CCR19-06 Sample A ++ ++ 0 A (Female) 100% 100% 0% resample A-1 0 ++ 0 P

Table 2-2. Continued USGS Manatee Sample plate C. Manati “LT” “ST” Visible ID skin remnants CCR19-06 resample A-2 ++ 0 0 P resample A-3 ++ ++ ++ P 75% 75% 25% CCR19-07 Sample A 0 + + P (Male) 0% 100% 100% resample A-1 +++ +++ +++ P resample A-2 +++ +++ +++ P resample A-3 +++ +++ +++ P 75% 100% 100% Estimated measurement of abundance was recorded for each morphospecies observed on each sample plate and categorized as 0 = absent, + = 1-15 nematodes visible, ++ = 15-99 nematodes visible, and +++ = 100 or more nematodes visible. Visible skin remnants were categorized as A = absent, or P = present. Samples under a specific manatee ID with a different letter are a different 5 cm X 5 cm mid-dorsal tail sampling on the same animal. All but CCR19-03 were sequentially sampled using increasing pressure. CCR refers to the USGS animal collection number. For example, Capture #07 at Crysal River in 2019 = CCR 19-07

CHAPTER 3 RESULTS AND DISCUSSION

Cutidiplogaster manati (Figure 3-1) was isolated from all of the wild sampled

Florida manatees from Crystal River, Florida from 2018-2019 (Tables 2-1 and 2-2). In addition, two other distinct diplogastrid nematode morphospecies were also identified from the dorsal mid-tail samplings (Tables 2-1 and 2-2). One of these has a somewhat long tail (Figure 3-2, Figure 3-3 and Figure 3-4), but not as long as C. manati (Figure 3-

1). The other new diplogastrid morphospecies has a very short tail (Figure 3-5). These diplogastrid morphospecies were referred to as Long Tail (“LT”) and Short Tail (“ST”) species and superficially resembled a Tylopharynx or Mononchoides in terms of their mouthparts with long and well-developed stegostomatal elements. Both appeared morphologically closer to each other, as far as mouthparts were concerned, compared with C. manati, although one does have a long tail. Molecular phylogenetic inferences depicted in the tree (Figure 3-6) revealed that the “LT” diplogastrid morphospecies is monophyletic with C. manati and apparently congeneric with it and three sequenced and two named Mononchoides species. These are Mononchoides sp. NK-2017 isolated from the palmetto weevil Rhynchophorus cruentatus (Kanzaki et al., 2017), M. macrospiculatum isolated from the red palm weevil R. ferrugineus (Troccoli et al., 2015),

M. compositicola (Steel et al., 2011) and M. striatus. “LT” is clearly a putative new species closest to or sister with C. manati that shares highly derived stomatal morphology and an association with the Florida manatee but should probably be reconsidered as a highly derived Mononchoides, not part of a separate genus (i.e.

Cutidiplogaster) (Figure 3-1). The ST diplogastrid morphospecies is probably also a putative new species that is monophyletic with three sequenced ‘Mononchoides’

species in GenBank (Mayer et al., 2009; Susoy et al., 2015) from scarabaeoid beetles

(RS5441, RS9007 and RS9008) that are more closely related to Tylopharynx,

Eudiplogasterium, Paroigolaimella and Sachsia than named Mononchoides (see Figure

3-4). This was determined through Bayesian analysis of concatenated LSU and SSU

results, and illustrated as a phylogenetic tree (Figure 3-6). Table 3-1 shows the

parameters for Bayesian inference using a GTR+G+I model as determined by MEGA X

software (Kumar et al., 2018) which was used to construct the tree. All of the GenBank

accession numbers and sequences compared in the phylogenetic analysis are listed in

Table 3-2.

Nematode population surveys (presence, absence, relative abundance of

different morphospecies) from the collected samples were observed on the freshly

collected plates under magnification and an abundance estimate for each

morphospecies in the sample was made. These were recorded for 2018 (Table 2-1) and

2019 (Table 2-2). All three diplogastrid nematode morphospecies, including C. manati,

were identified in samples from every manatee sampled during 2018, but not for every

sample (Table 2-1). For the single animal that was sequentially sampled with light and

then increased pressure, CCR18-20, there was no apparent difference between which

nematodes were found. Using minimal downward pressure, C. manati was located in

one of four samples, versus using normal downward pressure (comparable a firm

human back scratch) C. manati was located again on a plate from a different sampling

location among the four re-samples that were collected.

In 2019, all three diplogastrid nematode morphospecies were found on every

sampled manatee and on every sequentially sampled area. When C. manati was not

observed on every sequential sample from each sampled area during 2019, it was always absent from the biofilm layer but present when dead skin was collected. This

was true for animals CCR19-04, CCR19-05 and CCR19-07 (Table 2-2). This would suggest that C. manati has a preference for greater depth within the dead skin matrix.

However, for CCR19-01, CCR19-02, and CCR19-03, a high relative abundance of all

three diplogastrid morphospecies was found at every sampling level.

Ebibiont nematodes as previously observed by Bledsoe et. al. (2006) were re-

discovered in dorsal mid-tail skin scrapings of wild Florida manatees. It seemed unlikely

that these nematodes were associated with mammalian skin lesions, because most

Diplogastridae are predatory/free-living nematodes or are insect associates with no

known vertebrate parasitic members (Sudhaus & Fürst von Lieven, 2003; Kanzki &

Giblin-Davis, 2015). The lack of association of the nematodes with skin lesions was

confirmed by this study because none of the manatees sampled (Tables 2-1 and 2-2)

had skin lesions present, but all of the adult manatees had all three diplogastrid species present. The original species description for C. manati provided a clue concerning this observation (Fürst von Lieven et al., 2011), because the authors specify that they found the nematodes anchored to epidermal bumps, whereas an ulcerative papillomavirus

lesion like that sometimes found on captive or immunosupressed manatees (Bledsoe et

al., 2006; Halvorsen & Keith, 2008) inherently lacks epidermal bumps since it disrupts

the epidermis. While the authors did not specify the type of lesions found on the captive

manatees with C. manati in Japan, there is no record of skin lesions in manatees which

do not disrupt the epidermis (Bledsoe et al., 2006; Halvorsen & Keith, 2008). Other

forms of lesions which are non-ulcerative, such as those induced by Cold Stress

Syndrome or brevitoxicosis, also disrupt normal epidermal features because they manifest as warts or keratonic flat skin (Halvorsen & Keith, 2008). Papillomavirus can also manifest as similar non-ulcerative lesions on manatees, but still replace normal epidermal features (Halvorsen & Keith, 2008). It seems likely that the C. manati

collected from lesions by Fürst von Lieven et al. (2011) were in fact from the border of

healthy tissue surrounding the lesions and not the causative agent of the lesions.

The similarity between the 100% association rate of the three diplogastrid

morphospecies is somewhat analogous to the occurrence of human (Homo sapiens)

skin mites. Human skin mites Demodex spp. live and reproduce within the hair follicles

of terrestrial mammals and harmlessly feed on dead skin cells (Palopoli et al., 2015).

They are considered to be ubiquitous symbionts of humans acquired by horizontal

transmission (Palopoli et al., 2015). The manatee diplogastrids could be acquired by

juvenile manatees in a similar way from their mothers, and this would be supported by

the data on the young calf in 2019 which was the only individual sampled between

2018-2019 that did not have all three morphospecies present in our sampling. In a

recent study comparing the population genetics of human skin mites D. folliculorum, it

was found that different human populations had skin mites that were genetically distinct

from skin mites living on humans populations from other continents and could be

separated into four distinct clades (Palopoli et al., 2015). It was estimated that the last

common ancestor shared by the four different mite clades occurred more than three

million years ago. Since this is earlier than the first appearance of modern humans, the

different mite species are hypothesized to have co-evolved with their ancestral pre-

human hosts (Palopoli et al., 2015). While the ecology of these diplogastrids seems to

be very different from that of human skin mites, there may be a similar population genetics relationship with distinct populations of worms associated with distinct manatee populations.

Although the morphology appears identical, C. manati needs to be collected again from populations of the West Indian Manatee, T. m. manatus in order to confirm

that the species found on the Florida manatee, T. m. latirostris is genetically identical.

This is true because the sequences from the original species description for C. manati

isolated from a West Indian Manatee in an aquarium in Japan were suboptimal for inclusion in modern phylogenetic analyses and cannot be compared against the new

sequence data generated for C. manati from the Florida manatee (this study). A

comprehensive study comparing the population genetics of diplogastrid nematodes

found on different manatee subspecies and species from around the world could

potentially reveal a cladistic association like that found in the association of skin mites

from different human population groups.

Diplogastrid nematodes occur in other biofouling-like environments, such as

inside rotting palm trees (Gerber & Giblin-Davis, 1990), or as halophytes in brackish

wastewater conditions (Romeyn et al., 1983). Diplogastrids are also associated with

insects in terrestrial environments (Kanzaki et al., 2017), and this could also provide a

challenging osmotic environment, especially when the nematode is an associate of an

insect but will also spend much of its life-cycle living inside a fig fruit (Giblin-Davis et al.,

2006; Kanzaki et al., 2014). These qualities suggest that this group shares a tolerance

for the waste metabolites of other organisms as well as an ability to tolerate shifts in

osmotic pressure. This adaptation may have allowed diplogastrids to exploit a niche

within the sloughing dead skin layer of the manatee epidermis while intermittently being exposed to both saltwater and freshwater, but this will need to be investigated further with manatees in brackish/marine environments.

Figure 3-1. Left image is Cutidiplogaster manati laying an egg with thread attached from vulva. Very long tail is coiled and tangled between the egg and the nematode. Right side image is closeup view of head, showing simplified mouthparts compared to “ST” and “LT” morphospecies. November 17, 2014. Courtesy of Rafael Gonzalez.

Figure 3-2. Head of diplogastrid morphospecies “LT”, showing robust mouth parts compared to C. manati. November 17, 2014. Courtesy of Robin M. Giblin- Davis.

Figure 3-3. Tail of diplogastrid morphospecies “LT”. Head is out of focal plane to right side of image. Tail is visibly equal to or slightly less than length of body. November 17, 2014. Courtesy of Rafael Gonzalez.

Figure 3-4. Two diplogastrid morphospecies “LT” individuals are seen with their long tails entangled. The head of the second worm on the right side of image is not visible. November 17, 2014. Courtesy of Rafael Gonzalez.

Figure 3-5. Composite image of diplogastrid morphospecies “ST”. A, D and E show the robust head of “ST”. B and C both show the short tail, with this male specimen’s spicule and gubernaculum visible near the cloacal opening in image B and with the spicule emerging in image C. November 17, 2014. Courtesy of Robin M. Giblin-Davis.

Figure 3-6. The combined Bayesian tree inferred from near full length of SSU and D2- D3 LSU. GTR+G+I model was applied for both loci. The parameters are listed in Table 3-1. Posterior probability values >50% are shown.

Table 3-1. Parameters for Bayesian inference from GTR+G+I model. Parameter(LSU) Setting Parameter(SSU) Setting InL -27032.96896 InL -24768.25718 freqA 0.21 freqA 0.25 freqC 0.22 freqC 0.21 freqG 0.32 freqG 0.27 freqT 0.25 freqT 0.27 R(a) 0.43 R(a) 1.00 R(b) 1.50 R(b) 2.88 R(c) 0.79 R(c) 2.38 R(d) 0.36 R(d) 0.88 R(e) 3.07 R(e) 4.25 R(f) 1.00 R(f) 1.00 Pinva 0.20 Pinva 0.38 Shape 1.03 Shape 0.57 These are the values required for the Mr. Bayes software and tree generation, and are determined via MEGA X using the Akaike information criterion (AIC) model selection criterion.

Table 3-2. Molecular sequences compared in the phylogenetic analysis. Species 28S (LSU) 18S (SSU) Species 28S(LSU) 18S(SSU) Rhabditoides Neodiplogaster inermis (Outgroup EU195981 AF082996 LC107878 LC107877 acaloleptae species) Cutidiplogaster Neodiplogaster MT160762 MT160758 AB326309 AB326310 manati crenatae Diplogastridae sp. Neodiplogaster sp. MT160763 MT160759 AB478641 AB478640 “LT” RGD904 Diplogastridae sp. Neodiplogaster sp. MT160764 MT160760 KJ877265 KJ877212 “ST” RS9009 Acrostichus Neodiplogaster LC374587 LC374587 MH048998 MH049001 floridensis unguispiculata Neodiplogaster Acrostichus halicti AB455818 AB455817 MH048996 MH048999 unguispiculata Acrostichus Neodiplogaster AB477074 AB477077 MH048997 MH049000 megaloptae unguispiculata Acrostichus Oigolaimella LC374584 LC374584 KJ877276 KJ877219 palmarum RGD194 attenuata Oigolaimella sp. Acrostichus puri AB477076 AB477079 KJ877275 KJ877218 RS9010 Acrostichus Oigolaimella sp. LC374583 LC374583 AB478631 AB478630 rhynchophori RGD844 Acrostichus ziaelasi LC530735 LC530736 Oigolaimella sp. AB478633 AB478632 RGD884 Acrostichus sp. Parapristionchus LC530747 LC530748 JX163972 JX163981 "femorata" giblindavisi Allodiplogaster Parasitodiplogaster KJ877266 KJ877224 AY840555 AB901285 hylobii citrinema Allodiplogaster cf. AB597244 AB597233 lucani Allodiplogaster Parasitodiplogaster JX163970 JX163979 AB810253 AB901283 seani maxinema Allodiplogaster Parasitodiplogaster EU195999 EU196025 LC109318 LC109317 josephi nymphanema Parasitodiplogaster LC101737 LC101736 obtusinema Allodiplogaster Paroigolaimella KJ877267 KJ877226 KJ877259 KJ877207 sudhausi micrura Butlerius sp. VS- Paroigolaimella KJ877247 KJ877204 KJ877261 KJ877230 2014 stresemanni Demaniella sp. Pristionchus LC210628 LC210625 KJ705000 KJ704996 NKZ367 aerivorus Diplogasteriana Pristionchus KJ877246 KJ877203 KJ704999 KJ704995 schneideri americanus Diplogasteriana sp. Pristionchus KJ877245 KJ877202 KT188878 KT188848 RS9000 arcanus Diplogasteroides Pristionchus KJ877253 KJ877227 AB852582 AB852581 (Fuchsnema) halleri bucculentus Diplogasteroides Pristionchus (Fuchsnema) sp. KJ877254 KJ877228 KT188873 KT188843 entomophagus S5537 Diplogasteroides Pristionchus (Pseudodiplogaster) KJ877270 KJ877214 KT188879 KT188849 exspectatus magnus Diplogasteroides Pristionchus (Pseudodiplogaster) LC0276755 LC027674 KJ877273 KT188855 fissidentatus nasuensis Diplogasteroides Pristionchus (Pseudodiplogaster) KJ877271 KJ877215 KT188880 KT188850 japonicus sp. RS5444

Table 3-2. Continued Species 28S (LSU) 18S (SSU) Species 28S(LSU) 18S(SSU) Diplogasteroides (Rhabdontolaimus) AB808723 AB808722 Pristionchus lheritieri KT188876 KT188846 andrassyi Diplogasteroides (Rhabdontolaimus) LC027673 LC027672 Pristionchus marianneae KT188866 KT188836 asiaticus Diplogasteroides (Rhabdontolaimus) LC099974 LC099973 Pristionchus maupasi LC011449 LC011448 luxuriosae Diplogasteroides nix LC145090 LC145091 Pristionchus pacificus EU195982 U81584 Pristionchus racemosae KT188888 KT188859 Diplogastrellus gracilis KJ877249 KJ877216 Pristionchus sycomori KT188886 KT188857 Diplogastrellus EU419762 EU419758 Pristionchus triformis KT188884 KT188854 metamasius Diplogastrellus (Metadiplogaster) sp. KJ877248 KJ877205 Pristionchus uniformis KJ877272 KJ877236 RS5608 Diplogastrellus Pseudodiplogasteroides cf. (Metadiplogaster) sp. AB597250 AB597239 AB597248 AB597237 compositus "Tadami" Eudiplogasterium Pseudodiplogasteroides KJ877258 KJ877206 KJ877250 KJ877217 levidentus sp. SB257 Pseudodiplogasteroides Fictor sp. RS9001 KJ877280 KJ877233 AB597249 AB597238 sp. 'Luc8' Fictor sp. RS9002 KJ877281 KJ877234 Rhabditidoides aegus AB597251 AB597240 Fictor stercorarius KJ877282 KJ877235 Rhabditidoides humicolus AB440322 LC095813 Koerneria cf. luziae AB597243 AB597232 Rhabditidoides sp. RS5443 KJ877251 KJ877229 Rhabditolaimus Koerneria sp. RS9004 KJ877283 KJ877239 AB849949 AB849946 anoplophorae Leptojacobus dorci KJ877277 KF924399 Rhabditolaimus leuckarti JQ005870 JQ005865 Rhabditolaimus sp. Levipalatum texanum KJ877257 KJ877221 KJ877255 KJ877220 RS5442 Rhabditolaimus sp. Mehdinema alii KJ877285 KJ877213 JQ005871 JQ005866 RS5414 Rhabditolaimus sp. Micoletzkya buetschlii KJ877252 JX163973 JQ005872 JQ005867 RSA134 Rhabditolaimus sp. Micoletzkya calligraphi KJ531092 KJ531036 AB849950 AB849947 "Episcapha" Micoletzkya Rhabditolaimus sp. KJ531102 KJ531046 AB849951 AB849948 hylurginophila "Euwallacea" Micoletzkya inedia KJ531104 KJ531048 Sachsia zurstrasseni KJ877260 KJ877208 Micoletzkya japonica JX163967 JX163976 Sudhausia aristotokia KJ877278 KJ877231 Micoletzkya masseyi JX163968 JX163977 Sudhausia crassa KJ877279 KJ877232 Micoletzkya palliati JX163965 JX163974 Sudhausia floridensis LC214842 LC214841 Teratodiplogaster Micoletzkya sexdentati KJ531094 KJ531038 AB440311 AB440308 fignewmani Mononchoides Teratodiplogaster sp. 1 LN827617 LN827618 KJ877268 KJ877225 macrospiculum VS-2014 Mononchoides Teratodiplogaster sp. 2 - GU943511 KJ877269 KJ877223 compositicola VS-2014 Teratodiplogaster Mononchoides striatus - AY593924 LC004468 LC004467 variegatae Mononchoides sp. KJ877262 KJ877210 Tylopharynx foetidus - EU306343 RS5441 Mononchoides sp. KJ877263 KJ877209 RS9007 Mononchoides sp. KJ877264 KJ877211 RS9008

CHAPTER 4 CONCLUSION

A causative link between C. manati and skin lesion disease is hereby rejected: all sampled Florida manatees from Crystal River, FL had the nematode present in addition to two putative new species of diplogastrids, but none of the animals had skin lesions.

The mouthparts and movement of C. manati when observed alive most closely resembles a bacterial feeding nematode. It is likely that this nematode lives as an epibiont, grazing on epiphytic bacteria which develop in the biofouling outermost layer of manatee epidermis. It is not known whether this conveys any advantage to the manatee. However, since it has been asserted that biofouling organisms such as bacteria can interfere with optimal marine mammal health (Baum et al., 2002), then any organism which consumes biofouling organisms may help to minimize detrimental effects coincidental with overgrowth of biofouling organisms. Disease as a consequence of overgrowth of biofouling organisms was seen in the case of the outbreak of papillomavirus skin disease affecting captive manatees at the Homosassa Springs

Wildlife State Park in Florida during 2003 (Bledsoe et al., 2006). It is not known whether the feeding habits or population abundance of the nematode C. manati or the other two diplogastrid species is sufficient to affect the biofouling community composition found on manatees, but it is possible that their presence in the matrix of decaying skin actually benefits the manatees by acting as a cleaner of harmful microbes. Analysis of sequential skin sampling results demonstrated that diplogastrid nematodes were found in the biofilm layer coating the epidermis, even in the absence of sloughed skin, but that they were also found at every sampling pressure level. This showed that the nematodes

are distributed throughout the entire matrix of biofilm and dead skin layer, and not confined to either the dead skin or biofilm portions.

Molecular data corroborated morphological work establishing three distinct morphospecies of diplogastrid nematodes including the newly re-isolated C. manati to be present on the Florida manatee, but placed one of them (“LT”) very close to and a putative sister species to C. manati within a clade including named species of

Mononchoides. This challenges the generic status of Cutidiplogaster. The other putative

new diplogastrid species identified in this work (“ST”) is close to Tylopharynx,

Eudiplogasterium, Paroigolaimella and Sachsia and monophyletic with several species

superficially resembling “Mononchoides” morphology. These nematodes are

“Mononchoides” sp. “RS5441” (Mayer et al., 2009), “M”. sp “RS9007” (Susoy et al.,

2015) and “M”. sp “RS9008” (Susoy et al., 2015). Shown in Figure 3-6 to be grouped

closely with Diplogastridae “ST”, these were all found associated with beetles in the

superfamily scarbaeoidea (Mayer et al., 2009; Susoy et al., 2015).

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BIOGRAPHICAL SKETCH

Rafael Gonzalez earned his bachelor’s degree from the University of Florida in

2011, focusing on horticulture. He worked diagnosing palm trees using molecular tools as a technician, and then later as a nematode biologist at the University of Florida Fort

Lauderdale Research and Education Center. Rafael received his master’s degree in entomology and nematology in the spring of 2020.