Effects of Cymatocarpus solearis (Trematoda: Brachycoeliidae) on its second intermediate host, the Caribbean spiny lobster Panulirus argus

Tomás Franco-Bodek; Cecilia Barradas-Ortiz; Fernando Negrete-Soto; Rossanna Rodríguez-Canul; Enrique Lozano-Álvarez; Patricia Briones-Fourzán

doi:10.1371/journal.pone.0287097

. 2023 Sep 29;18(9):e0287097. doi: 10.1371/journal.pone.0287097

Effects of Cymatocarpus solearis (Trematoda: Brachycoeliidae) on its second intermediate host, the Caribbean spiny lobster Panulirus argus

Tomás Franco-Bodek ¹, Cecilia Barradas-Ortiz ², Fernando Negrete-Soto ², Rossanna Rodríguez-Canul ³, Enrique Lozano-Álvarez ², Patricia Briones-Fourzán ^2,^*

Editor: Hudson Alves Pinto⁴

PMCID: PMC10540948 PMID: 37773971

Abstract

Many digenean trematodes require three hosts to complete their life cycle. For Cymatocarpus solearis (Brachycoeliidae), the first intermediate host is unknown; the Caribbean spiny lobster Panulirus argus is a second intermediate host, and the loggerhead turtle Caretta caretta, a lobster predator, is the definitive host. Trophically-transmitted parasites may alter the behavior or general condition of intermediate hosts in ways that increase the hosts’ rates of consumption by definitive hosts. Here, we examined the effects of infection by C. solearis on P. argus by comparing several physiological and behavioral variables among uninfected lobsters (0 cysts) and lobsters with light (1–10 cysts), moderate (11–30 cysts), and heavy (>30 cysts) infections. Physiological variables were hepatosomatic index, growth rate, hemocyte count, concentration in hemolymph of cholesterol, protein, albumin, glucose, dopamine (DA) and serotonin (5-HT). Behavioral variables included seven components of the escape response (delay to escape, duration of swimming bout, distance traveled in a swimming bout, swim velocity, acceleration, force exerted, and work performed while swimming). There was no relationship between lobster size or sex and number of cysts. Significant differences among the four lobster groups occurred only in concentration of glucose (lower in heavily infected lobsters) and 5-HT (higher in heavily and moderately infected lobsters) in plasma. As changes in 5-HT concentration can modify the host’s activity patterns or choice of microhabitat, our results suggest that infection with C. solearis may alter the behavior of spiny lobsters, potentially increasing the likelihood of trophic transmission of the parasite to the definitive host.

Introduction

Parasitism is an important factor influencing the composition and structure of populations and communities [1, 2]. Parasites can affect many phenotypical characteristics of their hosts, such as growth rate, reproductive rate, nutritional condition, fecundity, immune response, and concentration in the hemolymph of metabolites or neuromodulators, among others [3–5]. Alterations in neuromodulators may in turn cause behavioral changes that increase susceptibility of hosts to predation [4, 6–8].

Digenean trematodes (Platyhelminthes) are widely distributed parasites that usually require three hosts to complete their life cycle: a definitive host and two intermediate hosts [9]. Trematode eggs released in the definitive host’s feces are ingested by the first intermediate host, which is usually a gastropod [10]. Eventually, cercariae are produced in the body of the first intermediate host and released into the water. Upon finding a second intermediate host, the cercariae penetrate its body and migrate to the appropriate tissue, where they encyst, becoming metacercariae. Metacercariae infect the definitive host via consumption of the second intermediate host [10].

Trophically-transmitted parasites may alter the behavior, appearance, or general condition of intermediate hosts in ways that increase their rates of consumption by predatory definitive hosts (review in [6]) and, in some cases, the effects may be multidimensional [4, 7]. It is important to determine which traits of a host are affected by a parasite because, even if the effects are subtle, they could change the host population dynamics and, by extension, the communities where they live [11, 12]. Trophically-transmitted parasites tend to have more dire consequences for the intermediate hosts than for the definitive hosts because the intermediate host needs to be eaten for the parasite to complete its life cycle in a definitive host, where it attains sexual maturity and reproduces [13].

A wide variety of trematodes use crustaceans as second intermediate hosts [14, 15]. This is the case for Cymatocarpus solearis (Brachycoeliidae) [16]. Definitive hosts for C. solearis are marine turtles, particularly loggerhead turtles Caretta caretta [17, 18]. The first intermediate host for C. solearis has not been identified yet but, to date, three species of decapod crustaceans have been reported as second intermediate hosts: the hermit crab Dardanus tinctor (formerly Pagurus tinctor) in the Persian Gulf [19], the channel crab Maguimithrax spinosissimus (formerly Mithrax spinosissimus) in Cuba [20], and the Caribbean spiny lobster, Panulirus argus, in the Mexican Caribbean and in Cuba [20–23].

Caribbean spiny lobsters constitute one of the most valuable fishing resources throughout the wider Caribbean region. Following a protracted larval phase that develops in oceanic waters, the postlarvae of P. argus settle in shallow marine vegetation habitats, where the juveniles remain for a few months. Subadults migrate to coral reef habitats where the adults live, although the latter may use a variety of habitats from patch reefs in shallow reef lagoons to deep reefs and rocky bottoms about 80 m in depth [24]. Caribbean spiny lobsters consume a variety of small marine invertebrates (mollusks, crustaceans, echinoderms, worms) and are consumed by many predators such as groupers, snappers, nurse sharks, stingrays, triggerfish, octopuses, dolphins, and loggerhead turtles [25, 26]. These lobsters are highly gregarious, with multiple individuals often sharing a single shelter. The social behavior of P. argus is mediated by conspecific chemical communication and is capitalized by some fisheries that are based on the extensive use of artificial shelters (casitas) that can harbor many lobsters [27].

Bahía de la Ascensión is a large bay in the Caribbean coast of Mexico with a successful casita-based fishery for spiny lobsters and where infection by C. solearis in P. argus was first discovered. Cysts of C. solearis appear as whitish spheres, ~1 mm in diameter, embedded in the muscles of infected lobsters. Cysts in the abdominal muscles are visible to the naked eye through the translucid membrane between the cephalothorax and abdomen, and the cuticle along the ventral wall of the abdomen [21]. Rapid field surveys based on visual assessment yielded estimates of prevalence of infection in the lobster population of the bay of 21% in 2011 [22] and 14% in 2016 [23], with the probability of infection increasing with size of lobsters. However, Cruz-Quintana [20] and Briones-Fourzán et al. [22] fully dissected infected lobsters in the laboratory and found more metacercariae in the muscles of the cephalothorax and the coxae than in the abdominal muscles (Fig 1), suggesting that visual assessment underestimates the prevalence of C. solearis infection in lobster populations, but whether and how C. solearis affects P. argus remained to be determined. In crustacean hosts, parasites may affect the nutritional condition, blood chemistry, or concentration of hemocytes in the hemolymph [28], the escape response [29–31], or the concentration of certain biogenic amines (neurotransmitters, neuromodulators, and neurohormones), causing behavioral changes that may increase their vulnerability to predators [6, 32]. Therefore, in species of commercial value, such as spiny lobsters, parasites may indirectly affect the fisheries if infected lobsters are more susceptible to predation [11].

Fig 1 — Anatomical parts of a spiny lobster mentioned in the text. The photo is of a large male. The body is composed of two main parts, cephalothorax (i.e., the fusion of head and thorax) and abdomen. The pereopods are the walking legs. The coxae are the first segments of the pereopods joining the legs to the thorax. Photo credit: F. Negrete-Soto.

In the present study, we assessed the effects of infection by C. solearis on several biochemical and physiological traits of lobsters. We examined whether infection affected the swimming performance of lobsters during the escape response, which consists of swimming backwards as a result of rapid abdominal flexions (“tail flips”) [33]. Because in many infections the energy normally used by the host to grow is diverted to combat the infection or to compensate for the problems caused by the infection [5, 34], we examined the effects of infection on growth rate and nutritional condition of lobsters. We also investigated whether infection with C. solearis altered the concentration of two biogenic amines in the host’s plasma: dopamine (DA), which is involved in gonad maturation, carbohydrate metabolism, and learning processes, and serotonin (5-HT), which modulates response to stress, sexual and agonistic behavior, physiological process (including control of energy balance), circadian rhythms, and neurogenesis [35–38]. We did this because changes in the concentration of biogenic amines may alter the host’s activity patterns or choice of microhabitat, potentially increasing exposure to predators [6, 8, 32, 39]. In crustaceans, the concentration of hemocytes in the hemolymph may increase as part of the immune response [40] or decrease as a result of heavy infections [41, 42], although the hemocyte counts may increase or decrease in the same host depending on the type of parasite [43–45]; therefore, we examined this trait in the P. argus-C. solearis system. Finally, we examined the relationship between number of parasites and host size and sex, and estimated sensitivity and specificity of visual assessment of infection in live lobsters versus assessment by full host dissection.

Materials and methods

Experimental lobsters

The study was conducted throughout 2018 and 2019. Live, legal-sized lobsters (~≥74 mm carapace length, CL) were purchased from the fishing cooperatives that catch lobsters from casitas in Bahía de la Ascensión (centered at 19° 40’ N, 87° 33’ W) and Bahía Espíritu Santo (centered at 19° 20’ N, 87° 34’ W), located on the Caribbean coast of Mexico. The lobsters were held in seawater tanks, 3 m- and 2 m-diameter and 1 m in height, with a water level of 80 cm, in UNAM’s facilities at Puerto Morelos (20°52’ N, 86° 52’ W). The tanks had an open seawater flow, pumped from the Puerto Morelos reef lagoon. Therefore, temperature and salinity were similar to ambient conditions (water temperature range for 2018–2019: 24.9–31.1°C; salinity range: 34.47–36.37 psu [46]). The lobsters were fed in excess three times a week with frozen (thawed) mussels and crabs. Any food remains were siphoned out the following day.

After two weeks of acclimatization, each lobster was measured with Vernier calipers (CL, from between the supraorbital horns to the posterior end of the cephalothorax). A digital photograph of the carapace was then taken and assigned a number. The photographs allowed post-molt identification of lobsters, because the body markings and patterns in the carapace of spiny lobsters are conserved after molting [47, 48]. Some lobsters (n = 107) were maintained for several weeks to months to estimate their growth rate; some (n = 80) were used to measure swimming performance (see below). The latter were returned to their holding tanks for several days to weeks prior to measuring the rest of the variables.

A sample of hemolymph was extracted from each lobster to estimate hemocyte density (n = 82) and the concentration of metabolites and biogenic amines (see below). The lobsters were measured again and weighed on a digital scale. The distal third of one pleopod was excised to determine the molt stage by observation under a microscope (see [49]), and the lobsters were euthanized by lowering their metabolism in iced seawater. Then the cephalothorax and abdomen were separated; the hepatopancreas was extracted, blotted to remove excess water, and weighed. The body parts of each lobster were completely dissected and examined for cysts of C. solearis, which are easily detected to the naked eye as small whitish spheres (~1 mm in diameter) embedded in muscle tissue [20, 21]. All cysts in each lobster were counted and a few were haphazardly selected to extract and examine the metacercariae under the microscope to ensure their identity. In all cases, the excysted metacercariae corresponded to the description of C. solearis [21].

Ethics statement

All experimental lobsters were purchased from registered Fishing Cooperatives, were maintained in good conditions until examination, and were euthanized quickly and humanely.

Comparison of lobster characteristics

Swimming performance (escape response)

To determine whether infection with C. solearis affects the swimming performance of P. argus, we measured three variables based on the protocol designed by Briones-Fourzán et al. [50]: (1) delay (s) to escape (how long before the lobster initiated the escape response), (2) duration of a swimming bout (s), i.e. the series of full tail flips between stimulation and rest, and (3) distance (m) traveled in a swimming bout. With these data, we further estimated (4) overall swimming velocity (m/s), (5) acceleration (m/s²), (6) force exerted (body weight in kg × acceleration, in newtons, N), and (7) work performed while swimming (acceleration × force, in joules, J) [50].

We used a concrete channel, 5 m long and 40 cm wide with a 50 cm water depth, marked every centimeter. A partition with a pulley formed a 50 × 60 cm “start compartment” at one end of the channel. One lobster was introduced into the compartment, which was covered to reduce disturbance. After the lobster stabilized for 2–3 min, a person would rapidly introduce the hands into the compartment and try to grab the lobster to simulate a predator attack while the partition of the compartment was lifted, allowing the lobster to swim backwards along the channel [50]. Two observers operating stopwatches were positioned one at each side of the channel. One of them measured the time elapsed from the simulation of the predator attach to the end of the swimming bout (Time A), while the other one measured the time elapsed between the moment the lobster exited the compartment and the end of the swimming bout (Time B). The delay to escape was estimated as Time A–Time B. Experimental runs in which a lobster did not swim (e.g., walked) or attempted to escape in the direction opposite to the wooden partition were not considered. In total, we used data from 78 lobsters used in this experiment, all of them in intermolt. Ovigerous females were excluded.

Growth rate

To measure the growth rate of lobsters, the holding tanks were checked daily for molts, which were extracted and contrasted with the photographs taken when the lobsters were first measured (CL₁) to identify the individual that molted. About 20 days later, once the exoskeleton had completely hardened, the molted individual was measured again (CL₂). The weekly growth rate was estimated as CL₂ –CL₁/number of days elapsed between molts × 7 [51].

Concentration of biogenic amines

To determine the concentration of DA and 5-HT in the lobster’s hemolymph, 1 ml of hemolymph was extracted and dissolved 2:1 in LIS to avoid clotting [52]. LIS is a “lobster isotonic solution” that uses EDTA (ethylene-diamine-tetracetic acid) as an anticoagulant, based on its chelant properties (NaCl 350 mM, KCl 10 mM, buffer HEPES 10 mM, EDTA-Na 10 mM, pH 7.3, approximate osmolarity 790 mOsm/kg). The hemolymph was extracted with a 3 ml syringe containing LIS, inserted at the basis of the fifth pereiopods. The hemolymph was immediately centrifuged at 4000 g to separate the plasma, which was frozen at -20° C until further processing.

The samples were processed using the ALPCO Dopamine quantification kit (17-DOPHU-E01.1) and the ALPCO DRG Serotonin FAST ELISA quantification kit (EIA-5061), following the manufacturer’s protocols. Absorbance readings for both neuromodulators were taken at 25° C at 450 nm (650 nm reference wavelength). Quantification of known samples (as μg ml⁻¹) was achieved by comparing their absorbance with a reference curve prepared with known standard concentrations [32].

Nutritional condition: Concentration of metabolites

To determine whether infection with C. solearis affected nutritional condition of lobsters, we measured the concentration in hemolymph of total protein, cholesterol, glucose, and albumin. SPINREACT colorimetric kits (Glucose-LQ 41010, Albumin 1001020, Total Protein 1001291, Cholesterol-LQ 41020) were used, following the manufacturer protocols with slight modifications. Because the readings were done in a spectrophotometer using 96-well plates, the protocol was modified using 200 μl of the kit’s reagent and 10 μl of the hemolymph sample dissolved in LIS. The cholesterol readings were measured at 505 nm after incubating the reaction for 5 min at 37° C. The glucose readings were measured at 505 nm after incubating the reaction for 10 min at 37° C according to the kit’s specifications. The albumin readings were measured at 630 nm, whereas the total protein readings were measured at 540 nm without incubation. As the concentration of metabolites in the hemolymph of crustaceans is influenced by molt stage [5], only samples from individuals in intermolt were used in these analyses.

Nutritional condition: Hepatosomatic index

The hepatosomatic index (HI) provides an indication of the nutritional condition of lobsters, including P. argus [53, 54]. The HI was estimated as wet weight of hepatopancreas/lobster weight × 100.

Total hemocyte count

The concentration of hemocytes in the hemolymph was estimated by resuspending in LIS the cellular pellet obtained upon extracting the plasma. An aliquot of 20 μl from the resuspended cellular pellet was put in a Neubauer chamber where the hemocytes were counted [55]. The following equation was used to estimate the density of hemocytes: (total count of hemocytes/8) × dilution factor × 1000, where 8 is the number of quadrants in the Neubauer chamber. The dilution factor varied between 1 and 4 depending on the sample. Only lobsters in intermolt were used for this estimation.

Statistical analyses

The effects of a parasite may vary with infection intensity [56–58]. Exploratory analyses of our results showed a wide dispersion in the results of almost all variables measured in infected individuals. Therefore, lobsters were categorized into four arbitrary groups based on the total number of cysts in their bodies as assessed after full dissection (i.e., after all variables had been measured): uninfected (0 cysts), lightly infected (1–10 cysts), moderately infected (11–30 cysts), and heavily infected (>30 cysts) [29]. This categorization rendered similar samples sizes for all groups and variables, and the mean size and weight of lobsters did not differ significantly among groups (see Results). However, as we measured some variables in some lobsters and other variables in other lobsters (see S1 Appendix), different analyses were done for different groups of variables. Two separate one-factor multivariate analyses of variance (MANOVA) with a general lineal model (GLM) approach [59] were used to test for a potential effect of parasite intensity (with four levels: uninfected, lightly infected, moderately infected, and heavily infected) on a combination of the four metabolites (albumin, cholesterol, glucose, and total protein) measured in the hemolymph of lobsters on the one side, and on a combination of the seven components of the escape response of lobsters on the other side. MANOVA results were followed by univariate analyses to examine the individual response variables. Data on the concentration of metabolites met all MANOVA assumptions, whereas in the case of the escape response variables, the assumption of homogeneity of the variance-covariance matrix was not met. However, departure from this assumption should not be an issue if sample sizes are equal or nearly equal [59, 60], and sample sizes were 19–21 for this particular group of variables.

To test for potential effects of parasite intensity on the remaining variables (concentration of dopamine, concentration of serotonin, hemocyte count, hepatosomatic index, and growth rate), data from each variable were individually subjected to an analysis of covariance (ANCOVA, also with a GLM approach), with parasite intensity as the factor (with four levels) and lobster size (CL) as the covariate. Significant results of GLMs were followed by Tukey’s HSD multiple comparisons test. Statistical analyses were done with the software Statistica v.10 (StatSoft, Inc., Tulsa, OK, USA).

Relationship between number of metacercariae and lobster size

Metacercariae may remain in a host’s body throughout its life, but their presence does not preclude the host from further reinfections. If parasites accumulate in a host as it grows without compromising its survival, then a positive linear relationship between the number of parasites and the size of the host would be expected, whereas if host mortality increases with number of parasites, then the relationship would be negative [58]. A non-linear relationship may indicate that the number of parasites increases with host size only to a point but then decreases due to the loss of highly parasitized individuals from the host population [3, 58, 61]. Also, in some decapods, intensity of infection varies with sex [58]. To examine whether number of cysts depended on lobster size and sex, we transformed the data to Log (number of cysts + 1) [58] and subjected these data to an ANCOVA, with sex as a categorical factor and CL as the covariate.

Validity measures for the visual assessment of infection by C. solearis

To date, prevalence of infection by C. solearis in wild lobsters has been estimated only by visual assessment, searching carefully for cysts in the abdominal muscles through the arthrodial membranes and the abdominal ventral wall [21–23]. If visual assessment provided a good approximation to real prevalence, it could constitute a rapid, non-invasive way of assessing infection in wild lobster populations, which would be of interest to both researchers and fishers. However, cysts may occur in muscles in the cephalothorax that are not evident to the naked eye [20, 22]. Therefore, visual assessment of infection needs to be validated by comparing it with a more precise test (“gold standard”).

Validity measures of a test are based on two criteria: sensitivity and specificity [62]. Sensitivity measures the proportion of correctly identified positives (e.g., the proportion of infected lobsters), whereas specificity measures the proportion of correctly identified negatives. For our purposes, the gold standard was the determination of the presence of metacercariae after full dissection of the entire lobster. Sensitivity of visual assessment was estimated as the number of lobsters classified as infected by visual inspection/number of lobsters classified as infected after full dissection. Specificity of the visual test was estimated as the number of lobsters without cysts/number of lobsters without cysts plus the number of false positive identifications [62]. We estimated 95% confidence intervals for proportions using the Wilson method with continuity correction [63].

Results

Size and weight of lobsters

The size range of lobsters was 72.8–142.5 mm CL. After separating the lobsters in four groups based on the presence of metacercariae and intensity of infection, no significant differences were found among the four groups of lobsters in either size (F_3,122 = 0.245, p = 0.865) (Fig 2A) or weight (F_3,119 = 0.235, p = 0.872) (Fig 2B). Means and 95% confidence intervals of all response variables of the four groups of lobsters are given in S1 Table.

Growth rate and hepatosomatic index

Neither size nor parasite intensity significantly affected the weekly growth rate of lobsters (Table 1, Fig 2C). The hepatosomatic index (HI) was significantly affected by lobster size, but not by parasite intensity (Table 1, Fig 2D). HI tended to decrease with increasing lobster size, but the relationship explained only 7% of the data variation.

Table 1. Effects of lobster size and parasite intensity on traits of Panulirus argus.

Variable	Effect	df	MS	F	p
Growth rate (mm/week)	Intercept	1	0.140	8.963	0.003
	Size	1	0.029	1.888	0.172
	Parasite intensity	3	0.015	0.982	0.405
	Error	102	0.016
Hepatosomatic index (HI)	Intercept	1	19.328	90.886	<0.001
	Size	1	1.881	8.847	0.004
	Parasite intensity	3	0.117	0.549	0.650
	Error	117	0.213
Dopamine (ng/ml)	Intercept	1	324133.4	0.351	0.556
	Size	1	12599.6	0.014	0.907
	Parasite intensity	3	281380.5	0.305	0.822
	Error	64	923589.6
Serotonin (ng/ml)	Intercept	1	8362.56	4.613	0.035
	Size	1	340.95	0.188	0.666
	Parasite intensity	3	20954.58	11.559	<0.001
	Error	78	1812.83
Hemocyte count (n/ml)	Intercept	1	2.46E+13	10.977	0.001
	Size	1	2.08E+12	0.931	0.338
	Parasite intensity	3	8.68E+11	0.388	0.762
	Error	77	2.24E+12

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Results of GLMs (α = 0.05) on data of several variables compared between four groups of lobsters differing in parasite intensity: uninfected (0 cysts), lightly infected (1–10 cysts), moderately infected (11–30 cysts), and heavily infected (>30 cysts), using lobster size as a covariate. N lobsters used for each variable: dopamine: 14–22 per group; serotonin: 20–22 per group; HI: 26–36 per group; growth rate: 16–25 per group; hemocyte count: 18–22 per group.

Swimming performance (escape response)

The presence and intensity of trematodes had no significant effects on the combination of escape response variables of individual lobsters (Wilk’s lambda = 0.753, F = 1.412; df = 21, 197; p = 0.504). All univariate tests confirmed a non-significant effect on each of the separate components (p-values: 0115–0.692) (Fig 3).

Fig 3 — Box plots of seven components of the swimming performance during the escape response: (A) delay to escape (s), (B) duration of swimming bout (s), (C) distance travelled during bout (m), (D) swimming velocity (m/s), (E) acceleration (m/s²), (F) force exerted (N), and (G) work performed (J), in lobsters *Panulirus argus* categorized into four grades of infection by metacercariae of *Cymatocarpus solearis*: uninfected (0 metacercariae), lightly infected (1–10 metacercariae), moderately infected (11–30 metacercariae), and heavily infected (>30 metacercariae). Central lines in boxplots correspond to medians, box extremities indicate interquartile range (IQR, 1st and 3rd quartiles), whiskers include all data within 1.5 times the IQR, white dots outside the whiskers denote outliers.

Concentration of metabolites in hemolymph

The presence and intensity of trematodes had no significant effects on the combination of concentrations of albumin, glucose, cholesterol, and total protein in plasma of individual lobsters (Wilk’s lambda = 0.806, F = 1.412; df = 12, 161; p = 0.330). The univariate tests confirmed a non-significant effect on the concentration of albumin, cholesterol, and total protein (p-values: 0.518–0.738) (Fig 4A, 4B and 4E), but revealed a significant effect of trematode intensity on concentration of glucose (F = 3.388; df = 3, 66; p = 0.023; power: 0.73) (Fig 4C).

Concentration of DA and 5-HT in hemolymph and hemocyte count

Lobster size and parasite intensity had no significant effects on concentration of DA in plasma (Table 1, Fig 4E). Concentration of 5-HT differed significantly among groups of lobsters (p < 0.001), with no significant effect of lobster size (Table 1) Values of serotonin in plasma were higher in heavily and moderately infected lobsters than in lightly infected and uninfected lobsters (Fig 4F). Total hemocyte count was not significantly affected either by lobster size or parasite intensity (Table 1, Fig 4G).

Validity measures of visual assessment of C. solearis

Of 208 lobsters examined, 28 (13.5%) had at least one visible cyst. However, upon full dissection, cysts were found in 110 lobsters (52.9%) (Table 2). Therefore, sensitivity of visual assessment was 25.4% {95% CI: 17.6%, 33.2%} (28 visually identified as infected/110 identified as infected by dissection). This result indicates that visual assessment detected about one fourth (25%) of all lobsters infected with metacercariae of C. solearis, i.e., that for each lobster visually identified as infected, there were three other lobsters also infected but with cysts in muscles not visible to the naked eye. Of the 98 lobsters that proved to have no cysts after full dissection, one had been erroneously identified as infected by visual inspection. Therefore, specificity of visual assessment was 99.1% {94.5%, 99.9%} (98 non-infected, but 1 false positive) (Table 2). The false positive was an individual in which the only visually detected “cyst” turned out to be a small scar in the muscle.

Table 2. Validity measures of visual assessment of infection.

	Full dissection (Reference test)
Visual assessment	Negative	Positive	Total
Negative	97 (true negatives)	82 (false negatives)	179
Positive	1 (false positives)	28 (true positives)	29
Total	98	110	208

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Relationship between results of visual assessment of presence of cysts of Cymatocarpus solearis infection and full dissection (reference test)

Number of cysts vs lobster size

In the 208 lobsters examined the total numbers of cysts ranged from 0 to 226, with a median abundance of 1 (IQR: 0–16.3). Considering only lobsters with 1 or more cysts (N = 110), the median intensity was 13.5 (3.3–41.0). Infected lobsters had less cysts in the abdomen (median: 2 (0–5.5), range: 0–32) than in the muscles of the coxae and cephalothorax (median: 12 (3–33.8), range: 1–199). The numbers of lobsters with 0 or ≥1 cysts in abdominal muscles were 128 and 80, respectively, whereas the numbers of lobsters with 0 or ≥1 cysts in cephalothoracic muscles were 101 and 107, respectively. These numbers differed significantly (Yates-corrected χ²₁ = 6.57, p = 0.01). On average, 16% of all cysts observed in all lobsters were in the abdomen and 84% in the cephalothorax. Results of ANCOVA revealed no significant effect of either size (F_{1, 205} = 0.566, p = 0.453) or sex (F_{1, 205} = 2.121, p = 0.147) on the number of cysts of C. solearis in lobsters (Fig 5).

Fig 5 — Relationship between size (carapace length, mm) of male and female *Panulirus argus* lobsters and Log (N + 1) of *Cymatocarpus solearis* cysts.

Discussion

The present study is the first examining effects of infection by C. solearis on P. argus by comparing several physiological and behavioral variables among groups of uninfected lobsters and lobsters in three different grades of infection (light, moderate, and heavy) based on the parasite load.

Overall, the trematode appears to have little pathological effects on lobsters. The condition of lobsters was generally not affected by the parasite, as the hepatopancreatic index and the concentration of cholesterol, protein, and albumin did not vary with grade of infection. The exception was the concentration of glucose, which was lower in heavily infected lobsters (i.e., those with ≥ 30 cysts) than in uninfected lobsters and lobsters with light and moderate infections. Muscle and connective tissue are rich in glucose, which serves as a primary molecule for energy exchange at the cellular level [64]. Cyst walls may be permeable to certain substances, including glucose [65, 66], and although the metabolism of metacercariae is not well understood [67], in some cases they appear to draw nutrition from the host’s glucose [7, 68]. This may be the case in the P. argus-C. solearis system, although the effect was relatively minor, suggesting that a potential metabolic cost would appear to occur only in severely infected lobsters.

Some parasites affect the swimming performance of their second intermediate hosts. This occurs in kyphosid fish Girella laevifrons infected with metacercariae of the bucephalid Prosorhynchoides sp., which encyst mainly in the tail fin [69]. However, the reduction in swimming performance of the fish is not proportional to the intensity of infection, suggesting that, rather than physical interference, the metacercariae may have other effects, such as causing inflammation, localized hemorrhage, or cell destruction, all of which are metabolically demanding [69]. In the grass shrimp Palaemonetes pugio, metacercariae of the microphallid Microphallus turgidus encyst mainly in the abdominal muscles, and although swimming of the shrimp can be severely affected, this effect is also independent of the intensity of infection [29]. Infected shrimp spend less time quiescent, more time outside their shelters, and have a shorter delay to first movement after disturbance, making them more easily identifiable by visual predators [31]. In contrast, none of the seven components of the escape response of lobsters varied with grade of infection by C. solearis. Not all infected lobsters had cysts in the abdominal muscles, but all except for one had cysts in the coxae of pereopods which, in conjunction with the thoracico-coxal joint, are rich in muscles [70] (see Fig 1). Regardless, cysts did not appear to physically interfere with the tail flips during the retrograde swim, as occurs with Norway lobsters (Nephrops norvegicus) infected with the dinoflagellate Hematodinium sp. [30]. Therefore, infection with C. solearis has no physiological or physical effects on the swimming performance of P. argus.

Parasite-induced changes in the concentration of neuromodulators in invertebrate hosts are common [6], and we found increased concentrations of 5-HT in moderately and heavily infected lobsters. In crustaceans, DA modulates activity [71–73] and is associated with increased aggressiveness [74], whereas 5-HT modulates intraspecific agonistic responses as well as swimming and escape behavior [32, 37, 38, 75, 76]. Pérez-Campos et al. [32] observed that most fiddler crabs Uca spinicarpa found outside their burrows during the day were infected with cystacanths of the acanthocephalan Hexaglandula corynosoma, potentially increasing their susceptibility to predation by herons, the definitive hosts. Infected crabs had a significantly higher concentration of 5-HT in hemolymph, but their concentration of DA did not differ from that of uninfected crabs [32]. We obtained similar results in the present study. The concentration of DA did not differ among our groups of lobsters, but was highly variable in all four groups. In contrast, clear differences occurred in the concentration of 5-HT, with higher values in heavily and moderately infected lobsters than in lightly infected and uninfected lobsters. Higher levels of serotonin might alter the behavior of lobsters, increasing their vulnerability to predators. For example, gammarid amphipods infected with metacercariae of M. papillorobustus exhibit an aberrant photophilic behavior [75] that can also be induced by directly injecting them with 5-HT [76, 77]. Subordinate individuals of Astacus astacus, Faxonius virilis (formerly Orconectes virilis), and Homarus americanus injected with a solution of 5-HT exhibited a “renovated will” to fight against a dominant conspecific that had previously defeated them [36]. They also exhibited an altered escape response [78] and an increase in fight duration [37, 79], mostly resulting from a decreased likelihood of retreat [80].

Although P. argus is mostly nocturnal, it is not uncommon for these lobsters to walk in the open during the day, when encounters with predators are more likely. Upon detecting a nearby predator, exposed lobsters “freeze” to avoid being visually detected. If this fails, then they use their long but stout spiny antennae to fend off attacks, often lunging towards the attacker. Ultimately, they can escape by rapid backwards swimming [50]. The swimming performance of P. argus was not affected by infection with C. solearis, but heavily infected lobsters had a slightly, but significantly, lower concentration of glucose in the hemolymph. Experiments in vertebrates have revealed that 5-HT might exert different effects on glucose metabolism depending on the species, as well as the hormonal and physiological context (review in [81]). However, the higher concentration of 5-HT in moderately and heavily infected lobsters may cause these lobsters to become more aggressive and attempt to fight an attacker beyond their capabilities, potentially affecting the response of lobsters in predator-prey encounters. It may also affect the social behavior of lobsters, with unknown consequences for casita-based fisheries. A comparison of defense and antipredator behaviors between uninfected and infected lobsters should be conducted in future studies.

In the lobster population of Bahía de la Ascensión, prevalence of infection by C. solearis was estimated at 21% and 14% by visual assessment [22, 23], whereas in the lobster population of the Gulf of Batabanó, Cuba, prevalence of infection by C. solearis was estimated at ~50% by full dissection of lobsters [21]. However, all these studies included juvenile lobsters in their estimates. Our results on sensitivity and specificity of visual assessment suggest that for every adult lobster visibly infected with C. solearis, there are three additional, not visibly infected adult lobsters, and that there is a 1% chance of confounding marks (such as scars) in muscles with cysts of C. solearis. Therefore, visual assessment is insufficient to determine real prevalence of infection with C. solearis. We used full dissection as the gold standard, but future studies should aim to develop a less invasive (e.g., molecular) test to determine prevalence of infection [23].

Cruz-Quintana [21] reported encapsulation of cysts of C. solearis in some infected lobsters, which is a common immune response in crustaceans [44, 82], but we did not detect any signs of melanization upon full dissection of lobsters, and the total hemocyte count in lobsters was not affected by infection with C. solearis. Also, in contrast with other host-parasite systems in which a positive, negative, or non-linear relationship between host size and number of parasites has been found [58, 61], we found no apparent relationship between number of encysted metacercariae of C. solearis and adult lobster size. Similarly, Kohler and Poulin [58] found a significant relationship in only 8 out of 21 instances where different species of parasites and crustacean hosts were examined.

In summary, of all the physiological and biochemical characteristics of P. argus that we analyzed as a function of infection with C. solearis, only two varied with intensity of infection: there was a lower concentration of glucose only in heavily infected lobsters, and a higher serotonin concentration in moderately and heavily infected lobsters relative to lightly infected and uninfected lobsters. These results suggest that infection with C. solearis may alter the behavior of spiny lobsters, which might increase the likelihood of trophic transmission of the parasite to the definitive host. Other important host traits that C. solearis may affect and warrant further investigation include concentration of other neurotransmitters (e.g., octopamine), female fecundity, and conspecific chemical communication.

Supporting information

S1 Table. Response variables of Panulirus argus lobsters in four grades of infection by Cymatocarpus solearis.

Means and [95% confidence intervals] of all variables compared among four groups of lobsters categorized by the presence and intensity of infection by C. solearis (number of metacercarial cysts).

(DOCX)

Click here for additional data file.^{(15.7KB, docx)}

S1 Appendix. Full dataset used in the article.

Sheet 1: Variables measured on lobsters (carapace length, total number of cysts, number of cysts in abdomen, number of cysts in cephalothorax, hemolymph components and neurotransmitters, components of the escape response, lobster weight, molt stage). Sheet 2: Number of cysts versus lobster size and sex.

(XLSX)

Click here for additional data file.^{(59.8KB, xlsx)}

Acknowledgments

We thank Edén Magaña-Gallegos for maintaining the seawater tank system and the lobsters well, and for his help in some of the experiments. We also thank Judith Sánchez-Rodríguez, Matteo Cazzanelli, Elisa Y. Chan-Vivas, Charlotte E. Davies, José A. López-Portillo Hurtado, Juan A. Pérez-Vega, Irma Pérez-García and Nancy Herrera-Salvatierra for helping in laboratory activities, and Miguel A. Gómez-Reali, Edgar Escalante-Mancera, and Gustavo Villarreal-Brito for providing technical support. Laura Celis-Gutiérrez helped with literature search. Water data were provided by Servicio Académico de Monitoreo Meteorológico y Oceanográfico, Puerto Morelos, Q.R.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study received funding from Universidad Nacional Autónoma de México (Program UNAM-DGAPA-PAPIIT, project IN206117 (https://dgapa.unam.mx/), granted to P.B.-F. The Consejo Nacional de Ciencia y Tecnología (CONACYT-México) (https://conacyt.mx/) provided a Master’s scholarship (2018-000012-01NACF-08432) for T.F.-B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0287097.r001

Decision Letter 0

Hudson Alves Pinto

24 Feb 2023

PONE-D-23-02958Effects of Cymatocarpus solearis (Trematoda: Brachycoeliidae) on its second intermediate host, the Caribbean spiny lobster Panulirus argusPLOS ONE

Dear Dr. Briones-Fourzán,

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Dear Authors,

Your MS received good reports from both Reviewers, and I think it has potential to be accepted after careful analysis of the corrections.

Best regards,

Hudson

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I have uploaded my full review as an attachment.

I believe the manuscript meets all PLOS ONE publication criteria. The work is scientifically sound, and I have no concerns about the experimental design. However, I would suggest that the authors implement some additional statistical tests to take full advantage of the data that they have painstakingly collected. For the most part, the authors limited themselves to using non-parametric hypothesis tests (Kruskal-Wallis) to test for significant differences. These tests can be useful, but they must be interpreted carefully because of problems with statistical vs. biological significance (i.e., Johnson 1995). More to the point, these tests have no real predictive abilities. Given the wealth of data collected for this project, I think the manuscript could be improved using glms and/or mixed models. These analyses would address the question: which variable(s) account for most of the variation in infection intensity? As a reader, I was left wondering about this. I have more detailed suggestions along these lines in the Methods/Results section of this review.

Reviewer #2: The authors didn't mention making their data available, but they might have somewhere and mention it when they submitted their work

General Comments:

Overall, I think this is a very informative study that adds to the current understanding of how parasites can alter crustaceans behavior and physiology, specifically, and the potential ecological and economic ramifications of parasites, at large. While I think my comments can greatly aid in the readability of this document, they are admittedly all relatively minor, as I think the authors work stands on its own.

Abstract

• In line 30, change “their” to the hosts’

• Split lines 34 – 38 into 2 sentences, one about the physiological parameters studied and one about the behavioral assays conducted

• Line 45- boldness and aggression are specific behavioral personalities that can be shown with repeated testing. It seems strange to include those terms here considering this paper isn’t about personalities

• Move the sentence in lines 44 – 45 up to right before the results

• Lines 44 – 47 don’t seem to fit in the abstract, or if the author feels they must be included, should be moved up to the methods part of the abstract instead of as the last sentence

Introduction:

• In general, the flow of the introduction seems a bit off. It immediately gives information about the host and parasite then go back to talking about parasite infection at large. All of the information is good, it’s just the order. Also, including transition sentences as the last sentence in each paragraph would greatly aid in flow for the reader

• Add a sentence at the end of the first paragraph to also include behavioral impact of parasites on hosts because this study includes physiological and behavioral impacts

• I found some studies (e.g. Gnanalingam and Bulter 2018) that say P. argus also eat gastropods, which could be important for parasite transmission, so should be included in their list of prey items in line 73

• Lines 75-78 don’t seem relevant to the paper, unless the parasite can be transmitted between 2 lobsters, which seems very unlikely

Materials and Methods:

• Lines 114-116, depth of tanks?

• Line 117- is there a known range for salinity and temperature during the time of the experiments that could be included here?

• Lines 124-126, include Ns for each

• Line 128, what is the N?

• Line 136 – 137- how were the cysts quantified?

• This response is called a tail flip in other crustaceans. Is it called that in lobsters?

• Were the lobsters used in these experiments fully intact?

• Lines 175 – 182 read like introduction material, not materials and methods, and should be moved accordingly. The information is pertinent to the paper

• Lines 212-214- the hepatopancreas also acts as a filter, removing toxins from the hemolymph

• Lines 218-227 also read as introduction material and should be moved

• Line 241 mentions these categories rendered samples of similar size. Does this mean the lobsters were of similar size or there was a similar N for all categories?

• No normality results are reported, so why was a Kruskal-Wallis test used?

• If lobsters were all dissected and metacercariae counted post-mortem anyway, why not just use these data for all analyses instead of using the visual tests of alive lobsters at all?

Results:

• While I have no doubt that the statistical analyses showed significant results for some of the escape response, figure 2 looks like there are no differences. Would bar graphs with error bars be more informative here?

• Same comment for figure 3

Discussion

• Lots of specific anatomical terminology is used, especially in lines 396-409. This terminology is definitely relevant to the findings, but adding another figure that is a labeled diagram of a lobster would greatly aid in readers’ understanding, especially if not familiar with crustacean anatomy

• Adding transition sentences throughout the discussion would also aid in flow, similar to the introduction

• Did that authors relate the fiddler crab behavior with 5-HT concentration for the example mentioned in 427-431? If so, that should be mentioned here because as written, this seems like 2 unrelated findings

• Line 439- Orconectes virilis has been reclassified as Faxonius virilis

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Reviewer #2: No

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PLoS One. 2023 Sep 29;18(9):e0287097. doi: 10.1371/journal.pone.0287097.r002

Author response to Decision Letter 0

1 May 2023

Manuscript Number: PONE-D-23-02958

Manuscript Title: Effects of Cymatocarpus solearis (Trematoda: Brachycoeliidae) on its second intermediate host, the Caribbean spiny lobster Panulirus argus

Dear Editor: Our responses to reviewers’ comments are in blue.

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2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

R: Grant number has been corrected.

R. Caption for Supporting Information has been added at the end of the manuscript. In-text citation matches (Line 249 of revised manuscript).

Reviewer 1.

Digenean trematode life cycles are fascinating and wonderfully complex. Many interesting ecological questions can be tested at the intermediate host-stage, as this is where behavioral manipulation of hosts is theorized to occur. In this paper, the authors quantified the effects of C. solearis infection on the Caribbean spiny lobster P. argus, a second-intermediate host for the parasite. There were clear differences in biomarkers of infection (e.g., concentrations of glucose and serotonin) and physical performance indicators (e.g., escape response) as a function of infection intensity in P. argus, findings that increase the likelihood of trophic transmission to an appropriate definitive host.

• Johnson, D. H. (1995). Statistical sirens: the allure of nonparametrics. Ecology, 76(6), 1998-2000.

R1: We appreciate the suggestion of using parametric analyses for the data. We have now used general linear models (GLM), i.e., conventional linear regression models for a continuous response variable given continuous and/or categorical predictors. We were worried about violating some assumptions of parametric tests, but this was not an issue since we used robust tests. Please see our response no. 7 (R7) below.

Introduction

Lines 79-89 -- why is visual assessment important? I think some additional context here would help. Is it just for researchers interested in the presence of the parasite, or do fishermen care as well? If appearance is important, this may be something to return to in the Discussion (beginning around line 460).

R2: If visual assessment of infection prevalence provided a good approximation to real prevalence, it would be a rapid, non-invasive way of assessing infection in the lobster populations, which would be of interest to both researchers and fishers. However, here we prove that visual assessment misses many infected lobsters and hence cannot be used for rapid assessments.

Lines 89-90 – the transition to the next paragraph about trophic transmission seemed a little abrupt. Perhaps another sentence is needed here?

R3. The Introduction has been completely rearranged to improve transition between paragraphs.

Lines 98-102 – The ideas here seemed a little vague. I think the reader may be left wondering why these parasites have more consequences for intermediate hosts. The intermediate host needs to be eaten for the parasite to complete its lifecycle in a definitive host. Perhaps this should be stated? Also, how would parasites affect the lobster fishery?

R4. Thank you for this suggestion. The paragraph has been revised.

Methods/Results

Line 182 – consider citing (and perhaps later discussing?) Shaw et al. 2009:

• Shaw, J. C., Korzan, W. J., Carpenter, R. E., Kuris, A. M., Lafferty, K. D., Summers, C. H., & Øverli, Ø. (2009). Parasite manipulation of brain monoamines in California killifish (Fundulus parvipinnis) by the trematode Euhaplorchis californiensis. Proceedings of the Royal Society B: Biological Sciences, 276 (1659), 1137-1146.

R5: Thanks for recommending this paper, which is relevant to our manuscript.

Suggestions for additional post-hoc testing:

It is possible to have a low p-value but a small effect size. In other words, the effect detected by the test that seemed so important (i.e., with the low p-value) is actually quite small. Cumming (2012) gives an excellent overview of this issue. To provide some additional context for your results from the Kruskal-Wallis tests, I might suggest testing the magnitude of effect for each significant p-value. The package “rstatix” is quite useful for basic statistical tests – I think Cohen’s D is preferred for parametric data and Wilcoxon effect size tests for non-parametric data.

• Cumming G. 2012. Understanding The New Statistics: Effect Sizes, Confidence Intervals, and Meta-Analysis (Multivariate Applications Series)

R6: After checking major assumptions for parametric tests, we have changed all statistical analyses to GLMs and do not use Kruskal Wallis tests in the revised manuscript.

Suggestions for model building using information theoretic approaches:

You probably have your own methods, but in the interests of being as helpful as possible I’ll share with you what packages I typically use for multimodel inference in R.

• glmmTMB – package for building and testing generalized linear mixed models that has a great suite of diagnostic tools for assessing model fit:

https://cran.r-project.org/web/packages/glmmTMB/index.html

• DHARMa – add-on package with helpful tests for assisting with model convergence issues:

https://cran.r-project.org/web/packages/DHARMa/vignettes/DHARMa.html

• AICcmodavg – package for ranking and comparing models using information theoretic approaches:

https://cran.r-project.org/web/packages/AICcmodavg/index.html

You could bin the data into infected (1 or more cysts) and uninfected (0 cysts) categories. The goal here would be to rank and compare models testing the likelihood of infection. What variable(s) best predict whether a lobster is infected or not? Your response variable would be infection status (yes or no), a binary outcome (1 or 0), and you would fit the data to a binomial error distribution. In models that incorporate swimming performance as a predictor, you could consider adding trial number as a random effect as this might account for some of the variation in your response variable but would not be of biological/ecological importance.

You could also build models using infection intensity as your response variable (0 cysts; 1-10 cysts; 11-30 cysts; > 30 cysts). What variable(s) predict the likelihood of infection intensity in each case? You would fit the data to a negative binomial distribution if they showed evidence of overdispersion, which may be the case for the final category – otherwise, a Poisson would probably fit the data better. For variables that had multiple trials, consider using trial number as a random effect in your model structure.

R7. Thank you for all this interesting and very useful information. However, in our study we were not trying to find variables predicting whether a lobster was infected or not, or using infection intensity as the response variable. Quite the opposite: we were trying to find how infection intensity (the predictor) affected a number of response variables of the lobsters (i.e., components of the escape response, growth rate, concentration of metabolites or biogenic amines, etc.). Originally, we had planned on measuring the entire group of variables in the same lobsters, but this was not possible due to time constraints, logistic problems, death of several lobsters, molting of lobsters, incompatibility of some experiments, recording errors, etc. However, it is true that parametric tests are more powerful, and many are robust to departure from some assumptions. Therefore, following your advice, we have now changed all statistical analyses. We used multivariate analyses of variance (MANOVA) with a general lineal model (GLM) approach to test for potential effects of parasite intensity (with four levels: uninfected, lightly infected, moderately infected, and heavily infected) on two groups of variables that were measured in the same lobsters: (a) the components of the escape response, and (b) the concentration of metabolites in hemolymph. In (b), all MANOVA assumptions were met, but in (a), the assumption of homogeneity of the variance-covariance matrix was not met. However, MANOVA is robust to departure from this assumption when sample sizes are equal or nearly equal (which was the case) (Tabacknik & Fidell 2012; Nimon 2012). The rest of the variables (concentration of dopamine, concentration of serotonin, hemocyte count, hepatosomatic index, and growth rate) were individually subjected to ANCOVAs, using size as a covariate.

Upon doing the MANOVA for the components of the escape response, I noticed an unfortunate mistake in the calculation of acceleration (m/s/s) in the group of uninfected lobsters due to the use of an erroneous column in the Excell sheet. The spurious results had led us to conclude that there were significant differences in acceleration (and in work performed and force exerted, which are calculated from acceleration) of uninfected lobsters relative to the three groups of infected lobsters, when in fact the differences were not significant. In view of this mistake, we double-checked again all data, and everything else was correct. Thus, although acceleration did not differ among lobster groups, these groups did differ significantly in the concentration of serotonin and glucose in the hemolymph.

Tabachnick BG, Fidell LS (2012) Using Multivariate Statistics, 6th edn. Pearson, London.

Nimon KF (2012) Statistical assumptions of substantive analyses across the general linear model: a mini-review. Front. Psychol. 3: 322.

Some thoughts on lobster size as a predictor variable:

I think you could make an argument for using lobster size as a fixed effect (predictor) in your models, or as a random effect since there doesn’t seem to be a clear linear relationship between size and infection status (lines 280-281). Admittedly, this result is a little surprising since larger (older) organisms tend to have more cysts, which you mention (lines 248-249). However, it may also be possible for crustaceans to “clear” their cysts over time. Older cysts can appear brown and withered (Blakeslee et al. 2020), a result of the long-term crustacean innate immune response (Lee and Soderhall 2002); however, you did not find evidence of melanization (line 472). One other intriguing possibility for this non-linear relationship is that larger adults are foraging in different areas compared to smaller individuals and so are not in proximity as often to the first-intermediate host. I have some unpublished data from stone crabs Menippe mercanaria that support this idea. Smaller individuals (up to approx. 40 mm carapace width) are sympatric with Panopeid mudcrabs in shallow water oyster reefs. These individuals often have trematode cysts. Larger stone crabs, on the other hand, tend to burrow more and seek out deeper habitats, meaning that they are not as close on average to the first-intermediate host, the mudsnail Ilyanassa obsoleta. I rarely find cysts in these larger stone crabs.

All in all, since the biological relationship between size and infection status seems unclear, I might argue for using size as a random effect since it is not an important predictor of infection but may still account for some variation in your response variables.

• Blakeslee, A., Ruocchio, M., & Moore, C. S. (2020). Altered susceptibility to trematode infection in native versus introduced populations of the European green crab.

• Lee, S. Y., & Söderhäll, K. (2002). Early events in crustacean innate immunity. Fish & shellfish immunology, 12(5), 421-437.

R8. In lines 249-249 of previous ms we were citing Koehler & Poulin (2010); ours is the first estimation of number of C. solearis cysts in P. argus lobsters. We found no relationship between lobster size and number of cysts by full dissection, so a larger number of cysts in larger organisms does not appear to apply to this system. Regardless, we included size as a covariate in the GLMs that we performed for the variables that were measured independently. We did this because most statistics books concur in that continuous variables such as size cannot be used as random effects but as covariates.

Discussion

I’ll mostly hold off on commenting on this section for now, as your results may change if you decide to incorporate my suggestions for additional analyses. Ultimately, I think what you have is great, but I think you could do a lot more with your data.

R9. Thank you very much for all your comments. The Discussion has indeed changed, mostly because of the detected mistake in the estimation of acceleration (and hence in force and work, which are derived from acceleration) for uninfected lobsters. Upon correcting the data, we found no significant effect of parasite intensity on any component of the escape response, and the Discussion was changed accordingly.

One note on the serotonin (5-HT) results:

Would elevated levels of 5-HT affect lobsters’ ability, or interest, to shelter together in casitas? How would this affect the fishery?

R10. It might, since in other decapods higher levels of 5-HT increase their “will to fight”. But whether this would affect the fishery remains to be determined. Therefore, we modified the Discussion like this (new Lines 464-469): “the higher concentration of 5-HT in moderately and heavily infected lobsters may cause these lobsters to become more aggressive and attempt to fight an attacker beyond their capabilities, potentially affecting the response of lobsters in predator-prey encounters. It may also affect the social behavior of lobsters, with unknown consequences for casita-based fisheries. A comparison of defense and antipredator behaviors between uninfected and infected lobsters should be conducted in future studies.”

WE THANK REVIEWER 1 FOR THE THOROUGH REVIEW AND HELPFUL COMMENTS.

Reviewer 2

General Comments:

R. We appreciate the positive feedback.

Abstract

• In line 30, change “their” to the hosts’

R. Done.

• Split lines 34 – 38 into 2 sentences, one about the physiological parameters studied and one about the behavioral assays conducted

R. Done.

R. The terms bolder and more aggressive have been deleted from the Abstract.

• Move the sentence in lines 44 – 45 up to right before the results

R. Done.

• Lines 44 – 47 don’t seem to fit in the abstract, or if the author feels they must be included, should be moved up to the methods part of the abstract instead of as the last sentence.

R. These lines were removed from the Abstract.

Introduction:

R. Thank you for your suggestions. The Introduction has been extensively rearranged.

• Add a sentence at the end of the first paragraph to also include behavioral impact of parasites on hosts because this study includes physiological and behavioral impacts

R. Thanks for the suggestion.

R. The diet of these lobsters is opportunistic and very diverse. They eat bivalves, gastropods, chitons (i.e. mollusks), cirripedes and all kinds of decapods, (crustaceans), ophiurids, urchins, sea cucumbers (echinoderms) and all kinds of annelids (worms). Because of this and because there is no trophic transmission of these trematodes from the first to the second intermediate host, we only mentioned the higher taxa.

• Lines 75-78 don’t seem relevant to the paper, unless the parasite can be transmitted between 2 lobsters, which seems very unlikely

R. These lines were intended to explain why casitas are used in some fisheries, such as the one from which we obtained the lobsters, and because, as suggested by Reviewer 1, the increase in serotonin in infected lobsters may alter their social behavior, which is now addressed in the Discussion.

Materials and Methods:

• Lines 114-116, depth of tanks?

R. Depth of tanks has been added.

• Line 117- is there a known range for salinity and temperature during the time of the experiments that could be included here?

R. Yes. The ranges have been added.

• Lines 124-126, include Ns for each

R. Done.

• Line 128, what is the N?

R. The sample size has been added.

• Line 136 – 137- how were the cysts quantified?

R. All cysts found in a lobster upon full dissection were counted.

• This response is called a tail flip in other crustaceans. Is it called that in lobsters?

R. Yes. This name has been added.

• Were the lobsters used in these experiments fully intact?

R. Yes, except for 4, which were lacking 1 appendix (leg or antenna), and 2 which lost it during the experiment.

• Lines 175 – 182 read like introduction material, not materials and methods, and should be moved accordingly. The information is pertinent to the paper

R. These lines were added here as an explanation of why we considered important to compare that characteristic. However, following your advice, we have moved a shortened version of all this material (and similar parts related to other characteristics) to the last part of the Introduction.

• Lines 212-214- the hepatopancreas also acts as a filter, removing toxins from the hemolymph

R. We have removed this piece of information.

• Lines 218-227 also read as introduction material and should be moved.

R. Same response as to Lines 175-182.

• Line 241 mentions these categories rendered samples of similar size. Does this mean the lobsters were of similar size or there was a similar N for all categories?

R. It means a similar N for all lobster groups. Sentence has been rephrased.

• No normality results are reported, so why was a Kruskal-Wallis test used?

R. We have changed all the statistical analyses to parametric tests. We now used MANOVAs and ANOVAs with a General Lineal Model (GLM) approach.

• If lobsters were all dissected and metacercariae counted post-mortem anyway, why not just use these data for all analyses instead of using the visual tests of alive lobsters at all?

R. In previous studies, prevalence of infection by C. solearis in wild lobsters was estimated only by visual assessment. We wanted to test how “accurate” these visual assessments are, because they could be used to conduct rapid assessments elsewhere. Therefore, we conducted the validity measures but find out that visual assessment missed many infected lobsters, so it is not a good method for rapid assessments.

Results:

R. These graphs have been changed because we found a mistake in the data processing, for which we apologize. Acceleration had been wrongly estimated (see response R7 to Reviewer 1). After correcting the mistake, no significant differences were found in any components of the escape response. Analyses and figures have now been corrected. However, we believe that, unlike column graphs with error bars, box plots show the actual distribution of the raw data, so they are a good choice when comparing groups.

• Same comment for figure 3.

R. Please see previous response.

Discussion

R. Thanks for this suggestion. We have added a photo of a lobster with the relevant names (Fig. 1 in revised ms).

• Adding transition sentences throughout the discussion would also aid in flow, similar to the introduction

R. The discussion has been changed to address the amended results. Care has been taken to enhance the flow between paragraphs.

R. Yes, it was the same authors. The sentence has been rephrased to improve clarity.

• Line 439- Orconectes virilis has been reclassified as Faxonius virilis

R. Thanks for catching this. The name has been corrected.

WE THANK REVIEWER 2 FOR THE THOROUGH REVIEW AND HELPFUL COMMENTS.

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PLoS One. doi: 10.1371/journal.pone.0287097.r003

Decision Letter 1

Hudson Alves Pinto

18 May 2023

PONE-D-23-02958R1Effects of Cymatocarpus solearis (Trematoda: Brachycoeliidae) on its second intermediate host, the Caribbean spiny lobster Panulirus argusPLOS ONE

Dear Dr. Briones-Fourzán,

Please submit your revised manuscript by Jul 02 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Hudson Alves Pinto, Ph.D

Academic Editor

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I congrats the authors for the excelent revision.

Some final interesing comments are presented by Reviewer 1.

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PLoS One. 2023 Sep 29;18(9):e0287097. doi: 10.1371/journal.pone.0287097.r004

Author response to Decision Letter 1

25 May 2023

Manuscript Number: PONE-D-23-02958

Manuscript Title: Effects of Cymatocarpus solearis (Trematoda: Brachycoeliidae) on its second intermediate host, the Caribbean spiny lobster Panulirus argus

Response to authors’ revisions:

I have read the authors’ response-to-reviewers document and the revised manuscript, which overall is much improved. The authors have done a thorough job with their revisions. In the process of reanalyzing the data, they uncovered and corrected a typo in the original dataset that would have affected their results and discussion. They also switched their primary method of analysis from hypothesis testing (i.e., Kruskal-Wallis) to regression-based methods, which are more comprehensive and informative. Parts of the Introduction and Discussion were reorganized as needed based on feedback from both reviewers.

At this point, I have only minor requests for clarification, primarily with how the stats are reported. I am trying to be as helpful as possible, as I think the work is interesting and very informative!

R. We appreciate the positive feedback.

• Methods

You mention that you used general linear models (line 314) as opposed to generalized linear models. The former assumes that the residuals from your dependent variable are normally distributed while the latter does not. Parasite count data is often non-normal…I believe your dependent variable was parasite intensity? (line 315). I think it would help to clarify that the residuals of your data were normally distributed and then report that prior to discussing how you used GLMs. Otherwise, you should switch to using generalized linear models, which are preferred for modeling data with non-normal residuals.

R. As previously explained, parasite intensity was not the dependent variable; it was the factor (independent variable) whose effect was tested on several response variables. That is, parasite intensity was considered as a categorical factor with four levels: uninfected, lightly infected, moderately infected, and heavily infected lobsters. A GLM MANOVA tested the effect of this factor (parasite intensity) on a combination of the four metabolites (albumin, cholesterol, glucose, and total protein) measured in the hemolymph of lobsters on the one side. Another tested the effect of parasite intensity on a combination of the seven components of the escape response of lobsters on the other side. The rest of the variables were individually compared between lobster groups with separate GLMs.

If you stick with the general linear models, I might suggest reporting a few other test statistics for your ANCOVA results. By themselves, p-values convey very little information about the magnitude of effect, i.e., how powerful, or significant, your results are. I would suggest including information on marginal means and their associated 95% confidence intervals and reporting this for each significant effect in your results. Here’s some additional information on reporting CIs and marginal means for ANCOVAs: https://www.medcalc.org/manual/analysis-of-covariance.php

R. Thanks for this suggestion. We have added a large supplementary table (S1 Table) with the means and their 95% confidence intervals for all the response variables that we measured and compared among the four groups of lobsters (uninfected, lightly infected, moderately infected, heavily infected).

• Other

I think it would help to have a final concluding sentence in your Abstract that kind of ties things together, a more general statement about how your results apply more broadly. You have something like this in your conclusion section (p. 29) around lines 626-629.

R. OK. We have changed the final sentence of the Abstract to the following: “As changes in 5-HT concentration can modify the host’s activity patterns or choice of microhabitat, our results suggest that infection with C. solearis may alter the behavior of spiny lobsters, potentially increasing the likelihood of trophic transmission of the parasite to the definitive host.”

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PLoS One. doi: 10.1371/journal.pone.0287097.r005

Decision Letter 2

Hudson Alves Pinto

30 May 2023

Effects of Cymatocarpus solearis (Trematoda: Brachycoeliidae) on its second intermediate host, the Caribbean spiny lobster Panulirus argus

PONE-D-23-02958R2

Dear Dr. Briones-Fourzán,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Hudson Alves Pinto, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I congrats the Authors for their efforts in the revision. The MS is now suitable for publication and will be is an important cientific contribution for the field.

PLoS One. doi: 10.1371/journal.pone.0287097.r006

Acceptance letter

Hudson Alves Pinto

1 Jun 2023

PONE-D-23-02958R2

Effects of <i>Cymatocarpus solearis<i/> (Trematoda: Brachycoeliidae) on its second intermediate host, the Caribbean spiny lobster Panulirus argus

Dear Dr. Briones-Fourzán:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Hudson Alves Pinto

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Response variables of Panulirus argus lobsters in four grades of infection by Cymatocarpus solearis.

Means and [95% confidence intervals] of all variables compared among four groups of lobsters categorized by the presence and intensity of infection by C. solearis (number of metacercarial cysts).

(DOCX)

Click here for additional data file.^{(15.7KB, docx)}

S1 Appendix. Full dataset used in the article.

(XLSX)

Click here for additional data file.^{(59.8KB, xlsx)}

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Effects of Cymatocarpus solearis (Trematoda: Brachycoeliidae) on its second intermediate host, the Caribbean spiny lobster Panulirus argus

Tomás Franco-Bodek

Cecilia Barradas-Ortiz

Fernando Negrete-Soto

Rossanna Rodríguez-Canul

Enrique Lozano-Álvarez

Patricia Briones-Fourzán

Roles

Abstract

Introduction

Fig 1. Ventral view of a male Panulirus argus.

Materials and methods

Experimental lobsters

Ethics statement

Comparison of lobster characteristics

Swimming performance (escape response)

Growth rate

Concentration of biogenic amines

Nutritional condition: Concentration of metabolites

Nutritional condition: Hepatosomatic index

Total hemocyte count

Statistical analyses

Relationship between number of metacercariae and lobster size

Validity measures for the visual assessment of infection by C. solearis

Results

Size and weight of lobsters

Fig 2. Body dimensions, growth rate, and hepatosomatic index of lobsters.

Growth rate and hepatosomatic index

Table 1. Effects of lobster size and parasite intensity on traits of Panulirus argus.

Swimming performance (escape response)

Fig 3. Escape response of lobsters.

Concentration of metabolites in hemolymph

Fig 4. Components in hemolymph of lobsters.

Concentration of DA and 5-HT in hemolymph and hemocyte count

Validity measures of visual assessment of C. solearis

Table 2. Validity measures of visual assessment of infection.

Number of cysts vs lobster size

Fig 5. Lobster size versus number of parasites.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Hudson Alves Pinto

Roles

Author response to Decision Letter 0

Decision Letter 1

Hudson Alves Pinto

Roles

Author response to Decision Letter 1

Decision Letter 2

Hudson Alves Pinto

Roles

Acceptance letter

Hudson Alves Pinto

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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