Infestation of two shrimp species of the genus Palaemon Fabricius , 1798 ( Decapoda , Palaemonidae ) by an isopod of the genus Probopyrus Giard & Bonnier , 1888 ( Bopyridae ) from the Brazilian southeast coast

We determined the infestation rate of Probopyrus sp. in populations of Palaemon pandaliformis (Stimpson, 1871) and P. northropi (Rankin, 1898) in the Ubatumirim River, localized in a mangrove ecosystem on Ubatumirim Beach, northern coast of the state of São Paulo, Brazil. Samplings were carried out monthly from April 2003 to March 2004. Monthly prevalence varied from 0 to 4.94 % for P. pandaliformis, and from 0 to 4.54 % for P. northropi. This is the first record of Probopyrus sp. infesting the studied species in this region. Species of Probopyrus (Giard and Bonnier, 1888) seem to have a high plasticity with regard to palaemonid hosts, as they can be parasites of shrimps in both Palaemon (Fabricius, 1798) and Macrobrachium (Spence Bate, 1868). The linear relationships between the parasite and host sizes suggest that the parasite infests both hosts early in their development. We concluded that the infestation of Probopyrus sp. has little impact on Palaemon populations, mostly due to the low prevalence of infestation. original arTiCle Nauplius, 26: e2018026 CORRESPONDING AUTHOR Bruno Gabriel Nunes Pralon pralonbra@gmail.com SUBMITTED 06 February 2018 ACCEPTED 09 July 2018 PUBLISHED 03 December 2018 DOI 10.1590/2358-2936e2018026 All content of the journal, except where identified, is licensed under a Creative Commons attribution-type BY. orcid.org/0000-0002-3733-3955 orcid.org/0000-0002-4434-779X orcid.org/0000-0002-8070-8089


inTroduCTion
Caridean shrimps of the genus Palaemon Fabricius, 1798 are components of estuarine food webs (Bass and Weis, 1999;Mortari and Negreiros-Fransozo, 2007).These crustaceans have their lives adversely affected in many ways when parasitized by isopods belonging to the family Bopyridae (Ocaña-Luna et al., 2009).
Bopyrid isopods are obligate hematophagous ectoand holoparasites of crustaceans (Chaplin-Ebanks and Curran, 2007;Boyko and Williams, 2009;Williams and Boyko, 2012).They have complex life cycles that require a decapod as a definitive host, and a calanoid copepod as an intermediate host (Anderson, 1990;Lester, 2005;Oliveira and Masunari, 2006;Boyko and Williams, 2009;Sherman and Curran, 2015).An adult female bopyrid is typically found attached to the gills inside the branchial chamber, while the dwarf adult male is often found attached to the ventral side of the female bopyrid (Beck, 1980;Choong et al., 2011;Sherman and Curran, 2015).Parasites induce visible lateral swellings on the carapace of the host and this deformation on the branchiostegite region characterizes the host-parasite relationship (Choong et al., 2011).
Bopyrids feed on the host's haemolymph and disrupt the normal mechanical functioning of the gills, impairing the respiration and metabolism of shrimps (Choong et al., 2011;Dale and Anderson, 1982).Shrimps parasitized by bopyrids may become reproductively impaired as both male and female individuals may become sexually sterile or show reduced fecundity, and male individuals can become somewhat become feminized due to their reduced chelae length (Bass and Weis, 1999).Female shrimps often suffer parasitic castration by having the full maturation of their ovaries inhibited (Rocha and Bueno, 2000).Thus, the deleterious effects of bopyrid infestation may act as an important factor regulating some decapod populations (Varisco and Vinuesa, 2011).
Among caridean shrimps, the family Palaemonidae is the main group with species acting as hosts for bopyrid isopods of the genus Probopyrus Giard and Bonnier, 1888, whose distribution is therefore tied to that of the Palaemonidae (Masunari et al., 2000).According to Lemos de Castro and Brasil-Lima (1974), on the Brazilian coast there are three species of Probopyrus: Probopyrus bithynis Richardson, 1904, P. palaemoni Lemos de Castro and Brasil-Lima, 1974and P. floridensis Richardson, 1904.As the same authors emphasize, P. bithynis infests several palaemonid species, including Palaemon pandaliformis (Stimpson, 1871) and Palaemon northropi (Rankin, 1898), while P. palaemoni has been recorded parasitizing P. pandaliformis.Nevertheless, despite the wide geographical distribution and phylogenetic divergencies showed by P. pandaliformis and P. northropi (see Carvalho et al., 2017 for details), extending from Central America to Southern South America (Holthuis, 1952), these species have never been recorded carrying bopyrid parasite on the northern coast of São Paulo State, Brazil.
Palaemon pandaliformis and P. northropi are abundant in the estuaries located along the southeastern Brazilian coast (Anger and Moreira 1998).They play an important role in the food web serving as prey for numerous predators.Presence of bopyrid ectoparasites in populations of those shrimps can potentially affect local reproduction.Thus, our study aimed to report for the first time the infestation of Probopyrus sp. in populations of P. pandaliformis and P. northropi from the Ubatuba region, northern coast of the state of São Paulo, Brazil.In addition, we commented on some aspects of their host-parasite relationships.Colpo et al. (2011).We conducted the samplings in the morning, during low tide, collecting samples within a 10 m stretch along the riverbank.We used a sieve (3 mm mesh), passing it under the submerged vegetation.For further information about sampling procedures see Mortari et al. (2009).

Specimens
We utilized plastic bags to pack samples, which were labeled with the date and site of the collection.Specimens were taken to the laboratory where they were identified according to Melo (2003), and preserved in 70% ethanol for later analysis.Voucher specimens were deposited in the crustacean collection of the Department of Zoology, IB, UNESP, Botucatu (NEBECC AD # 0001; AD # 0002).Carapace length (CL = from the posterior margin of the orbit to the posterior margin of the carapace) of each shrimp was measured under a stereomicroscope provided with a micrometer scale (mm).Sex evaluation was based on the presence (male) or the absence (non-ovigerous female) of an appendix masculina on the endopods of the second pair of pleopods.
Parasites were identified under a dissecting microscope, according to available literature (Lemos de Castro and Brasil-Lima, 1974;Richardson, 1904).Photographs of the ectoparasite and its host were taken with a Zeiss Stemi SV6 stereoscopic microscope.Parasites (female and male) were measured for total length (TL) and width (TW) to the nearest 0.01 mm with a stereoscopic microscope, provided with a micrometer scale.Male parasites are symmetrical in shape and were measured from the anterior edge of the cephalon to the posterior edge of the pleotelson, while females are asymmetrical in shape and were measured along the longest side from the anterior margin of the body to the end of the pleotelson (Cash and Bauer, 1993).Immature and mature parasites were differentiated according to Conner and Bauer (2010).The use of the term "prevalence" followed Margolis et al. (1982), and expressed the percentage of infested shrimps present in the sampled population.Linear regressions of parasite body size (TL, dependent variable) vs. host body size (CL, independent variable) were calculated by using the least square method, with a significance level of 5% (Wilkinson, 1988).

resulTs
We examined 2,878 P. pandaliformis (CL ranging from 1.5 to 5.8 mm) and 486 P. northropi (CL ranged from 1.9 to 9.6 mm), from which seventeen were from the former species (CL: 3.12 -4.49mm) and five from the latter one (CL: 3.75 -5.74 mm) that were parasitized by Probopyrus sp.(Figs.1A-B; Tab. 1).The monthly prevalence was low throughout the study and varied from 0 to 4.94% (mean value = 0.8 ± 1.39) for P. pandaliformis and from 0 to 4.54% (mean value = 0.8 ± 1.63) for P. northropi (Tab.2).All bopyrid isopods removed from their hosts matched the morphological characteristics described by Richardson (1904) for Probopyrus sp. (Fig. 1C).Only four parasites were breeding (females with eggs in the marsupium).Two of those specimens, obtained in November 2003, were parasitizing P. pandaliformis, and the other two, obtained in August 2003 and November 2003, were parasitizing P. northropi (Tab.1).Unfortunately, most eggs of the breeding females were lost, so the determination of fecundity was not possible.However, we could observe that the eggs were still at an early stage of development, as they were full of yolk and with no evidence of developing embryos.Each female had a dwarf male of yellow coloration attached to its pleon (Fig. 1D).In the present study, infestation by Probopyrus sp. was observed mostly in males of P. pandaliformis (Tab.1).We found a positive correlation between the TL of the parasites and the CL of the shrimp hosts (P.northropi n = 5; R 2 = 0.97; p < 0.01; P. pandaliformis n = 17; R 2 = 0.54; p < 0.01) (Fig. 2).We also found the proportion of parasitized specimens varied during the study period for both host populations.There were no bopyrids for at least five months of the year for either host species (see Tab.2).We record here, for the first time, the infestation of P. pandaliformis and P. northropi by Probopyrus sp. on the southeast coast of Brazil.According to Ocaña-Luna et al. (2009), bopyrids of the genus Probopyrus typically infest palaemonid prawns from the following genera: Macrobrachium Spence Bate, 1868 and Palaemon.In South America, the association of P. floridensis with the host Palaemon paludosus (Gibbes, 1850) (as Palaemonetes exilipes) has been reported by Lemos de Castro and Brasil-Lima (1974), and the same parasite infested Macrobrachium potiuna (Muller, 1880) according to Masunari et al. (2000), and Rocha and Bueno (2000).Two more palaemonid species are here recorded as hosts of a Probopyrus species.Information on host specificity of bopyrids and their geographic distribution are scarce for most species (Romero-Rodríguez et al., 2017).Even when studies have addressed ecological information of the hosts, reports about their parasites were often neglected or omitted (Romero-Rodríguez et al., 2017).Williams and Boyko (2012) mentioned that the absence of some parasitic groups, in certain areas, might be related to the lack of sampling.
Ectoparasitism in palaemonid shrimps by Probopyrus sp. is not host species-specific, or even host genusspecific, since infestations by species of Probopyrus have been reported for five different host species (Tab.3).The same pattern was verified for Probopyrus bithynis (as P. pandalicola Packard, 1879) that was recognized to infest several Macrobrachium species (Markham, 1985).Infestation on Palaemon species has been previously reported for P. pandaliformis by both P. palaemoni and P. bithynis (Dale and Anderson, 1982;Markham, 1985).The latter parasite also infests P. northropi, as mentioned by Lemos de Castro and Brasil-Lima (1974).
We cannot accurately identify the species of the Probopyrus specimens obtained, mainly because we only had adult individuals.Dale and Anderson (1982) described Probopyrus larvae from northwest Atlantic and found that it is possible to distinguish three species based on larval features: P. pandalicola, P. floridensis and P. bithynis.According to Markham (1985), these species cannot be distinguished in the adult stage.
Although the relationship of Probopyrus with a palaemonid host has already been reported in the state of São Paulo (Rocha and Bueno, 2000), our study expanded the distribution of this bopyrid genus, adding a new locality on the southeast region of Brazil, in the Ubatumirim River.In general, the range of distribution of Probopyrus sp. is narrower than that of its host species (Ocaña-Luna et al, 2009).In the present study, the distribution area of Probopyrus sp. was extended to the northern coast of São Paulo State, although the parasite distribution remains more limited than that of its hosts.
The mean prevalence of Probopyrus sp. in both Palaemon species hosts during monthly samplings was relatively low, as it was also found by Rocha and Bueno (2000).On the other hand, Masunari et al. (2000) found a higher value of prevalence for P. floridensis in M. potiuna.According to these authors, monthly infestation varied from 1.1% to 91.7% throughout the year, when taking into account all life cycle stages of the parasites, in Paranaguá Bay, state of Paraná, Brazil.The differences between our results and those from other studies in the South Atlantic area might be related to numerous factors, such as density of cryptoniscid larvae, massive recruitment of hosts during specific periods of the year, natural mortality of shrimps, and the annual life cycle of the parasites.Further studies should be done to make a detailed examination of this host/ parasite relationship.We found that the proportion of parasitized specimens varied during the study period for both host populations.There was an absence of bopyrids for at least five months of the year for both host species.As noted by Masunari et al. (2000), we suspect that the reproduction of Probopyrus sp.occurs once a year in the Ubatumirim estuary.However, our data on monthly prevalence do not support the conclusion that the parasite's reproduction is seasonal for both shrimp species from the study area.
In the present study, infestation by Probopyrus sp.occurred mostly in males of P. pandaliformis, differently from studies conducted on other carideans, in which parasite prevalence was either higher in female hosts (Chaplin-Ebanks and Curran, 2007;Rasch and Bauer, 2015) or equivalent for both sexes ( Jarrin and Shanks, 2008).With respect to P. northropi, it is not possible to conclude on its host sex-specific prevalence due to the low number of parasites found in the collected specimens.
In other decapod species with sexual dimorphism associated with body size, bopyrid infestation can be higher in larger hosts ( Jordá and Roccatagliata, 2002).When there are no apparent morphological differences, the parasites may infest both sexes at the same proportion (Blower and Roughgarden, 1988).Thus, in the case of crustacean hosts, bias for males in a host-parasite system could be attributed to differences in sexual selection pressure.An experimental test with a copepod revealed a male-biased susceptibility to helminth infection attributed to sexual selection (Wedekind and Jakobsen, 1998).Although sexual selection on Palaemon species has not been detailed yet, a possible explanation for male bias is that males experience higher intra-sexual selection.Thus, their energetic resources are used up more rapidly, and this can cause a reduced ability to avoid infestation (Wedekind and Folstad, 1994).
Our data indicate that specimens of P. pandaliformis and P. northropi may be parasitized in the juvenile stage.The size of the hosts correlated positively with the size of the parasites (Fig. 2); the largest specimens of Probopyrus sp.(larger than 1 mm TL) were found in larger shrimps (larger or equal to 3.97 mm CL).Cryptoniscid larvae usually infest shrimps during the host's early developmental stages and grow together with the hosts, although this is not always the case; in rare cases, bopyrids may infest older and larger shrimp hosts (Conner and Bauer, 2010).Video observations of P. pandalicola infesting Palaemon pugio (Holthuis, 1949) suggest that most bopyrids must survive through the ecdysis of the host (Cash and Bauer, 1993).
In summary, our results indicate that there is a low and variable prevalence of Probopyrus sp. on both sympatric host populations.Thus, it is suggested that these infestations may not negatively affect the presently studied Palaemon species.However, for better understanding the role played by this bopyrid in Palaemon population dynamics, further studies in the field and laboratory are still necessary.aCKnowledgMenTs

Table 1 .
Number of specimens and carapace length (CL) of males (M) and females (F) of Palaemon pandaliformis and P. northropi infested by Probopyrus sp.Developmental stage, total length (TL) and total width (TW) of males and females of Probopyrus sp.

Table 2 .
Prevalence of Probopyrus sp. in Palaemon species at the Ubatumirim River estuary, southeast coast of Brazil.