Morphology and distribution of two epizoic diatoms ( Bacillariophyta ) in Brazil

(Morphology and distribution of two epizoic diatoms (Bacillariophyta) in Brazil). The epizoic diatoms Pseudohimantidium pacificum and Falcula hyalina, which live on copepods, were investigated using light and electron microscopes, based on material gathered from different marine environments along the Brazilian coast. Pseudohimantidium pacificum is reported for the first time for the Southwestern Atlantic Ocean, significantly enlarging its range of geographic distribution. This species usually covers the entire body surface of the copepods Corycaeus amazonicus and Euterpina acutifrons, and of cypris larvae of Cirripedia. Falcula hyalina uses a higher number of copepod hosts, particularly Oithona oswaldocruzii, Pseudodiaptomus richardii and Acartia spp. The valve morphology and biometrical data of both diatoms were within the range limits recorded in the literature, including the original publications. Both species occurred in all the sampling stations along the Brazilian coastline stretching from 12°S down to 28°S. Falcula hyalina had already been found as far as latitude 31°S in the Southwestern Atlantic Ocean.


Introduction
The exoskeleton of crustacean copepods constitutes a convenient habitat for a variety of epibiont microorganisms such as bacteria, microalgae and protozoans (Carman & Dobbs 1997;Walkusz & Rolbiecki 2007).Among these organisms, pennate diatoms have been commonly reported growing on different parts of the body of copepods (Carman & Dobbs 1997).Originally revealed by Giesbrecht (1892) in the Adriatic Sea, epizoic diatoms have since then been recorded in different regions around the world.They are widespread in the Pacific Ocean along the Sea of Japan to the coasts of South America (Hiromi et al. 1985;Rivera et al. 1986).Several sparse records were made from both margins of the United States and Mexico, Caribbean, Mediterranean Sea and East Africa (Voigt 1960;1961;Gibson 1979a;1979b;Navarro 1982;Prasad et al. 1989;Garate-Lizarraga & Muñeton-Gomez 2009).
In this report, we describe two species of epizoic diatoms and their occurrence on the surface of the body of copepods, which were detected in samples from different environments along the Brazilian coast.Pseudohimantidium pacificum and Falcula hyalina were examined using light and electron microscopes to detect possible morphological and metric differences compared to other specimens described in the literature.

Material and methods
The sample collection (n=30) used in this study was gathered in different localities along the Brazilian coastline (12°S to 28°S), between 1994 and2011 (Tab.1).Only the material sampled in Paranaguá Bay, Paraná State, had the host copepods identified to the species level.Zooplankton was collected using plankton nets with a 300μm mesh size.Each sample was screened using a stereomicroscope to detect copepods with epizoic diatoms.Individuals were picked up with a micropipette and placed in 15 ml centrifuge tubes for further processing of the diatom frustules.
The diatom cells and the copepods were washed in distilled water followed by cleaning of frustules from the organic matter according to the technique of Hasle & Fryxell (1970).Permanent slides were mounted using Naphrax (refractive index = 1.74) as a mounting medium.For light microscopy (LM), specimens were measured and photographed using an Olympus BX-51 microscope.Samples for scanning electron microscopy (SEM) were prepared by adding a drop of sample onto aluminum stubs, which were air-dried and sputter-coated with gold.Samples were examined using a Jeol-JSM 6360LV scanning electron microscope at an accelerating voltage of 20 kV.Terminology followed Ross et al. (1979), with the additions of Round et al. (1990).A literature search was facilitated by consulting Gaul et al. (1993) and Henderson & Reimer (2003).
The species occurred in all the samples examined (Tab.1).In the samples collected in Paranaguá Bay, the cells of P. pacificum appeared in high abundance on the copepods Corycaeus amazonicus Dahl F. and Euterpina acutifrons Dana; a few cells were also detected living on a cypris larva of Cirripedia.Diatoms occupied almost all the available copepod exoskeleton, especially the locomotory appendages and antennae, though a few cells were attached on the dorsal surface.

Discussion
Epizoic diatoms have been recorded from different oceans around the world, despite the peculiar habitat they grow in.Surprisingly, only a few publications have reported these interesting diatoms for the South Atlantic Ocean.This is the first report of Pseudohimantidium pacificum from the Southwestern Atlantic Ocean, found along the Brazilian coast (12°S to 28°S).The species was recorded in the North Atlantic from the Caribbean Sea to the east coast of North America, the Pacific Ocean and the Mediterranean Sea (Gibson 1979a;Hiromi et al. 1985;Garate-Lizarraga & Muñeton-Gomez 2009).
The other species found in this study, Falcula hyalina, had been reported only for the Pacific Ocean (Japan and Western Australia) and the North Atlantic Ocean (Gulf of Maine, Florida) (Takano 1983;Hiromi et al. 1985;Prasad et al. 1989).Regarding its distribution in Brazil, F. hyalina was previously known only from the Bay of Tijucas in the state of Santa Catarina (Souza-Mosimann et al. 1989) and the Lagoon of Peixe (31°00'46"S, 51°09'51"W) in the state of Rio Grande do Sul (Donadel & Torgan 2010), the latter corresponding to the most austral report of F. hyalina in the Atlantic Ocean.In both of these places, the authors reported F. hyalina living on the copepod Acartia lilljeborgii Giesbrecht.In our material, both species were found in all the samples examined from the Brazilian coastline, encompassing a wide variety of environments, such as estuaries, bays and coastal waters, thus suggesting that their geographic distribution might be wider than previously recorded.Regarding the material gathered from Paranaguá Bay, Paraná, where the copepods and other crustaceans were identified to the species level, the epizoic diatoms presented some degree of preference in relation to the copepod hosts.Pseudohimantidium pacificum was on Corycaeus amazonicus and Euterpina acutifrons, confirming what has been reported in the literature already.However, Falcula hyalina was growing on a higher number of hosts, such as Acartia lilljeborgii, Acartia tonsa, Oithona oswaldocruzii and Pseudodyaptomus richardi.From these copepods, only A. tonsa had been recorded as a host for F. hyalina; thus, our findings add three new hosts to the list of Hiromi et al. (1985).To date, the copepods mentioned above correspond to the most abundant species of the year-round zooplankton community of Paranaguá bay and adjacencies (Lopes et al., 1998).
Biometrical data of P. pacificum from the Brazilian material agree well with the original (Krasske 1941) and other later publications (Simonsen 1970;Gibson 1978;Rivera et al. 1986).To date, in Krasske's (1941) original material the apical axis varied from 44 μm to 78 μm, while the transapical axis was 9-11 μm.However, the number of striae (30-32 in 10 μm) was lower than in Brazilian specimens (30-40 in 10 μm).Furthermore, Rivera et al. (1986) found a larger range for the transapical axis, 9.8 μm to 19.0 μm, and the number of striae as low as 24 in 10 μm.Several discrete morphological differences were found in the Brazilian specimens compared with what has been published in the literature.For instance, there were 4-10 rimoportulae in the valves from Brazil, while other authors recorded a maximum of nine (Russel & Norris 1971;Rivera et al. 1986).Moreover, some valves of our material presented the external opening of rimoportulae almost orthogonal to the ventral margin instead of being parallel as usually observed elsewhere.
The valves of Falcula hyalina examined in this work fit well into the dimensions published for other regions (Gibson 1978;Hiromi et al. 1985;Prasad et al. 1989).Measurements reported in the original description of Takano (1983) are 20-38 μm apical axis, 3.9-5.5 μm transapical axis and 20-23 striae in 10 μm.Regarding the earlier reports from Brazil, our material has similar dimensions to the specimens investigated by Souza-Mosimann et al. (1989) and Donadel & Torgan (2010) from Southern Brazil.