ABSTRACT

This study aimed to assess population and reproductive aspects of the ampithoid amphipod Cymadusa filosa Savigny, 1816 in a region with pollution by petrogenic hydrocarbons. Sampling of Sargassum furcatum Kützing, 1843 fronds and the associated fauna was conducted in March, September and December 2007 and February 2008, on four rocky shores located on São Sebastião Island, southeastern Brazil. A total of 1372 specimens of C. filosa were recorded, including 934 juveniles, 274 females (60 ovigerous) and 164 males. Population mean density ranged from 1.27 to 6.40 ind/g with higher mean values in March 2007 and lower in December 2007 and February 2008. The total body length of males varied from 4.11 to 20.75 mm and of females from 3.58 to 20.22 mm. No significant difference was detected between male and female body length. The overall sex ratio significantly differed from 1:1 proportion and was skewed toward females (0.60:1). Ovigerous females occurred in higher proportion in February 2008 and presented mean fecundity of 26.01 ± 1.84 eggs/female (mean ± se), with great variation in the number of eggs found in the marsupium (between 1 and 56). Fecundity increased significantly with increasing female body length. The C. filosa population structure and reproduction were similar to those of populations in other locations, in Brazil and abroad indicating that pollution by petrogenic hydrocarbons in the São Sebastião Channel did not affect the population parameters of C. filosa in this region.

KEYWORDS:
Peracarida; amphipod; Ampithoidae; reproduction; petroleum

RESUMO

Este estudo teve como objetivo avaliar aspectos populacionais e reprodutivos do anfípode ampitoídeo Cymadusa filosa Savigny, 1816 em uma região com poluição por hidrocarbonetos petrogênicos. Amostragens de frondes de Sargassum furcatum e da fauna associada foram realizadas em março, setembro e dezembro de 2007 e fevereiro de 2008, em quatro costões rochosos localizados na Ilha de São Sebastião, sudeste do Brasil. Um total de 1372 espécimes de C. filosa foi registrado, incluindo 934 juvenis, 274 fêmeas (60 ovígeras) e 164 machos. A densidade populacional média variou de 1,27 a 6,40 ind/g, com valores médios maiores em março de 2007 e menores em dezembro de 2007 e fevereiro de 2008. O comprimento corporal total dos machos variou de 4,11 a 20,75 mm e das fêmeas de 3,58 a 20,22 mm. Nenhuma diferença significativa foi detectada entre o tamanho do corpo de machos e fêmeas. A razão sexual geral diferiu significativamente da proporção de 1:1 e foi desviada para as fêmeas (0,60:1). Fêmeas ovígeras ocorreram em maior proporção em fevereiro de 2008 e apresentaram fecundidade média de 26,01 ± 1,84 ovos/fêmea (média ± erro padrão), com grande variação no número de ovos encontrados no marsúpio (entre 1 e 56). A fecundidade aumentou significativamente com o aumento do comprimento corporal das fêmeas. A estrutura populacional e a reprodução de C. filosa foram semelhantes às de populações de outras localidades no Brasil e no exterior, indicando que a poluição por hidrocarbonetos petrogênicos no Canal de São Sebastião não afetou os parâmetros populacionais de C. filosa nesta região.

PALAVRAS-CHAVE:
Peracarida; anfípode; Ampithoidae; reprodução; petróleo

Amphipods are the most representative group of peracarids among the invertebrates that are found associated with different species of macrophytes including macroalgae and seagrasses (Mukai, 1971Mukai, H. 1971. The phytal animals on the thalli of Sargassum serratifolium in the Sargassum region, with reference to their seasonal fluctuations. Marine Biology 8:170-182.; Edgar, 1983a Edgar, G. J. 1983a. The ecology of south-east Tasmanian phytal animal communities. II. Seasonal change in plant and animal populations. Journal of Experimental Marine Biology and Ecology 70:159-179.; Norton & Benson, 1983Norton, T. A. & Benson, M. R. 1983. Ecological interactions between the brown seaweed Sargassum muticum and its associated fauna. Marine Biology 75:169-177.; Gunnill, 1985Gunnill, F. C. 1985. Growth, morphology and microherbivore faunas of Pelvetia fastigiata (Phaeophyta, Fucaceae) at La Jolla, California, USA. Botanica Marina 28:187-199.) in all the coastal regions of the world. Associated with brown algae of the genus Sargassum C. Agardh, 1820, which occur in both temperate and tropical regions, amphipods and molluscs generally represent the group with the highest species richness and abundance (Edgar, 1983aEdgar, G. J. 1983a. The ecology of south-east Tasmanian phytal animal communities. II. Seasonal change in plant and animal populations. Journal of Experimental Marine Biology and Ecology 70:159-179.; Leite & Turra, 2003Leite, F. P. P & Turra, A. 2003. Temporal variation in Sargassum biomass, Hypnea epiphytism and associated fauna. Brazilian Archives of Biology and Technology 46(4):665-671.; Jacobucci et al., 2009Jacobucci, G. B.; Tanaka, M. O. & Leite, F. P. P. 2009. Temporal variation of amphipod assemblages associated with Sargassum filipendula (Phaeophyta) and its epiphytes in a subtropical shore. Aquatic Ecology 43:1031-1040.; Leite, 2011Leite, F. P. P. 2011. Biodiversidade - Amphipoda. In: Amaral, A. C. Z. & Nallin, S. A. H. org. Biodiversidade e Ecossistemas bentônicos Marinhos do Norte do Estado de São Paulo - Sudeste do Brasil. Campinas, UNICAMP/IB, p. 133-143.<Available at https://econtents.bc.unicamp.br/omp/index.php/ebooks/catalog/book/13 >. Accessed on 04 May 2021
https://econtents.bc.unicamp.br/omp/inde... ; Longo et al., 2019Longo, P. A. D. S.; Mansur, K. F. R.; Leite, F. P. P. & Passos, F. D. 2019. The highly diverse gastropod assemblages associated with Sargassum spp. (Phaeophyceae: Fucales) habitats. Journal of the Marine Biological Association of United Kingdom 99:1295-1307.). Amphipods associated with Sargassum and other marine macroalgae represent an important link between these producers and higher trophic levels. They are an important food source for many coastal fishes including species of economic importance (Nakamura et al., 2003Nakamura, Y.; Horinouchi, M.; Nakai, T.; Sano, M. 2003. Food habits of fishes in a seagrass bed on a fringing coral reef at Iriomote Island, southern Japan. Ichthyological Research 50:15-22.).

In Brazil, especially on the north coast of state of São Paulo in southeast Brazil, there are extensive beds of different species of Sargassum (Széchy & Paula, 2000Széchy, M. T. M. & Paula, E. J. 2000. Padrões estruturais quantitativos em bancos de Sargassum (Phaeophyta, Fucales) do litoral dos Estados do Rio de Janeiro e São Paulo, Brasil. Revista Brasileira de Botânica 23:121-132.) where about 30 species of associated amphipods have already been found, with high abundances and different life and feeding habits (Wakabara et al., 1983Wakabara, Y.; Tararam, A. S. & Takeda, A. M. 1983. Comparative study of the amphipod fauna living on Sargassum of two Itanhaém shores, Brazil. Journal of Crustacean Biology 3:602-607.; Leite et al., 2000Leite, F. P. P.; Güth, A. Z. & Jacobucci, G. B. 2000. Temporal comparison of gammaridean amphipods of Sargassum cymosum on two rocky shores in southeastern Brazil. Nauplius 8(2):227-236, 2007Leite, F. P. P.; Tanaka, M. O. & Gebara, R. S. 2007. Structural variation of the brown alga Sargassum cymosum and its effects on associated amphipod assemblages. Brazilian Journal of Biology 67(2):215-221.; Tanaka & Leite, 2003Tanaka, M. O. & Leite, F. P. P. 2003. Spatial scaling in the distribution of macrofauna associated with Sargassum stenophyllum (Mertens) Martius: analyses of faunal groups, gammarid life habits, and assemblage structure. Journal of Experimental Marine Biology and Ecology 293(1):1-22.; Jacobucci et al., 2009Jacobucci, G. B.; Tanaka, M. O. & Leite, F. P. P. 2009. Temporal variation of amphipod assemblages associated with Sargassum filipendula (Phaeophyta) and its epiphytes in a subtropical shore. Aquatic Ecology 43:1031-1040.).

Among the amphipods associated with Sargassum, representatives of the Ampithoidae family are generally represented by larger species that use algae as food and to build their shelters for reproduction (Appadoo & Myers, 2003Appadoo, C. & Myers, A. A. 2003. Observations on the tube-building behavior of the marine amphipod Cymadusa filosa Savigny (Crustacea; Ampithoidae). Journal of Natural History 37(18):2151-2164.; Jacobucci & Leite, 2008Jacobucci, G. B.; Güth, A. Z. & Leite, F. P. P. 2008. Experimental evaluation of amphipod grazing over biomass of Sargassum filipendula (Phaeophyta) and its dominant epiphyte. Nauplius 16:65-71; Tavares et al., 2013Tavares, M. R.; Grande, H. & Jacobucci, G. B. 2013. Habitat and food selection by herbivorous amphipods associated with macroalgal beds on the southeast coast of Brazil. Nauplius 21:9-15.; Machado et al., 2017Machado, G. B. O.; Siqueira, S. G. L. & Leite, F. P. P. 2017. Abundance, performance, and feeding preference of herbivorous amphipods associated with a host alga-epiphyte system. Journal of Experimental Marine Biology and Ecology 486:328-335.). Due to their predominantly herbivorous diet, these organisms can exert a strong influence on the structuring of Sargassum bed communities, as they feed on this macroalgae and associated epiphytes (Cruz-Rivera & Hay, 2001Cruz-Riviera, E. & Hay, M. E. 2001. Macroalgal traits and the feeding and fitness of an herbivorous amphipod: the roles of selectivity, mixing, and compensation. Marine Ecology Progress Series 218:249-266.; Jacobucci et al., 2009Jacobucci, G. B.; Tanaka, M. O. & Leite, F. P. P. 2009. Temporal variation of amphipod assemblages associated with Sargassum filipendula (Phaeophyta) and its epiphytes in a subtropical shore. Aquatic Ecology 43:1031-1040.).

In Brazil, 15 species of ampithoids have already been recorded (Andrade & Senna, 2017a Andrade, L. F. & Senna, A. R. 2017a. Four new species of Cymadusa Savigny, 1816 (Amphipoda: Ampithoidae) and new records of C. filosa Savigny, 1816 from Brazilian coast. Zootaxa 4226(3):359-389.; Iwasa-Arai et al., 2021Iwasa-Arai, T.; Siqueira, S. G. L.; Segadilha, J. L. & Leite, F. P. P. 2021. The unique Amphipoda and Tanaidacea (Crustacea: Peracarida) associated with the brown algae Dictyota sp. from the oceanic Trindade Island, Southwestern Atlantic, with biogeographic and phylogenetic insights. Frontiers in Marine Science 8:1-30.) associated with different species of algae (Ampithoe ramondi Audouin, 1816, A. robustimana Andrade & Senna, 2017, A. seticoxae Serejo & Licínio, 2002, A. divisura Shoemaker, 1938, A. suapensis Correia et al., 2016, A. thaix Siqueira & Iwasa-Arai, 2021, Peramphithoe conlanae Souza-Filho et al., 2016, Sunampithoe pelagica (Milne Edwards, 1830), Cymadusa filosa Savigny, 1816, C. peartae Andrade & Senna, 2013, C. icapui Andrade & Senna, 2017; C. trindadensis Andrade & Senna, 2017; C. rasae Andrade & Senna, 2017; C. ygara Andrade & Senna, 2017 and C. tartarugae Andrade & Senna, 2017). Cymadusa filosa is a widely distributed species, occurring in shallow coastal regions of North and South America, northwest and southeast Africa, the Mediterranean Sea, Indian Ocean (Madagascar) and Pacific Ocean (New Caledonia) (Andrade & Senna, 2017aAndrade, L. F. & Senna, A. R. 2017a. Four new species of Cymadusa Savigny, 1816 (Amphipoda: Ampithoidae) and new records of C. filosa Savigny, 1816 from Brazilian coast. Zootaxa 4226(3):359-389.). In Brazil, C. filosa was recorded on the coast of the states of Pernambuco to Santa Catarina (Andrade & Senna, 2017bAndrade, L. F. & Senna, A. R. 2017b. Two new species of Ampithoidae (Crustacea: Amphipoda) from northeastern Brazil. Zootaxa 4282 (3):487-500.) associated with different macroalgae (Jacobucci et al. 2009Jacobucci, G. B.; Tanaka, M. O. & Leite, F. P. P. 2009. Temporal variation of amphipod assemblages associated with Sargassum filipendula (Phaeophyta) and its epiphytes in a subtropical shore. Aquatic Ecology 43:1031-1040.; Tavares et al., 2013Tavares, M. R.; Grande, H. & Jacobucci, G. B. 2013. Habitat and food selection by herbivorous amphipods associated with macroalgal beds on the southeast coast of Brazil. Nauplius 21:9-15.).

Characteristics related to the algal morphology, hydrodynamics and presence of natural enemies, in addition to the occurrence of environmental contaminants, can influence the population and reproductive parameters of amphipods in different ways (Borowsky, 1983Borowsky, B. 1983. Reproductive behavior of three tube-building peracarid crustaceans: the amphipods Jassa falcata and Ampithoe valida and the tanaid Tanais cavolinii. Marine Biology 77:257-263.; Leite,1996Leite, F. P. P. 1996. Fecundidade de sete espécies de gamarídeos (Crustacea, Amphipoda, Gammaridea) associados à alga Sargassum cymosum. Iheringia, Série Zoologia 80:39-45; Ford et al., 2003Ford, A. T.; Fernandes, T. F.; Rider, S. A.; Read, P. A.; Robinson, C. D. & Davies, I. M. 2003. Measuring sublethal impacts of pollution on reproductive output of marine Crustacea. Marine Ecology Progress Series 265:303-309.). On the north coast of the state of São Paulo, the São Sebastião channel region has an intense flow of ships that transport oil and its derivatives, as this is the largest oil terminal in the country. Historically, the São Sebastião channel has been affected by oil spills (Poffo et al., 2001Poffo, I. R. F.; Xavier, J. C. M. & Serpa, R. R. 2001. A história dos 27 anos de vazamento de óleo no litoral norte do estado de São Paulo (1974-2000). Revista Meio Ambiente Industrial 30:98-104.; Zanardi-Lamardo et al., 2013Zanardi-Lamardo, E.; Bicego, M. C. & Weber, R. R. 2013. The fate of an oil spill in São Sebastião channel: a case study. Brazilian Journal of Oceanography 61(2):93-104.). Apparently, these pollutants are incorporated by different marine organisms that occur in the region, since petrogenic hydrocarbons have already been detected in fronds of Sargassum and in amphipods in that region, including ampithoids (Lourenço et al., 2019Lourenço, R. A.; Magalhães, C. A.; Taniguchi, S.; Siqueira, S. G. L.; Jacobucci, G. B.; Leite, F. P. P. & Bícego, M. C. 2019. Evaluation of macroalgae and amphipods as bioindicators of petroleum hydrocarbons input into the marine environment. Marine Pollution Bulletin 145:564-568.).

Laboratory studies that evaluated the effect of petrogenic hydrocarbons on amphipods recorded behavioural changes (Satbhai et al., 2017Satbhai, K. M.; Louka, F. R. & Klerks, P. L. 2017. Individual and combined effects of petroleum hydrocarbons phenanthrene and dibenzothiophene on reproductive behavior in the amphipod Hyalella azteca. Water, Air and Soil Pollution 228:91.) and increased mortality (Driscoll et al., 1998Driscoll, S. B. K.; Schaffner L. C. & Dickhut, R. M. 1998. Toxicokinetics of fluoranthene to the amphipod, Leptocheirus plumulosus, in water-only and sediment exposures. Marine Environmental Research 45(3):269-284. ), which can potentially affect populations that occur in contaminated areas. Reproductive patterns and life cycles of ampithoid amphipods are still poorly known and may vary according to different environmental conditions (Leite, 1996Leite, F. P. P. 1996. Fecundidade de sete espécies de gamarídeos (Crustacea, Amphipoda, Gammaridea) associados à alga Sargassum cymosum. Iheringia, Série Zoologia 80:39-45; Appadoo & Myers, 2004Appadoo, C. & Myers, A. A. 2004. Reproductive bionomics and life history traits of three gammaridean amphipods, Cymadusa filosa Savigny, Ampithoe laxipodus Appadoo and Myers and Mallacoota schellenbergi Ledoyer from the tropical Indian Ocean (Mauritius). Acta Oecologica 26:227-238.; Jacobucci & Leite, 2006Jacobucci, G. B. & Leite, F. P. P. 2006. Biologia populacional das espécies de Ampithoidae (Crustacea, Amphipoda) associadas a Sargassum filipendula (Phaeophyta, Fucales) na Praia da Fortaleza, Ubatuba, São Paulo, Brasil. Revista Brasileira de Zoologia 23(4):1207-1216.), including contamination by petrogenic hydrocarbons. In this context, this study aimed to make an initial exploratory survey, in order to determine the population and reproductive aspects of C. filosa in a region with pollution by petrogenic hydrocarbons and to compare the data obtained to works carried out in other locations of Brazil and abroad.

MATERIALS AND METHODS

Study area. Sampling was conducted along the rocky shoreline of São Sebastião Island, one of the largest coastal islands in Brazil, located in the northern coast of state of São Paulo, southeastern of the country (Fig. 1). The coast in the region has alternating rocky shores and sandy beaches, with many other coastal islands. The climate is subtropical, and water temperatures range from 12°C at the bottom (50 m deep) to 28^oC on the surface in summer. The infralittoral rocky shore communities are dominated by algal beds of Sargassum furcatum Kützing, 1843 (Phaeophyceae).

In the São Sebastião Channel is located a large international harbour which includes the Dutos and Terminais Centro Sul (DTCS), formerly the Almirante Barroso Petroleum Terminal (TEBAR), responsible for intense traffic of ships transporting petroleum products. The São Sebastião Channel has also an intense movement of touristic and fishing boats (Marques et al., 2013Marques, A. C.; Klôh, A. dos S.; Migotto, A. E.; Cabral, A. C.; Rigo, A. P. R.; Bettim, A. L.; Razzolini, E. L.; Matthews-Cascon, H.; Bardi, J.; Kremer, L. P.; Vieira, L. M.; Bezerra, L. E. A.; Haddad, A. M. A.; Filho, R. R. R. de O.; Gutierre, S. M. M.; Miranda, T. P.; Franklin Jr, W. & Rocha, R. M. da. 2013. Rapid assessment survey for exotic benthic species in the São Sebastião Channel, Brazil. Latin American Journal of Aquatic Research 41(2):265-285. ). The presence of aliphatic and polycyclic aromatic hydrocarbons (PAHs) was detected in sediments, algae and amphipods collected in the São Sebastião Channel in January, August and November 2008. Total PAHs ranged from 33.4 to 2010 ng g−1 dry weight of Sargassum with the predominance of low molecular weight PAHs, mostly of naphthalene and alkyl-naphthalenes. In ampithoid amphipods PAHs concentrations ranged from 71.2 to 989 ng g^-1 dry weight of amphipods. The concentrations of PAHs in ampithoids obtained from São Sebastião Channel were higher than those reported in studies with amphipods in other regions (Silva & Bícego, 2010Silva, D. A. M. & Bícego, M. C. 2010. Polycyclic aromatic hydrocarbons and petroleum biomarkers in São Sebastião Channel, Brazil: assessment of petroleum contamination. Marine Environmental Research 68(5):277-286.; Lourenço et al., 2019Lourenço, R. A.; Magalhães, C. A.; Taniguchi, S.; Siqueira, S. G. L.; Jacobucci, G. B.; Leite, F. P. P. & Bícego, M. C. 2019. Evaluation of macroalgae and amphipods as bioindicators of petroleum hydrocarbons input into the marine environment. Marine Pollution Bulletin 145:564-568.).

Fig. 1.
Location of the sampling stations in the São Sebastião Channel, state of São Paulo, southeastern coast of Brazil (DTCS- Dutos and Terminais Centro Sul, Almirante Barroso Petroleum Terminal).

Sampling and processing. Sampling was conducted in March, September and December 2007 and February 2008, on four rocky shores located on São Sebastião Island (permit SISBIO no. 29765). Among the rocky shores sampled, two are located to the northeast and two to the southeast of the Dutos and Terminais Centro Sul (DTCS) (Fig. 1). On these rocky shores, the collections were performed by means of boat incursions; material was collected via snorkeling between 1 and 2 m depth. In the Sargassum furcatum beds of each rocky shore, a 50 m transect was delimited, in which 10 algal fronds were randomly collected. The fronds were covered with 0.2 mm mesh fabric bags to prevent the organisms from escaping and were transported to the Marine Biology Centre laboratory at the University of São Paulo (CEBIMAR -USP).

In the laboratory, the algae were kept in the freezer at -20^o C until the animals showed no vital signs. After this cooling, the algae were subjected to four successive washes with sea water and the water resulting from this process was filtered through a 0.2 mm mesh to retain the macrofauna, then was subsequently fixed in 70% ethanol. The dry biomass of the Sargassum fronds was obtained after drying in an oven at 60^o C for 72 hours (Jacobucci et al., 2009Jacobucci, G. B.; Tanaka, M. O. & Leite, F. P. P. 2009. Temporal variation of amphipod assemblages associated with Sargassum filipendula (Phaeophyta) and its epiphytes in a subtropical shore. Aquatic Ecology 43:1031-1040.).

With the aid of a stereoscopic microscope, all individuals of C. filosa were separated from the macrofauna, identified, and counted. The sexual differentiation between males and females was performed due to secondary sexual characteristics such as the size of the gnathopods (Figs 2-5) the presence of oostegites (Jacobucci & Leite, 2006Jacobucci, G. B. & Leite, F. P. P. 2006. Biologia populacional das espécies de Ampithoidae (Crustacea, Amphipoda) associadas a Sargassum filipendula (Phaeophyta, Fucales) na Praia da Fortaleza, Ubatuba, São Paulo, Brasil. Revista Brasileira de Zoologia 23(4):1207-1216.). Females carrying eggs or embryos were considered ovigerous and individuals with no secondary sexual traits were considered juveniles until the minimum size of ovigerous females (Jacobucci & Leite, 2006Jacobucci, G. B. & Leite, F. P. P. 2006. Biologia populacional das espécies de Ampithoidae (Crustacea, Amphipoda) associadas a Sargassum filipendula (Phaeophyta, Fucales) na Praia da Fortaleza, Ubatuba, São Paulo, Brasil. Revista Brasileira de Zoologia 23(4):1207-1216.). The eggs of the females were classified into four stages of development: stage I, eggs with an oval shape without indications of body structures; stage II, eggs with a marked oval shape with the appearance of the caudal cleft and cephalic appendages; stage III, differentiation and appearance of the head, body segments and appendages; stage IV, free juveniles at the marsupium.

Figs 2-5.
Sexual differentiation between male and female of Cymadusa filosa Savigny, 1816: 2, female; 3, gnathopods 1 and 2 of the female; 4, male; 5, gnathopods 1 and 2 of the male (bars: 1 mm)

For ovigerous females with intact marsupium, the total number of eggs was counted (Jacobucci & Leite, 2006Jacobucci, G. B. & Leite, F. P. P. 2006. Biologia populacional das espécies de Ampithoidae (Crustacea, Amphipoda) associadas a Sargassum filipendula (Phaeophyta, Fucales) na Praia da Fortaleza, Ubatuba, São Paulo, Brasil. Revista Brasileira de Zoologia 23(4):1207-1216.). The amphipods were photographed with a digital camera (Nikon Coolpix 938 3.1 megapixel) coupled with a Zeiss SV11 stereoscopic microscope. The images were analysed using the UTHSESA Image Tool for Windows (Version 3.0), determining the individuals’ total body length from the distance between the insertion of the first pair of antennas and the telson.

Data analysis. The individuals of C. filosa collected from the four rocky shores were grouped by sampling periods and organized into size classes. Size distributions were analysed adopting the 1.8 mm class interval according to the Sturges formula (K = 1+3.322( log 10 N) ); where K = number of classes and N = total number of individuals analysed) (Sturges, 1926Sturges, H. A. 1926. The choice of a class interval. Journal of the American Statistical Association 21:65-66.).

The density of associated amphipods in each frond was obtained by dividing the total number of individuals by the dry weight of the frond in grams.

To compare the Sargassum biomass between sampling periods one-way ANOVA was used, followed by the a posteriori Newman-Keuls test. The number of eggs of the ovigerous females at different stages of development was also compared using one-way ANOVA, followed by the a posteriori Newman-Keuls test. Amphipod density was compared using Kruskal Wallis followed by the a posteriori Dunn test. The sizes of males and females were compared through the analysis of variance (ANOVA) of two factors (sex and period), where the size of the individuals was considered the response variable. Prior to analysis, the data were assessed for normality and homogeneity of variance (Zar, 2010Zar J. H. 2010. Biostatistical Analysis. 5th ed. Upper Saddle River, Prentice-Hall. 944p.).

The sex ratio was estimated as the quotient between the number of males and the total number of individuals in the population (males plus females) (Wilson & Hardy, 2002Wilson, K. & Hardy, I. C. W. 2002. Statistical analysis of sex ratios: an introduction. In: Hardy, I. C. W. ed. Sex ratios: concepts and research methods. Cambridge, Cambridge University Press, p. 48-92.). Deviations from a 1:1 sex ratio were tested using (² test (p <0.05). Sex proportion values higher or lower than 0.5 indicated populations skewed toward males or females, respectively.

The reproductive period of C. filosa was assessed by the relative frequency of ovigerous to non-ovigerous females. The minimum, maximum and mean values of fecundity were determined. The fecundity was evaluated through the relationship between the number of eggs, classified in the 1st stage of development, and the size of the females, using simple linear regression (Zar, 2010Zar J. H. 2010. Biostatistical Analysis. 5th ed. Upper Saddle River, Prentice-Hall. 944p.).

For all analyses, a significance level of 5% (α = 0.05) was considered. Tests for homoscedasticity and normality of the contrasted data sets (after ln-ln transformation) were performed and found to be satisfactory (Zar, 2010Zar J. H. 2010. Biostatistical Analysis. 5th ed. Upper Saddle River, Prentice-Hall. 944p.).

RESULTS

The biomass of Sargassum furcatum fronds was significantly lower in September 2007 compared to the other sampling periods (F = 15.73, df = 3, p < 0.001) (Fig. 6). A total of 1372 specimens of Cymadusa filosa were recorded, including 934 juveniles, 274 females (60 ovigerous) and 164 males. Population mean density of C. filosa ranged from 1.27 to 6.40 ind/g with higher mean values in March 2007 compared to the other sampling periods (H = 25.59, df = 3, p < 0.001) (Fig. 7).

Figs 6, 7.
Dry weight in grams (±standard error) of Sargassum furcatum Kützing, 1843 fronds (Fig. 6) and mean density (±standard error) (number of individuals/gram of algal dry weight) of Cymadusa filosa Savigny, 1816 (Fig. 7) in the São Sebastião Channel, state of São Paulo, southeastern coast of Brazil. Bars with the same letter indicate absence of significant difference (p > 0.05).

The mean total body length (mean ± se) recorded for the sampled population was 5.64 ± 0.10 mm. The length of males varied from 4.11 to 20.75 mm (9.87 ± 0.25 mm) and of females from 3.58 to 20.22 mm (9.09 ± 0.21 mm). No significant difference was detected between male and female body length (F = 0.68, df = 3, p = 0.55).

The overall sex ratio significantly differed from a 1:1 proportion and was skewed toward females (0,60 male: 1 females) ((² = 11.78, df = 3; p < 0.01). The same pattern was observed in each sampling period (Tab. I).

The population was dominated by juveniles in March and September 2007 and February 2008, whereas in December 2007 there was a predominance of adult individuals, more specifically females. Juveniles were distributed in the size classes 1.32-3.11 to 4.91-6.71 mm. Males and females were recorded in all size classes except the first. Ovigerous females were recorded in intermediate size classes from 4.91-6.71 to 17.52-19.31 mm. (Fig. 8).

Fig. 8.
Histograms of total body length (mm) of males, females and juveniles of Cymadusa filosa Savigny, 1816 in the São Sebastião Channel, state of São Paulo, southeastern coast of Brazil.

Sixty ovigerous females were recorded, with a higher proportion in relation to the total females in February 2008 ((² = 15.32, df = 3; p < 0.01) (Tab. I; Fig. 9).

Fig. 9.
Relative frequency of ovigerous (light grey) and non-ovigerous (dark grey) females of Cymadusa filosa Savigny, 1816 in the São Sebastião Channel, state of São Paulo, southeastern coast of Brazil.

Ovigerous females showed great variation in the number of eggs found in the marsupium (between 1 and 56) with 26.01 ± 1.84 eggs/ovigerous female (mean ± se). Females with eggs were found in the four stages of development. Significant difference in the number of eggs occurred only between the 1^st and 4^th stages of development, with a lower average in the last stage (F = 3.84, df = 3, p < 0.05) (Tab. II).

Fecundity showed that the number of eggs increased with female size (y = -28.0812 + 4.1551x, R² = 0.6458, p <0.001) (Fig. 10).

Fig. 10.
Regression analyses between ovigerous females’ length (mm) and brood size (number of eggs in the marsupium) of Cymadusa filosa Savigny, 1816, in the São Sebastião Channel state of São Paulo, southeastern coast of Brazil.

DISCUSSION

We compared population data of Cymadusa filosa in regions with high organic and heavy metal contamination in Ubatuba (Vicente et al., 2021Vicente, V. S.; Mansur, K. F. R.; Longo, P. A. S.; Olivino, A. L. L. & Leite, F. P. P. 2021. Variation in population and reproductive parameters of the amphipods, Cymadusa filosa Savigny, 1816 and Sunamphitoe pelagica (H. Milne Edwards, 1830), associated with Sargassum beds in an historically impacted bay. Nauplius 29:e2021041.) and other sites with reduced contamination (Appadoo & Myers 2003Appadoo, C. & Myers, A. A. 2003. Observations on the tube-building behavior of the marine amphipod Cymadusa filosa Savigny (Crustacea; Ampithoidae). Journal of Natural History 37(18):2151-2164.; Jacobucci & Leite, 2014Jacobucci, G. B. & Leite, F. P. P. 2014. The role of epiphytic algae and different species of Sargassum in the distribution and feeding of herbivorous amphipods. Latin American Journal of Aquatic Research 42:353-363.) to the present study. A summary of biological characteristics in the different sites are present in Tab. III.

No clear variation of density, adult size, proportion of ovigerous females and number of eggs in brood were verified considering the data available in those studies.

Amphipods in tropical regions are characterized by continuous reproduction and multivoltinism (Sainte-Marie, 1991Sainte-Marie, B. 1991. A review of the reproductive bionomics of aquatic gammaridean amphipods: variation of life history traits with latitude, depth, salinity and superfamily. Hydrobiologia 223:189-227.). Appadoo & Myers (2004Appadoo, C. & Myers, A. A. 2004. Reproductive bionomics and life history traits of three gammaridean amphipods, Cymadusa filosa Savigny, Ampithoe laxipodus Appadoo and Myers and Mallacoota schellenbergi Ledoyer from the tropical Indian Ocean (Mauritius). Acta Oecologica 26:227-238.) studying the life history of three amphipods in the Indian Ocean, including that of Cymadusa filosa, indicated that this species had continuous reproduction and multivoltinism, also observed by Jacobucci & Leite (2006Jacobucci, G. B. & Leite, F. P. P. 2006. Biologia populacional das espécies de Ampithoidae (Crustacea, Amphipoda) associadas a Sargassum filipendula (Phaeophyta, Fucales) na Praia da Fortaleza, Ubatuba, São Paulo, Brasil. Revista Brasileira de Zoologia 23(4):1207-1216.) in Ubatuba in the coast of state of São Paulo.

In the present study, the presence of young C. filosa in all months of the study and ovigerous females in three of the four months indicates that this amphipod follows the pattern observed for tropical species. However, a reproductive peak was observed in March 2007 and February 2008, indicated by the high presence of juveniles and ovigerous females. According to Sastry (1983Sastry, A. N. 1983. Ecological aspects of reproduction. In: Vernberg, F. J. & Vernberg, W. B. eds. The Biology of Crustacea: Enviromental adaptations. Vol. 8. New York, Academic Press, p. 179-270.), reproductive peaks are frequent in different species and locations in tropical regions in response to small environmental variations as observed by Appadoo & Myers (2004Appadoo, C. & Myers, A. A. 2004. Reproductive bionomics and life history traits of three gammaridean amphipods, Cymadusa filosa Savigny, Ampithoe laxipodus Appadoo and Myers and Mallacoota schellenbergi Ledoyer from the tropical Indian Ocean (Mauritius). Acta Oecologica 26:227-238.). In other study conducted in the northern coast of the state of São Paulo in a nearby region, density peaks of the ampithoids A. ramondi, S. pelagica and C. filosa also occurred in warmer months and coincided with high values of epiphytic algae biomass (Jacobucci et al., 2009Jacobucci, G. B.; Tanaka, M. O. & Leite, F. P. P. 2009. Temporal variation of amphipod assemblages associated with Sargassum filipendula (Phaeophyta) and its epiphytes in a subtropical shore. Aquatic Ecology 43:1031-1040.). The increase in epiphyte biomass favours detritus accumulation in Sargassum fronds and tube-building ampithoids, like C. filosa that can use detritus for tube construction (Appadoo & Myers, 2003Appadoo, C. & Myers, A. A. 2003. Observations on the tube-building behavior of the marine amphipod Cymadusa filosa Savigny (Crustacea; Ampithoidae). Journal of Natural History 37(18):2151-2164.), can be favoured. Epiphytes are also an import food resource for C. filosa (Jacobucci & Leite, 2008Jacobucci, G. B. & Leite, F. P. P. 2008. Effect of temporal variation and size of herbivorous amphipods on consumption levels of Sargassum filipendula (Phaeophyta, Fucales) and their main epiphyte, Hypnea musciformis. Neotropical Biology and Conservation 3:78-85.). These factors could favour the sexual differentiation and female maturation.

A common characteristic among amphipods is the difference in terms of body sizes between males and females, as occurs, for example, in amphipods Apohyale media Bousfield & Hendrycks, 2002, Orchestia gammarellus Pallas, 1766, Ptilohyale crassicornis Haswell, 1879 (Leite & Wakabara, 1989Leite, F. P. P. & Wakabara, Y. 1989. Aspects of marsupial and post-marsupial development of Hyale media (Dana) 1853 (Hyalidae, Amphipoda). Bulletin of Marine Science 45(1):85-97; Dias & Sprung, 2003Dias, N. & Sprung, M. 2003. Population dynamics and production of the amphipod Orchestia gammarellus (Talitridade) in a Ria Formosa saltmarsh (Southern Portugal).Crustaceana76(9):1124-1141.; Tsoi & Chu, 2005Tsoi, K. & Chu, K. 2005. Sexual dimorphism and reproduction of the amphipod Hyale crassicornis Haswell (Gammaridea: Hyalidae). Zoological Studies 44(3):382-392.) with larger males. As most of male energy is allocated for growth, they are generally larger than females. Females, in contrast, use much of their energy for egg growth and production, which slows down their growth rate (Cardoso & Veloso, 2001Cardoso, R. S. & Veloso, V. G. 2001. Embryonic development and reproductive strategy of Pseudorchestoidea brasiliensis (Amphipoda: Talitridae) at Prainha Beach, Brazil. Journal of Natural History 35:201-211.). Although this pattern has already been recorded for C. filosa (Appadoo & Myers, 2004Appadoo, C. & Myers, A. A. 2004. Reproductive bionomics and life history traits of three gammaridean amphipods, Cymadusa filosa Savigny, Ampithoe laxipodus Appadoo and Myers and Mallacoota schellenbergi Ledoyer from the tropical Indian Ocean (Mauritius). Acta Oecologica 26:227-238.), in the present study there was no significant difference in body length between males and females.

The small proportion of individuals recorded in classes of body size greater than 12 mm, in which higher representation of males would be expected, may explain the absence of difference in size between males and females. As indicated by Appadoo & Myers (2004Appadoo, C. & Myers, A. A. 2004. Reproductive bionomics and life history traits of three gammaridean amphipods, Cymadusa filosa Savigny, Ampithoe laxipodus Appadoo and Myers and Mallacoota schellenbergi Ledoyer from the tropical Indian Ocean (Mauritius). Acta Oecologica 26:227-238.) and Jacobucci & Leite (2006Jacobucci, G. B. & Leite, F. P. P. 2006. Biologia populacional das espécies de Ampithoidae (Crustacea, Amphipoda) associadas a Sargassum filipendula (Phaeophyta, Fucales) na Praia da Fortaleza, Ubatuba, São Paulo, Brasil. Revista Brasileira de Zoologia 23(4):1207-1216.), this may result from selective predation by fish, with preference for larger amphipods (Edgar, 1983b Edgar, G. J. 1983b. The ecology of south-east Tasmanian phytal animal communities. IV. Factors affecting the distribution of ampithoid amphipods among algae. Journal of Experimental Marine Biology and Ecology 70:205-225. ). In fact, the impacts of herbivorous and visually oriented fish are well known on amphipod fauna (Nelson, 1979Nelson, W. G. 1979. Experimental study of selective predation on amphipods: consequences for amphipod distribution and abundance. Journal of Experimental Marine Biology and Ecology 38:225-245.) and may be a structuring factor in the population in the São Sebastião Channel, because there is a high diversity of fish in this region (Lamas et al., 2016Lamas, R. A.; Rossi-Wongtschowski, C. L. D. B. & Contente, R. F. 2016. Checklist of the fish fauna of the Araçá Bay, São Sebastião Channel, northern coast of São Paulo, Brazil. Check List 12(6):2004.). Further evidence of size restrictions in natural populations is that individuals of C. filosa reared in the laboratory often reach body sizes larger than animals collected in the field (unpubl. data).

The sex ratio deviation observed in the present study in relation to the 1:1 ratio, with higher frequency of females, may be related to factors dependent on the life cycle of the species (Hamilton, 1967Hamilton, W. D. 1967. Extraordinary sex ratios. A sex-ratio theory for sex linkage and inbreeding has new implications in cytogenetics and entomology. Science 156(3774):477-488.). Females are generally more frequent than males, a result also observed for other amphipods (Kevrekidis, 2005Kevrekidis, T. 2005. Life history, aspects of reproductive biology and production of Corophium orientale (Crustacea: Amphipoda) in Monolimni lagoon (Evros Delta, north Aegean Sea). Hydrobiologia 537(1):53-70.; Castiglioni et al., 2016Castiglioni, D. S.; Ozga, A. V.; Rodrigues, S. G. & Bueno, A. A. P. 2016. Population dynamics of a freshwater amphipod from South America (Crustacea, Amphipoda, Hyalellidae). Nauplius 24:e2016028.) including C. filosa (Appadoo & Myers, 2004Appadoo, C. & Myers, A. A. 2004. Reproductive bionomics and life history traits of three gammaridean amphipods, Cymadusa filosa Savigny, Ampithoe laxipodus Appadoo and Myers and Mallacoota schellenbergi Ledoyer from the tropical Indian Ocean (Mauritius). Acta Oecologica 26:227-238.). This pattern could be related with differences in the mortality rate, longevity, growth rate, behaviour and habitat use between the sexes (Wenner, 1972Wenner, A. M. 1972. Sex ratio as a function of size in marine Crustacea. American Naturalist 106(949):321-350.; Gable & Crooker, 1977Gable, M. F. & Crooker, R. A. 1977. The salt marsh amphipod Gammarus palustris Bousfield, 1969 at the northern limit of its distribution. I. Ecology and life cycle. Estuarine, Coastal and Shelf Science 5(1):123-134.). Variation in mortality can occur due to the higher susceptibility of one of the sexes to predation, due to the reproductive behaviour of the species (differential migration). Borowsky (1983Borowsky, B. 1983. Reproductive behavior of three tube-building peracarid crustaceans: the amphipods Jassa falcata and Ampithoe valida and the tanaid Tanais cavolinii. Marine Biology 77:257-263.) experimentally studying the reproductive behaviour of the amphipods Jassa falcata Montagu, 1808, Ampithoe valida Smith, 1873 and the tanaid Tanais cavolini Milne Edwards, 1829, observed that males are more active than females and often look for new females for mating. Leite et al. (2003Leite, F. P. P; Turra, A. & Souza, E. C. F. 2003. Population biology and distribution of the tanaid Kalliapseudes schubarti Mañé-Garzon, 1949, in an intertidal flat in southeastern Brazil. Brazilian Journal of Biology 63(3):469-479.) suggested that males of the tanaid Kalliapseudes schubarti Mané-Garzon, 1949, in search of new copulation opportunities, could leave their tubes and be the target of predators, which would result in a larger number of females in the population. This is likely to occur for the C. filosa population investigated, as males were seen swimming more frequently than females in laboratory cultures (pers. obs.). However, other factors probably affect sex ratio since Jacobucci & Leite (2014Jacobucci, G. B. & Leite, F. P. P. 2014. The role of epiphytic algae and different species of Sargassum in the distribution and feeding of herbivorous amphipods. Latin American Journal of Aquatic Research 42:353-363.), and Vicente et al. (2021Vicente, V. S.; Mansur, K. F. R.; Longo, P. A. S.; Olivino, A. L. L. & Leite, F. P. P. 2021. Variation in population and reproductive parameters of the amphipods, Cymadusa filosa Savigny, 1816 and Sunamphitoe pelagica (H. Milne Edwards, 1830), associated with Sargassum beds in an historically impacted bay. Nauplius 29:e2021041.) recorded a higher proportion of males in the populations studied.

The mean number of eggs (26 eggs) produced by C. filosa found on the shores of the São Sebastião Channel was similar to that of the population of C. filosa (25 eggs) of Flamengo cove in Ubatuba (Leite, 1996Leite, F. P. P. 1996. Fecundidade de sete espécies de gamarídeos (Crustacea, Amphipoda, Gammaridea) associados à alga Sargassum cymosum. Iheringia, Série Zoologia 80:39-45), and higher than the number of eggs, 20.6, produced by this species in Mauritius in the Indian Ocean (Appadoo & Myers, 2004Appadoo, C. & Myers, A. A. 2004. Reproductive bionomics and life history traits of three gammaridean amphipods, Cymadusa filosa Savigny, Ampithoe laxipodus Appadoo and Myers and Mallacoota schellenbergi Ledoyer from the tropical Indian Ocean (Mauritius). Acta Oecologica 26:227-238.). However, Jacobucci & Leite (2006Jacobucci, G. B. & Leite, F. P. P. 2006. Biologia populacional das espécies de Ampithoidae (Crustacea, Amphipoda) associadas a Sargassum filipendula (Phaeophyta, Fucales) na Praia da Fortaleza, Ubatuba, São Paulo, Brasil. Revista Brasileira de Zoologia 23(4):1207-1216.) found slightly higher mean fecundity (29 eggs) on the rocky shore of Fortaleza beach, also located in the municipality of Ubatuba. Fecundity of C. filosa was positively related to the body size of ovigerous females. This positive relationship was found in populations of C. filosa studied by Leite (1996Leite, F. P. P. 1996. Fecundidade de sete espécies de gamarídeos (Crustacea, Amphipoda, Gammaridea) associados à alga Sargassum cymosum. Iheringia, Série Zoologia 80:39-45) and Appadoo & Myers (2004Appadoo, C. & Myers, A. A. 2004. Reproductive bionomics and life history traits of three gammaridean amphipods, Cymadusa filosa Savigny, Ampithoe laxipodus Appadoo and Myers and Mallacoota schellenbergi Ledoyer from the tropical Indian Ocean (Mauritius). Acta Oecologica 26:227-238.) but was not recorded by Jacobucci & Leite (2006Jacobucci, G. B. & Leite, F. P. P. 2006. Biologia populacional das espécies de Ampithoidae (Crustacea, Amphipoda) associadas a Sargassum filipendula (Phaeophyta, Fucales) na Praia da Fortaleza, Ubatuba, São Paulo, Brasil. Revista Brasileira de Zoologia 23(4):1207-1216.), possibly due to the small number of ovigerous females collected in this study. This positive relationship between fecundity and female body size is common among malacostracan crustaceans (Jensen, 1958Jensen, J. P. 1958. The relation between body size and number of eggs in marine malacostracans. Meddel. Danmarks Fisk-og Havundersog 2:1-25.) and has already been identified in other species of amphipods (Borowsky, 1986Borowsky, B. 1986. Laboratory observations of the pattern of reproduction of Elasmopus levis (Crustacea: Amphipoda). Marine Behaviour and Physiology 12 (4): 245-256 .; Fonseca et al., 2000Fonseca, D. B.; Veloso, V. G. & Cardoso, R. S. 2000. Growth, mortality and reproduction of Excirolana braziliensis Richardson, 1912 (Isopoda, Cirolanidae) on the Prainha beach, Rio de Janeiro, Brazil. Crustaceana 73(5):535-545.; Jacobucci & Leite, 2006Jacobucci, G. B. & Leite, F. P. P. 2006. Biologia populacional das espécies de Ampithoidae (Crustacea, Amphipoda) associadas a Sargassum filipendula (Phaeophyta, Fucales) na Praia da Fortaleza, Ubatuba, São Paulo, Brasil. Revista Brasileira de Zoologia 23(4):1207-1216.).

The reduction in the number of eggs in the fourth stage of development may reflect the active removal of unviable eggs from the marsupium, as suggested for the amphipod Apherusa jurinei Milne-Edwards, 1930 (Dick et al., 2002Dick, J. T. A.; Bailey, R. J. E. & Elwood, R. W. 2002 Maternal care in the rockpool amphipod Apherusa jurinei: developmental and environmental cues. Animal Behaviour 63:707-713.). With advanced stages of egg development, there is an increase in their volumes; the space in the marsupium becomes restricted, making the development of all eggs unfeasible (Cardoso & Veloso, 2001Cardoso, R. S. & Veloso, V. G. 2001. Embryonic development and reproductive strategy of Pseudorchestoidea brasiliensis (Amphipoda: Talitridae) at Prainha Beach, Brazil. Journal of Natural History 35:201-211.).

Therefore, it was expected that striking differences in the number of eggs, individual body length and population density were found but the population structure and reproduction presented by C. filosa in the studied region were similar to those of populations in other locations, indicating that pollution by petrogenic hydrocarbons in the São Sebastião Channel did not affect the population and reproductive parameters investigated in this region.

Acknowledgments.

The authors thank CNPq (Proc. #474143/2006-6) for financial suport for FPPL, and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2007/50488-9) for the fellowship to SGLS. We wish to thank the Centro de Biologia Marinha da Universidade de São Paulo (CEBIMar/USP), for their help and support during the sampling process and the members of Laboratório de Interações em Comunidades Marinhas (LICOMAR) in the separation of the fauna.

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