Introduction

Sea cucumbers (Holothuroidea) are echinoderms found in all regions of the oceans, from intertidal regions to the deep-sea and from polar regions to the tropics. With approximately two thousand species they represent important components of marine ecosystems (^{Belbachir & Mezali, 2018}; ^{Marquet et al., 2017}; ^{Purcell et al., 2016}; ^{Tolon & Engin, 2019}). In addition to their ecological significance, the body walls of sea cucumbers are considered highly nutritional and valuable seafood (bêche-de-mer or trepang) that is particularly demanded by Asian consumers (^{Hamel et al., 2001}; ^{Purcell et al., 2013}; ^{Ramofafia et al., 2003}; ^{Robinson & Lovatelli, 2015}), reaching high prices and considered a commodity in the international seafood market (^{Benítez-Villalobos et al., 2013}; ^{Purcell & Eriksson, 2015}). Moreover, holothurians possess a wide range of bioactive compounds that can be used in the production of pharmaceutical, nutraceutical and cosmetics products (^{Egloso & Delantar, 2018}; ^{Marrugo-Negrete et al., 2021}; ^{Mezali et al., 2014}; ^{Purcell, 2014}). The strong demand for these organisms has depleted stocks worldwide, and has led to the collapse of natural populations, classifying some species as threatened (^{Carleton et al., 2013}; ^{Diupotex-Chong et al., 2022}; ^{Laguerre et al., 2020}; ^{Lewerissa et al., 2021}; ^{Purcell et al., 2012}; ^{Santos et al., 2015}; ^{Shedrawi et al., 2019}; ^{Tolon & Engin, 2019}).

Holothuria (Halodeima) grisea Selenka, 1867 is one of approximately 120 large and conspicuous sea cucumbers species of the genera that occur in shallow tropical or subtropical waters (^{Hamel et al., 2001}). It has been recognized as amphi-Atlantic species, from the Gulf of Mexico to Brazil and found in the Eastern Atlantic off West Africa (^{Pawson et al., 2010}). Along the Brazilian coast H. (H.) grisea represents a very common sea cucumber usually, living under rocks or burrowing into sandy substrata, inhabiting the intertidal and subtidal habitats protected from wave action (^{de Lima-Bueno et al., 2018}; ^{Sabino-Rupp et al., 2024}).

A fundamental barrier to improvements in the management of sea cucumber fisheries is a lack of data regarding the basic biological parameters of the most exploited species (^{Laguerre et al., 2020}; ^{Ramos-Miranda et al., 2017}; ^{Tolon & Engin, 2019}; ^{Venâncio et al., 2022}). Studies on the reproduction of marine invertebrates are an important source of information for the potential sustainable exploitation of these resources for those species already exploited, not only for proposing management plans and closed seasons for commercial catches but also to analyze the potential use of this species in an aquaculture production (^{de Lima-Bueno et al., 2015}; ^{Muthiga et al., 2009}; ^{Sabino-Rupp et al., 2021}). The knowledge of the reproductive characteristics of H. (H.) grisea is essential because this species is already subject of indiscriminate catch and commercialization in Brazil (^{Zacagnini-Amaral et al., 2008}; ^{Rodrigues-Ponte & Vieira-Feitosa, 2019}; ^{Sabino-Rupp & Cacciatori-Marenzi, 2021}).

Southern Brazil is the high latitude limit of geographic distribution of H. (H.) grisea along with two other holothuroids: Isostichopus badionotus and Parathyone braziliensis (^{de Lima-Bueno et al., 2018}; ^{Sabino-Rupp et al., 2023}; ^{Slivak et al., 2022}; ^{Ramos-Xavier, 2010}). Due to its great abundance (^{Mendes et al., 2006}; ^{Sabino-Rupp et al., 2023}; ^{Sabino-Rupp et al., 2024}) and potential for aquaculture development (^{Sabino-Rupp et al., 2021}) this work aimed to study for the first time the reproductive biology H. (H.) grisea in Santa Catarina coast. Aiming to generate information about its reproductive characteristics and to contribute to aquaculture development, this study focuses on important aspects such as sex ratio, gonad (tubule development) and maturity indices, macro and microscopical analysis of the tubules in different periods of the year.

Materials and methods

Study site: South Brazil is in a subtropical region, which is considered a transition area between the tropical marine biogeographic province and the warm-temperate province (^{Briggs & Bowen, 2013}). Santa Catarina coastline comprises around 550 km, which includes hundreds of beaches, several coastal islands, mangroves, coastal lagoons, bays and estuaries (^{Sabino-Rupp et al., 2023}). Large cities are also located in the coastal zone holding a population of more than 1 million people, which is increasing pressure on marine ecosystems (^{Ventura-de Souza et al., 2022}).

The regional climate is dominated by the Atlantic Tropical air mass, with average monthly temperatures between 15 °C and 18 °C in winter and between 24 °C and 26 °C in summer. Average monthly rainfall varies between 100 and 350 mm, with a tendency for rain to be concentrated in the spring and summer months, and relative humidity can reach up to 85 % (^{Rodrigues-Filho et al., 2016}). The prevailing winds are Northeasterly throughout the year and Southwesterly in winter (^{de Araújo et al., 2006}). The water masses that occur near the continental shelf are the Tropical Water (summer and fall) influenced by the Brazil Current and, eventually, the Central Water of the South Atlantic (ACAS), in summer, in the lower layers of the water column in coastal areas (^{Resgalla-Jr & França-Schettini, 2006}; ^{Sabino-Rupp et al., 2005})

Armação do Itapocoroy Bay is placed on the North-central coast of Santa Catarina State (Penha municipality) in Southern Brazil (26º47’S & 48º 36’W). It is a sheltered bay with low-hydrodynamic wave action, gently sloping bedrock, and a sandy substrate consisting of coarse grain-sized sediment (^{Mendes et al., 2006}). Along the coast H. (H.) grisea was common in lower intertidal rocky shores in the Northern and central portions of the littoral (^{Sabino-Rupp et al., 2023}).

Sampling: Twenty adult individuals per month (the largest sizes observed in the field) were randomly collected by hand from a rocky-sandy shore where the intertidal region was enlarged and had low slope. Specimens were sampled for over one year (August and October 2019; February, March, May and October 2020). This study was carried out under a license (number 68215-1) to collect native fauna from the Brazilian Biodiversity Authorization (^{Instituto Chico Mendes de Conservação da Biodiversidade, 2007}) and Information System and registration (number A61927E) of the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (^{Alves et al., 2018}).

All individuals were transported to the Centro de Maricultura of CTTMar/UNIVALI laboratory where they were placed in buckets with 7 % magnesium chloride in seawater to relax until processing. Gonads were sexed through visual observation of the color and subsequent confirmation by histological examination. Gonads that could not be sexed macroscopically were recorded as indeterminate.

Measurements of total fresh weight (g) and total length (cm) were recorded with Mark M223 digital analytical balance (0.01 g) and a manual vernier caliper (0.02 mm) After that an incision was made along the ventral surface to remove the organs, including gonads; the gonad wet weight (g ± 0.01) was also recorded. Gonad indexes (GI) were calculated using the ratio between the gonad weight (GW), total wet weight (TWW) and gutted body wet weight (GBW) for comparisons of their effectiveness as proposed by the literature (^{Arsad et al., 2017}; ^{Gaudron et al., 2008}; ^{Tahri et al., 2019}): GI₁ (GW × 100) / TW) and GI₂ (GW × 100) / GBW.

Histological analysis: The gonads were immediately fixed in a buffered 4 % formalin solution for 48 h and subsequently transferred to Davidson solution or formalin solution until the histological procedures. The samples were then processed with sequential submersions in graded ethanol for dehydration followed by xylene for clarification and impregnation with paraffin wax at 60 °C. After the gonad samples were embedded in 100 % (v / v) paraffin, they were cut with a thickness of 7 µm using a manual rotary microtome (Minot type) and stained with Harris’ Haematoxylin solution and alcoholic Eosin Y (yellowish) solution.

Only a section of a gonad tubule was used to obtain definitive preparations. Digital images of the histological sections were captured for sex diagnosis and the gonadal development scale (GDS) under a light optical microscope (Olympus CX43) coupled with a camera (Olympus EP50). GDS was classified for each sex were based on the most outstanding histological characteristics specified for the stages used in other following previous works for the genera (^{Benítez-Villalobos et al., 2013}; ^{de Lima-Bueno et al., 2015}; ^{Leite-Castro et al., 2016}; ^{Ramofafia et al., 2003}; ^{Navarro et al., 2012}; ^{Venâncio et al., 2022}).

To quantify reproductive status the maturity index (MI) is also calculated by the formula:

MI = Σ (ni × si) / N

Where ni is the number of sea cucumbers at each tubule development stage (i.e. GDS), si is the numerical score attributed to that stage, and N is the total number of sea cucumbers collected monthly (^{Arsad et al., 2017}; ^{Benítez-Villalobos et al., 2013}; ^{Venâncio et al., 2022}). Each MI value indicates its respective GDS throughout the collecting period.

Oocyte size: Measurements of female gametes (i.e. non-vitellogenic ‘primary/secondary’ and vitellogenic oocytes) were performed along the major axis and only those whose nucleus was visible. The diameter of all oocytes (per female) was recorded at least five areas in each histological preparation using Feret’s diameter measurement (the longest distance between any 2 points along the selection perimeter) (^{Benítez-Villalobos et al., 2013}) with the image analysis package ImageJ 2 (^{Rueden et al., 2017}). The absolute frequencies of all oocyte diameters were grouped into size ranges each month.

The intervals of the size classes of the diameter average (Da) of the oocyte were determined by the Sturges’ rule (^{Sturges, 1926}), the main cohorts of the oocytes were determined using the Bhattacharya method (^{Bhattacharya, 1967}). The development of oogenesis was analyzed through the evolution of oocyte size in relation to time. ^{Bhattacharya’s (1967)} method was utilized to separate oocyte cohorts (^{Benítez-Villalobos et al., 2013}; ^{Stakowian et al., 2020}).

Data analysis: A theoretical 1 : 1 sex ratio (female : male) was tested using Chi-square test (χ²) considering the total sample over the study period. Differences in Gonad Index (GI) and mature oocyte diameter by months were analyzed using Kruskal-Wallis non-parametric test. All differences were considered statistically significant at the significance level of 5 % (p-value < 0.05). All statistical analyses were performed in R (^{R Core Team, 2021}). Additional packages used included MASS (^{Venables & Ripley, 2002}), vegan (^{Oksanen et al., 2020}) and tidyverse (^{Wickham et al., 2019}).

Results

Sex ratio: A total of 83 H. (H.) grisea were sampled for histological analyses, with an average length (± standard deviation) of 13.7 ± 2.5 cm (from 8.8 to 20.2 cm) and an average weight of 52.2 ± 22.1 g (from 28.9 to 200.9 g). In total, 45 males (54.2 %), 36 females (43.4 %) and 2 undetermined (2.4 %) were sampled. The sex ratio of H. (H.) grisea not differs significantly from the 1:1 ratio. Macroscopic sexing is based on visual analysis of the gonads diagnosed correctly 85.5 % of the total organisms sexed by histological analysis and most of the misdiagnosed individuals were those visually classified as indeterminate, due to the absence of gonad coloration.

Gonad indexes: GI₁ values ranged from 2.76 ± 1.81 to 5.83 ± 5.12 and GI₂ from 4.12 ± 2.46 to 10.77 ± 15.46 with no significant differences detected between sexes or months (GI₁: χ² = 0.09, d.f. = 5, p = 0.999; GI₂: χ² = 0.23, d.f. = 5, p = 0.998) (SMF1A, SMF1B). The maximum values were recorded in October 2019 and 2020 (spring) and the lowest GI mean were pointed in February and May 2020 (summer and autumn months).

Gametogenic events: Histological diagnosis of gametogenic events were described and were made by some features as tubules and lumen aspects, degree of development of gametes and presence of nutritive phagocytes (SMT1, SMF2). Qualitative and/or quantitative analysis allowed us to characterize five different stages of gametogenic development: growing, premature, mature, spawning and post-spawning).

Biometric oocyte analysis: A total of 5 860 oocytes with diameter average (Da) ranging from 5.3 to 135.3 μm were measured. The Sturges’ method distributed the population into 14 size classes of 10 μm mm o μm / class. Bhattacharya’s method identified 2 main cohorts and then established three cytometric intervals for oocyte population: (1) growing: Da < 2 5.3 μm, (2) premature: 25.3 < Da < 85.3 μm and (3), mature: Da > 85.3 μm. Vitellogenic oocytes (completely mature), were observed with average diameters from 65.0 to 85.3 µm.

Throughout the study period, most oocyte population consisted of premature interval (65 %) followed completely mature (19 %) and growing (16 %) (SMF3). The latter were observed in August 2019 (35 %), an important gonial trigger, and in October 2019 (16 %) and October 2020 (23 %). Premature gametes were observed in all months with most cells measured in February/March and all in May 2020. Complete cell maturation made up 25 to 30 % of the cells in March and October 2020.

The females were categorized in four stages based on size ranges: growing, when an remarkable amount of primary oocytes was observed (1 / 3 of cells were above to 25.3 μm) and 2 / 3 are premature (in the process of vitellogenesis); mature, when 1 / 3 of oocytes were up to 85.3 μm and 2 / 3 are premature; spawning with proliferation when primary, premature and mature cells are in 1 : 2 : 1 ratio; and depletion or post spawning when all cells (the lowest quantities observed over the period) were only in intermediated diameter (SMF4).

Kruskal-Walli’s test comparisons of mature (vitelogenic) oocytes (Da > 65 mm) showed significant differences (H = 266.73, d.f.= 5 / 1677, p = 0.0001) between October 2019 (Da = 85.3 μm; n = 757) and February, March and May 2020 that were grouped with a mean oocyte diameter ranging from 71.0 to 75.8 μm (17 > n > 447) (SMF5). Averages values close to 80 μm were recorded in August 2019 (n = 67) and October 2020 (n = 310).

Tubule development stages (GDS) frequency and Maturity Index (MI): Growing and premature stages were usually observed in August 2019 and October 2019 (winter and spring months) (SMF6). Maturation and spawning occurred in February, May and October 2020 (summer, fall and spring months). Post spawning stage was observed mainly in February 2020. The maximum MI values were associated with maturation and spawning stages from March to May 2020.

In males, the growing stage was not observed. Premature stage was observed in August 2019, October 2019 and 2020 (winter and spring) but growing cells could be seen in the same individual. Maturation and spawning events were frequent in all months mainly February, March and May 2020 (summer, autumn and spring). The maximum MI values were associated with spawning stage in March and May 2020.

A summary of the macro-microscopic (sex, GDS and tubules colors) diagnosis monthly records was presented with seven colors categories: transparent, transparent-pink, white, transparent-white, creamy-white, cream and pink with a large overlap between sex and GDS (SMT2).

Discussion

The reproductive pattern of H. (H.) grisea from Santa Catarina population was characterized by a constancy of mature and releasing gametes for most part of the year. The females of this population showed a period of great investment in cell growth (winter and spring months) which allowed the development of multiple cohorts of oocytes observed in subsequent seasons and thus supported the great reproductive period in practically three seasons of the year (spring, summer and autumn). Post spawning stage observed mainly in summer end probably reflects a greater effort to release gametes. It is speculated that favorable conditions for the supply of energy resources from feeding during the winter months were responsible for this scenario.

The highest intensity of the reproductive events occurring between the spring and summer months was also observed by ^{de Lima-Bueno et al. (2015)} in a population from the coast of Paraná located around 100 km North from Armação do Itapocoroy, in both locations the months with lowest temperatures were characterized by a reduction in reproductive activity: growing (autumn) and post-spawning (winter). The presence of underdeveloped gonads or almost non-existent gonads described by ^{de Lima-Bueno et al. (2015)} probably refers to a large reduction in the size of the organ. This process of resorption and disappearance of gonads is considered a common phenomenon in species of the order Aspidochirotida during post-spawning stage (^{Hamel et al., 1993}; ^{Hoareau & Conand, 2001}; ^{Ramofafia et al., 2000}; ^{Rasolofonirina et al., 2005}).

In comparison to Paraná population, it is agreed that winter may be a recovering period after the last reproductive effort during autumn. Although both locations are geographically close and within the same Spalding's ecoregion (^{Spalding et al., 2007}). The identification of a longer period of reproductive activity in the present study may be due to interpopulation variations (^{Arsad et al., 2017}; ^{Marquet et al., 2017}; ^{Pasquini et al., 2022}; ^{Tahri et al., 2019}) where a series of environmental parameters that are acting successively or in combination to orchestrate spawning in the sea cucumbers (^{Marquet et al., 2017}; ^{Tolon & Engin, 2019}) or even inter-annual variation (^{Marquet et al., 2017}; ^{Venâncio et al., 2022}) as observed in this study with the difference frequency of stages between October 2019 and 2020.

Another factor to be considered is the low sampling frequency (^{de Lima-Bueno et al., 2015}), as pointed by ^{Leite-Castro et al. (2016)} which can compromise the diagnosis of gametogenic stages and possibly explain why only three stages of tubules development were found in Paraná. We also believe that a major point to be discussed is how to quantify gonad maturity, since tubule recruitment growth model can occur (see ^{Smiley, 1988}). If no synchronization was observed in tubules development in each gonad, we could expect a high variability among the same individual (^{Hamel et al., 1993}; ^{Marquet et al., 2017}; ^{Mercier & Hamel, 2009}; ^{Rasolofonirina et al., 2005}).

The present study reveals that the spring and summer seasons are the mainly reproductive period over Brazilian coast. Besides small differences between Southern (present study) and Northeast populations (^{Leite-Castro et al., 2016}) of H. (H.) grisea along to Brazilian coast, individuals have a long reproductive period (mainly spring-summer) which suggests the existence of a similar pattern between these tropical and subtropical environments. Holoturia species have a wide geographical range and when exposed to different environmental factors follow a biannual or continuous reproductive pattern with variations in the onset of gametogenesis and spawning (^{Abdel-Razek et al., 2005}; ^{Dissanayake & Stefansson, 2010}; ^{Mercier et al., 2007}; ^{Muthiga & Kawaka, 2008}; Muthiga et al., 2009; ^{Rogers et al., 2018}).

A review of publications on reproduction in sea cucumbers indicates that several species of Holothuria both near the equator and at higher latitudes showed reproductive periodicity at least a half of the year with patterns which reflect the seasonal differences in the environment they inhabit (^{Benítez-Villalobos et al., 2013}) (SMT3). Generally, the size of the organism, in particular body length and weight, can be associated with gonad growth and in Holothuria, the body size is highly variable (SMT3). In the present study the sizes of H. (H.) grisea can be considered intermediate to small, compared to the other species of the same genus.

In the present study fluctuation of both gonadal indices (GI₁ and GI₂) could be associated with the longer period of highest variability of cells ratios in the females (immature and mature in oocytes) and the continuous reproductive activity of males during the year. Although GI₁ has been widely used as a good indicator of gonad maturity of sea cucumber (^{Arsad et al., 2017}; ^{Bahida et al., 2022}; ^{Gaudron et al., 2008}; ^{Rogers et al., 2018}; ^{Tolon & Engin, 2019}), many times this index could be influenced by gonad recovery period (^{Benítez-Villalobos et al., 2013}) or by resorption process (^{Drumm & Loneragan, 2005}; ^{Rasolofonirina et al., 2005}).

We also agree with the use of GI relationship with the wet weight of the body wall consider a more reliable parameter for sea cucumber size for some authors (^{Muthiga et al., 2009}; ^{Ramofafia et al., 2000}; ^{Rasolofonirina et al., 2005}). Furthermore, in reproductive season large-sized individuals could have less developed gonads or, inversely, or even gonads not developed at all or missing. This phenomenon in holothuroids could be also influenced by evisceration and subsequent viscera regeneration (^{Ramos-Miranda et al., 2017}).

For most benthic marine species with complex life cycles, the larval stage is the dominant dispersal stage, this being directly correlated to the temporal extension of the larval phase (^{Rakaj et al., 2018}). ^{Moran et al. (2013)}, ^{Stakowian et al. (2020)} and ^{Venâncio et al. (2022)} pointed out that development time is inversely proportional to egg size. Larval period of H. (H.) grisea in laboratory conditions was about 20 days, but it may be influenced by temperature and diet conditions (^{Sabino-Rupp et al., 2021}).

This reproductive strategy matches what we observed in Holothuria species that presents an intermediate egg size-less than 200 μm (^{Benítez-Villalobos et al., 2013}; ^{Huang et al., 2018}; ^{Navarro et al., 2012}; ^{Rasolofonirina et al., 2005}; ^{Rogers et al., 2018}) with H. (H.) grisea turned out to be considerably smaller than that reported for other congeneric species which might also reflect its rapid larval development as a life history strategy (^{Rakaj et al., 2018}). In laboratory condition carried out by ^{Sabino-Rupp et al. (2021)} at Armação do Itapocoroy the vitellogenic oocytes had an average diameter of 135.8 μm (± 6.7 μm) for this species and it may reach 160 μm (Rupp pers. obs.). Thus, it is important to bear in mind that in studies where histological techniques are used, we are referring to a gamete that has undergone retraction after the fixation techniques and is not considered a cell under fresh conditions.

The allocation of reproductive energy into a large versus a small number of offspring based on egg size is an important life-history strategy for any species. When comparing the three Brazilian populations of sea cucumbers of H. (H.) grisea we can hypothesize that the smaller size ranges of the oocytes in southern Brazil would be associated with differences in the metabolic demands of each population under local environmental conditions.

The present study integrated qualitative (macroscopical diagnosis and microscopic assessment) and quantitative tools (oocyte size scale and gonad and maturity indexes) to evaluate the reproductive activity and the gamete (oocyte) investment of H. (H.) grisea from Santa Catarina population. Integrative use of quantitative and qualitative analyses for gonadal developmental stage determination proved to be insightful, since qualitative methods alone impair an adequate comparison due to the lack of standardization. Quantitative tools such as oocyte diameter distribution, tubule development and maturity index provided strong evidence to indicate that the species exhibits a continuous reproductive pattern with a reduction in winter season.

Most studies on the reproduction of holothuroids use the coloration of the gonads for sexing and the degree of development of the gonad (tubules). It is common to indicate that creamy-colored gonads are testes and that pink-colored gonads are ovaries (^{Arsad et al., 2017}; ^{de Lima-Bueno et al., 2015}; ^{Muthiga et al., 2009}; ^{Navarro et al., 2012}; ^{Pasquini et al., 2022}; ^{Rogers et al., 2018}; ^{Santos et al., 2015}; ^{Venâncio et al., 2022}). In our study, the visual assessment of sexual identification was mostly correct but inconsistent for diagnosing the animal maturity. Although no photographic records of the colors were made in this study, recommended that these are taken for further studies.

Aquaculture and fisheries remarks: As sea cucumber fisheries along the Brazilian coast is an unreported and unregulated activity (^{Rodrigues-Ponte & Vieira-Feitosa, 2019}; ^{Sabino-Rupp & Cacciatori-Marenzi, 2021}), it is urgent to carry out biological and ecological studies to develop effective management measures before the overexploitation of sea cucumber populations. In Brazil, aquaculture efforts have been attempted in the Northeast region (^{Souza-Junior et al., 2017}) and more recently in the South (Santa Catarina). The availability of H. (H.) grisea natural stocks is one of the main reasons for the selection of this species for aquaculture studies (^{Sabino-Rupp et al., 2021}; ^{Sabino-Rupp et al., 2023}; ^{Sabino-Rupp et al., 2024}). The information from this study may support spawning induction by knowing when to capture broodstock but it does not support larval culture or juvenile production. Therefore, it is suggested that the activities to induce spawning of H. (H.) grisea should not be attempted during late autumn and winter months. It is also recommended to carry out further studies on growth, recruitment and survivorship on longer time-scales, on determination of the first sexual maturity and on fecundity per size distribution. Such features would allow a better understanding of the population ecology and the development of aquaculture and stock management tools, as well as fishing regulations based on precise information for conservation of the Santa Catarina ecosystems.

In fact, this study is the first to describe the reproductive behavior of H. (H.) grisea on the coast of Santa Catarina, and specifically in Armação de Itapocoroy. This can offer a baseline for future research, providing various qualitative and quantitative description tools for greater effectiveness in understanding the maturity cycle of populations and managing the sea cucumber fishery in Brazil. Finally, it is suggested that additional reproductive studies be carried out over a longer period of years, as inter-annual variation is likely to occur, and ongoing climate change, along with an above historical average increase in rainfall and heat in Southern Brazil make this recommendation imperative.

Ethical statement: The authors declare that they all agree with this publication and made significant contributions; that there is no conflict of interest of any kind; and that we followed all pertinent ethical and legal procedures and requirements. All financial sources are fully and clearly stated in the acknowledgments section. A signed document has been filed in the journal archives.

See supplementary material

A34v73n1-suppl1