Ecological observations on the colonial ascidian Didemnum sp. in a New England tide pool habitat

The colonial ascidian Didemnum sp. has colonized northwestern Atlantic coastal habitats from southern Long Island, New York, to Eastport, Maine. It is also present in offshore habitats of the Georges Bank fishing grounds. It threatens to alter fisheries habitats and shellfish aquacultures.Observations in a tide pool at Sandwich, MA from December 2003 to February 2006 show that Didemnum sp. tolerates water temperatures ranging from ≤ 1 to > 24 °C, with daily changes of up to 11 °C. It attaches to pebbles, cobbles, and boulders, and it overgrows other tunicates, seaweeds, sponges, and bivalves. From May to mid July, colonies appear as small patches on the bottoms of rocks. Colonies grow rapidly from July to September, with some growth into December, and they range in color from pink to pale yellow to pale orange. Colony health declines from October through April, presumably in response to changes in water temperatures, and this degenerative process is manifested by color changes, by the appearance of small dark brown spots that represent clumps of fecal pellets in the colony, by scavenging by periwinkles, and by a peeling-away of colonies from the sides of cobbles and boulders. At Sandwich, colonies died that were exposed to air at low tide. The species does not exhibit this seasonal cycle of growth and decline in subtidal habitats (40-65 m) on the Georges Bank fishing grounds where the daily climate is relatively stable and annual water temperatures range from 4 to 15 °C. Experiments in the tide pool with small colony fragments (5 to 9 cm²) show they re-attach and grow rapidly by asexual budding, increasing in size 6- to 11-fold in the first 15 days. Didemnum sp. at Sandwich has no known predators except for common periwinkles (Littorina littorea) that graze on degenerating colonies in the October to April time period and whenever colonies are stressed by desiccation.The tendencies of the ascidian (1) to attach to firm substrates, (2) to rapidly overgrow other species, (3) to tolerate a wide temperature range, (4) to be free from predation, and (5) to spread by colony fragmentation combine to make it a potential threat to benthic marine habitats and aquacultures. Didemnum sp. is known to overgrow mussels, oysters, and sea scallops, and it likely envelops other bivalves too.     

The colonial ascidian Didemnum sp. A: Current distribution, basic biology and potential threat to marine communities of the northeast and west coasts of North America

Didemnum sp. A is a colonial ascidian with rapidly expanding populations on the east and west coasts of North America. The origin of Didemum sp. A is unknown. Populations were first observed on the northeast coast of the U.S. in the late 1980s and on the west coast during the 1990s. It is currently undergoing a massive population explosion and is now a dominant member of many subtidal communities on both coasts. To determine Didemnum sp. A's current distribution, we conducted surveys from Maine to Virginia on the east coast and from British Columbia to southern California on the west coast of the U.S. between 1998 and 2005. In nearshore locations Didemnum sp. A currently ranges from Eastport, Maine to Shinnecock Bay, New York on the east coast. On the west coast it has been recorded from Humboldt Bay to Port San Luis in California, several sites in Puget Sound, Washington, including a heavily fouled mussel culture facility, and several sites in southwestern British Columbia on and adjacent to oyster and mussel farms. The species also occurs at deeper subtidal sites (up to 81 m) off New England, including Georges, Stellwagen and Tillies Banks. On Georges Bank numerous sites within a 230 km² area are 50-90% covered by Didemnum sp. A; large colonies cement the pebble gravel into nearly solid mats that may smother infaunal organisms. These observations suggest that Didemnum sp. A has the potential to alter marine communities and affect economically important activities such as fishing and aquaculture.     

Variation in the ability of Didemnum sp. to invade established communities

Over the past 30 years southern New England, USA has been invaded by several species of ascidians, including Botrylloides violaceus, Diplosoma listerianum, Styela clava, and Ascidiella aspersa. These species have become dominate in coastal embayments and marinas but are usually absent from more open water coastal areas. A colonial ascidian, Didemnum sp. has invaded southern New England during the past 10 years and we first observed this species in eastern Long Island Sound in 2000. It has become the dominant at several field sites while remaining in low abundance at others. We conducted an experiment at two places, a protected marina and an open coast site, to examine its ability to compete with the established fouling community. Small colonies of Didemnum were transplanted onto panels with communities that varied in age from one to four weeks old and these treatments along with controls with only Didemnum were exposed at both sites. In most treatments Didemnum became a dominant species in the communities at both sites but it reached higher abundances at the open coast site. Potential causes of the observed differences are predation on other species of ascidians at the open coast site reducing recruitment of these species and competition, lower tolerance for elevated temperatures at the marina site, or other environmental parameters that might affect growth rates.     

Distribution and long-term temporal patterns of four invasive colonial ascidians in the Gulf of Maine

Invasive ascidians are a growing concern for ecologists and natural resource managers, yet few studies have documented their short- and long-term temporal patterns of abundance. This study focuses on the invasion of the Gulf of Maine by the colonial ascidians Botryllus schlosseri, Botrylloides violaceus, Diplosoma listerianum and Didemnum sp. A. We examined the time of arrival and potential vectors for these four invasive ascidians using survey data (collected from 1969 onwards) and literature documentation. We also compared the dominance and seasonal patterns of abundance of these species using data from two identical panel studies; one conducted from 1979 to 1980, the other from 2003 to 2005. Didemnum and Botrylloides were most likely first introduced into the Damariscotta River, Maine in the early 1970's through oyster aquaculture while Botryllus and Diplosoma were probably transported by commercial and recreational vessels. The overall abundance of colonial ascidians has increased since 1979 and 1980. Botryllus was the only invasive colonial ascidian present during the 1979 to 1980 study and accounted for an average of 6.16% cover on panels. From 2003 to 2005, the more recently arrived colonial ascidians Botrylloides and Didemnum accounted for 7.38% and 2.08% cover respectively, while Botryllus covered only 1.16%. Our results reveal a shift in seasonal abundance between 1979 to 1980 and 2003 to 2004. In 1979 and 1980, colonial ascidians had the highest percent cover in fall and winter while in 2003 and 2005 they had highest percent cover in summer and fall. Seasonal patterns of space occupation by colonial ascidians were correlated with seasonal changes in seawater temperature.     

Cold activity of Beauveria and Metarhizium, and thermotolerance of Beauveria

Heat and cold are environmental abiotic factors that restrict the use of entomopathogenic fungi as agents for biological control of insects. The thermotolerance and cold activity of 60 entomopathogenic fungal isolates, including five species of Beauveria and one isolate of Engyodontium albus (=Beauveria alba) were examined as to tolerance of temperatures that might be encountered during field use. In addition, cold activity of eight Metarhizium spp. isolates was evaluated. The isolates were from various geographic regions, arthropod hosts or substrates. High variability in conidial thermotolerance was found among the Beauveria spp. isolates after exposure to 45 °C for 2 h, as evidenced by low (0-20%), medium (20-60%), or high germination (60-80%). The thermal death point (0% germination) for three rather thermotolerant B. bassiana isolates (CG 138, GHA and ARSEF 252) was 46 °C for 6 h. At low temperatures (5 °C), with few exceptions (e.g. CG 66, UFPE 479, CG 227, CG 02), most of the B. bassiana isolates germinated well (ca. 100%). On the other hand, only one isolate of Metarhizium sp. was cold-active (i.e. ARSEF 4343 from Macquarie Island, 54.4°S, Australia). This probably is a M. frigidum isolate. The E. albus isolate (UFPE 3138) was the most susceptible isolate to both heat and cold stress. Isolates ARSEF 252 and GHA of B. bassiana, on the other hand, presented exceptionally high thermotolerance and cold activity. Some isolates with high cold activity, however, were thermosensitive (e.g. ARSEF 1682) and others with high thermotolerance had low cold activity (e.g. CG 227). An attempt to correlate the latitude of origin with thermotolerance or cold activity indicated that B. bassiana isolates from higher latitudes were more cold-active than isolates from nearer the equator, but there was not a similar correlation for heat.     

Interacting effects of temperature and salinity on Bonamia sp. parasitism in the Asian oyster Crassostrea ariakensis

The proposition to introduce the Asian oyster Crassostrea ariakensis to the mid-Atlantic region of the USA is being considered with caution, particularly after the discovery of a novel microcell haplosporidian parasite, Bonamia sp., in North Carolina. Although this parasite was found to be pathogenic in C. ariakensis under warm euhaline conditions, its persistence in C. ariakensis exposed to various temperature and salinity combinations remained unresolved. In this laboratory experiment, we tested the influence of temperature in combination with a wide range of salinities (10, 20 and 30 psu) on Bonamia sp. Temperature was either changed from warm (>20 °C) to cold (6 °C for 6 weeks) and back to warm or maintained constant and warm. Warm temperature was associated with higher host mortality than cold temperature, suggesting that temperature influenced Bonamia sp. pathogenicity. The effect of salinity was revealed under warm temperature with highest mortality levels observed in infected C. ariakensis exposed to 30 psu. When temperature was increased following low-temperature exposure, Bonamia sp. was not detected; however sub-optimal experimental conditions may have contributed to this result, making it difficult to draw conclusions regarding the reemergence of the parasite after low-temperature exposure. Although the overwintering of Bonamia sp. in C. ariakensis will need to be further investigated, the results presented here suggest that Bonamia sp. may be able to persist in C. ariakensis under a combination of low temperature and meso- to euhaline salinities.     

Larsenia salina gen. nov., sp. nov., a new member of the family Halomonadaceae based on multilocus sequence analysis

Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18^T and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0–7.0) and at temperatures between 15 and 40 °C (optimal at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985^T (95.2–94.8%) and Chromohalobacter salexigens DSM 3043^T (95.0–94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA–DNA hybridization between both strains was 82%, whereas the values between strain M1-18^T and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C_18:1ω7c/C_18:1ω6c, C_16:0, and C_16:1ω7c/C_16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18^T (= CCM 8464 = CECT 8192^T = IBRC-M 10767^T = LMG 27461^T).     

Himantura tutul sp. nov. (Myliobatoidei: Dasyatidae), a new ocellated whipray from the tropical Indo-West Pacific,described from its cytochrome-oxidase I gene sequence

It has been previously established that the Leopard Whipray, Himantura leoparda, consists of two genetically isolated, cryptic species, provisionally designated as ‘Cluster 1’ and ‘Cluster 4’ (Arlyza et al., Mol. Phylogenet. Evol. 65 (2013) [1]). Here, we show that the two cryptic species differ by the spotting patterns on the dorsal surface of adults: Cluster-4 individuals tend to have larger-ocellated spots, which also more often have a continuous contour than Cluster-1 individuals. We show that H. leoparda's holotype has the typical larger-ocellated spot pattern, designating Cluster 4 as the actual H. leoparda. The other species (Cluster 1) is described as Himantura tutul sp. nov. on the basis of the nucleotide sequence of a 655-base pair fragment of its cytochrome-oxidase I gene (GenBank accession No. JX263335). Nucleotide synapomorphies at this locus clearly distinguish H. tutul sp. nov. from all three other valid species in the H. uarnak species complex, namely H. leoparda, H. uarnak, and H. undulata. H. tutul sp. nov. has a wide distribution in the Indo-West Pacific, from the shores of eastern Africa to the Indo-Malay archipelago. H. leoparda under its new definition has a similarly wide Indo-West Pacific distribution.     

Temperature and salinity effects on growth, survival, reproduction, and potential distribution of two non-indigenous botryllid ascidians in British Columbia

Two non-indigenous botryllid ascidian species - Botryllus schlosseri (golden star tunicate) and Botrylloides violaceus (violet tunicate) - have become established in British Columbia (BC), Canada. One species, B. schlosseri, is native to Europe while the other, B. violaceus, is native to Asia. Environmental tolerances of both species are poorly understood. We examined the effects of temperature and salinity on growth, survival, and reproduction of these species in the laboratory in order to characterize their environmental tolerances and preferences. Laboratory-raised juvenile colonies were studied using a two-factorial experimental design with five levels of temperature (5, 10, 15, 20, 25 °C) and five levels of salinity (14, 20, 26, 32, 38‰). Both B. schlosseri and B. violaceus possessed broad temperature and salinity tolerances, but B. schlosseri was slightly more euryhalinal than B. violaceus. Generally, B. schlosseri survived environmental conditions of 10-25 °C and 14-38‰, exhibited positive growth at 10-25 °C and 20-38‰, and attained its largest colony sizes at 15-20 °C and 20-38‰. Botrylloides violaceus tolerated environmental conditions between 5-25 °C and 20-38‰, demonstrated positive growth at 15-25 °C and 26-38‰, and attained its largest colony sizes at 20-25 °C and 26-38‰. Results from the laboratory experiment were then used in a modeling exercise to determine the coastal areas of BC that these organisms might be likely to exist in or invade, based on near-surface temperatures and salinities. The model predicted that no areas were totally unsuitable for survival and growth of either species (based solely on temperature and salinity tolerances), with the most suitable locations being along the west coast of Vancouver Island, a region with significant shellfish aquaculture activity.     

Respiration rate and oxy-regulatory capacity in cold stenothermal chironomids

The effects of temperature and oxygen saturation on the respiration rate of two cold stenothermal chironomids, Diamesa insignipes and Pseudodiamesa branickii were investigated. Fourth instar larvae were collected in winter in a glacio-rhithral stream (1300 m a.s.l., Alps, NE-Italy) and their respiration rate was measured with a Clark's electrode in the range 0-14 °C. The respiration rate was significantly higher in D. insignipes than in P. branickii at low temperatures (≤4 °C), higher in P. branickii between 8 and 12 °C and comparable at 14 °C. Higher values of R (regulation value), R_25% (respiration rate at 25% oxygen saturation) and b₁/b₂ (slope ratio in piecewise linear regression), and lower values of P_c (critical pressure) and I (initial decrease) were recorded in P. branickii than in D. insignipes. These values are compatible with oxy-regulatory behaviour in P. branickii, whereas D. insignipes appeared to be almost an oxy-conformer. On the basis of this autoecological information, new implications regarding survival of species from cold, high altitude habitats under changing climatic conditions are made.     

Identification and characterization of a novel spermidine/spermine acetyltransferase encoded by gene ste26 from Streptomyces sp. 139

Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which can bind IL-1R specifically and exhibits anti-rheumatic arthritis activity in vivo. With the Ebosin biosynthesis gene cluster (ste) consisting of 27 ORFs identified previously the focus of this study was to characterize the protein encoded by ste26 gene. After cloning and expressing ste26 in Escherichia coli BL21, we purified the recombinant Ste26 protein and revealed its ability of transferring the acetyl group from AcCoA to spermidine and spermine, with spermine being the preferred substrate. Therefore Ste26 has been determined to be a spermidine/spermine acetyltransferase which can use spermine (Km of 72.1 ± 7.4 μM), spermidine (Km of 147.2 ± 11 μM), AcCoA (Km of 45.7 ± 2.5 μM) and poly-l-lysine (Km of 99.7 ± 11 μM) as substrates. The optimum pH, temperature and time for the activity have been shown to be 7.5, 37°C and 10 min, respectively. This is the first spermidine/spermine acetyltransferase characterized in Streptomyces and its function in Ebosin biosynthesis is discussed.     

Effects of marine organisms extracts on microtubule integrity and cell cycle progression in cultured cells

Marine organisms have been reported to be a rich source of biologically active compounds, but when compared with plants, their use is much more restrict in popular medicine. Among marine animals, sponges and ascidians are two of the most prominent sources of new compounds with cytotoxic potential. In this study, we tested 40 extracts of marine sponges and ascidians from southeastern Brazilian coast aiming to evaluate their anti-proliferative effects on cultured cells. The extracts of Amorphinopsis sp., Arenosclera brasilensis, Cystodytes dellechiajei, Cliona aff. celata, Didemnum sp., Hadromerida and Scopalina ruetzleri showed an IC₅₀≤30 μg/mL and produce strong effects on microtubules organization and on the cell cycle progression.     

Natural Product Antifoulants from the octocorals of Indian waters

Natural Product Antifoulants (NPAs) have been proposed as one of the best alternatives for the globally banned toxic biocide -TBT-in antifouling coatings. In search of NPAs from Indian waters twenty nine species of Octocorals, collected from Gulf of Mannar and Lakshadweep islands, were screened for their antifouling potential against the cyprids of the cosmopolitan biofouler, Balanus amphitrite. The crude extracts of 8 species of these corals (Cladiella krempfi, Lobophytum irregulare, Lobophytum sarcophytoides, Sarcophyton ehrenbergi, Sarcophyton glaucum, Sinularia kavarattiensis, Melitodes sp. and Subergorgia reticulata) exhibited relatively high settlement inhibition properties. The bioassay-guided purification of the crude extracts of 3 active and abundant species-Cladiella krempfi, Sinularia kavarattiensis and Subergorgia reticulata-yielded five NPAs, (l’E,5′E)-2-(2′,6′-dimethylocta-l’,5′,7′-trienyl)-4-furoic acid 1, (−)-6-??-hydroxy polyanthellin A 2, (+)-(7R,10S)-2-methoxy calamenene 3, (+)-(7R,10S)-2,5-dimethoxy calamenene 4 and (+)-(7R,10S)-2-methoxy,5-acetoxy calamenene 5). Among these, 5 exhibited high future prospects on account of its low EC₅₀ value (0.0335 ??g/ml) and high therapeutic ratio (799).     

The environmental effects of salinity and temperature on the oxygen consumption and total body osmolality of the marine flatworm Procerodes littoralis

The present study examined the effect of salinity and temperature on the rate of oxygen consumption and total body osmolality of the triclad turbellarian Procerodes littoralis, a common marine flatworm normally found in areas where freshwater streams run out over intertidal areas. Extremes in environmental factors encountered by P. littoralis were recorded at the study site. These were salinity (0-44 psu), temperature (2.7-24.9 °C) and oxygen concentration (2.8-16.1 mg l⁻¹). Respirometry experiments showed minimal oxygen consumption rates at the salinity extremes encountered by the study species (0 and 40 psu). Further experiments showed relatively constant oxygen consumption rates over the temperature range 5-20 °C and elevated consumption rates at temperatures above 25 °C. Total body osmolality of P. littoralis increased with increasing salinity. The study illustrates how a marine flatworm uses integrated physiological and behavioural mechanisms to successfully inhabit an environment that is predominantly freshwater for up to 75% of the tidal cycle.     

Isolation of a heavy metal-resistant 4-Chloronitrobenzene degrader Cupriavidus sp. D4 and cloning of its cnb genes

Strain D4 was isolated from the sludge of the wastewater treating system of a 4-Chloronitrobenzene (4-CNB) manufacturer. It was able to utilize 4-CNB as the sole carbon and nitrogen source for growth. Strain D4 was preliminarily identified as Cupriavidus sp. based on its physiological & biochemical characteristics and 16S rRNA gene sequence analysis. It could completely degrade 300 mg L⁻¹ of 4-CNB within 25 h under the condition of 30 °C and pH 7.0. Strain D4 could also degrade 4-CNB in presence of heavy metals including Co²⁺, Cd²⁺, Pb²⁺, Zn²⁺, Mn²⁺and so on, therefore it was an excellent candidate for the bio-treatment of 4-CNB and heavy metals co-contaminated environments. The main 4-CNB degrading related genes (cnb A, B, Cab, D, G, Z) and arsenate resistance gene fragment of strain D4 were cloned, sequenced and analyzed, which showed high similarity with the corresponding genes of a reported 4-CNB-degrader, strain CNB-1. The cnb genes of strain D4 were located on two plasmids. This is the first report on the degradation of 4-CNB by the strain from the genus of Cupriavidus sp.     

Gaiella occulta gen. nov., sp. nov., a novel representative of a deep branching phylogenetic lineage within the class Actinobacteria and proposal of Gaiellaceae fam. nov. and Gaiellales ord. nov

Two isolates, with an optimum growth temperature of about 35-37 °C and an optimum pH for growth between 6.5 and 7.5, were recovered from a deep mineral water aquifer in Portugal. Strains form rod-shaped cells and were non-motile. These strains were non-pigmented, strictly aerobic, catalase and oxidase positive. Strains F2-233^T and F2-223 assimilated carbohydrates, organic acids and amino acids. Major fatty acids were novel iso internally branched such as 17:0 iso 10-methyl, 17:0 iso and 15:0 iso 8-methyl. The peptidoglycan contained meso-diaminopimelic acid and menaquinone MK-7 was the major respiratory quinone. Analysis of the 16S rRNA gene shows the strains to cluster with species of the genera Thermoleophilum, Patulibacter, Conexibacter and Solirubrobacter to which they have pairwise sequence similarity in the range 87-88%. Based on 16S rRNA gene sequence analysis, physiological and biochemical characteristics we describe a new species of a novel genus represented by strain F2-233^T (=CECT 7815^T = LMG 26412^T) for which we propose the name Gaiella occulta gen. nov., sp. nov. We also propose that this organism represents a novel family named Gaiellaceae fam. nov. of a novel order named Gaiellales ord. nov.     

Northward expansion of a marine parasite: Testing the role of temperature adaptation

The known range of the eastern oyster (Crassostrea virginica) parasite, Perkinsus marinus, expanded into the northeastern United States in the early 1990s. We used both in vitro and in vivo data to test the hypothesis that the northward expansion was associated with a low-temperature adapted strain of the parasite. In vitro proliferation of nine P. marinus isolates from three geographic sites, Massachusetts and New Jersey in the new range, and South Carolina in the historic southern range, was measured at seven temperatures (5 to 35 °C) using a tetrazolium blue dye assay. We wanted to determine if there were between- and within-geographic location differences in the P. marinus proliferation rate, and if so, whether they were associated with temperature. We found no evidence of low-temperature adaptation based on the fact that net proliferation rates for isolates from all three geographic locations were similar at temperatures from 5 to 20 °C. On the other hand, at temperatures of 25 to 35 °C, the South Carolina isolates exhibited higher proliferation rates than the northern isolates suggesting possible high-temperature adaptation of parasite strains that are routinely exposed to higher temperatures. Although there was significant within-location variation among isolates, the data tended to group together by geographic location supporting the hypothesis that there is an important regional component to the proliferation rate of P. marinus isolates. A survey of published data showed that the temperature at which in vivo proliferation was first observed in oysters at sites from the Gulf of Mexico to Massachusetts was typically between 20 and 23 °C with no evidence of a geographic cline. These results lend support to the hypothesis that the recent warming trend in the northeastern US is the most likely explanation for the P. marinus range extension.     

Temperature-induced oviposition in the brachyuran crab Cancer setosus along a latitudinal cline: Aquaria experiments and analysis of field-data

Ovigerous females of Cancer setosus are present year-round throughout most of its wide range along the Peruvian/Chilean Pacific coast (2°S-46°S). However, their number of egg-masses produced per year remains speculative and as such has neither been considered in latitudinal comparisons of reproduction, nor for its fisheries management. In order to reveal the effect of temperature on egg-mass production and egg-development, female C. setosus were held in through-flow aquaria under natural seasonal temperature conditions (16-23 °C) in Antofagasta (23°S), Northern Chile (05/2005-03/2006; 10 months), and at three constant temperatures (12, 16, 19 °C) in Puerto Montt (41°S), Central Southern Chile (09/2006-02/2007; 5 months). Female crabs uniformly produced up to 3 viable egg-masses within 4 1/2 months in Antofagasta and in Puerto Montt (at 19 °C). The second egg-mass was observed 62.5 days (± 7.6; N = 7) after the oviposition of the first clutch and a third egg-mass followed 73.5 days (± 12.5; N = 11) later in Antofagasta (at 16-23 °C). Comparably, a second oviposition took place 64.4 days (± 9.8, N = 5) after the first clutch and a third, 67.0 days (± 2.8, N = 2), thereafter, at 19 °C in Puerto Montt. At the two lower temperatures (16 and 12 °C) in Puerto Montt a second egg-mass was extruded after 82.8 days (± 28.9; N = 4) and 137 days (N = 1), respectively. The duration of egg-development from oviposition until larval hatching decreased from 65 days at 12.5 °C to 22.7 days at the observed upper temperature threshold of 22 °C. Based on the derived relationship between temperature and the duration of egg-development (y = 239.3175e^{− 0.107x}; N = 21, r² = 0.83) and data on monthly percentages of ovigerous females from field studies, the annual number of egg-masses of C. setosus was calculated. This analysis revealed an annual output of about one egg-mass close to the species northern and southern distributional limits in Casma (9°S) and Ancud (43°S), respectively, while at Coquimbo (29°S) about two and in Concepción (36°S) more than 3 egg-masses are produced per year.