Determining the optimum temperature and salinity for larval culture,and describing a culture protocol for the conservation aquaculture for European smelt Osmerus eperlanus (L.)

Populations of anadromous European smelt Osmerus eperlanus (L.) are declining across its geographical range in northern Europe, but no practical culture techniques exist to develop stock enhancement programmes for this species. In this study, a culture protocol is described to rear fish from fertilised eggs to mature adults in 2 years involving the use of ‘green water’, live feed and artificial diets. The sequence of embryonic development for eggs incubated at 10°C/0 ppt was described and photographed. To determine the optimum conditions for larval culture, fertilised eggs were reared at a range of salinities (0–20 ppt) and temperatures (5–18°C) until first feeding. Best hatching success (ca. 97%), size at hatch (ca. 0.8 mm) and survival to first feeding (ca. 96%) of larvae were achieved under combined conditions of low salinity (0–0 ppt) and temperature (5–10°C). No larvae survived a salinity of 20 ppt. The time taken from fertilisation to hatch (FtH) and hatching duration (HD) were temperature-dependent ranging from 42 days FtH and 10 days HD at 5ºC, to 10 days FtH and 2 days HD at 18°C irrespective of salinity. The results indicate that conservation programmes could utilise existing salmonid hatchery facilities (i.e. freshwater, ≤10°C water temperature) for stock enhancement. Since on-growing of smelt involves the logistical and technical problems of live feed production, it is recommended that smelt enhancement programme utilise freshwater hatchery facilities to rear fish until hatching, and then stock out onto known spawning grounds in rivers allowing hatched larvae to drift into estuaries to complete the larval and juvenile phases. This approach would minimise the time spent in the hatchery post-hatching, eliminate the need for live food production, prevent the development of predator-naïve fish, and hence would mimic the natural life cycle of the species as closely as possible.     

Evaluation of an improved RNA/DNA quantification method in a common carp (Cyprinus carpio Linnaeus 1758) larval feeding trial with Artemia,two nematodes (Panagrellus redivivus Linnaeus 1758, Panagrolaimus sp. Fuchs 1930) and dry feed

The RNA/DNA ratio commonly used as proxy for the nutritional condition of fish larvae is affected by RNA degradation during analysis. For evaluation of two strategies to improve RNA integrity, a three‐week feeding trial was carried out to assess the suitability of two nematode species (fam. Panagrolaimidae) as feed for newly hatched carp larvae (Cyprinus carpio) in comparison to Artemia nauplii (Artemia sp.) and a commercial dry feed. Aiming for an increased reproducibility of RNA/DNA determination, a high‐salt inactivation (RNA later) as well as a targeted approach with a recombinant RNase inhibitor were compared to the classical protocol using lab chip technology. Improved RNA integrity was observed with high‐salt inactivation when compared with a strategy applying a specific RNase inhibitor or the classic protocol. Carp larvae fed Artemia for 2 weeks and then dry feed for 1 week revealed the best overall growth performance as well as survival [83.0 ± 35.2 mg fresh weight (FW), 20.0 ± 2.4 mm total length (TL), 86.6 ± 11.7% survival]. Larvae fed the nematode species Panagrellus redivivus for 1 week and subsequently dry feed for 2 weeks (37.4 ± 29.1 mg FW, 14.7 ± 2.8 mm TL, 76.0 ± 6.0% survival) performed better than larvae fed with dry feed alone (28.2 ± 29.6 mg FW, 14.3 ± 2.9 mm TL, 54.3 ± 14.2% survival) or those receiving Panagrellus for 2 weeks. Between both nematode species, Panagrellus was a better feed with regard to growth performance and survival. RNA/DNA ratios ranged between 0.65 ± 0.27 (8 days post‐hatch) and 1.96 ± 0.63 (22 days post‐hatch) and were in the same treatment order as the other growth parameters. RNA/DNA ratios were significantly correlated with the growth rate, and decreasing RNA/DNA ratios in larger larvae may reflect decreasing growth rates with size rather than decreased nutritional status. Here, an improved RNA/DNA ratio protocol is presented in a feeding trial that reveals the suitability of nematodes as a first feed for common carp larvae.     

Sitting ducklings: Timing of hatch,nest departure,and predation risk for dabbling duck broods

For ground‐nesting waterfowl, the timing of egg hatch and duckling departure from the nest may be influenced by the risk of predation at the nest and en route to wetlands and constrained by the time required for ducklings to imprint on the hen and be physically able to leave the nest. We determined the timing of hatch, nest departure, and predation on dabbling duck broods using small video cameras placed at the nests of mallard (Anas platyrhynchos; n = 26), gadwall (Mareca strepera; n = 24), and cinnamon teal (Anas cyanoptera; n = 5). Mallard eggs began to hatch throughout the day and night, whereas gadwall eggs generally started to hatch during daylight hours (mean 7.5 hr after dawn). Among all species, duckling departure from the nest occurred during daylight (98%), and 53% of hens typically left the nest with their broods 1–4 hr after dawn. For mallard and gadwall, we identified three strategies for the timing of nest departure: (a) 9% of broods left the nest the same day that eggs began to hatch (6–12 hr later), (b) 81% of broods left the nest the day after eggs began to hatch, and (c) 10% of broods waited 2 days to depart the nest after eggs began to hatch, leaving the nest just after the second dawn (27–42 hr later). Overall, eggs were depredated at 10% of nests with cameras in the 2 days prior to hatch and ducklings were depredated at 15% of nests with cameras before leaving the nest. Our results suggest that broods prefer to depart the nest early in the morning, which may best balance developmental constraints with predation risk both at the nest and en route to wetlands.     

Age and growth of early-life-stage Atlantic tarpon (Megalops atlanticus) from the northcentral Gulf of Mexico

Age and growth of early-life-stage Atlantic tarpon Megalops atlanticus collected from Mississippi coastal waters in the northcentral Gulf of Mexico (GOM) are described using otolith microstructure analysis. Tarpon leptocephali (n = 95, 16.0—27.8 mm standard length, L_S) collected from June throughOctober 2013—2018, ranged in age from 22 to 43 days (mean = 30.9 ± 0.5 days). Leptocephalus somatic growth rates ranged 0.46—1.24 mm day⁻¹ (mean = 0.76 ± 0.02 mm day⁻¹), and leptocephalus otolith growth rates ranged 1.78—3.97 μm day⁻¹ (mean = 2.58 ± 0.04 μm day⁻¹). Growth rates were inversely correlated to leptocephalus age, indicating the shrinkage phase associated with leptocephalus metamorphosis. Juvenile tarpon (n = 358, 50—359 mm fork length, L_F) were collected from August through December 2007—2018. Juveniles exhibited a positive allometric relationship (adjusted R² = 0.99, P < 0.001) between length and mass. The age of 100 juveniles (71—277 mm L_F) ranged from 76 to 174 days. Juvenile growth rate was estimated as 1.56 ± 0.11 mm day⁻¹. Significant (P < 0.001) linear relationships were found between juvenile age and otolith metrics, including otolith mass (R² = 0.81) and radius (R² = 0.68). Evaluation of the backcalculated hatch dates suggests that specimens in the collection hatched from late May through mid-September with slight peaks during July and August. A Rao's Spacing Test of Uniformity indicates the presence of significant lunar periodicity in leptocephalus hatch dates (n = 95, U = 250.1, P < 0.05), with 50% of the leptocephali hatched within 5 days (before or after) of the full moon. This study fills critical gaps in the scientific knowledge of tarpon and provides estimates of early-life-history metrics for an iconic game fish at the northernmost extent of its GOM range.     

Improved survival and growth of silver therapon Leiopotherapon plumbeus early juveniles through co-feeding with Artemia and commercial feeds

This study examined the effects of co-feeding Artemia and commercial feeds on survival, growth and fatty acid composition of silver therapon Leiopotherapon plumbeus early juveniles. Triplicate groups of 36 days post hatch (DPH) early juveniles (17.09 ± 1.69 mm; 0.07 ± 0.02 g) were stocked in nine glass aquaria at 25 individuals per aquarium and reared for 60 days on three feeding regimes: (A) Artemia + powdered commercial tilapia feed (35% crude protein (CP)); (B) Artemia + powdered commercial prawn feed (38% CP); and (C) Artemia nauplii only as the control group. Early juveniles co-fed Artemia and commercial feeds had significantly higher survival (97%) than those fed Artemia alone (86%). Except for the condition factors that were similar to the control group, higher mean total length (30.2 ± 1.3 mm and 27.6 ± 1.2 mm), body weight (401 ± 64 mg and 339 ± 46 mg), length- (SGRL; 0.95 ± 0.07%/day and 0.80 ± 0.07%/day) and weight-specific growth rates (SGRW; 2.85 ± 0.27%/day and 2.58 ± 0.22%/day) were also observed in the co-feeding groups, independent of protein, fat and other nutrient levels in commercial feeds. Higher levels of long-chain polyunsaturated fatty acids were reflected in early juveniles co-fed Artemia and commercial feeds than those fed exclusively on Artemia contributing, in part, to the higher growth and survival observed in the co-feeding groups. Together, these results suggest that co-feeding strategy showed best results in terms of growth and survival, and that commercial feed with 35% protein and 6% crude fat levels may be beneficial in supplementing live feed with essential nutrients to optimize production of silver therapon fry during nursery culture.     

Nematicidal activity of fervenulin isolated from a nematicidal actinomycete, <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CMU-MH021, on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

An isolate of the actinomycete, Streptomyces sp. CMU-MH021 produced secondary metabolites that inhibited egg hatch and increased juvenile mortality of the root-knot nematode Meloidogyne incognita in vitro. 16S rDNA gene sequencing showed that the isolate sequence was 99% identical to Streptomyces roseoverticillatus. The culture filtrates form different culture media were tested for nematocidal activity. The maximal activity against M. incognita was obtained by using modified basal (MB) medium. The nematicidal assay-directed fractionation of the culture broth delivered fervenulin (1) and isocoumarin (2). Fervenulin, a low molecular weight compound, shows a broad range of biological activities. However, nematicidal activity of fervenulin was not previously reported. The nematicidal activity of fervenulin (1) was assessed using the broth microdilution technique. The lowest minimum inhibitory concentrations (MICs) of the compound against egg hatch of M. incognita was 30 μg/ml and juvenile mortality of M. incognita increasing was observed at 120 μg/ml. Moreover, at the concentration of 250 μg/ml fervenulin (1) showed killing effect on second-stage nematode juveniles of M. incognita up to 100% after incubation for 96 h. Isocoumarin (2), another bioactive compound produced by Streptomyces sp. CMU-MH021, showed weak nematicidal activity with M. incognita.     

Age,growth and hatch dates of ingressing larvae and surviving juveniles of Atlantic menhaden Brevoortia tyrannus

Ages, growth and hatch dates of ingressing Brevoortia tyrannus larvae were determined in a 3 year sampling survey at the mouth of the Chesapeake Bay, U.S.A. To determine if otolith‐aged cohorts had variable relative survival, hatch dates of summer‐caught young‐of‐the‐year (YOY) juveniles collected throughout the Chesapeake Bay were compared with hatch dates of ingressing larvae. Modal total length of ingressing larvae was similar among years: 28 mm in 2005–2006 and 2007–2008, and 30 mm in 2006–2007. Ages of ingressing larvae ranged from 9 to 96 days post hatch (dph); mean ages were similar among years, but significantly older in 2006–2007 (50 dph) than in 2005–2006 (44 dph) and 2007–2008 (46 dph). Larval growth rates differed among years. Earliest growth, when larvae were offshore (0–20 dph), was faster in 2006–2007 (0·62 mm day^?1), than in 2005–2006 and 2007–2008 (0·55 mm day^?1 in these years). Subsequently, from 30 to 80 dph, growth was slowest in 2006–2007. Hatch dates of ingressing larvae occurred from September to March and 90% (2007–2008) to 98% (2006–2007) had hatched prior to 31 December. In contrast, most surviving YOY juvenile B. tyrannus had hatched in January to February, suggesting selective mortality of early‐hatched individuals, apparently during the overwinter, larval to juvenile transition period.     

Effects of feeding rate on the growth performance of gynogenetic albino sterlet,Acipenser ruthenus (Linnaeus, 1758) larvae

The feeding rate effects were studied on the growth performance of gynogenetic diploid larvae of sterlet Acipenser ruthenus during the first 4 weeks of exogenous feeding. The experimental rearing was conducted from 7 to 38 days post‐hatch (dph) in a circulation system. This was set up in four groups with three replicates (440 individuals/replicate), viz: AC‐control larvae fed Artemia sp., CFC‐control larvae fed compound feed, AG‐gynogenetic larvae fed Artemia sp., and CFG‐gynogenetic larvae fed compound feed. The larvae were reared in glass tanks (44 L volume, 10 individuals/L) with the temperature maintained at 18 ± 0.5°C, photoperiod of 12L:12D and water flow regime of 1‐L/min and fed 50%, 25%, 25%, and 9% of their total biomass/day during feeding. Highest TL and WBW of gynogenetic diploid larvae (AG) were observed with 50.6 ± 1.2 mm and 607.3 ± 36.1 mg (n = 30) at 38 dph. Highest TL and WBW of control larvae (AC) were recorded with 49.5 ± 3.8 mm and 600.8 ± 88.0 mg (n = 30), respectively, with 73.1% ± 11.4% survival; the lowest survival rate was at 46.4% ± 7.1% (n = 30) for the CFG group. The results indicate that the gynogenetic and normal larvae of sterlet fed with live food (Artemia nauplii) from 7 dph can achieve higher growth and survivability compared to the larvae fed with formulated test feed. Results of this study suggest that the effective rearing of sterlet larvae from 7 to 38 dph strongly depends upon the types of feed rather than the genome manipulation performed.     

An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

Effects of live food and formulated diets on survival, growth and protein content of first-feeding larvae of Plelteobagrus fulvidraco

In recent years, much progress has been made in the rearing of fish larvae fed only artificial diets. A preliminary study was made in an attempt to evaluate the effects of live food and formulated diets on survival, growth and body protein content of first‐feeding larvae of Plelteobagrus fulvidraco. Three test diets varying in protein level were formulated: Feed 1 containing 45% protein, Feed 2 with 50% protein and Feed 3 with 55% protein. Larvae fed live food (newly hatched Artemia, unenriched) were the control. The experiment started 3 days post‐hatch and lasted for 23 days. At the end of the 23‐day trial, survival was best in the control group (65.6%) whereby the final body weight and specific growth rate (SGR) were significantly lower than those in the test feed groups. At the same time, coefficients of variation for SGR and final body weight in the test groups were significantly higher than those in the control. Whole body protein content in all treatments showed a similar tendency during development: significantly higher 3 days post‐hatch, then decreasing significantly, and then increasing unstatistically 10 days post‐hatch. All results suggest that live food is still better for first‐feeding larvae of P. fulvidraco, since live food leads to healthier larvae growth.     

Intraspecific diversity and distribution of the cosmopolitan species Pseudo‐nitzschia pungens (Bacillariophyceae): morphology,genetics, and ecophysiology of the three clades

Three clades of Pseudo‐nitzschia pungens, determined by the internal transcribed space (ITS) region, are distributed throughout the world. We studied 15 P. pungens clones from various geographical locations and confirmed the existence of the three clades within P. pungens, based on ITS sequencing and described the three subgroups (IIIaa, IIIab, and IIIb) of clade III. Clade III (clade IIIaa) populations were reported for the first time in Korean coastal waters and the East China Sea. In morphometric analysis, we found the ultrastructural differences in the number of fibulae, striae, and poroids that separate the three clades. We carried out physiological tests on nine clones belonging to the three clades growing under various culture conditions. In temperature tests, only clade III clones could not grow at lower temperatures (10°C and 15°C), although clade I and II clones grew well. The estimated optimal growth range of clade I clones was wider than that of clades II and III. Clade II clones were considered to be adapted to lower temperatures and clade III to higher temperatures. In salinity tests, clade II and III clones did not grow well at a salinity of 40. Clade I clones were regarded as euryhaline and clade II and III clones were stenohaline. This supports the hypothesis that P. pungens clades have different ecophysiological characteristics based on their habitats. Our data show that physiological and morphological features are correlated with genetic intraspecific differentiation in P. pungens.     

Impact of increasing temperatures and exposure duration on egg hatch in the hemlock looper,Lambdina fiscellaria

As the growing season is expected to begin earlier under climate change, insects should initiate reproduction several days or weeks earlier than they used to. In eastern Canada, hemlock looper (HL) Lambdina fiscellaria (Guenée) (Lepidoptera: Geometridae) females generally oviposit in September, with eggs entering an obligatory diapause quickly after their deposition. We therefore simulated an early start of the HL reproduction cycle of 2, 4, 6, or 8 weeks to examine the extent to which freshly laid eggs from two populations (island and mainland) can withstand exposure to four temperature conditions (15, 20, 25 °C, or fluctuating temperature in an outdoor insectary), with all treatments ending on 1 September 2007. On this date, half the eggs from each population were immediately incubated at 15 °C, while the rest were stored in an outdoor insectary until their incubation at 15 °C the following spring. In a separate experiment, the effect of temperature on pre‐diapause duration was determined from the number of days required for eggs to change colour after oviposition. The pre‐diapause phase was completed faster as temperature increased. Regardless of incubation date and population, percent hatch decreased significantly after 6‐8 weeks of exposure to 25 °C or in the outdoor insectary. Under most treatments, the odds of dying as pharate larvae increased with exposure duration. When eggs were incubated at 15 °C immediately after treatment, time to hatch and diapause duration remained constant over treatments, except at 25 °C when they both decreased. After 8 weeks of exposure to 15 or 20 °C, eggs transferred outdoors were more likely to hatch precociously than those exposed to 25 °C or insectary conditions. Globally, mortality seemed greater among eggs stored outdoors than among those kept indoors. Most eggs that survived the winter hatched synchronously after incubation in spring. Overall, larger eggs from the island population survived better than smaller eggs from the mainland population.     

Effects of plant growth substances on the conchocelis phase of Alaskan Porphyra (Bangiales,Rhodophyta) species in conjunction with environmental variables1

The effect of plant growth substances (PGSs) on conchocelis growth of Alaskan Porphyra (P. abbottiae V. Krishnam., P. pseudolanceolata V. Krishnam., P. pseudolinearis Ueda) was investigated. Growth was measured under different combinations of PGS concentrations (0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 ppm), PGS type (gibberellic acid, kinetin, and indole‐3‐acetic acid), temperature (7, 11, and 15°C), and photoperiod (16:8 light:dark [L:D] cycle and 8:16 L:D cycle). Plant growth substances effectively promoted the growth of Porphyra conchocelis. Depending on culture conditions, growth rates were increased relative to controls 6.9%–31.7% for P. abbottiae, 4.7%–25.7% for P. pseudolanceolata, and 8.9%–35.1% for P. pseudolinearis. Maximal growth of P. abbottiae occurred with 0.8 ppm kinetin, 15°C, and short‐day conditions (8:16 L:D). Porphyra pseudolanceolata exhibited maximal growth with 0.4 ppm indole‐3‐acetic acid, 7°C, and long days (16:8 L:D). Indole‐3‐acetic acid also effected maximal growth of P. pseudolinearis at 0.4 ppm, 15°C, and long‐day conditions (16:8 L:D). For P. abbottiae and P. pseudolinearis, intermediate PGS concentrations (0.4–1.6 ppm) had the greatest growth‐stimulating effects, whereas for P. pseudolanceolata, higher growth generally occurred at lower concentrations (0.1–0.8 ppm). Kinetin and indole‐3‐acetic acid had more influence on the conchocelis phase than gibberellic acid. The PGS concentrations greater than 1.6 ppm had a diminishing effect on growth, especially in P. pseudolanceolata. For P. abbottiae and P. pseudolinearis, higher temperatures resulted in higher growth rates, in contrast to P. pseudolanceolata, which grew faster at the lower temperatures.     

Production of the immunosuppressant cyclosporin A by a new soil isolate,Aspergillus fumigatus,in submerged culture

Cyclosporin A (CyA) has received meticulous attention owing to its immunosuppressive and biological activities. In this study, a soil isolate, capable of producing CyA, was named Zag1 strain and identified as Aspergillus fumigatus based on macroscopic and microscopic characteristics, 18S rDNA sequence, and phylogenetic characteristic analysis. To maximize the production of CyA, the fungal culture was grown under various fermentation conditions including selection of the cultivation medium, agitation rate, fermentation time, incubation temperature, pH value, inoculum nature, and medium volume. A simple medium (pH 5.0) containing 5% maltose as a carbon source and 2% potassium nitrate as a nitrogen source favored the highest CyA production when the fermentation process was maintained at 120 rpm for 9 days and at 30 °C using 3% standard inoculum of 5-day-old. The final CyA titer under these conditions was intensified to 2.23–3.31-fold, as compared with the amount obtained with seven types of basal media. A. fumigatus Zag1 appears to possess a good biotechnological potential for CyA production under favorable culture conditions.

     

Morpho‐colorimetric analysis and seed germination of Brassica insularis Moris (Brassicaceae) populations

Brassica insularis is a perennial plant growing on both coastal and inland cliffs. Three seed lots from Sardinia were analysed using an image analysis system to detect differences in seed morphology, both within and among populations. Germination requirements at constant (5–25 °C) and alternating temperatures (25/10 °C), both in light and in darkness, were evaluated for all populations. In addition, the effect of a dry after‐ripening period (90 days at 25 °C) was also investigated. Morpho‐colorimetric analysis clearly identified seeds from different populations and discriminated three chromatic categories for seeds belonging to the Isola dei Cavoli coastal population, but not for the inland Masùa and the coastal Planu Sartu. Inter‐population variability was also observed in germination behaviour. B. insularis seeds germinated, with percentages up to 60%, in a wide range of temperatures (5–25 °C), and neither light nor dry after‐ripening affected final germination percentages. Moisture content measurements were made for seeds of each colour, but there were no particular differences among colours. Inter‐populational variability in germination behaviour may be a survival strategy for species growing under unpredictable environmental conditions, such as under Mediterranean climate, while heteromorphy may be due to independent evolutionary divergence processes of the Isola dei Cavoli population.     

Lethal activity of beauvericin,a Beauveria bassiana mycotoxin,against the two‐spotted spider mites,Tetranychus urticae Koch

The entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin is known to produce a broad range of secondary metabolites. Beauvericin, a cyclic hexadepsipeptide, is the best known mycotoxin produced by B. bassiana; however, reports discussing the insecticidal activity of beauvericin per se are limited. In this study, we assessed the lethal activity of beauvericin against Tetranychus urticae Koch (Acarina: Tetranychidae). In addition, screening for suitable application of the mycotoxin against T. urticae on greenhouse strawberries is discussed. Beauvericin was able to control successfully T. urticae where concentrations of 10, 100 and 1,000 µg/g recorded mortalities of 84%, 100% and 100%, respectively, against motile stages. Furthermore, beauvericin inhibited egg hatching up to 83.3%, 69.3% and 53.3%, respectively, using the same concentrations under laboratory conditions. Under greenhouse conditions, the efficacy recorded was 52.6%, 85.7%, 72.4% and 72.4% at 1, 3, 7 and 10 days post‐inoculation, respectively. Beauvericin was efficacious under greenhouse conditions since the application increased strawberry yields while showing no phytotoxicity and ecotoxicological risk. Resistance to beauvericin was not detected initially at the unselected strain of T. urticae. Yet, the laboratory selection of populations of T. urticae exposed to beauvericin resulted in relatively resistant T. urticae strain that displayed no cross‐resistance to cyflumetofen and bifenazate. The acaricidal activity of beauvericin documented in this study would increase the efficacy of integrated pest management strategies.     

Embryonic development of the threatened freshwater pipefish Microphis deocata (Hamilton, 1822)

The present study provides the first comprehensive embryonic development of the freshwater Syngnathid fish species, Microphis deocata (Hamilton), a Near Threatened pipefish endemic to the Brahmaputra River drainage in Northeast India and Bangladesh. Microphis deocata is a Gastrophori species as the males develop an abdominal brood pouch. Mature individuals were collected and maintained in well-aerated aquaria under controlled conditions to induce natural spawning. The number of eggs within the males' brood pouch ranged from 17 to 22 (for n = 10), measuring 0.7–1.0 mm in diameter. A total of 10 developmental stages could be recognized under four developmental periods namely, early embryogenesis, eye development, snout formation and juvenile. However, sensitivity, and therefore mortality, while handling of this species restricted the study from reporting the exact time intervals for stages following the blastodisc formation ~48 hr post fertilization. A newborn larvae measures ~14 mm and is free-swimming with distinct dorsal fin (with 31–32 rays) and a sector-shaped caudal fin (with 8–9 rays). The study aims to provide baseline information on the embryology of M. deocata in culture condition which will be helpful for future studies on conservation biology, population status and management of this species.     

Effect of rearing temperature on sex ratio in juvenile Atlantic halibut, Hippoglossus hippoglossus

Several flatfish species exhibit temperature-dependent sex determination. This research investigated the effects of rearing temperature on sex ratio in Atlantic halibut, Hippoglossus hippoglossus, a species in which females grow larger and faster than males under culture conditions. Previous research has shown that ovarian differentiation occurs in Atlantic halibut in the size interval of 38–50 mm, and precedes the differentiation of testes. In the current study, triplicate groups of juvenile Atlantic halibut were reared at each of three temperatures (7, 12 and 15°C) from an initial mean size of 21 mm to a final mean size of 80 mm (total length). The sex of each fish was then determined by macroscopic and histological examination of the gonads. Sex ratios were not significantly different from 1:1 in any group, suggesting that sex in this species is not influenced by temperature.     

Larval development of the invasive charru mussel,Mytella strigata (Bivalvia: Mytilidae)

ABSTRACT

The South American charru mussel, Mytella strigata, was recently recorded in Singapore waters, possibly introduced into Southeast Asia through shipping. The mussels have rapidly spread across estuarine coastal mudflats. Adult mussels were collected, spawned in aquaria and larvae were successfully cultured to the juvenile stage in the laboratory. The larval morphology and development of M. strigata is described in this paper. D-shaped veligers were produced within 20 h of fertilization and were approximately 75 µm in shell length. These larvae were capable of settlement two weeks post fertilization. Given an adequate amount of food, they were able to grow up to 1 mm in shell length within 30 days. The larval shell of M. strigata possesses anterodorsal G2 hinge teeth as distinct wavy ledges, with a pitted resilial ridge clearly evident in the juvenile shell.     

Waiting for a sign: extended incubation postpones larval stage in the beach spawning California Grunion <Emphasis Type="Italic">Leuresthes tenuis</Emphasis> (Ayres)

Unlike most embryos that hatch on a predetermined timetable, California Grunion Leuresthes tenuis can prolong the embryonic period up to three times longer than the time required for hatching readiness. L. tenuis are teleosts that spawn tidally around the highest spring tides of spring and summer, incubating eggs above the water line. Embryos are competent to hatch in 10 days, however they do not hatch until triggered by an environmental cue, agitation in seawater, as the next spring tides rise. This study examined the growth and survival of L. tenuis embryos and larvae that were all fertilized on the same day, then triggered to hatch after different durations of incubation, up to 35 days post fertilization. L. tenuis embryos that survive extended incubation had decreased yolk reserves and did not advance appreciably in morphological development, even when incubation time was extended to its upper limit. After extended incubation, length of hatchlings was significantly longer than hatchlings from the primary incubation time. Regardless of the duration of incubation, larvae provided food ad libitum grew rapidly and were not significantly different in length at three weeks post hatch. Dry mass increased over time and was not significantly different between larval groups within any post-hatch age. Larval growth and survival after one additional tidal cycle of incubation are not adversely affected, but longer incubation significantly decreases embryonic and larval survival. Large reproductive output, environmentally cued hatching, and plasticity in incubation duration enable L. tenuis to reproduce successfully in the unpredictable sandy intertidal ecosystem.