Heterosigma akashiwo in central California waters

Heterosigma akashiwo (Hada) gives rise to red tides along the Atlantic and Pacific coasts and is known to produce brevetoxins. This investigation establishes baseline information showing the presence of H. akashiwo along the central California coast based on water samples collected from the Santa Cruz pier in Monterey Bay (on the open coast) and the Berkeley pier in San Francisco Bay. Light and electron microscopy as well as two species-specific DNA probe methods based on cell homogenates preparations were employed to detect H. akashiwo during the 2001–2002 field study. The DNA probe methods consisted of a sandwich hybridization assay (SHA), which targets ribosomal RNA (rRNA), and an end-point polymerase chain reaction (PCR) assay, which targets internal transcribed spacer (ITS) sequences of rRNA genes. The SHA was used to provide semi-quantitative data showing the intermittent presence of the species during a 13-month period in Monterey Bay. Samples that showed a variety of responses in the SHA (negative as well as the highest) were then subjected to the PCR assay in an attempt to confirm species identification using an independent DNA probe method that employs cell homogenates; samples included those from Monterey Bay and one from a red tide event in San Francisco Bay. SHA and PCR assays agreed on the presence or absence of H. akashiwo. Gene products from two field samples positive for H. akashiwo by PCR were cloned and sequenced and found to be identical to those of that species in GenBank. When the same samples were viewed by light microscopy, however, H. akashiwo cells were only seen in the sample with the highest abundance of that species, as evidenced by SHA. It was extremely difficult to recognize naturally occurring H. akashiwo using light microscopy in field samples that had been preserved with Lugol's iodine, including samples that gave positive results by cell homogenate methods. Results of this study indicate that H. akashiwo is present along the open California coast and could easily be missed in routine phytoplankton surveys. Despite its presence, H. akashiwo does not appear to routinely bloom with sufficient densities to cause harmful outbreaks of the frequency and severity documented in some other coastal environments. Molecular identification techniques may be the preferred approach over light microscopy when there is a need to rapidly screen many samples for fragile, harmful species and those that are otherwise problematic to identify based on their gross morphology alone.     

Alga-lytic activity of Pseudomonas fluorescens against the red tide causing marine alga Heterosigma akashiwo (Raphidophyceae)

A bacterial strain, HAK-13, exhibited strongest activity against Heterosigma akashiwo and was capable of controlling this bloom forming phytoplankton. Based on 16S rDNA sequences and biochemical and morphological characteristics, the strain HAK-13 was determined to be Pseudomonas fluorescens on the basis of 99.9% similarity with reference strains in the DNA databases. The growth of H. akashiwo was strongly suppressed by HAK-13 in all growth phases, with the strongest alga-lytic activity noted against harmful bloom-forming species in the exponential stage (6–22 days). Host range tests showed that HAK-13 also significantly inhibited the growth of Alexandrium tamarense and Cochlodinium polykrikoides but could not destroy Gymnodinium catenatum. P. fluorescens HAK-13 indirectly attacked H. akashiwo by alga-lytic substances that might be located at the compartment of cytoplasmic membrane of the bacterium at a level of 45.86 units/mg of specific activity. The results indicated that P. fluorescens HAK-13 caused cell lysis and death of H. akashiwo, A. tamarense, and C. polykrikoides dramatically and Prorocentrum dentatum slightly. Therefore, P. fluorescens HAK-13 has potential for use as a selective biocontrol of harmful algal blooms.     

A novel Heterosigma akashiwo (Raphidophyceae) bloom extending from a South Carolina bay to offshore waters

In April 2003, a novel Heterosigma akashiwo bloom was observed that extended from Bulls Bay, South Carolina USA, to approximately 8 km offshore. The bloom was associated with a fish kill of approximately 10⁴ fish. The bloom coincided with salinities anomalously low for the region and optimal for H. akashiwo growth. The low salinities were related to the rediversion of freshwater a month earlier from the Cooper River into the Santee River, which partially feeds into Bulls Bay. H. akashiwo identification was confirmed using a species-specific real-time PCR assay modified for the direct amplification of target DNA from the bloom sample. Because this H. akashiwo bloom was associated with a fish kill, and exposure to bloom waters caused sublethal toxic effects on oysters, the resolution of the cause and potential recurrence of the bloom are of importance to fishery management.     

Nitrogen utilization by the raphidophyte Heterosigma akashiwo: Growth and uptake kinetics in laboratory cultures

The nitrogen uptake and growth capabilities of the potentially harmful, raphidophycean flagellate Heterosigma akashiwo (Hada) Sournia were examined in unialgal batch cultures (strain CCMP 1912). Growth rates as a function of three nitrogen substrates (ammonium, nitrate and urea) were determined at saturating and sub-saturating photosynthetic photon flux densities (PPFDs). At saturating PPFD (110 μE m⁻² s⁻¹), the growth rate of H. akashiwo was slightly greater for cells grown on NH₄⁺ (0.89 d⁻¹) compared to cells grown on NO₃⁻ or urea, which had identical growth rates (0.82 d⁻¹). At sub-saturating PPFD (40 μE m⁻² s⁻¹), both urea- and NH₄⁺-grown cells grew faster than NO₃⁻-grown cells (0.61, 0.57 and 0.46 d⁻¹, respectively). The N uptake kinetic parameters were investigated using exponentially growing batch cultures of H. akashiwo and the ¹⁵N-tracer technique. Maximum specific uptake rates (V_max) for unialgal cultures grown at 15 °C and saturating PPFD (110 μE m⁻² s⁻¹) were 28.0, 18.0 and 2.89 × 10⁻³ h⁻¹ for NH₄⁺, NO₃⁻ and urea, respectively. The traditional measure of nutrient affinity—the half saturation constants (K_s) were similar for NH₄⁺ and NO₃⁻ (1.44 and 1.47 μg-at N L⁻¹), but substantially lower for urea (0.42 μg-at N L⁻¹). Whereas the α parameter (α = V_max/K_s), which is considered a more robust indicator for substrate affinity when substrate concentrations are low (<K_s), were 19.4, 12.2 and 6.88 × 10⁻³ h⁻¹/(μg-at N L⁻¹) for NH₄⁺, NO₃⁻ and urea, respectively. These laboratory results demonstrate that at both saturating and sub-saturating N concentrations, N uptake preference follows the order: NH₄⁺ > NO₃⁻ > urea, and suggests that natural blooms of H. akashiwo may be initiated or maintained by any of the three nitrogen substrates examined.     

Monitoring for Heterosigma akashiwo using a sandwich hybridization assay

Field testing of a ribosomal RNA (rRNA)-targeted sandwich hybridization assay (SHA) for Heterosigma akashiwo (Raphidophyceae) in Puget Sound, WA, USA, has showed that the lower limit of detection is well below the level at which cells pose a danger to fish. Moreover, the assay has proven to be both rapid and easy-to-use. Isolates of H. akashiwo from Australia, Japan, New Zealand, South Korea, Spain and USA were correctly identified using the SHA, indicating that this diagnostic tool could be deployed globally. Samples containing H. akashiwo can be preserved for subsequent SHA analysis using several methods: fixation with acidic Lugol’s iodine followed by room temperature storage, collection onto Durapore filters followed by storage at −70 °C or, alternatively, the filters are mixed with a lysis solution buffer and the sample lysate stored at −70 °C. Additionally, we sought to determine whether the SHA could successfully detect H. akashiwo in the presence of clay that might some day be used to mitigate the impacts of natural H. akashiwo blooms. Results from preliminary laboratory trials indicate that clay at the maximum proposed dosage rate does not interfere with the assay. Thus, it may be possible to use the SHA as a simple means of following the fate of H. akashiwo cells during larger-scale clay mitigation trials.     

Extracellular organic compounds from the ichthyotoxic red tide alga Heterosigma akashiwo elevate cytosolic calcium and induce apoptosis in Sf9 cells

Toxin(s) from the ichthyotoxic red tide alga Heterosigma akashiwo have been responsible for the destruction of millions of dollars of finfish aquaculture around the globe. Mechanisms of toxicity may include the production of reactive oxygen species (ROS) or organic toxins. The purpose of this study was to investigate the bioactivity of extracellular organic compounds from cultures of H. akashiwo. Cytosolic free calcium levels ([Ca²⁺]_i) in Spodoptera frugiperda (Sf9) insect cells infected with baculoviruses encoding the M1 muscarinic receptor were monitored.Exposure of cells to Heterosigma organics increased [Ca²⁺]_i up to 120 nM above basal levels (two-fold increase). Within minutes following exposure of the cells to the organics, the increase in [Ca²⁺]_i peaked and was followed by a slightly reduced, yet sustained plateau. This plateau was maintained for the duration of an experiment (>15 min) and was inhibitable by lanthanum. Furthermore, stimulation of Ca²⁺ release from intracellular stores by carbachol (muscarinic agonist) or thapsigargin (sarco-endoplasmic reticulum Ca²⁺-ATPases, SERCA inhibitor) potentiated the [Ca²⁺]_i response induced by the organics resulting in a maximal increase of >250 nM above basal levels (three-fold increase). However, the [Ca²⁺]_i response to Heterosigma organics was strictly dependent on the presence of extracellular calcium. Flow cytometric analyses revealed that these organics induced apoptosis of these same cells. Collectively, our data indicate that extracellular organics from cultures of H. akashiwo acutely increase [Ca²⁺]_i in cells by inhibiting the plasma membrane Ca²⁺-ATPase transporter and ultimately induce apoptotic cell death. These organics may play a significant role in the ichthyotoxic and allelopathic behaviour of this alga.     

Sublethal effects of the toxic alga Heterosigma akashiwo on the southeastern oyster (Crassostrea virginica)

Over the last three years, several blooms of Heterosigma akashiwo (Raphidophyceae) were documented in South Carolina (SC) brackish waters, including areas containing extensive oyster (Crassostrea virginica) beds. This study examined the sublethal effects of H. akashiwo on C. virginica, based on cellular biomarker responses after exposure to laboratory cultures of H. akashiwo isolated from SC waters, and to water collected from two SC H. akashiwo blooms. Exposure to laboratory cultures or blooms of H. akashiwo significantly increased oyster hepatopancreas lysosomal destabilization rates, but had little effect on gill p-glycoprotein (p-gp) expression. Lysosomal destabilization in oysters continued to increase even after a 7-day recovery period in clean seawater, suggesting that H. akashiwo toxin or other cellular byproducts continued to damage the hepatopancreas. These results suggest that even short-term exposures of oysters to high cell densities of H. akashiwo could have long-term adverse physiological effects, and imply that oyster health may be compromised in areas where repetitive H. akashiwo blooms occur.     

Pelagic-benthic transition of the harmful alga, Heterosigma akashiwo: Changes in swimming and implications for benthic cell distributions

Many harmful algal blooming (HAB) species transition between a vegetative, motile phase in the water column and a dormant, non-motile resting phase in the sediments. These life history transitions potentially regulate the timing, location and persistence of bloom events. Motility promotes aggregation and influences vertical distributions in the water column. However, the contribution of this behavior to benthic distributions of resting cells is currently unknown. We used video-tracking techniques to test the hypothesis that algal cells use active down-swimming during pelagic-benthic transition to favorably influence benthic distributions. In an experimental water column, we monitored cell swimming trajectories of Heterosigma akashiwo for 14 days after cells were signaled to enter the benthic resting stage. Using the statistical characteristics of individual cell trajectories, we developed a video-based motion assay to assign each tracked Heterosigma cell to one of three cell states known to occur during pelagic-benthic transition: induced motile, transitional and resting. The primary swimming characteristic influencing benthic distribution, net vertical velocity, was essentially the same for all three cell states. Hence, we found no evidence that active down-swimming influences benthic distributions. Our data suggest that benthic distributions of Heterosigma resting cells are similar to distributions of slowly sedimenting passive particles. These observations suggest that Heterosigma benthic resting cell distributions can be predicted by modeling the effects of cell sedimentation rates combined with geophysical flow patterns.     

Bottom-up controls on a mixed-species HAB assemblage: A comparison of sympatric Chattonella subsalsa and Heterosigma akashiwo (Raphidophyceae) isolates from the Delaware Inland Bays, USA

Recent novel mixed blooms of several species of toxic raphidophytes have caused fish kills and raised health concerns in the highly eutrophic Inland Bays of Delaware, USA. The factors that control their growth and dominance are not clear, including how these multi-species HAB events can persist without competitive exclusion occurring. We compared and contrasted the relative environmental niches of sympatric Chattonella subsalsa and Heterosigma akashiwo isolates from the bays using classic Monod-type experiments. C. subsalsa grew over a temperature range from 10 to 30 °C and a salinity range of 5–30 psu, with optimal growth occurring from 20 to 30 °C and 15 to 25 psu. H. akashiwo had similar upper temperature and salinity tolerances but also lower limits, with growth occurring from 4 to 30 °C and 5 to 30 psu and optimal growth between 16 and 30 °C and 10 and 30 psu. These culture results were confirmed by field observations of bloom occurrences in the Inland Bays. Maximum nutrient-saturated growth rates (μ_max) for C. subsalsa were 0.6 d⁻¹ and half-saturation concentrations for growth (K_s) were 9 μM for nitrate, 1.5 μM for ammonium, and 0.8 μM for phosphate. μ_max of H. akashiwo (0.7 d⁻¹) was slightly higher than C. subsalsa, but K_s values were nearly an order of magnitude lower at 0.3 μM for nitrate, 0.3 μM for ammonium, and 0.2 μM for phosphate. H. akashiwo is able to grow on urea but C. subsalsa cannot, while both can use glutamic acid. Cell yield experiments at environmentally relevant levels suggested an apparent preference by C. subsalsa for ammonium as a nitrogen source, while H. akashiwo produced more biomass on nitrate. Light intensity affected both species similarly, with the same growth responses for each over a range from 100 to 600 μmol photons m⁻² s⁻¹. Factors not examined here may allow C. subsalsa to persist during multi-species blooms in the bays, despite being competitively inferior to H. akashiwo under most conditions of nutrient availability, temperature, and salinity.     

Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo.     

Synchronous division and the pattern of diel vertical migration of Heterosigma akashiwo (Hada) Hada (Raphidophyceae) in a laboratory culture tank

Heterosigma akashiwo (Hada) Hada (Raphidophyceae) causes red tides in Osaka Bay (Japan). A clonal culture of the alga was grown in a 2 m tall culture tank on a 12: 12 LD cycle to determine patterns of vertical migration and cell division. A specific growth rate of 0.43 ln unit · day⁻¹ was obtained during complete mixing conditions. Under weakly stratified conditions (≈ΔT = 3–4°C/1.5 m), H. akashiwo in the tank grew and showed a similar pattern of vertical migration to that observed in the field for at least 6 days. Cell concentration, mean cell volume, and photosynthetic capacity, estimated by DCMU-induced fluorescence increase of H. akashiwo, were monitored in the stratified tank at 2-h intervals over 24 h at three levels in the water column. Cell ascent began shortly before the light period and vertically swimming cells were smaller in size than those sampled near the bottom of the tank. The cell division cycle and the pattern of vertical migration were phased individually by the light regime and were well synchronized with each other. This synchrony must be due to the interrelation between these two processes or the existence of a clock which controlled endogenous rhythms of both processes and was entrained by a light: dark cycle. The relative increase of fluorescence with DCMU was higher for migrating cells than for non-migrating cells.     

Algicidal activity of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa

The algicidal activity of the rhamnolipid biosurfactants (the mixture of Rha-Rha-C₁₀-C₁₀ and Rha-C₁₀-C₁₀) produced by Pseudomonas aeruginosa was investigated in the present paper. The results indicated that the biosurfactants had potential algicidal effects on the harmful algal bloom (HAB) species, Heterosigma akashiwo. The growth of H. akashiwo was strongly inhibited in medium containing rhamnolipids (0.4–3.0 mg L⁻¹); moreover, the rhamnolipids showed strong lytic activity toward H. akashiwo at higher concentrations (≥4.0 mg L⁻¹). In addition, the effects of the rhamnolipids on the growth of Gymnodinium sp. and Prorocentrum dentatum, another two kinds of HAB species, were also studied. Compared with the dramatic algicidal effect on H. akashiwo, the cells of P. dentatum were inhibited or lysed at higher concentrations (1.0–10.0 mg L⁻¹), while the cells of Gymnodinium sp. were not suppressed with the same treatment, indicating the rhamnolipids had the potential for the selective control of HABs.Morphometric analysis at ultrastructural level by transmission electron micrographs indicated that the extent of ultrastructural damage of the alga was severe at high concentrations of rhamnolipids and during extended periods of contact. The first response occurred in the plasma membrane which partly disintegrated. The lack of membrane facilitated the rhamnolipid biosurfactants into the cells and allowed damage to other organelles, which resulted in the injury of chloroplast, vacuolization of mitochondria and deformation of the cristae, disruption of nuclear membrane and condensation of chromatin in nucleus, suggesting that the lytic activity of rhamnolipids was mainly due to their powerful surfactivity and their tendency to cohere on the surface of phospholipids bimolecular layer of the cells and further destroyed the layers, and then the structure of quasi-membrane configurations inside the cells was disintegrated, following by the irreversible damage to the ultrastructure and the loss of the function of organelles, consequently leading the cells to lyse.     

Electron-opaque annular structure girdling the constricting isthmus of the dividing chloroplasts ofHeterosigma akashiwo (Raphidophyceae, Chromophyta)

Summary The plastokinesis (kinesis of chloroplasts) of a raphidophyte alga,Heterosigma akashiwo, was studied by electron microscopy using rapid freezing and freeze-substitution techniques. The chloroplasts are enveloped by two pairs of tightly appressed double membranes, the inner and the cytoplasmic outer pair. The inner pair constricts to divide in advance of the outer pair. By observation of serial sections an electron-opaque, annular structure (plastid-dividing ring) was observed at the isthmus of constricting chloroplasts, girdling the periplastidal outer surface of the inner pair of the four surrounding membranes. These observations suggest that the mechanisms underlying the constriction of the inner and outer pair may differ from each other. The localization of the annular structure (plastid-dividing ring) suggests that the inner pair of the surrounding membranes may be homologous to the double envelope membranes of the chloroplasts of Chlorophyta and Rhodophyta. In addition these findings provide a new evidence supporting the secondary endosymbiosis hypothesis for the origin of the chloroplasts in chromophyte algae.     

Diurnal appearance,fine structure,and chemical composition of fatty particles inHeterosigma akashiwo (Raphidophyceae)

Summary In cells ofHeterosigma akashiwo cultured under a photoperiod of 1212 LD, one or several fatty particles have appeared, grew in size approximately the second hour of the dark period, diminished in size from the tenth hour of the dark period, and completely disappeared by the seventh hour of the light period. The particles, usually located on the side of the nucleus away from the flagellar bases, were partially surrounded by endoplasmic reticulum, but not bounded by a membrane. Lipids comprised 80% of the isolated particles fraction, proteins only 1%.Triacyl-glycerol occupied 80% of the acyllipids in the lipid fraction. Tetradecanoic, hexadecanoic, and hexadecenoic acids comprised 80% of the total fatty acids composition.     

High toxicity of the novel bloom-forming species Chattonella ovata (Raphidophyceae) to cultured fish

A toxicological study of an axenic cell line of novel species Chattonella ovata Y. Hara et Chihara (Raphidophyceae) revealed that cultured species of sea bream (Pagrus major), horse mackerel (Trachurus japonicus), and yellowtail (Seriola quinqueradiata) were killed by 4.1–6.8 × 10³, 5.4 × 10³, and 2.8 × 10³ cells/mL, respectively. The sensitivity of the gill lamellae to C. ovata differed among the fish species tested. This finding revealed that C. ovata was highly toxic to the cultured fish. Histological examination showed that edema and hyperplasia of the secondary gill lamellae of red sea bream and horse mackerel occurred when exposed to, or killed by C. ovata, whereas severe damage in the gill lamellae was not observed in yellowtail. Chattonella produced high amounts of superoxide anion radicals and hydrogen peroxide, possibly responsible for the fish death observed. Based on the results of this study and occurrence of a red tide by this organism in China in 2001, we consider this organism to be one of the harmful algae in coastal waters. This is the first report demonstrating that C. ovata is highly toxic to fish, and that it produces superoxide and hydrogen peroxide.     

Effects of temperature and light on cyst germination and germinated cell survival of the noxious raphidophyte Heterosigma akashiwo

The effects of temperature and light on the germination of Heterosigma akashiwo cysts were examined using bottom sediments collected from Hakata Bay, Japan. In a suspension of mixed sediment and seawater in the temperature range of 5–30 °C, motile cells emerged within 3 weeks, but at ≤12 °C the cell numbers were markedly lower and the emergence of motile cells delayed. When suspension samples incubated at various temperatures were moved to 20 °C and incubated, only a few additional motile cells emerged. The number of motile cells germinated in the dark was significantly lower than under light conditions. When suspension samples incubated in the dark were exposed to light, only a few additional motile cells emerged. These results indicate that the initiation of germination in Heterosigma cysts suspended in seawater is not dependent on temperature and light conditions, although the speed of the germination process is affected by temperature, and cell survival just after germination is strongly affected by temperature and light.     

Anticariogenic activity of macelignan isolated from Myristica fragrans (nutmeg) against Streptococcus mutans

The occurrence of dental caries is mainly associated with oral pathogens, especially cariogenic Streptococcus mutans. Preliminary antibacterial screening revealed that the extract of Myristica fragrans, widely cultivated for the spice and flavor of foods, possessed strong inhibitory activity against S. mutans. The anticariogenic compound was successfully isolated from the methanol extract of M. fragrans by repeated silica gel chromatography, and its structure was identified as macelignan by instrumental analysis using 1D-NMR, 2D-NMR and EI-MS. The minimum inhibitory concentration (MIC) of macelignan against S. mutans was 3.9 microg/ml, which was much lower than those of other natural anticariogenic agents such as 15.6 microg/ml of sanguinarine, 250 microg/ml of eucalyptol, 500 microg/ml of menthol and thymol, and 1000 microg/ml of methyl salicylate. Macelignan also possessed preferential activity against other oral microorganisms such as Streptococcus sobrinus, Streptococcus salivarius, Streptococcus sanguis, Lactobacillus acidophilus and Lactobacillus casei in the MIC range of 2-31.3 microg/ml. In particular, the bactericidal test showed that macelignan, at a concentration of 20 microg/ml, completely inactivated S. mutans in 1 min. The specific activity and fast-effectiveness of macelignan against oral bacteria strongly suggest that it could be employed as a natural antibacterial agent in functional foods or oral care products.     

Phylogenetic relationships in the harmful dinoflagellate Cochlodinium polykrikoides (Gymnodiniales, Dinophyceae) inferred from LSU rDNA sequences

Phylogenetic relationships among chain-forming Cochlodinium species, including the harmful red tide forming dinoflagellate Cochlodinium polykrikoides, were investigated using specimens collected from coastal waters of Canada, Hong Kong, Japan, Korea, Malaysia, México, Philippines, Puerto Rico, and USA. The phylogenetic tree inferred from partial (D1–D6 regions) large subunit ribosomal RNA gene (LSU rDNA) sequences clearly differentiated between C. polykrikoides and a recently described species, Cochlodinium fulvescens. Two samples collected from the Pacific coasts of North America (British Columbia, Canada and California, USA) having typical morphological characters of C. fulvescens such as the sulcus located in the intermediate region of the cingulum, were closely related to C. fulvescens from western Japan in the phylogenetic tree. Cochlodinium polykrikoides formed a monophyletic group positioned as a sister group of the C. fulvescens clade with three well-supported sub-clades. These three clades were composed of (1) East Asian, including specimens collected from Hong Kong, western Japan, and southern Korea, (2) Philippines, from Manila Bay, Philippines and Omura Bay, Japan, and (3) American/Malaysian, from the Atlantic coasts of USA, the Pacific coast of México, Puerto Rico, and Borneo Island, Malaysia. Each of these clades is considered to be a so-called “ribotype” representing the population inhabiting each region, which is distinguished based on ribosomal RNA gene sequences in the species despite similarities in their morphological characters.     

Acaricidal activity of the essential oil from Tetradenia riparia (Lamiaceae) on the cattle tick Rhipicephalus (Boophilus) microplus (Acari; Ixodidae)

Tetradenia riparia (Lamiaceae) is a well-known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of T. riparia essential oil (EO) against engorged females of Rhipicephalus (Boophilus) microplus (Acari; Ixodidae). For this purpose, nine serial concentrations (12.50%, 6.25%, 3.75%, 1.80%, 0.90%, 0.45%, 0.22%, 0.11%, and 0.056% w/v) of T. riparia were used for the adult immersion test (AIT). For the larval packet test (LPT), we used 14 serial concentrations (100.00%, 50.00%, 25.00%, 12.50%, 6.25%, 3.65%, 1.82%, 0.91%, 0.45%, 0.228%, 0.114%, 0.057%, 0.028%, and 0.014% w/v). The results for AIT showed 100.00% and 2.05% mortality, 19.00 and 90.20% for the total number of eggs, egg-laying inhibition of 0.00% and 90.20%, hatchability inhibition of 0.00% and 70.23%, and product effectiveness of 100.00% and 2.89%, respectively. The AIT indicated that the LC₅₀ and LC_99.9, calculated using the Probit test, were for mortality (%) 0.534 g/mL (0.436–0.632) and 1.552 g/mL (1.183–1.92); for total number of eggs were 0.449 g/mL (0.339–0.558) and 1.76 g/mL (1.27–2.248); and for hatchability inhibition were 0.114 g/mL (0.0–0.31) and 2.462 g/mL (1.501–3.422), respectively. Larvae between 14 and 21 days old were fasted and placed in each envelope. Bioassays were performed at 27° ± 1 °C, RH ? 80%. Larval mortality was observed 24 h after treatment and showed 10.60–100% mortality in the LPT bioassay. The LPT showed that the LC₅₀ and LC_99.9 were 1.222 g/mL (0.655–1.788) and 11.382 g/mL (7.84–14.91), respectively. A positive correlation between T. riparia EO concentration and tick control, was observed by the strong acaricidal effects against R. (B.) microplus, and the mortality rate of ticks was dose-dependent. Our results showed that T. riparia is a promising candidate as an acaricide against resistant strains of R. (B.) microplus.