Application of ecotoxicological tools to evaluate the quality status of mangroves under restoration in the Yucatán Peninsula, Mexico.

Ecotoxicological tools, namely biomarkers and bioassays, may provide insights on the ecological quality status of mangroves under restoration. We investigated how 1) physicochemical parameters and water bioassays using Artemia franciscana; and 2) quantification of sublethal (osmoregulatory capacity, biochemical, and oxidative stress) and individual biomarkers (density, length-weight relationship [LWR], parasitic prevalence) in the sentinel fiddler crab Minuca rapax, can improve restoration indicators in mangroves from the Yucatán Peninsula, Southern Gulf of Mexico. We showed that water quality was improved with restoration, but still presented toxicity. Regarding sublethal biomarkers, M rapax from restored areas lower osmotic regulatory capacity, higher oxidative stress, and showed lipid peroxidation. As to the individual biomarkers, the density, LWR, and the prevalence of parasites in M. rapax was higher in restored areas. The use of bioassays/biomarkers were useful as early warning indicators to better assess the health of mangroves under restoration.

ArHsp90 is important in stress tolerance and embryo development of the brine shrimp, Artemia franciscana.

Females of the extremophile crustacean, Artemia franciscana, either release motile nauplii via the ovoviviparous pathway or encysted embryos (cysts) via the oviparous pathway. Cysts contain an abundant amount of the ATP-independent small heat shock protein that contributes to stress tolerance and embryo development, however, little is known of the role of ATP-dependent molecular chaperone, heat shock protein 90 (Hsp90) in the two processes. In this study, a hsp90 was cloned from A. franciscana. Characteristic domains of ArHsp90 were simulated from the deduced amino acid sequence, and 3D structures of ArHsp90 and Hsp90s of organisms from different groups were aligned. RNA interference was then employed to characterize ArHsp90 in A. franciscana nauplii and cysts. The partial knockdown of ArHsp90 slowed the development of nauplius-destined, but not cyst-destined embryos. ArHsp90 knockdown also reduced the survival and stress tolerance of nauplii newly released from A. franciscana females. Although the reduction of ArHsp90 had no effect on the development of diapause-destined embryos, the resulting cysts displayed reduced tolerance to desiccation and low temperature, two stresses normally encountered by A. franciscana in its natural environment. The results reveal that Hsp90 contributes to the development, growth, and stress tolerance of A. franciscana, an organism of practical importance as a feed source in aquaculture.

Comparative Microbiome Analysis of Artemia spp. and Potential Role of Microbiota in Cyst Hatching.

Brine shrimp (Artemia spp.) is a significant factor in determining aquaculture production. Since the microbiota of Artemia can colonize the gut in larvae, various microorganisms transmitted from Artemia can affect host larval health. Although the microbiota composition of Artemia would be essential in determining aquaculture productivity, our understanding on microbiome of Artemia is still insufficient. Through our study, we identified the species of Artemia cysts supplied by three different manufacturers (P1, P2, and P3) with investigation of size and hatching efficiency. The species of Artemia from P1 was identified as A. tibetiana, and P2 and P3 was A. franciscana. A. tibetiana hatched from the P1 cysts had the largest body size with the lowest hatching rate. Furthermore, we conducted a comprehensive analysis of the microbiome present in the rearing water and the nauplius whole body from each product. We observed specific microbiota compositions, both beneficial and harmful, depending on the product types and the sample types. Additionally, we found that the microbiota composition in the rearing water was associated with the manufacturing environment, while the compositions in the nauplius whole body were species-specific. Notably, we discovered that an extract containing microbiota from the nauplius sample of P3 increased the hatching rate of A. tibetiana, indicating a positive role in Artemia culture. These findings demonstrate that the microbial communities present in Artemia vary according to the product and/or species, underscoring their significance in aquaculture production.

Isolation of 4-nerolidylcatechol from leaves of Piper peltatum L., and evaluation of larvicidal activity in mosquito vectors, with emphasis on Aedes aegypti (Diptera: Culicidae)

ABSTRACT Synthetic chemical insecticides are widely used in population control of pests. Aedes aegypti is the primary vector of dengue, Zika, chikungunya and yellow fever to humans, and has proven resistance to chemical insecticides. As an alternative vector control method, the ethanolic extract of the leaves of Piper peltatum L. (Piperaceae) showed larvicidal activity against Ae. aegypti. Despite the wide medicinal use of this plant, the biological activity of its isolated constituents remains unexplored. In this sense, we isolated, identified and evaluated the larvicidal activity of 4-nerolidylcatechol (4-NC) from P. peltatum against Ae. aegypti, Culex quinquefasciatus and Anopheles darlingi, focusing on the larvicidal, adulticidal and genotoxic potential of 4-NC on Ae. aegypti. Larvae were captured in the city of Manaus, Amazonas state, Brazil. 4-NC was isolated from the extract of the leaves of P. peltatum via silica gel chromatography. This was identified using nuclear magnetic resonance spectroscopy and tested in Artemia franciscana (6.25, 12.5, 25, 50, 100 and 200 µg/mL). In the toxicity bioassay, Ae. aegypti larvae were exposed to 30, 50, 70, 90, and 110 µg/mL of 4-NC, while Cx. quinquefasciatus and An. darlingi were exposed to 6.25, 12.5, 25, 50 and 100 µg/mL. Ae. aegypti larvae were also subjected to 40 and 60 µg/mL of 4-NC (genotoxicity bioassay), and adult females to 62.5 to 1,000 µg/mL (adulticidal bioassay). The results of the 4-NC toxicity assays showed that there was 100% mortality in larvae of Ar. franciscana at the concentration of 200 µg/mL, with an LC50 of 8.0 μg/mL. In the larvae of Ae. aegypti, mortality was 100%, with an LC50 of 62 μg/mL. In larvae of Cx. quinquefasciatus, 97% mortality occurred, with an LC50 of 52.3 μg/mL, and in An. darlingi larvae there was an 83% mortality rate, with an LC50 of 55.8 μg/mL. In adults of Ae. aegypti, however, there was no adulticidal activity. In the larvae of Ae. aegypti, the genotoxic effect of 4-NC (40 and 60 µg/mL) showed significant frequency (p < 0.05) of cellular abnormalities (micronuclei, budding and nuclear bridges) of interphasic nuclei of neuroblasts and oocytes in relation to the negative control. This result may be associated with a decrease in oviposition of females, which was observed in two generations. We can confirm that 4-NC has larvicidal activity against Ae. aegypti, Cx. quinquefasciatus and An. darlingi. Although it does not present adulticidal activity in Ae. aegypti, it reduced the oviposition of females. Therefore, 4-NC seems to be a strong candidate for the development of an alternative method for the control of these mosquitoes in the immature phase.

Evaluation of Black Soldier Fly Hermetia illucens as Food for Pink-Spotted Lady Beetle Coleomegilla maculata.

The discovery of new and improved factitious and artificial diets is necessary for cost-effective rearing of predatory arthropods. This study evaluated Hermetia illucens black soldier fly (BSF) as a suitable alternative food source for rearing the predatory coccinellid Coleomegilla maculata (Cmac). The hypothesis that BSF larval powder was suitable food to support the growth, development, and reproduction of Cmac was tested in the laboratory. When compared to a standard in-house diet containing brine shrimp egg powder plus Chlorella vulgaris green algae and myristic acid (BSE+CM), the BSF and BSF+CM diets reduced immature growth and development. Immatures successfully reared to teneral adults were smaller when fed BSF or BSF+CM rather than BSE+CM. Combining BSF with a powdered artificial diet (AD), i.e., BSF+AD, did not improve predator growth or development, compared to Cmac reared on BSE+CM. Cmac oviposition responses, i.e., egg clutch production, to BSF vs. BSE+CM or BSF+AD vs. BSE+CM did not differ significantly. In conclusion, BSF has the potential to be food that supports Cmac oviposition behavior. Future research is necessary to discover an ideal mixture of BSF, BSE+CM, or AD that supports Cmac growth, development, and reproduction over multiple generations.

Effects of salinity on growth and survival in five Artemia franciscana (Anostraca: Artemiidae) populations from Mexico Pacific Coast

Salinity is an important factor influencing growth and survival of aquatic organisms such as Artemia, a valuable aquaculture species. This study evaluated the effects of salinity on A. franciscana populations from different water bodies in Mexico’s Pacific Coast. With this purpose, five autochthonous bisexual Artemia populations were tested to assess their survival and growth values against salinities of 40, 60, 80, 100 and 120g/l, under laboratory conditions (25±2ºC; pH 8-10; constant light and aeration). The organisms were fed with 100mL of rice bran and 2L of Tetraselmis suecica (500 000cel/ml). The culture experiments were made in 200L plastic tanks, and survival and growth final values were obtained after 21 culture days. Survival and growth curves were determined by a regression analysis (R²). The significant differences between salinities were determined by ANOVA test (p<0.05). The best survival and growth rates were found at salinities of 100-120g/l. When the Mexican Artemia populations were cultivated at 40g/l of salinity, 100% mortality was observed in the juvenile stage. This study determined that survival and growth values of A. franciscana populations increased with salinity. The five A. franciscana populations presented significant differences in their survival rate under various salinity regimes. The studied populations experienced high mortality at salinities under 60g/l and over 200g/l, and especially during the metanauplius stage. The present study confirms that growth rates in Mexican A. franciscana populations from Pacific Coast habitats are not inversely proportional to salinity. These A. franciscana populations should be cultured at 100-120g/l of salinity to obtain better survival and growth rates. This data is useful to improve culture systems in aquaculture biomass production systems. Rev. Biol. Trop. 59 (1): 199-206. Epub 2011 March 01.

La salinidad es el factor más importante que modifica el crecimiento y la sobrevivencia de las poblaciones mexicanas de Artemia en el mundo. El efecto de este factor en las poblaciones costeras del Pacífico Mexicano ha sido poco estudiado. Cinco poblaciones bisexuales de Artemia provenientes de cuerpos de aguas costeras del Pacífico Mexicano, se estudiaron para determinar el efecto que tiene la salinidad de 40, 60, 80, 100 y 120g/l sobre la sobrevivencia y el crecimiento, en condiciones de laboratorio (25±2ºC, pH 8-10; luz y aireación continua). Los experimentos de cultivo fueron realizados en tanques de plástico de 200l. Los valores finales de sobrevivencia y crecimiento fueron determinados después de 21 días de cultivo. Los organismos fueron alimentados con 100ml de salvado de arroz y 2l de Tetraselmis suecica (500cel/ml). Las curvas de sobrevivencia y crecimiento fueron determinadas usando un análisis de regresión (R²). Las diferencias significativas entre los valores promedio a las distintas salinidades fueron determinadas por una prueba de ANDEVA (p<0.05). Las mejores tasa de sobrevivencia y crecimiento se encontraron a la salinidad de 100-120g/l. Cuando las poblaciones mexicanas fueron cultivadas a 40 g/l de salinidad, hubo una mortalidad del 100% de los organismos en estadio juvenil. A la salinidad de 80g/l, los valores de sobrevivencia y crecimiento, fueron menores que cuando se cultivaron entre 100 y 120g/l. Las cinco poblaciones bisexuales de A. franciscana presentaron diferencias significativas en sus tasas de sobrevivencia bajo las salinidades estudiadas. Las tasas de sobrevivencia fueron afectadas por la salinidad. Las poblaciones estudiadas experimentaron alta mortalidad a salinidades por debajo de 60 g/l y por arriba de 200g/l. Es importante mencionar que las altas mortalidades se observaron en el estadio metanauplios. El presente trabajo confirma que las tasas de crecimiento de las poblaciones que costeras del Pacífico Mexicano de A. franciscana no son inversamente proporcionales a la salinidad. Las poblaciones de A. franciscana deben de ser cultivadas a una salinidad de 100-120g/l para obtener mejores tasas de sobrevivencia y crecimiento. Estos datos permiten realizar mejoras en los sistemas de cultivo de este crustáceo para la obtención de biomasa para la acuicultura.

Bioactive dihydroxyfuranonaphthoquinones from the bark of Tabebuia incana A.H. Gentry (Bignoniaceae) and HPLC analysis of commercial pau d'arco and certified T.incana bark infusions

Tabebuia incana A.H. Gentry (Bignoniaceae) is a tree from the Brazilian Amazon having medicinal uses and is one several Tabebuia spp. known as pau d'arco or palo de arco in this region. Fractionation of the bark ethanolic extract afforded a mixture of 5 and 8-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-diones (1 and 2, respectively) identified on the basis of nuclear magnetic resonance (NMR), infrared (IR) and mass (MS) spectra, whose in vitro antimalarial and antitumor activity have been shown previously. This is the first study on T. incana bark, and 2 are described in this species for the first time. Also, high performance liquid chromatography (HPLC) analysis of T. incana bark tea revealed the presence of the 1 + 2 mixture peak corresponding to a concentration in the range 10-6-10-5 M. The chromatograms of teas prepared from commercial pau d' arco and T. incana bark were also studied and the presence of the 1 + 2 peak has potential for quality control of commercial plant materials.

Tabebuia incana A.H. Gentry (Bignoniaceae) é uma árvore da Amazônia brasileira com usos medicinais. É uma de várias espécies de Tabebuia conhecidas como pau d'arco ou palo de arco nesta região. O fracionamento do extrato etanólico das cascas resultou no isolamento da mistura de 5 e 8-hidróxi-2-(1-hidroxietil)nafto[2,3-b]furano-4,9-dionas (1 e 2, respectivamente), identificadas com base em seus espectros de ressonância magnética nuclear (RMN), infravermelho (IV) e massa (EM), e cujas atividades antimalárica e antitumoral in vitro foram mostradas previamente. Este é o primeiro estudo das cascas de T. incana e a primeira vez que o composto 2 é descrito nesta espécie. Análises por cromatografia liquida de alto empenho (CLAE) do chá das cascas de T. incana revelaram a presença de um pico correspondente à mistura de 1 + 2, permitindo inferir uma concentração na faixa de 10-6-10-5 M desses componentes no chá. Os cromatogramas de chás (infusões) preparados a partir das cascas de pau d' arco commercial and T. incana certificada também foram estudados. A verficação da presença do pico das substâncias 1 + 2 nos cromatogramas tem potencial contribuição para o controle de qualidade de material vegetal comercial.

Screening of plants found in Amazonas State for lethality towards brine shrimp

226 methanol and water extracts representing 74 mainly native plant species found in Amazonas State, Brazil, were tested at a standard concentration of 500 g/mL for lethality towards larvae of the brine shrimp species Artemia franciscana. Several cytotoxic plant species were identified in this work: Aspidosperma marcgravianum, A. nitidum, Croton cajucara, Citrus limetta, Geissospermum argenteum, Minquartia guianensis, Piper aduncum, P. amapense, P. capitarianum, P. tuberculatum and Protium aracouchini. The results were analyzed within the context of the available traditional knowledge and uses for these plants.

Foram testados 226 extratos metanólicos e aquosos de 74 espécies vegetais (a maioria nativas) encontradas no Estado do Amazonas, Brasil, para letalidade às larvas da espécie microcrustácea Artemia franciscana na concentração teste de 500 g/mL. Várias espécies citotóxicas foram identificadas nesse estudo: Aspidosperma marcgravianum, A. nitidum, Citrus limetta, Croton cajucara, Geissospermum argenteum, Minquartia guianensis, Piper aduncum, P. amapense, P. capitarianum, P. tuberculatum e Protium aracouchini. Os resultados foram analisados levando em consideração as informações populares, quando disponíveis.