Larval Ontogenesis Research Articles

Unveiling the early life core microbiome of the sea cucumber Apostichopus japonicus and the unexpected abundance of the growth-promoting Sulfitobacter

BackgroundMicrobiome in early life has long-term effects on the host’s immunological and physiological development and its disturbance is known to trigger various diseases in host Deuterostome animals. The sea cucumber Apostichopus japonicus is one of the most valuable marine Deuterostome invertebrates in Asia and a model animal in regeneration studies. To understand factors that impact on host development and holobiont maintenance, host-microbiome association has been actively studied in the last decade. However, we currently lack knowledge of early life core microbiome during its ontogenesis and how it benefits the host’s growth.ResultsWe analyzed the microbial community in 28 sea cucumber samples from a laboratory breeding system, designed to replicate aquaculture environments, across six developmental stages (fertilized eggs to the juvenile stage) over a three years-period to examine the microbiomes’ dynamics and stability. Microbiome shifts occurred during sea cucumber larval ontogenesis in every case. Application of the most sophisticated core microbiome extraction methodology, a hybrid approach with abundance-occupancy core microbiome analyses (top 75% of total reads and > 70% occupation) and core index calculation, first revealed early life core microbiome consisted of Alteromonadaceae and Rhodobacteraceae, as well as a stage core microbiome consisting of pioneer core microbe Pseudoalteromonadaceae in A. japonicus, suggesting a stepwise establishment of microbiome related to ontogenesis and feeding behavior in A. japonicus. More interestingly, four ASVs affiliated to Alteromonadaceae and Rhodobacteraceae were extracted as early life core microbiome. One of the ASV (ASV0007) was affiliated to the Sulfitobactor strain BL28 (Rhodobacteraceae), isolated from blastula larvae in the 2019 raring batch. Unexpectedly, a bioassay revealed the BL28 strain retains a host growth-promoting ability. Further meta-pangenomics approach revealed the BL28 genome reads were abundant in the metagenomic sequence pool, in particular, in that of post-gut development in early life stages of A. japonicus.ConclusionRepeated rearing efforts of A. japonicus using laboratory aquaculture replicating aquaculture environments and hybrid core microbiome extraction approach first revealed particular ASVs affiliated to Alteromonadaceae and Rhodobacteraceae as the A. japonicus early life core microbiome. Further bioassay revealed the growth promoting ability to the host sea cucumber in one of the core microbes, the Sulfitobactor strain BL28 identified as ASV0007. Genome reads of the BL28 were abundant in post-gut development of A. japonicus, which makes us consider effective probiotic uses of those core microbiome for sea cucumber resource production and conservation. The study also emphasizes the importance of the core microbiome in influencing early life stages in marine invertebrates. Understanding these dynamics could offer pathways to improve growth, immunity, and disease resistance in marine invertebrates.

Molecular expression of digestive enzymes during the ontogeny of the Mayan cichlid ( Cichlasoma urophthalmus )

The Mayan cichlid (Cichlasoma urophthalmus), is a freshwater fish of the Cichlidae family, native to the south east of Mexico, it has potential for extensive cultivation due to its excellent meat quality and local demand. However, the nutrigenomic aspects and molecular expression of genes involved in its digestion that could contribute to its growth during the larval stage are unknown. Some enzymes such as Pepsin (PEP), pancreatic such as trypsin (TRY), Chymotrypsin [CHY] and intestinal enzymes such as Leucine aminopeptidase [ANPEP], function during larval ontogenesis and activity patterns of the key factors involved in the digestive system. For this study, 30–50 larvae were collected, which were processed with mRNA extraction techniques and cDNA synthesis by RT-PCR using qPCR oligos designed from Oreochromis niloticus, measuring molecular expression during the initial ontogeny of C. urophthalmus. The results obtained show the molecular expression of these enzymes occurs through epigenetic factors such as changes from endogenous to exogenous feeding, presenting the activation of genes for the formation of organs of the digestive tract that allow the secretion of specific enzymes for processing and digestion of food, that can activate these genes to increase the digestion efficiency and the optimal use of nutrients.

Influence of temperature on larval ontogenesis of Geophagus brasiliensis (Quoy & Gaimard, 1824) (Pisces: Cichlidae).

Studies on the larval development of fish are essential for conservation and improvements in cultivation techniques. Geophagus brasiliensis popularly known as Cará has potential as a fish of interest in ornamental aquaculture. Wild adults of G. brasiliensis were kept in an aquarium for spontaneous reproduction. Newly hatched larvae were transferred to 5-litre aquaria at 22, 26 and 30°C until total yolk sac resorption. Histological slides were used for biometric analysis and monitoring of larval ontogenesis at different temperatures. Histologically, from the first to the fourth days it was possible to identify myomeres, optic vesicle, yolk syncytial layer, brain, heart and differentiation of the eye layers. From the fourth to the seventh days, it was possible to identify mandibular and gill cartilages, swim bladder, liver, prismatic epithelium with striated border in intestine and renal epithelium. All biometric measurements increased over the days, except height and length of the yolk sac, which gradually decreased until the complete resorption of the yolk sac that occurred on the fifth day at a temperature of 30°C, the sixth day at 26°C and the seventh day at 22°C. Morphological events at 30°C such as the reabsorption of the yolk sac, the appearance of cartilage in the branchial arches and differentiation of the layers of the eyes occurred faster compared with the other temperatures tested. Opening of the mouth and digestive tract occurred at a similar time on the fourth day in all temperatures.

Состав жирных кислот в трохофорах мидий Mytilus galloprovincialis, выращенных в условиях загрязнённости полихлорбифенилами

Состояние черноморских популяций Mytilus galloprovincialis в естественной среде обитания напрямую зависит от развития мидии на всех стадиях, в том числе на начальных стадиях личиночных форм, наиболее чувствительных к загрязнению окружающей среды. Поллютанты органического происхождения оказывают негативное влияние на личинки моллюска, проявляющееся в торможении их роста и развития. Закономерности размножения мидии хорошо изучены, что даёт возможность получать в контролируемых лабораторных условиях личинки из искусственно оплодотворённых яйцеклеток этого вида моллюсков. В работе впервые исследован жирнокислотный состав общих липидов, выделенных из биомассы тканей личинок M. galloprovincialis на стадии трохофоры в контроле и после их трёхдневной экспозиции в среде с добавлением различных концентраций полихлорбифенилов. Жирнокислотный состав суммарных липидов в биомассе личинок, полученных на третьи сутки эксперимента, исследовали методом хромато-масс-спектрометрии. Всего идентифицировано 14 жирных кислот: 59 % из них относились к насыщенным, 24 % — к моноеновым, 17 % — к полиеновым. Для статистического анализа использовали программу MATLAB (версия 8.2). В условиях проведённого эксперимента в липидах трохофор M. galloprovincialis достоверно отличались значения суммы мононенасыщенных и полиненасыщенных жирных кислот. Сумма насыщенных жирных кислот статистически значимо не изменялась. Основными насыщенными жирными кислотами во всех исследуемых трохофорах мидий являлись пальмитиновая (C16:0) и стеариновая (C18:0). Их концентрации значительно не изменялись под действием полихлорбифенилов. Наиболее значимые мононенасыщенные жирные кислоты — олеиновая (C18:1ω9), пальмитолеиновая (C16:1ω7) и вакценовая (C18:1ω7). Содержание мононенасыщенных жирных кислот понижалось вдвое при действии полихлорбифенилов с концентрациями 0,1 и 1 мкг·л−1; при концентрации полихлорбифенилов 10 мкг·л−1 суммарное содержание этих кислот было равно таковому в контроле. Среди полиненасыщенных жирных кислот, обладающих положительной биологической активностью, были идентифицированы арахидоновая (C20:4ω6), эйкозапентаеновая (C20:5ω3) и докозагексаеновая (C22:6ω3). Суммарное содержание Омега-3 и Омега-6 кислот в личинках мидий в контрольном опыте не превышало 12,8 %. С увеличением концентрации полихлорбифенилов в среде выращивания трохофор с 0,1 до 1 мкг·л−1 концентрация полиненасыщенных жирных кислот повышалась в 2,5 раза. При концентрации полихлорбифенилов 10 мкг·л−1 и в пробе с ацетоном суммарное содержание полиненасыщенных жирных кислот было сопоставимо с таковым в контрольном опыте. Результаты исследования свидетельствуют о том, что жирнокислотный отклик трохофор мидий M. galloprovincialis максимален при воздействии концентраций полихлорбифенилов от 0,1 до 1 мкг·л−1. При концентрации загрязнителей 10 мкг·л−1 и выше биохимические процессы в личинках, по-видимому, замедляются. Результаты данного исследования могут способствовать лучшему пониманию перестроек, позволяющих моллюскам на личиночных стадиях развития адаптироваться к условиям загрязнения среды обитания органическими поллютантами.

Ontogenesis and embryogenesis of Megaleporinus elongatus larvae (Characiformes: Anostomidae) from the Jequitinhonha River Basin

Five females and five males of Megaleporinus elongatus captured in the Jequitinhonha River were transferred to hypophysation tanks with a constant water flow at 26°C at the Machado Mineiro Fish Farm. They were submitted to induced reproduction by the hypophysation method. Crude carp pituitary extract (Cyprinus carpio) was injected into the coelomic cavity in two doses (2.5 and 5.0 mg/kg) for males and 3 doses (2.5, 5.0, and 2.5 mg/kg) for females. The doses were applied with a 24-hr interval between the 1st and 2nd doses and a 12-hr interval between the 2nd and 3rd ones. Oocytes and semen were obtained by manual extrusion, and the dry method was used for fertilisation. The eggs were kept in funnel type 20-litre incubators with constant water flow at 23°C. Egg samples were collected every 10 min and photographed under a stereomicroscope for analysis of embryonic development until hatching. The main events of embryogenesis and their respective duration times were: cleavage (45 min), blastula (3 hr 14 min), gastrula (5 hr 25 min), closure of blastopore (6 hr 34 min), differentiation of somites (13 hr 20 min), tail release (17 hr 50 min), and hatching (24 hr 50 min). During the larval ontogenesis, the yolk sac gradually decreased until its complete reabsorption on the 7th day after hatching. The opening of the mouth and digestive tract occurred from the 5th to the 7th day, indicating that larvae could feed exogenously. These results provide information to improve the techniques of induced spawning and cultivation of M. elongatus.

Solea senegalensis vasa transcripts: molecular characterisation, tissue distribution and developmental expression profiles

The Vasa protein is an RNA helicase belonging the DEAD (Asp-Glu-Ala-Asp)-box family. The crucial role played by the vasa gene in the germ-cell lineage of both vertebrates and invertebrates has made this gene a useful molecular marker for germinal cells and a useful tool in surrogate broodstock production using primordial germ cell transplantation. With the aim of establishing a novel approach to improving Solea senegalensis broodstock management, the vasa gene in this species was characterised. Four S. senegalensis vasa transcripts were isolated: Ssvasa1, Ssvasa2, Ssvasa3 and Ssvasa4. Their phylogenetic relationship with other vasa homologues was determined confirming the high degree of conservation of this helicase throughout evolution. Our qPCR results showed that S. senegalensis vasa transcripts are prevalently expressed in gonads, with ovary-specific expression for Ssvasa3 and Ssvasa4. During embryonic and larval development, a switch between the longest and the shortest transcripts was observed. While Ssvasa1 and Ssvasa2 were maternally supplied, Ssvasa3 and Ssvasa4 depended on the de novo expression program of the growing juveniles, suggesting that vasa mRNA could be involved in Senegalese sole gonad differentiation. In situ hybridisation and immunohistochemical analysis performed in 150-days after hatching (DAH) larvae showed vasa product expression in the germinal region of early gonads. In our work we demonstrated the usefulness of Ssvasa mRNAs as molecular markers for primordial germ cells and germinal cells during embryonic development, larval ontogenesis and gonad differentiation. Furthermore, our results confirmed the potential of vasa to help investigate germinal cell biotechnology for Senegalese sole reproduction.

Development of Labrus Bergylta (Ascanius 1767) Larvae from Hatching to Metamorphosis

Ballan wrasse, Labrus bergylta (Ascanius, 1767), has commercial interest as a cleaner fish in aquaculture, and culture of juveniles may be a good alternative to catching wild fish. However, basic knowledge of early development is limited. To study hatching and larvae ontogenesis, eggs were collected from a broodstock of wild caught ballan wrasse held in captivity. Larvae were fed rotifers for 27 days; Artemia was introduced at day 20 and fed larvae to day 49 after hatching. Age at hatching, and larval ontogenesis based on external morphological features were linked to the chronological age of larvae as days post hatching (DPH) and physiological age as degree day (°C.day) and standard length (SL). The ballan wrasse egg diameter was 1.05 ± 0.04 mm and 0.87 ± 0.05 mm with and without the gelatinous layer, respectively, and hatched at a SL of 3.64 ± 0.05 mm, 7 days, i.e., 72°C.day, after fertilization. The ontogeny of larvae from hatching to metamorphosis was divided into four stages. Yolk sac larva, 0 to 9 DPH (SL 4.28 ± 0.11 mm), where mouth opens and pigmentation of the eyes is seen. Dilated swim bladder is visible. Preflexion larva, 10 to 25 DPH (SL 5.35 ± 0.30 mm); yolk sac has disappeared, and initial formation of caudal fin rays occurs. Gaseous inflation of swim bladder is observed. Flexion larva, 26 to 33 DPH (SL 5.9 ± 0.78 mm), initial resorption of primordial fin fold is seen. Postflexion larva, 34 to 49 DPH (SL 10.52 ± 0.82 mm) by the end of this stage (age 686°C.day), the dorsal, anal, caudal and pelvic fins were developed. The ontogenetic development, linked to SL, chronological and physiological age, provides a baseline reference and makes it possible to compare developmental levels in future studies of ballan wrasse in culture and wild populations.

Transcriptomics for understanding marine fish larval development1This review is part of a virtual symposium on current topics in aquaculture of marine fish and shellfish.

The larval phase is a crucial period in the life of marine fish. During this phase, the organism will acquire the phenotype of an adult fish through the development of tissues and organs and the maturation of some of the principal physiological functions. Many biological processes (differentiation, cellular proliferation, growth, etc.) are regulated during this period. These regulations take place at different biological levels and particularly concern the expression of genes involved in larval ontogenesis processes. The development of bioinformatic resources (DNA or cDNA sequences) and molecular tools enabling high throughput gene expression analysis (microarrays) have allowed the transcriptome of marine fish species to be studied. In the present review, we summarize the main findings from transcriptomic investigations of development of marine fish larvae. Special attention is paid to investigations of transcriptomic patterns during postembryonic development and to the impact of environmental or nutritional factors on the transcriptome of marine fish larvae. Transcriptomic approaches will be especially useful in the future for investigating the effect of temperature and water acidification (or pH) on the development of different fish species in the context of global climate change.

External proprioceptors of locust locomotor organs and their change in the course of early larval ontogenesis

This work studies topography and structure of such important insect external proprioceptors as campaniform sensillae (CS). These mechanoreceptors are essential components of insect posture and locomotion regulation and participate in control of various forms of insect motor behavior (walking, jump, flight). There are traced their quantitative changes as well as differences in distribution of groups of these leg receptors at consecutive stages (from the 1st to the 4th instar) of ontogenetic development of larva of the locust Locusta migratoria L. The presence of groups of CS in proximal parts of extremities has been noted as early as in the 1st instar larvae. The CS groups in the wing pads were revealed only in the 4th instar larvae. The presented data allow connecting changes in structure and distribution of these proprioceptors with differences in locomotion of imagoes and larvae. A possible effect of these insect larval proprioceptors on central generators of the locomotion rhythms is discussed.

Early development of the silver catfishRhamdia quelen(Quoy & Gaimard, 1824) (Pisces:Heptapteridae) from the São Francisco River Basin, Brazil

The silver cat¢sh, Rhamdia quelen, is endemic to North, Central and South America with high aquaculture potential and wide acceptance in the market. Breeder ¢sh were subjected to induced reproduction through hypophysation using a crude common carp pituitary extract. Egg characteristics, oocyte surface ultrastructure and histology of larval ontogenesis until whole yolk resorption were described for the ¢rst time for this species. Oocytes and semen were obtained by manual extrusion, and fertilization was conducted using the dry method. After fertilization, eggs were kept in incubators at 241C. The embryonic development was monitored using a stereomicroscope every 10 min until hatching. To analyse the larval development, larvae samples were collected from incubators daily until the ¢fth day, ¢xed in Bouin’s £uid and subjected to routine histological techniques. The oocyte extrusion occurred 8 h after the second hormone dose at 261C. The oocytes were spherical, non-adhesive and yellow, with a diameter of 1471.75 � 47.63 mm. Scanning electron microscopy revealed a thin jelly coat covering the zona radiata in the animal pole around the micropyle. The blastopore closure occurred within 8 h after fertilization, and the fertilization rate was 79.9 � 5.2% at 24 1C. Embryonic development was completed within 25 h 30 min after fertilization. The complete resorption of the yolk and the formation of the digestive system organs and the mouth opening occurred on the ¢fth day, indicating a need for exogenous feeding. The results of this study provide information important for improvement in R. quelen culture and management.

Patchiness of eggs, larvae and juveniles of European hake Merluccius merluccius from the NW Mediterranean

AbstractWe estimated patchiness for different life stages (egg, larvae and juveniles) of European hake (Merluccius merluccius) from two ichthyoplankton surveys (September and November 1999) and four bottom‐trawl surveys (November 1998, February, June and September 1999). Lloyd's patchiness P = 1 + 1/k, where k is the dispersion parameter of the negative binomial distribution, was calculated. Juveniles showed lower indices of patchiness than early ontogenetic stages. In juveniles, patchiness was highest for smaller size classes (3.5–5 cm TL) settling to their near‐bottom habitat and was very low for those larger than 5 cm up to the 30–35 cm size class (approximately 3 yr of age, 50% mature individuals). Patchiness of settling juveniles was especially high in the June and September surveys, coinciding with peak recruitment to the bottom. Both eggs and yolk sac larvae showed patchiness values <10, increasing in flexion stage larvae from 12 to 20. This pattern is different to that observed in other studies for pelagic species, where patchiness of early‐stage eggs is very high, but coincides with results obtained for other demersal species with pelagic eggs. Factors that may explain these patterns are discussed in light of oceanographic conditions, adult distribution and larval ontogenesis.

Development of Some Elements of the Peripheral Nervous System of the Locust Locusta migratoria during Larval Ontogenesis

Using supravital Methylene blue staining, development of elements of the peripheral nervous system was traced in the 1st–5th instar larvae of the locust Locusta migratoria L. Data were obtained on sensory cells of the I and II types in larvae of different instars, as well as on character of their changes in the course of larval development of the insect. The studied elements of the peripheral nervous system have been shown to be essentially transformed in the course of larval ontogenesis. The data are presented about the sensory cells of the proximal parts of the nerve trunks; they indicate that in the course of larvae development the cell number in these trunks near the ganglia of the nerve chain decreases, which might be due to their partial degeneration. With growth of muscle, changes in their sensory innervation take place. Subepithelial nerve plexus in larvae is largely represented in areas with hard and soft cuticles.

Teneur en ADN de la larve deDicentrarchus labrax: évolution ontogénétique et effet de la privation de nourriture

Sea bass Dicentrurchus labrax larvae were hatched and reared for 34 days in the laboratory to test a recent biochemical index (DNA/C) of larval fish nutritional condition. Variations in DNA/C ratio values were monitored during the whole larval ontogenesis of fed individuals and the effect of starvation was assessed at three developmental stages. Three main periods in the relative DNA content variations were determined in close connection with growth patterns during larval ontogenesis.

Food selection (size and flavor) during development of echinoderm larvae

Summary Food preferences (flavor and size) by two echinoderm plutei, Paracentrotus lividus and Arbacia lixula, were studied in two experiments. These included selection between polystyrene spheres (flavored and non-flavored) of different sizes in short-term (15 min) feeding experiments and selection among food algae of varying sizes in long-term (24 h) feeding experiments. Clearance and ingestion rates change during larval ontogenesis and are dependent on the size and the quality of available particles. In general, clearance rates were higher for larvae fed on a mixture of three sizes of polystyrene spheres or algae than for those fed separate foods. Young larvae (4- and 6-arm stage) had a higher clearance rate with larger-sized spheres (18.5 μm) and larger-sized alga Prorocentrum micans (26.0 μm). For older larvae (8-arm stage with rudiment), the preference was divided between medium- and larger-sized particles. Retention capability of small-sized spheres (2.9 μm) and algae (Isochrysis galbana), compared...

Mitochondrial calcium flux in temperature-sensitive mutants of [formula omitted

The mechanisms of mitochondrial calcium flux in normal and temperature-sensitive mutants of Drosophila were investigated. Adult mitochondria from all stocks, when analysed with an oxygen electrode, gave respiration rates which exhibited normal responses to adenosine diphosphate and uncoupling agents but no stimulation by calcium. In contrast, calcium stimulates the respiration rate of normal larval mitochondria particularly those of the second instar. This is not evident in second instar mitochondria from the temperature sensitive mutants. There is a rapid accumulation of mitochondrial calcium during normal larval ontogenesis. The levels in temperature-sensitive second instar mitochondria are higher than those of any of the normal larval stages. Adult mitochondria in all cases contain very low levels of calcium. The amount of calcium taken up by mitochondria of second instar temperature-sensitive mutants is lower than that of normal. This may reflect the higher endogenous levels already present in the former. The implications of these variations in calcium metabolism for the behavioural defects of the temperature sensitive mutants is discussed.