The chromosome level genome assembly of the Asian green mussel, Perna viridis.

The Asian green mussel, Perna viridis is an important aquaculture species in the family Mytilidae contributing substantially to molluscan aquaculture. We generated a high-quality chromosome level assembly of this species by combining PacBio single molecule sequencing technique (SMRT), Illumina paired-end sequencing, high-throughput chromosome conformation capture technique (Hi-C) and Bionano mapping. The final assembly resulted in a genome of 723.49 Mb in size with a scaffold N50 of 49.74 Mb with 99% anchored into 15 chromosomes. A total of 49654 protein-coding genes were predicted from the genome. The presence of 634 genes associated with the cancer pathway and 408 genes associated with viral carcinogenesis indicates the potential of this species to be used as a model for cancer studies. The chromosome-level assembly of this species is also a valuable resource for further genomic selection and selective breeding for improving economically important aquaculture traits and augmenting aquaculture productivity.

Mitogenomic recognition of incognito lineages in the mud spiny lobster Panulirus polyphagus (Herbst, 1793): A tale of unique genetic structuring and diversification.

This study provides the first documentation of three deep conspecific lineages within Panulirus polyphagus in the Indian Ocean, bridging the gap in genetic research. Comparative mitogenomics between lineages (L) at both species and family levels, evolutionary relationships and heterogeneity of sequence divergence within Decapoda, and divergence time estimation were performed. The characterized mitogenomes ranged from 15,685-15,705 bp in size and exhibited a typical pancrustacean pattern. Among the three lineages, L1 predominated the Bay of Bengal, L2 the Arabian Sea, and L2.a, a less common lineage genetically closer to L2, was restricted to the latter region. A minor lineage L1.a, was observed in the Coral Triangle area. All PCGs displayed evidence of purifying selection across species and family levels. The largest genetic distance (K2P) between lineages was 9 %, notably between L1.a and L2.a. The phylogenetic tree subdivided the Achelates into Palinuridae and Scyllaridae, and the topology demonstrated a distinct pattern of lineage diversification within P. polyphagus. AliGROOVE analysis revealed no discernible divergence in Decapoda. The diversification of P. polyphagus appears to have occurred during Miocene, with further diversification in Pliocene. Furthermore, genetic stocks and population connectivity recognized here will provide valuable insight for spatial management planning of this dwindling resource.

Mechanistic insights into the early life stage microbiota of silver pompano (Trachinotus blochii).

Introduction: Deep investigations of host-associated microbiota can illuminate microbe-based solutions to improve production in an unprecedented manner. The poor larval survival represents the critical bottleneck in sustainable marine aquaculture practices. However, little is known about the microbiota profiles and their governing eco-evolutionary processes of the early life stages of marine teleost, impeding the development of suitable beneficial microbial management strategies. The study provides first-hand mechanistic insights into microbiota and its governing eco-evolutionary processes in early life stages of a tropical marine teleost model, Trachinotus blochii. Methods: The microbiota profiles and their dynamics from the first day of hatching till the end of metamorphosis and that of fingerling's gut during the routine hatchery production were studied using 16S rRNA amplicon-based high-throughput sequencing. Further, the relative contributions of various external factors (rearing water, live feed, microalgae, and formulated feed) to the microbiota profiles at different ontogenies was also analyzed. Results: A less diverse but abundant core microbial community (~58% and 54% in the whole microbiota and gut microbiota, respectively) was observed throughout the early life stages, supporting 'core microbiota' hypothesis. Surprisingly, there were two well-differentiated clusters in the whole microbiota profiles, ≤10 DPH (days post-hatching) and > 10 DPH samples. The levels of microbial taxonomic signatures of stress indicated increased stress in the early stages, a possible explanation for increased mortality during early life stages. Further, the results suggested an adaptive mechanism for establishing beneficial strains along the ontogenetic progression. Moreover, the highly transient microbiota in the early life stages became stable along the ontogenetic progression, hypothesizing that the earlier life stages will be the best window to influence the microbiota. The egg microbiota also crucially affected the microbial community. Noteworthily, both water and the feed microbiota significantly contributed to the early microbiota, with the feed microbiota having a more significant contribution to fish microbiota. The results illustrated that rotifer enrichment would be the optimal medium for the early larval microbiota manipulations. Conclusion: The present study highlighted the crucial foundations for the microbial ecology of T. blochii during early life stages with implications to develop suitable beneficial microbial management strategies for sustainable mariculture production.

Unraveling the spawning and reproductive patterns of tomato hind grouper, Cephalopholis sonnerati (Valenciennes, 1828) from south Kerala waters.

The objective of this study is to provide information on the reproductive biology of tomato hind grouper, Cephalopholis sonnerati (Valenciennes, 1828) for conservation and management purposes. Fish caught by artisanal fishermen from September 2019 to August 2021 were analysed. A total of 280 females, 31 males, and 4 transitional and 178 sex-undetermined fish were analysed. The female to male sex proportion was 9:1, and the fish reached a maximum total body length of 38.5 and 54.5 cm for females and males, respectively. The following microscopic stages were identified: immature, developing, ripe, running ripe/releasing, and spent in both males and females. Several asynchronous development patterns were observed in the studied gonads, including multiple oocyte stages and early and advanced stages of sexual transition. High gonadosomatic index (GSI) for both males and females was recorded in March, May, and November. Running ripe and releasing stages in females were identified in the months from March to June, which indicates the spawning season. The absolute and relative fecundity of the species ranged from 162,723 ± 207,267 and 239 ± 285, respectively. An exponential relationship was found between fecundity and total body length (TL), fecundity and total body weight (TW), and fecundity and gonad weight (GW).

Population genetic structure of Randall's threadfin bream Nemipterus randalli in Indian waters based on mitochondrial and nuclear gene sequences.

Nemipterus randalli, commonly known as Randall's threadfin bream, is a commercially important marine finfish. Understanding its genetic structure is critical to effective management and conservation efforts. Previous investigations on population structure in this species were limited by geographic coverage. In this study, we utilized the mitochondrial Cytochrome b gene and nuclear Ribosomal protein gene intron Rp S7 sequences to investigate the population genetic structure, demography and genetic diversity of N. randalli along Indian waters. Our results revealed high haplotype diversity but low nucleotide diversity. AMOVA revealed that the variation among the population was highly significant. Hierarchical AMOVA provided further evidence of significant genetic differentiation between the west and east coasts, which was corroborated by the Bayesian tree and the median-joining network diagram. The mtDNA sequences revealed significant genetic structure between populations based on fixation index analysis following the isolation-by-distance model. Furthermore, the neutrality test and mismatch analysis suggest that N. randalli populations may have experienced a population expansion. However, nuclear marker RpS7, showed a high level of polymorphism, which obscured the population structuring observed with the mitochondrial marker. Consequently, concordant results were not obtained when comparing the mitochondrial and nuclear DNA sequences. The strong genetic differentiation between the east and west coast observed using mitochondrial marker could be attributed to a combination of geographic and environmental factors. These findings lay the groundwork for developing effective conservation and management strategies for N. randalli, considering its genetic structure.

De novo transcriptome for Chiloscyllium griseum, a long-tail carpet shark of the Indian waters.

Sharks have thrived in the oceans for 400 million years, experienced five extinctions and evolved into today's apex predators. However, enormous genome size, poor karyotyping and limited tissue sampling options are the bottlenecks in shark research. Sharks of the family Orectolobiformes act as model species in transcriptome research with exceptionally high reproductive fecundity, catch prominence and oviparity. The present study illustrates a de novo transcriptome for an adult grey bamboo shark, Chiloscyllium griseum (Chondrichthyes; Hemiscyllidae) using paired-end RNA sequencing. Around 150 million short Illumina reads were obtained from five different tissues and assembled using the Trinity assembler. 70,647 hits on Uniprot by BLASTX was obtained after the transcriptome annotation. The data generated serve as a basis for transcriptome-based population genetic studies and open up new avenues in the field of comparative transcriptomics and conservation biology.

In vitro and in silico analysis unravelled clinically desirable attributes of Bacillus altitudinis L-asparaginase.

AIMS: To identify a marine L-asparaginase with clinically desirable attributes and characterize the shortlisted candidate through in silico tools. METHODS AND RESULTS: Marine bacterial strains (number = 105) isolated from marine crabs were evaluated through a stepwise strategy incorporating the crucial attributes for therapeutic safety. The results demonstrated the potential of eight bacterial species for extracellular L-asparaginase production. However, only one isolate (Bacillus altitudinis CMFRI/Bal-2) showed clinically desirable attributes, viz. extracellular production, type-II nature, lack of concurrent L-glutaminase and urease activities, and presence of ansZ (functional gene for clinical type). The enzyme production was 22.55 ± 0.5 µM/mg protein/min within 24 h without optimization. The enzyme also showed good activity and stability in pH 7-8 and temperature 37°C, predicting the functioning inside the human body. The Michealis-Menten constant (Km) was 14.75 µM. Detailed in silico analysis based on functional gene authenticating the results of in vitro characterization and predicted the nonallergenic characteristic of the candidate. Docking results proved the higher affinity of the shortlisted candidate to L-asparagine than L-glutamine and urea. CONCLUSION: Comprehensively, the study highlighted B. altitudinis type II asparaginase as a competent candidate for further research on clinically safe asparaginases.

ACTION APAC: Understanding perceptions, attitudes and behaviours in obesity and its management across south and Southeast Asia.

To identify perceptions and attitudes among people with obesity (PwO) and healthcare professionals (HCPs) toward obesity and its management in nine Asia-Pacific (APAC) countries, a cross-sectional online survey was conducted among adult PwO with self-reported body mass index of ≥25 kg/m2 (≥27 kg/m2, Singapore), and HCPs involved in direct patient care. In total, 10 429 PwO and 1901 HCPs completed the survey. Most PwO (68%) and HCPs (84%) agreed that obesity is a disease; however, a significant proportion of PwO (63%) and HCPs (41%) believed weight loss was the complete responsibility of PwO and only 43% of PwO discussed weight with an HCP in the prior 5 years. Most respondents acknowledged that weight loss would be extremely beneficial to PwO's overall health (PwO 76%, HCPs 85%), although nearly half (45%) of PwO misperceived themselves as overweight or of normal weight. Obesity was perceived by PwO (58%) and HCPs (53%) to negatively impact PwO forming romantic relationships. HCPs cited PwOs' lack of interest (41%) and poor motivation (37%) to lose weight as top reasons for not discussing weight. Most PwO (65%) preferred lifestyle changes over medications to lose weight. PwO and HCPs agreed that lack of exercise and unhealthy eating habits were the major barriers to weight loss. Our data highlights a discordance between the understanding of obesity as a disease and the actual behaviour and preferred approaches to manage it among PwO and HCPs. The study addresses a need to align these gaps to deliver optimal care for PwO.

Genetic analyses reveal panmixia in Indian waters and population subdivision across Indian Ocean and Indo-Malay Archipelago for Decapterus russelli.

The Indian Scad, Decapterus russelli is an important pelagic carangid widely distributed throughout the Indian Ocean and the Indo-West Pacific. Despite being widely distributed in the Indian Ocean, the information regarding genetic structuring and diversity has been lacking compared to its Indo Malay Archipelago counterparts. The present study was conducted to investigate the genetic stock structure of D. russelli based on mitochondrial (Cyt b) and nuclear (DrAldoB1) markers along Indian waters. The results indicated the presence of a single panmictic stock across the Indian Ocean region. High haplotype diversity associated with low nucleotide diversity suggested a population bottleneck followed by rapid population growth. Phylogenetic analysis revealed the absence of geographical clustering of lineages with the most common haplotype distributed globally. The pelagic life style, migratory capabilities, and larval dispersal may be the contributing factors to the observed spatial homogeneity of D. russelli. However, significant genetic differentiation was observed between the populations from Indian Ocean and Indo-Malay Archipelago. Hierarchical molecular variance analysis (AMOVA), pairwise FST comparisons and SAMOVA showed existence of two distinct genetic stocks of D. russelli in the Indian Ocean and IMA. The observed interpopulation genetic variation was high. A plausible explanation for the genetic differentiation observed between the Indo-Malay Archipelago and the Indian Ocean regions suggest the influence of historic isolation, ocean surface currents and biotic and abiotic features of the ocean. Also, there was a significant relationship between genetic distance and geographical distance between population pairs in a manner consistent with isolation-by-distance. These resulted in the evolution of a phylogeographic break for this species between these regions. The findings of these results suggest that D. russelli from the Indian Ocean shall be managed in its entire area of distribution as a single stock. Further, the Indian Ocean and Indo-Malayan stocks can be managed separately.

Ajay Kumar Parida (1963-2022), an eminent plant biotechnologist with a passion for mangrove biology.

We remember Dr Ajay Parida, a leading plant biotechnologist, whose premature passing has deprived the Indian plant science community of a committed scientist and an able administrator. Born on 12 December 1963 in Bhagabanpur, Cuttack District (now Jajpur district), Odisha, he passed away in Guwahati on 19 July 2022. A collegial scientist, his down-to-earth and approachable nature, as well as his resourcefulness were instrumental in advancing the cause of Indian science and harnessing frontier biotechnological tools as vehicles of social consciousness. His expertise in quantitative DNA variation and molecular marker analysis, paved the way for subsequent research on mangrove molecular diversity at the M. S. Swaminathan Research Foundation (MSSRF), Chennai. His contributions to mangrove biology, genetics and genomics as well as extremophile plant species in the Indian context over two decades are a benchmark in his field. He also provided commendable leadership in his capacity as Director, Institute of Life Sciences (ILS), Bhubaneshwar during the COVID-19 pandemic.

The sequence and de novo assembly of the genome of the Indian oil sardine, Sardinella longiceps.

The Indian oil sardine, Sardinella longiceps, is a widely distributed and commercially important small pelagic fish of the Northern Indian Ocean. The genome of the Indian oil sardine has been characterized using Illumina and Nanopore platforms. The assembly is 1.077 Gb (31.86 Mb Scaffold N50) in size with a repeat content of 23.24%. The BUSCO (Benchmarking Universal Single Copy Orthologues) completeness of the assembly is 93.5% when compared with Actinopterygii (ray finned fishes) data set. A total of 46316 protein coding genes were predicted. Sardinella longiceps is nutritionally rich with high levels of omega-3 polyunsaturated fatty acids (PUFA). The core genes for omega-3 PUFA biosynthesis, such as Elovl 1a and 1b,Elovl 2, Elovl 4a and 4b,Elovl 8a and 8b,and Fads 2, were observed in Sardinella longiceps. The presence of these genes may indicate the PUFA biosynthetic capability of Indian oil sardine, which needs to be confirmed functionally.

Monitoring of Harmful Algal Bloom (HAB) of Noctiluca scintillans (Macartney) along the Gulf of Mannar, India using in-situ and satellite observations and its impact on wild and maricultured finfishes.

In the Gulf of Mannar, Noctiluca scintillans blooms have been observed three times in September 2019, September and October 2020, and October 2021. It was determined and measured how the bloom period affects ichthyo-diversity. Noctiluca cell density varied slightly from year to year, ranging from1.8433 × 103 cells/L to 1.3824 x 106cells/L. In surface and sea bottom waters, high ammonia levels and low dissolved oxygen levels were noted. During the bloom period a significant increase in chlorophyll concentration was found. The amount of chlorophyll in GOM was extremely high, according to remote sensing photos made using MODIS-Aqua 4 km data. Acute hypoxia caused the death of wild fish near coral reefs and also in fish reared in sea cages. The decay of the bloom resulted in significant ammonia production, a dramatic drop in the amount of dissolved oxygen in the water, and ultimately stress, shock, and mass mortality of fishes.

Full-length transcriptome from different life stages of cobia (Rachycentron canadum, Rachycentridae).

Cobia (Rachycentron canadum, Rachycentridae) is one of the prospective species for mariculture. The transcriptome-based study on cobia was hampered by an inadequate reference genome and a lack of full-length cDNAs. We used a long-read based sequencing technology (PacBio Sequel II Iso-Seq3 SMRT) to obtain complete transcriptome sequences from larvae, juveniles, and various tissues of adult cobia, and a single SMRTcell generated 99 gigabytes of data and 51,205,946,694 bases. A total of 8609435, 7441673 and 9140164 subreads were generated from the larval, juvenile, and adult sample pools, with mean sub-read lengths of 2109.9, 1988.2 and 1996.2 bp, respectively. All samples were combined to increase transcript recovery and clustered into 35661 high-quality reads. This is the first report on a full-length transcriptome from R. canadum. Our results illustrate a significant increase in the identified amount of cobia LncRNAs and alternatively spliced transcripts, which will help improve genome annotation. Furthermore, this information will be beneficial for nutrigenomics and functional studies on cobia and other commercially important mariculture species.

A Global-Scale Ecological Niche Modeling of the Emerging Pathogen Serratia marcescens to Aid in its Spatial Ecology.

Serratia marcescens is a big emerging concern for human health and coral biodiversity. Spatial ecology and the influencing factors on pathogen ecology, however, remain unknown. The study forms the first global risk assessment of S. marcescens. MaxEnt niche modeling was applied using two biotic and sixteen abiotic variables. The world was classified into five risk-level categories based on the pathogen ecology, and the world population exposed to S. marcescens infection was then quantified. The prepared model showed an area under the curve value of 0.918 ± 0.028, implying excellent prediction ability. The highly and moderately suitable areas occupied around 0.52% and 17.9% of the total global land area. The order of probability of having S. marcescens-related infections was Asia > North America > South America > Europe > Africa > Australia. Human population density and temperature were the most influential factors in the distribution. The moderate to high transmission risk zones contained 0.20% (1.61 billion people) of the human population. In brief, these results give novel insights into its spatial ecology and provide the risk maps that can be utilized to plan targeted strategic control measures against future invasions of this emerging pathogen.

Cochlear implantation in a profoundly deaf child with cystic leukoencephalopathy without megalencephaly.

BACKGROUND: Cochlear implantation candidacy criteria have continued to evolve over the years, and cochlear implantation is possible with many inner-ear and brain anomalies with good hearing and linguistic outcomes. Cystic leukoencephalopathy without megalencephaly is a rare disease in children, with only 30 cases reported in the literature, but it is associated with hearing loss in only three cases. Radiological investigations can help in diagnosing this rare entity before proceeding with cochlear implantation. CASE REPORT: A four-year-old female child born out of consanguinity with normal psychomotor development, bilateral sensorineural hearing loss and an incidental magnetic resonance imaging finding of cystic leukoencephalopathy without megalencephaly underwent successful cochlear implantation. Her post-operative period was uneventful with successful mapping of the cochlear implant. CONCLUSION: This is the first reported case of cystic leukoencephalopathy without megalencephaly and with sensorineural hearing loss in which cochlear implantation was performed successfully. White matter and temporal lobe abnormalities should not deter paediatric cochlear implantation.

Evaluation of the inhibitory efficacy of quaternary ammonium compounds on in vitro growth of Theileria equi parasite in MASP culture.

Equine piroplasmosis has become a global problem of the equine husbandry sector. Haemoprotozoans evolved very quickly and developed resistance against most of the current available drugs. Phospholipid membrane synthesis by choline kinase enzyme is vital for propagation of intra-erythrocytic protozoa parasites. This pathway was targeted in the present study. Quaternary ammonium salts (QAS) and their analogues act against choline and hamper the biosynthesis process for phosphatidylcholine. We analysed anti-T. equi activity of three QAS - decamethonium bromide (DMB), decyl trimethyl ammonium bromide (DTAB) and dodecyl trimethyl ammonium bromide (DDTAB). Theileria equi parasites in vitro treated with different concentrations of DMB, DDTAB and DTAB. Drug treated T. equi failed to multiply further in the viability test. The IC50 value of DMB, DDTAB and DTAB for growth inhibition of T. equi was 14.0 µM, 469.51 nM and 558.40 nM, respectively. DMB, DDTAB and DTAB treated T. equi parasites were observed to be devoid of internal structures, showing pyknotic and degenerative appearances. Various concentration of DMB, DDTAB and DTAB were analysed for their cytotoxicity and haemolytic activity on horse's PBMCs and RBCs. DMB was less than 10% cytotoxic to PBMCs, while DDTAB and DTAB were 40%-50% cytotoxic at 1000 µM concentrations. The respective CC50 values were 7202.96 µM, 1026.26 µM and 1263.95 µM. DMB and DTAB showed least haemolytic activity (<3%); whereas DDTAB was more haemolytic to RBCs at highest concentration of 2000 µM. The respective CC50 values of these drugs were 224495.3 µM, and 39101.35 µM; 713.54 µM. Specific selective index for DMB, DDTAB and DTAB values with respect to host's PBMC and RBC cells, were 514.50, 2185.81, 2263.52 and 16035.38, 1519.75, 70023.91, respectively. These data indicated its non-toxicity to host's cells and selective potential of anti-T. equi in vitro activity.

A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

Besides its functions in the skeletomuscular system, vitamin D is known to alleviate viral-inflicted pathologies. However, the mechanism underlying protective vitamin D function remains unclear. We examined the role of vitamin D in controlling cellular infections by Chandipura virus, an RNA virus implicated in human epidemics. How immune signaling pathways, including those regulating NF-κB and IFN regulatory factors (IRFs), are activated in virus-infected cells has been well studied. Our investigation involving human- and mouse-derived cells revealed that vitamin D instructs the homeostatic state of these antiviral pathways, leading to cellular resilience to subsequent viral infections. In particular, vitamin D provoked autoregulatory type 1 IFN-IRF7 signaling even in the absence of virus infection by downmodulating the expression of the IFN-inhibitory NF-κB subunit RelB. Indeed, RelB deficiency rendered vitamin D treatment redundant, whereas IRF7 depletion abrogated antiviral vitamin D action. In sum, immune signaling homeostasis appears to connect micronutrients to antiviral immunity at the cellular level. The proposed link may have a bearing on shaping public health policy during an outbreak.

Comparative evaluation of fish larval preservation methods on microbiome profiles to aid in metagenomics research.

Applications of microbiome research through metagenomics promise to generate microbiome manipulation strategies for improved larval survival in aquaculture. However, existing lacunae on the effects of sample preservation methods in metagenome profiles hinder the successful application of this technique. In this context, four preservation methods were scrutinized to identify reliable methods for fish larval microbiome research. The results showed that a total of ten metagenomics metrics, including DNA yield, taxonomic and functional microbiome profiles, and diversity measures, were significantly (P < 0.05) influenced by the preservation method. Activity ranking based on the performance and reproducibility showed that three methods, namely immediate direct freezing, room temperature preservation in absolute ethanol, and preservation at - 20 °C in lysis, storage, and transportation buffer, could be recommended for larval microbiome research. Furthermore, as there was an apparent deviation of the microbiome profiles of ethanol preserved samples at room temperature, the other methods are preferred. Detailed analysis showed that this deviation was due to the bias towards Vibrionales and Rhodobacterales. The microbial taxa responsible for the dissimilarity across different methods were identified. Altogether, the paper sheds light on the preservation protocols of fish larval microbiome research for the first time. The results can help in cross-comparison of future and past larval microbiome studies. Furthermore, this is the first report on the activity ranking of preservation methods based on metagenomics metrics. Apart from methodological perspectives, the paper provides for the first time certain insights into larval microbial profiles of Rachycentron canadum, a potential marine aquaculture species. KEY POINTS: • First report on effects of preservation methods on fish larval microbiome profiles. • First report on activity ranking of preservation methods based on metagenomics metrics. • Storage methods influenced DNA yield, taxonomic and functional microbiome profiles.

Comparative mitogenomics and phylogenetics of the family Carangidae with special emphasis on the mitogenome of the Indian Scad Decapterus russelli.

Carangids are abundant and commercially important marine fish that contribute to a significant portion of the fisheries in many parts of the world. In the present study, we characterized the complete mitogenome of the Indian scad, Decapterus russelli and performed a comprehensive comparative mitogenomic analysis of the family Carangidae. The comparative mitogenomics provided valuable insights into the structure, variability, and features of the coding and non-coding regions that evolved across species over millions of years. The structural features of tRNAs revealed changes in the frequency of mismatched and wobble base pairs, which is reflected in the base composition of H and L strands. The highly conserved sequence motif of the mTERF binding site in carangids over the ~ 400 MYA of their divergence demonstrated the functional importance of these sites. The control region of carangids was characterized by the presence of discontinuous repeat units with a high rate of sequence divergence in the form of base substitutions, insertions, and deletions. The maintenance of secondary structures in the control region independent of the rapid evolution of primary structure suggested the effect of selective constraints on their maintenance. Maximum likelihood (ML) and Bayesian inference (BI) phylogeny revealed a similar topology consistent with previous taxonomic studies. The extant carangids diverged through the evolutionary events experienced during the Cretaceous, Paleogene, and Neogene periods.