UVB radiation exposure modulates mitophagy in embryonic cells of freshwater prawn Macrobrachium olfersii: Exploring a protective organelle quality control mechanism.

Aquatic environments are subject to ultraviolet B (UVB) radiation incidence, and its effects on organisms are dose-dependent. Besides DNA, mitochondria are an important target of this radiation that causes structural damage and impairs its functional dynamics. Here, we hypothesize that mitophagy acts as an organelle quality control mechanism to mitigate UVB impacts in embryonic cells. Then, freshwater prawn Macrobrachium olfersii embryos was used as a model to investigate the effects of UVB on genes (Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3) and proteins (TOM20, PINK1, p62 and LC3B) involved in mitophagy modulation. The choice of genes and proteins was based on the identification of mitochondrial membrane (Tomm20, Opa1 and TOM20), mediation of mitophagy (Pink1, Prkn and PINK1), and recognition of mitochondria by the autophagosome membrane (Sqstm1, Map1lc3, p62 and LC3B). First, the phylogeny of all genes presented bootstrap values >80 and conserved domains among crustacean species. Gene expression was inherently modulated during development, with transcripts (Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3) overexpressed in the initial and final stages of development. Moreover, UVB radiation induced upregulation of Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3 genes at 6 h after exposure. Interestingly, after 12 h, the protein content of PINK1, p62, and LC3B increased, while TOM20 was not responsive. Despite UVB radiation's harmful effects on embryonic cells, the chronology of gene expression and protein content indicates rapid activation of mitophagy, serving as an organelle quality control mechanism, given the analyzed cells' integrity.

Ultraviolet-B radiation induces transcriptional modulation of components associated with the extracellular matrix in embryos of decapod Macrobrachium olfersii.

The extracellular matrix (ECM) is a non-cellular and three-dimensional structure, constituted by a macromolecular dynamic network that involves the cells in all animal tissues, including embryonic ones. Several studies with vertebrates and cell cultures have reported deleterious effects of ultraviolet-B (UVB) radiation on the components associated with the ECM. However, studies focusing on the UVB radiation effects on ECM components of crustaceans during embryonic development are very scarce. Thus, the aim of this study was to identify the coding sequences of components associated with the ECM and to evaluate the effect of UVB radiation on embryos of the ecologically-important decapod Macrobrachium olfersii. To evaluate the modulation of these ECM components during embryonic development, the transcript levels of Col4α1, Itgß, Lamα, Mmp1 and Timp in M. olfersii embryos were analyzed at early developmental stages (E1, E3 and E4), intermediate developmental stage (E7) and late developmental stages (E10 and E14). In addition, embryos at E7, which correspond to a landmark of crustacean development, were analyzed after 12 h of UVB exposure to verify UVB effects on the ECM components. The ECM component sequences were similar to other decapods, suggesting conservation of these genes among crustaceans. The results showed modulations of the ECM components of M. olfersii embryos that reflect the need for each component in the cellular mechanisms, necessary for normal embryonic development. After UVB exposure, embryos showed opacity of embryonic tissues and it was found the overexpression of Col4α1, Itgß, Mmp1 and Timp transcript levels (1.82-, 1.52-, 2.34- and 6.27-fold, respectively). These impairments can compromise important events for normal embryonic development, such as growth of optic lobes, caudal papilla, ramification of appendages and differentiation of organic systems. The results presented here, together with the effects on morphology, cell proliferation, differentiation, and apoptosis demonstrated previously, strengthen the knowledge of the complex impacts of UVB radiation on freshwater embryos. Nevertheless, our results encourage further investigations focusing on the assessment of UVB effects on different organisms in order to better understand the myriad of UVB effects on ECM components.

Evaluation of the acute toxicity, phototoxicity and embryotoxicity of a residual aqueous fraction from extract of the Antarctic moss Sanionia uncinata.

BACKGROUND: Ultraviolet (UV) radiation is the main exogenous inductor of skin damage and so photoprotection is important to control skin disorders. The Antarctic moss Sanionia uncinata is an important source of antioxidants and the photoprotective activity of its organic extracts has been investigated. This study aimed to evaluate the potential photoprotection, cytotoxicity and embryotoxicity of residual aqueous fraction (AF) from the moss S. uncinata. METHODS: UV-visible spectrum and SPF (sun protection factor) were determined by spectrophotometry. Embryotoxicity potential was evaluated by Fish embryo-larval toxicity test using zebrafish (Danio rerio) as organism model. Cell death assays by water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) were investigated using HaCaT keratinocyte cell line cultured in monolayers and three dimensions (3D). Phototoxicity and association with UV-filters were performed by 3T3 neutral red uptake test. RESULTS: The AF showed sharp absorption bands in the UV region and less pronounced in the visible region. The SPF was low (2.5 ± 0.3), but the SPF values of benzophenone-3 and octyl-methoxycinnamate increased ~ 3 and 4 times more, respectively, in association with AF. The AF did not induce significant lethal and sublethal effects on zebrafish early-life stages. In monolayers, the HaCaT cell viability, evaluated by WST-1, was above 70% by ≤0.4 mg AF/mL after 48 and 72-h exposure, whereas ≤1 mg AF/mL after 24-h exposure. The LDH assay showed that the cell viability was above 70% by ≤0.4 mg AF/mL even after 72-h exposure, but ≤1 mg/mL after 24 and 48-h exposure. In 3D cell culture, an increased cell resistance to toxicity was observed, because cell viability of HaCaT cell by WST-1 and LDH was above ~ 90% when using ≤1 and 4 mg AF/mL, respectively. The AF demonstrated values of photo irritation factor < 2 and of photo effect < 0.1, even though in association with UV-filters. CONCLUSIONS: The residual AF absorbs UV-vis spectrum, increased SPF values of BP-3 and OMC and does not induce embryotoxicity to zebrafish early life-stage. The cell death assays allowed establishing non-toxic doses of AF and phototoxicity was not detected. AF of S. uncinata presents a good potential for skin photoprotection against UV-radiation.

Radiosensitizer effect of usnic acid on Biomphalaria glabrata embryos.

PURPOSE: Some phytochemicals have shown the potential of being radiomodifiers, especially phenolic compounds, such as lichenic secondary metabolites. To evaluate the phytochemical usnic acid as a radiomodifier, embryonic cells of molluscs have been used due to their ease of collection, high sensitivity to physical and chemical agents, well-known embryology and low cost for analysis. MATERIALS AND METHODS: This study aimed to assess the radiosensitizing action of usnic acid on Biomphalaria glabrata embryos. Samples were irradiated with 4 Gy of gamma rays from a 60Co source (dose rate 2.906 Gy/h). An acute toxicity test was performed using B. glabrata embryos in the blastula stage, in order to determine the toxicity of usnic acid and to establish the lethal Concentration for 50% (LC50). Subsequently, the radiomodifing capacity of usnic acid was estimated using assays with B. glabrata embryos. RESULTS: Irradiation increased the number of non-viable embryos compared to unirradiated controls. Additionally, it was observed that embryos exposed to a non-toxic concentration of usnic acid (0.6 µg/mL) before irradiation showed a further enhancement in non-viable embryos when compared with exposure to ionizing radiation alone. CONCLUSION: The results presented here indicate that usnic acid makes cells more sensitive to the damaging effects of radiation.

Effect of UVB radiation exposure in the expression of genes and proteins related to apoptosis in freshwater prawn embryos.

Our previous studies showed that embryos of the freshwater prawn Macrobrachium olfersii exposed to ultraviolet B (UVB) radiation exhibited DNA damage, excessive ROS production, mitochondrial dysfunction and increased hsp70 expression, which are able, independently or together, to induce apoptosis. Thus, we attempted to elucidate some key apoptosis-related genes (ARG) and apoptosis-related proteins (ARP) and their expression during different stages of embryonic development, as well as to characterize the chronology of ARG expression and ARP contents after UVB radiation insult. We demonstrate that p53, Bax and Caspase3 genes are active in the embryonic cells at early embryonic developmental stages, and that the Bcl2 gene is active from the mid-embryonic stage. After UVB radiation exposure, we found an increase in ARP such as p53 and Bak after 3h of exposure. Moreover, an increase in ARG transcript levels for p53, Bax, Bcl2 and Caspase3 was observed at 6h after UVB exposure. Then, after 12h of UVB radiation exposure, an increase in Caspase3 gene expression and protein was observed, concomitantly with an increased number of apoptotic cells. Our data reveal that ARG and ARP are developmentally regulated in embryonic cells of M. olfersii and that UVB radiation causes apoptosis after 12h of exposure. Overall, we demonstrate that embryonic cells of M. olfersii are able to active the cell machinery against environmental changes, such as increased incidence of UVB radiation in aquatic ecosystems.

Are embryonic developing modes determinant in the acquisition and levels of photoprotective compounds in slipper limpets of the Crepipatella genus?

The type of embryonic development (mixed and direct) and its influence on the accumulation and translocation of photoprotective compounds from the mother to the encapsulated embryo was studied in the intertidal gastropods Crepipatella peruviana and Crepipatella dilatata during their reproductive peak. HPLC/MS was used to determine type and levels of sunscreen compounds (total carotenoids; TC/and mycosporine-like amino acid; MAA) in brooding females, capsule walls and early and pre-hatching embryos of both species. Photoprotective compounds were only quantified in nurse eggs of C. dilatata. Our results indicate that females of both species can accumulate TC and MAA at different levels, and they are able to transfer them selectively to capsule walls, embryos and nurse eggs. Palythine-serine (MW=244Da; λmax=320nm) and MAA-330 (MW=234Da; λmax=330nm) constitute total MAA pool in brooding females, whereas brooded embryos incorporate palythine (MW=244Da; λmax=320nm) to the MAA pool. Although TC was transferred from the mother to the embryo through the yolk in both species, MAA trespass showed differences. Females of C. peruviana transfer MAA to their embryos through the embryonic yolk; C. dilatata can transfer MAA only through their nurse eggs, which are consumed by embryos during the terminal stages of intracapsular development. Differences between mixed and direct embryonic development, as well as environmental UV-R levels, which the recently hatched larvae and juveniles of C. peruviana and C. dilatata are exposed to, would determine levels of sunscreen compounds in each species. Higher TC and MAA levels in pre-hatching larvae of C. peruviana compared to C. dilatata, indicate a necessity of C. peruviana for protection against UV-R radiation during approximately 15days when their veliger larvae remain in the water column before metamorphosis is complete. Conversely, low photoprotective levels in pre-hatching juveniles of C. dilatata could be related to low UV-R exposure levels due to the direct incorporation to the benthos and the presence of a protective shell.

Developmental effects of exposure to ultraviolet B radiation on the freshwater prawn Macrobrachium olfersi: Mitochondria as a target of environmental UVB radiation.

In South America, increased UVB radiation has become an important environmental issue that is potentially threatening aquatic ecosystems. Considering that species exhibit different degrees of sensitivity to UVB radiation and that embryos are more sensitive than organisms at later life stages, the aim of this study was to characterize the effects of UVB radiation on subcellular compartments of embryos of the freshwater prawn Macrobrachium olfersi. This species lives and reproduces in clear and shallow waters, where UV radiation can fully penetrates. Embryos were irradiated with a UVB 6W lamp for 30min and examined after 1h, 12h, 24h and 48h of exposure. The irradiance of the UVB used simulates the UV radiation that embryos receive in the natural environment. The subcellular compartment most affected by the UVB radiation was the mitochondria, which exhibited a circular shape, a decrease in mitochondrial cristae, rupture of membranes and a morphology compatible with fission. These impairments were observed simultaneously with increased ROS production, just after 1h of UVB exposure. Thus, we investigated proteins related to mitochondrial fission (Drp-1) and fusion (Mfn-1), which are essential to cell maintenance. We found a significant increase in Drp-1 expression at all analyzed time-points and a significant decrease in Mfn-1 expression only after 24h of UVB exposure. Additionally, a decrease in embryonic cell viability was verified via the mitochondrial integrity assay. To conclude, we observed important mitochondrial dysfunctions against the environmental stress caused by UVB radiation. Moreover, the cellular responses found are critical and should not be disregarded, because they impact embryos that can potentially compromise the aquatic ecosystems.

Multifractal dimension and lacunarity of yolk sac vasculature after exposure to magnetic field.

Several studies have reported about the effects of magnetic fields (MFs) on vascular tissue. Extremely low frequency magnetic fields (ELF-MFs) can promote either inhibition or stimulation of vasculogenesis and angiogenesis, depending upon the intensity and time of exposure to the MF. To investigate the possible effects of ELF-MF on vascular processes, it is necessary to employ methods that allow parameterization of the vascular network. Vascular network is a structure with fractal geometry; therefore, fractal methods have been used to evaluate its morphometric complexity. Here, we used the lacunarity parameter (complementary method of fractal analysis) and multifractal analyses to investigate angiogenesis and vasculogenesis in the embryonic yolk sac membrane (YSM) of Japanese quails (Coturnix japonica) with and without exposure to an external MF of 1 mT and 60 Hz. Lacunarity results showed that the vascular density was lower for the group exposed to the magnetic field for 9 h/day. In addition, multifractal analysis showed reduced vascularization in the experimental groups (6 h/day and 9 h/day of exposure to MF). Furthermore, multifractal analysis showed difference between the groups exposed for 12 and 24 h/day. Using multifractal methods (generalized dimensions and singularity spectrum), it was possible to characterize the vascular network of the quail embryo YSM as a multifractal object, therefore proving this method to be a more appropriate application than the traditional monofractal methods.

Fractal analysis of extra-embryonic vascularization in Japanese quail embryos exposed to extremely low frequency magnetic fields.

Magnetic fields (MF) can alter the dynamic behavior of vascular tissue and may have a stimulatory or inhibitory effect on blood vessel growth. Fractal geometry has been used in several studies as a tool to describe the development of blood vascular networks. Due to its self-similarity, irregularity, fractional dimension, and dependence on the scale of vessel dimensions, vascular networks can be taken as fractal objects. In this work, we calculated the fractal dimension by the methods of box counting (D(bc)) and information dimension (D(inf)) to evaluate the development of blood vessels of the yolk sac membrane (YSM) from quail embryos exposed to MF with a magnetic flux density of 1 mT and a frequency of 60 Hz. The obtained results showed that when the MF was applied to embryos aged between 48 and 72 h, in sessions of 2 h (6 h/day) and 3 h (9 h/day) with exposure intervals between 6 and 5 h, respectively, blood vascular formation was inhibited. Exposure sessions shorter than 2 h or longer than 3 h had no observable change on the vascular process. In contrast, the magnetic field had no observable change on the YSM vascular network for embryos aged between 72 and 96 h, irrespective of the exposure time. In conclusion, these results show a "window effect" regarding exposure time.

Effects of environmental and artificial UV-B radiation on freshwater prawn Macrobrachium olfersi embryos.

The recent decrease of the stratospheric ozone has resulted in an increase of ultraviolet-B (UV-B) radiation reaching the Earth's surface. In freshwater ecosystems with transparent water, UV-B rays easily penetrate and potentially cause harmful effects to organisms. In this study, embryos of the prawn Macrobrachium olfersi were used to evaluate the impact of UV-B rays in freshwater environments. We observed three groups of embryos: the first was to assess whether UV-B radiation produced morphological defects and/or biochemical impairments in the laboratory. The second was to check whether embryos with the same impairments as those observed in the laboratory were found in their environment, under natural solar radiation. The third group was the non-irradiated control. The embryos irradiated with 310 mW cm(-2) UV-B for 30 min showed morphological alterations similar to those observed in embryos from the environmental control group. The most important effects of the UV-B radiation observed in M. olfersi embryos were morphological (1.2% of the total number of embryos from the environment and 2.8% of the total number of irradiated embryos), pigmentation changes in the eyes (78.0% of the total number of embryos from the environment and 98.9% of the total number of irradiated embryos), and disruption of the chromatophores (46.9% of the total number of embryos from the environment and 95.5% of the total number of irradiated embryos). We also observed an increase in egg volume, which was accompanied by a significant increase in water content in UV-B irradiated groups when compared with aquaria control embryos. In addition, a significant decrease in the mitotic index in eggs exposed to UV-B radiation was detected (0.17 for the embryos from the aquaria control, 0.10 for the embryos of the environmental control, and 0.04 for the irradiated groups). The low levels of NPSH and high levels of TBARS indicated that UV-B rays directly compromised the antioxidant function of the embryonic cells, leading to oxidative stress. Our combined morphological and biochemical analyses revealed important effects induced by UV-B on M. olfersi embryos, and the results suggest that the recent changes in global conditions may have injurious effects, at least on the embryos of freshwater prawns.

Stage-dependent teratogenic and lethal effects exerted by ultraviolet B radiation on Rhinella (Bufo) arenarum embryos.

The adverse effects of ultraviolet B radiation from 547.2 to 30,096 J/m2 on morphogenesis, cell differentiation, and lethality of amphibian embryos at six developmental stages were evaluated from 24 up to 168 h postexposure. The ultraviolet B radiation lethal dose 10, 50, and 90 values were obtained for all developmental stages evaluated. The lethal dose 50 values, considered as the dose causing lethality in the 50% of the organisms exposed, in J/m2 at 168 h postexposure, ranged from 2,307 to 18,930; gill circulation and blastula were the most susceptible and resistant stages, respectively. Ultraviolet B radiation caused malformations in all developmental stages but was significantly more teratogenic at the gill circulation and complete operculum stages. Moreover, at the gill circulation stage, even the lowest dose (547.2 J/m2) resulted in malformations to 100% of embryos. The most common malformations were persistent yolk plug, bifid spine, reduced body size, delayed development, asymmetry, microcephaly and anencephaly, tail and body flexures toward the irradiated side, agenesia or partial gill development, abnormal pigment distribution, and hypermotility. The stage-dependent susceptibility to ultraviolet B radiation during amphibian embryogenesis could be explained in the framework of evoecotoxicology, considering ontogenic features as biomarkers of environmental signatures of living forms ancestors during the evolutionary process. The stage-dependent susceptibility to ultraviolet B radiation on Rhinella (Bufo) arenarum embryos for both lethal and teratogenic effects could contribute to a better understanding of the role of the increased ultraviolet B radiation on worldwide amphibian populations decline.

Effect of UV-B exposure on eggs and embryos of patagonian anurans and evidence of photoprotection.

In Patagonia, enhanced levels of ultraviolet radiation (UVR) are affecting not only the southernmost territories but the northern as well, and there is a growing body of evidence indicating effects on terrestrial and aquatic communities. Anurans occur along a wide geographical gradient in Patagonia and are common in shallow waters, usually breeding during spring when seasonal UV-B radiation is at its highest level. In this paper we analyse the effective irradiances (I) experienced in nature by eggs and embryos of the patagonian anurans Pleurodema bufoninum, Pleurodema thaul and Bufo spinulosus papillosus, considering water and egg jelly optical properties. Besides, we determine photoprotective pigments in eggs and embryos of the different species, and ontogenetic shifts in pigment concentration. Finally, we studied the effects of natural and enhanced levels of UV-B on eggs of P. bufoninum aiming to evaluate the role of the jelly envelope in screening the damaging radiation. The evidence gathered in this work shows that natural and enhanced UV-B levels did not induce significant changes on the survivorship but incremented the occurrence of malformations. Also, the species experiencing high levels of exposure presented high concentrations of melanin suggesting that exposure to sunlight triggers photoprotection by pigments. Collectively our results suggest that the studied species bear certain levels of adaptation to cope with high ambient UVR conferred by environmental and biological factors.

Dominant lethal effect of 60Co gamma radiation in Biomphalaria glabrata (SAY, 1818).

The dominant lethal effects of gamma radiation of 60Co in the snail Biomphalaria glabrata were studied. Three groups of 13 wild-type snails were irradiated with single doses of 2.5; 10 and 20 Gy. Crossings were carried out at intervals of 7, 17, 23, 30 and 36 days after irradiation. The dominant lethal effect was observed only at the first crossing occurring 7 days after irradiation with 2.5 Gy. With 10 and 20 Gy, the induction of lethal mutations was detected at 7, 17 and 23 days after irradiation; a dose-response effect was observed. The effect was stronger 7 days after irradiation, decreasing in the succeeding crossings up to 30 days. Cell-killing effects on germ cells were detected in the crossings at 23 days and 30 days after irradiation with 20 Gy. After 36 days, frequencies of malformations resumed background levels; crossing rates partially recovered. These results show that gamma radiation affected all the stages of spermatogenesis. Germ cells at later phases were more sensitive to the mutagenic effect of radiation and the cell killing effects were observed on the youngest cells. This response was similar to the highly homogeneous pattern observed in widely different species and allowed us to estimate some parameters of spermatogenesis in B. glabrata.

Photodynamic toxicity and its prevention by antioxidative agents in Bufo arenarum embryos.

In this work we describe an experimental model to evaluate the photodynamic toxicity on amphibian embryos, as well as the protective effect of antioxidants against the lethal oxidative stress induced by photosensitization. Bufo arenarum embryos were treated with 10 mg/l methylene blue (MB) in AMPHITOX solution for 72 h and then irradiated with a red laser or white light for variable times. Both light sources affected the survival of MB-treated animals and lethal effects occurred within the initial 12 h post-irradiation. For white light irradiation, the most effective phototoxic condition in our study, the LD10, 50 and 90 at 6 h post-irradiation corresponded to 13.57, 19.87 and 29.10 J/cm2, respectively. To explore the action of antioxidants against the photogenerated oxidative stress, MB-treated embryos were incubated with 1mM glutathione (GSH) or ascorbic acid (AA) during 48 h before irradiation. For GSH and 21.6 J/cm2 irradiation, the survival increased from 20 to 90%, whereas 100% survival was achieved with AA even after 43.2 J/cm2 irradiation. These results indicate that both the lethal photodynamic effect and its prevention by antioxidants can be evaluated by means of a simple toxicity test employing amphibian embryos.

Effect of 60Co gamma radiation on Biomphalaria glabrata (Mollusca, Gastropoda) embryos: mortality, malformation and hatching.

A study was carried out on the radiosensitivity of Biomphalaria glabrata embryos submitted to doses of 5, 10, 15, 20 and 25 Gy of 60Co during the cleavage, blastula, gastrula, young trochophore and trochophore stages. Mortality, malformation and hatching were the parameters used to evaluate the damage induced by ionizing radiation. Estimated LD50 values (15 days) showed that the cleavage stage (4.3 Gy) was approximately four times more radiosensitive than the trochophore stage (17.0 Gy). Susceptibility to malformation induction was higher in the blastula, gastrula and young trochophore stages. Several types of morphogenetic malformations were observed, such as head malformations, exogastrulas, shell malformations, and embryos with everted stomodeum, with nonspecific malformations being the most frequent. The types of malformation induced by radiation probably are not radiation-specific and do not depend on the dose applied. The dose of 15 Gy was sufficient to greatly reduce the number of hatching snails regardless of the embryonic stage irradiated. We conclude that the effect of 60Co gamma radiation on B. glabrata embryos presented a specific pattern.

Effect of 60CO gamma radiation on Biomphalaria glabrata (Mollusca, Gastropoda) embryos: mortality, malformation and hatching

A study was carried out on the radiosensitivity of Biomphalaria glabrata embryos submitted to doses of 5, 10, 15, 20 and 25 Gy of 60Co during the cleavage, blastula, gastrula, young trochophore and trochophore stages. Mortality, malformation and hatching were the parameters used to evaluate the damage induced by ionizing radiation. Estimated LD(50) values (15 days) showed that the cleavage stage (4.3 Gy) was approximately four times more radiosensitive than the trochophore stage (l7.0 Gy). Susceptibility to malformation induction was higher in the blastula, gastrula and young trochophore stages. Several types of morphogenetic malformations were observed, such as head malformations, exogastrulas, shell malformations, and embryos with everted stomodeum, with nonspecific malformations being the most frequent. The types of malformation induced by radiation probably are not radiation-specific and do not depend on the dose applied. The dose of 15 Gy was sufficient to greatly reduce the number of hatching snails regardless of the embryonic stage irradiated. We conclude that the effect of (60)Co gamma radiation on B.glabrata embryos presented a specific pattern.

Mortality staging of dominant lethals induced in the F1 generation of the sugarcane borer; Diatraea saccharalis (f.).

Comparative egg maturation was observed between fertile eggs laid by unirradiated females, by unirradiated females mated with irradiated males, by irradiated females mated with normal males, and by virgin unirradiated females. Haploid eggs turn yellow and dry by the fifth day. Eggs from irradiated parents develop more slowly than normal eggs. Most embryos from parents irradiated as P generation adults at 16.8 kilorads for males and 12.0 kR for females die early in embryonic development. A description of egg development is included.