*3.2. Delphi Analysis Results*

The Delphi survey among 13 experts found that the issues that the participants agreed upon most were that URAS should have the right to work, the right to buy MOPH health insurance or insurance provided by the private sector, the right to access basic education, the right to live outside the detention centers, and the right to receive medical benefits that cover treatment for public health threats and conditions (e.g., tuberculosis and influenza). All of these issues received the median score of five. Issues that most experts rated the least (median = two) were; 'Overall, the Thai government presents appropriate policy direction towards URAS', 'The medical benefit for URAS should focus on emergency illnesses and accidents only', and 'The medical charge of URAS should be mainly shouldered by NGOs', Figures 3–7. *Int. J. Environ. Res. Public Health* **2021**, *182*, 566 9 of 15 *Int. J. Environ. Res. Public Health* **2021**, *182*, 566 9 of 15

**Figure 3.** Delphi survey asking about the main responsible authority that should cover medical charges of URAS. Note: The square on the line denotes the median score. The left and right ends of the line denote the score at 25th and 75th percentile, respectively. MOPH = 3, NHSO = 3, URAS = 3, UNHCR = 4, NGO = 2. **Figure 3.** Delphi survey asking about the main responsible authority that should cover medical charges of URAS. Note: The square on the line denotes the median score. The left and right ends of the line denote the score at 25th and 75th percentile, respectively. MOPH = 3, NHSO = 3, URAS = 3, UNHCR = 4, NGO = 2. **Figure 3.** Delphi survey asking about the main responsible authority that should cover medical charges of URAS. Note: The square on the line denotes the median score. The left and right ends of the line denote the score at 25th and 75th percentile, respectively. MOPH = 3, NHSO = 3, URAS = 3, UNHCR = 4, NGO = 2.

**Figure 4.** Delphi survey asking about the benefit package that should cover for URAS. Note: The Scheme 25. th and 75th percentile, respectively. Basic disease = 4, emergency and accident = 2, communicable disease = 5, similar to Thai citizens = 4, similar to migrant workers = 4, mental disease = 4. **Figure 4.** Delphi survey asking about the benefit package that should cover for URAS. Note: The Scheme 25. th and 75th percentile, respectively. Basic disease = 4, emergency and accident = 2, communicable disease = 5, similar to Thai citizens = 4, similar to migrant workers = 4, mental disease = 4. **Figure 4.** Delphi survey asking about the benefit package that should cover for URAS. Note: The Scheme 25th and 75th percentile, respectively. Basic disease = 4, emergency and accident = 2, communicable disease = 5, similar to Thai citizens = 4, similar to migrant workers = 4, mental disease = 4.

center = 5.

*Int. J. Environ. Res. Public Health* **2021**, *182*, 566 10 of 15

**Figure 5.** Delphi survey asking about the main health insurance provider that should cover for URAS. Note: The square on the line denotes the median score. The left and right ends of the line denote the score at 25th and 75th percentile, respectively. HICS = 5, SSS = 4, NHSO = 4, Private **Figure 5.** Delphi survey asking about the main health insurance provider that should cover for URAS. Note: The square on the line denotes the median score. The left and right ends of the line denote the score at 25th and 75th percentile, respectively. HICS = 5, SSS = 4, NHSO = 4, Private organization = 5. URAS. Note: The square on the line denotes the median score. The left and right ends of the line denote the score at 25th and 75th percentile, respectively. HICS = 5, SSS = 4, NHSO = 4, Private organization = 5.

**Figure 6.** Delphi survey asking about the other aspects of well-being that should urban refugees should have rights. Note: The square on the line denotes the median score. The left and right ends of the line denote the score at 25th and 75th **Figure 6.** Delphi survey asking about the other aspects of well-being that should urban refugees should have rights. Note: The square on the line denotes the median score. The left and right ends of the line denote the score at 25th and 75th percentile, respectively. Enjoy basic education = 5, get work permit = 5, register to civil profile = 4, live outside detention center = 5. **Figure 6.** Delphi survey asking about the other aspects of well-being that should urban refugees should have rights. Note: The square on the line denotes the median score. The left and right ends of the line denote the score at 25th and 75th percentile, respectively. Enjoy basic education = 5, get work permit = 5, register to civil profile = 4, live outside detention center = 5. *Int. J. Environ. Res. Public Health* **2021**, *182*, 566 11 of 15

percentile, respectively. Enjoy basic education = 5, get work permit = 5, register to civil profile = 4, live outside detention

**Figure 7.** Delphi survey asking about position of Thai government towards URAS. Note: The square on the line denotes the median score. The left and right ends of the line denote the score at 25th and 75th percentile, respectively. Ratify to the 1951 Refugee Convention = 3, appropriate position of Thai government = 2. **4. Discussion Figure 7.** Delphi survey asking about position of Thai government towards URAS. Note: The square on the line denotes the median score. The left and right ends of the line denote the score at 25th and 75th percentile, respectively. Ratify to the 1951 Refugee Convention = 3, appropriate position of Thai government = 2.

#### **4. Discussion**

organization = 5.

policy and the attitudes of all relevant stakeholders towards the well-being of URAS. The interviews and Delphi-survey suggested that the ideology of national security and international relations influenced the concept of human rights for URAS. The concept The study is among the very first studies in Thailand, which focus on the country policy and the attitudes of all relevant stakeholders towards the well-being of URAS.

The study is among the very first studies in Thailand, which focus on the country

of URAS health has been tightly woven with foreign policy issues over many years. Health

in many other parts of the world. For example, according to a study by Klaus, the campaign for the restriction of rights for immigrants and refugees in Poland was used by the populist political parties to win the election in 2015 [18]. By late 2015, the Polish government launched the Antiterrorist Act stipulating that every foreign citizen would be put under surveillance without any court control with an ultimate aim to stop a refugee influx

With regard to URAS problem solving mechanism, the UN agencies usually puts emphasis on the "relief" of URAS' suffering rather than addressing the structural problems that compromise the well-being of migrants. The same situation often occurs with NGOs or charitable organizations that most of the time serve as humanitarian support for URAS. This problem reflects the limitation of UN agencies that do not have solid legal mechanisms to force or even encourage any particular nation to re-orientate its health system by making it more "inclusive" for all people on its soil. Therefore, a more practical approach for UN agencies and NGOs is to comply with the local government. However, this approach has a setback as most of the NGOs' (or even UNHCR's) activities usually end up with a humanitarian relief for URAS (or any activities that make URAS more "resilient" with the status quo health system) as it is less disputable compared with shaking the structural problems or recognizing URAS as part of the society on equitable grounds

Although Thailand is not a signatory to the 1951 Refugee Convention, the country has long been a key member of ASEAN [20], which has its own agreement among member states to guarantee human rights, including the right to health for everybody in the region. Therefore, Thailand cannot deny its responsibility to protect the health of URAS. However, it appears that current Thai laws are not developed to protect URAS from the outset. The majority of byelaws for non-Thais mostly concern migrant workers, but not URAS.

into the Polish territory.

with the nationals [19].

The interviews and Delphi-survey suggested that the ideology of national security and international relations influenced the concept of human rights for URAS. The concept of URAS health has been tightly woven with foreign policy issues over many years. Health issues are still considered "low politics", while foreign policy is deemed to be "high politics" in decision making [16,17]. This situation has occurred not only in Thailand, but also in many other parts of the world. For example, according to a study by Klaus, the campaign for the restriction of rights for immigrants and refugees in Poland was used by the populist political parties to win the election in 2015 [18]. By late 2015, the Polish government launched the Antiterrorist Act stipulating that every foreign citizen would be put under surveillance without any court control with an ultimate aim to stop a refugee influx into the Polish territory.

With regard to URAS problem solving mechanism, the UN agencies usually puts emphasis on the "relief" of URAS' suffering rather than addressing the structural problems that compromise the well-being of migrants. The same situation often occurs with NGOs or charitable organizations that most of the time serve as humanitarian support for URAS. This problem reflects the limitation of UN agencies that do not have solid legal mechanisms to force or even encourage any particular nation to re-orientate its health system by making it more "inclusive" for all people on its soil. Therefore, a more practical approach for UN agencies and NGOs is to comply with the local government. However, this approach has a setback as most of the NGOs' (or even UNHCR's) activities usually end up with a humanitarian relief for URAS (or any activities that make URAS more "resilient" with the status quo health system) as it is less disputable compared with shaking the structural problems or recognizing URAS as part of the society on equitable grounds with the nationals [19].

Although Thailand is not a signatory to the 1951 Refugee Convention, the country has long been a key member of ASEAN [20], which has its own agreement among member states to guarantee human rights, including the right to health for everybody in the region. Therefore, Thailand cannot deny its responsibility to protect the health of URAS. However, it appears that current Thai laws are not developed to protect URAS from the outset. The majority of byelaws for non-Thais mostly concern migrant workers, but not URAS. The most relevant law relating to URAS is the 'Regulation of the Office of the Prime Minister: Screening process for aliens entering the Kingdom of Thailand and incapable of returning to their home country' B.E. 2562 (2019). It establishes a screening mechanism for groups of aliens in line with the nature of Thai society and international situations in order to reach sustainable solutions for Thailand's alien management problem [21].Yet, the regulation still lacks operational details. Whether Thailand will become a party of the 1951 Refugee Convention or not may not be so important, what may be more important is to have a clear stance on the Thai government's responsibility to take care of URAS.

As long as there is no clear direction from central authorities, there will always be variation in day-to-day operations of the treatment of URAS among street-level bureaucrats [22]. A clear example can be noticed in the recent COVID-19 pandemic in Thailand. Though the Thai government publicly announced to the wider public that all COVID-19 patients (regardless of the nationalities) are able to access free treatment of COVID-19. The hospitals are able to reimburse the healthcare cost from the MOPH and the National Health Security Office (NHSO). However, in practice, there are also administrative problems in reimbursing the healthcare cost for undocumented migrants, not to mention URAS [23,24].

If the Thai government steadfastly rejects the accession of the 1951 Refugee Convention, the domestic mechanisms to take care of URAS health should be strengthened. Moreover, the Thai government should learn from international experience, especially from countries that accepted a refugee influx while refraining from the 1951 Refugee Convention. Janmyr raised the case of Lebanon as an example of a country that is not a party of the 1951 Refugee Convention, but is hailed by the international community for its generosity towards refugees as probably hosting the highest number of refugees in the world in proportion to its population size [25]. Lebanon already has human rights obligations towards refugees on its territory by virtue of membership of the United Nations and its ratification

of a number of core human rights instruments [25]. Another interesting issue is the URAS right to work and eligibility to be enrolled in public health insurance in Thailand. For the right to work, Brown et al. suggested that the lack of legal instruments to allow URAS to work legitimately has caused URAS to face a greater risk of being arrested or detained [26].

Additionally, the provision of the right to work is likely to lead to better quality of life for URAS because they will be able to access the labor market and gain sustainable livelihood opportunities. The right to work will also allow URAS to gain increased selfreliance and dignity and improve mental health [27,28]. Fleay and Hartley cited a case study in Australia, suggesting that without the right to work, asylum seekers in Australian communities faced exacerbated feelings of anxiety, sadness, and fear [29]. Most of this study's interviewees, including those who participating in the Delphi survey, indicated that the right to work should be implemented as soon as possible with no critical disagreement from the wider public. The law to promulgate this policy—Royal Ordinance Concerning the Management of Employment of Foreign Workers, B.E.2560 (2017)—is in place [30] and only a Cabinet Resolution is required to implement the policy concretely.

Giving URAS the right to work also means that Thailand will benefit from an increased labor force (and some URAS are quite well educated). The opening of employment opportunities helps improve the registration data of URAS and positively affects the Thai economy. Additionally, to deal with the refugee crises, it has been recommended that, while poorer countries accede to host refugees, richer countries should help provide financial support to those host countries in order to protect refugees' health and well-being [31]. Since Thailand host numbers of refugees from various nations, this may benefit Thailand as well.

High healthcare costs and financial difficulties are also a key barrier faced by refugees and asylum-seekers [32]. According to Elsouhang et al. possessing health insurance was significantly associated with increased utilization of medical services among Iraqi refugees in the United States of America [33]. In the Thai context, the financial difficulties that URAS face are not just a matter concerning the work performance of an individual, but these also intertwine with the legal design of a system that does not allow URAS to work legitimately. Moreover, the inclusion of URAS in public health insurance schemes may benefit not only URAS, but the health system as a whole. A substantial volume of literature shows that the exclusion of non-national populations (such as migrants, refugees, and asylum seekers) from official primary health care might save costs early on, but this effect might be lost as costs are shifted to healthcare providers in secondary care or community settings [34]. Again, the promotion of the right to public health insurance for URAS is interlinked with the affordability of insurance, which is also linked to the right to work. It is also linked to the regulation of the state which needs to be clearly specified in laws or official state regulations in order to avoid haphazard interpretation by local healthcare providers [35].

Concerning methodological approaches, some limitations remain in this study. Firstly, this study did not encompass all types of refugees and asylum seekers. Those who were detained in detention centers and those living in the temporary shelter areas along the country border were excluded. A discussion on policies to take care of the health of these populations necessitates a more extensive review on the relevant laws and perhaps requires a wider range of interviewees (including police and state prosecutors). Secondly, the issue of healthcare for URAS is relatively sensitive. According to research ethics, in the fieldwork, the researchers needed to disclose their own work status as persons working with the MOPH, which may have created an unfavorable feeling among the URAS interviewees. For example, some URAS might feel uncomfortable disclosing their life stories or unpleasant experiences with state officials to the researchers. In contrast, state official interviewees may have tended to answer the interview questions in a way that tried to meet the researchers' expectation (social desirability bias). However, the researchers addressed these issues by using methodological triangulations (such as contrasting the interview findings with the review on policy documents). Lastly, suggestions from the interviews or Delphi survey (for instance, the provision of rights to employment and public health insurance enrollment)

does not mean final consensus from policy experts and of course does not mean that these suggestions can be implemented without societal disagreement. In reality, to implement such policies, there needs a much wider consultative process from all angles of the political sphere. Further studies that explore policy feasibility for URAS are of great value.


**Acknowledgements**

Author González-Pérez Julio Emmanuel (https://orcid.org/0000-0002-0218-6158) gratefully acknowledges financial support for his PhD studies from Universidad de las Americas Puebla (UDLAP) and the National Council for Science and Technology (CONACyT) of Mexico.


**1.4 Linking the Ecdysone pulse to cell cycle**

In *Drosophila*, cell growth and cell cycle progression are regulated by a number of key genes, which have been shown to control the cell cycle in an analogous manner in all multicellular organisms. These include the *Drosophila* orthologue of the mammalian *c-myc* transcription factor and oncogene, dMyc, which drives growth and progression through G1 to S-phase (Johnston et al. 1999), the essential G1 to S-phase Cyclin complex, Cyclin E (CycE) and its Cyclin-dependent-kinase (Cdk) partner Cdk2, which triggers S-phase by promoting DNA replication (Knoblich et al. 1994; Neufeld et al. 1998; Richardson et al. 1995), and the *Drosophila* orthologue of the Cdc25 phosphatase, String (Stg), which is required for G2/M progression and promotes mitotic entry by activating the Cdk1/Cyclin B complex (Edgar and Datar 1996). CycE and Stg are the rate limiting factors for S-phase and mitosis, respectively, and both are activated by the *Drosophila* orthologue of human E2F1 protein, dE2F1 (Neufeld et al. 1998). dE2F1 responds to the relevant Cdk-Cyclin complex (CycE/Cdk2 for S-phase and CycB/Cdk1 for mitosis) to coordinate cell cycle progression from G1 to S-phase and G2 into mitosis (Reis and Edgar 2004).

During metamorphosis, following removal of the obsolete larval structures, proliferation of the remaining tissue occurs in an ecdysone-dependent manner to produce adult structures. For example, during pupal development the larval midgut is removed by apoptosis and is replaced through proliferation of the remaining tissue to form the adult midgut (Jiang, Baehrecke, and Thummel 1997). Microarray analysis has revealed that the ecdysone signal is associated with the activation of key cell cycle genes, including *Cyclin B, Cdc2* and *Cyclin D*, during the initiation of midgut metamorphosis (Li and White 2003). Analysis of *EcR* null mutants also revealed that EcR function was necessary for the cell cycle and growth genes to be activated in the larval midgut, suggesting that the ecdysone pathway is required for cell division control. The body of this chapter will discuss how the ecdysone pulse achieves changes to cell growth and cell cycle progression. First we will describe how ecdysone levels dictate body size cell extrinsically by controlling developmental timing. Then we will discuss how ecdysone works with its receptors, in a tissue autonomous manner to control transcription of cell cycle genes, which most likely occurs indirectly by modifying the activity of developmental signalling pathways.


**4. Discussion**

The results of our study do not support the hypothesis that Neotropical katydid species specialize their diet by host plant. By extracting plant DNA from the digestive systems of katydids, we were able to use DNA barcoding to identify which plant species and/or families were recently consumed. Individual katydids of the same species often had multiple and different plant families in their digestive systems, indicating that katydid species were not specializing on single host plants. In addition, the use of multiple primers sometimes recovered different plant species from the same katydid, providing evidence that katydids would feed on a diversity of plants even at short timescales. Consequently, dietary specialization on a specific host plant is not providing these species with a means of facilitating mate localization in the face of intense predation on signaling males and mate-searching females.

In contrast, the katydid species studied here were consuming a wide variety of plant families (16) and orders (12) comprising over half the orders of plants found on BCI. While many plant families and orders appeared in the katydid diet, some were particularly well-represented. Within the katydid samples that yielded a single identified plant order, 27% of the samples were Laurales and 23% were Fabales. No other plant family or order comprised more than 8% of the samples (Table 1). The abundance of Laurales and Fabales in the diet appears to be consistent with the relative abundance of stems of these plant orders on Barro Colorado Island, although species diversity in several other lineages, such as Gentianales, Myrtales, Rosales, and Malpighiales, is equally high or higher, suggesting that katydids may avoid some lineages of plants in favor of others [47–49]. Furthermore, it should be noted that the katydid specimens were collected primarily in January when certain trees may be in the young leaf stage and hence easier to digest than other species. If this is the case, then our results may in part be dependent on tree phenology. Further sampling at different times of the year in different seasons is certainly warranted.

The dietary composition of katydids provides one-directional information about the height at which katydids are foraging. Because even canopy emergent tree species start as saplings, the presence of canopy species in the katydid diet does not necessarily mean that the species was consumed in the canopy. In contrast, when katydids are eating shrubs and understory trees, it is strong evidence that the species is foraging low in the forest. While sample sizes are small for some katydid species, katydid species that are observed at ground level are in fact consuming understory vegetation (e.g., *Docidocercus gigliotosi* [22]) (Figure 4).

One notable finding was that many plant families were detected in only one year. In part, the year-to-year differences likely reflect the fact that there are many plant families and most were represented with relatively low frequency. However, the fact that some plant families are well-represented in one year and rare or absent in others suggests that there may be times when particular plant families or individual plants are especially palatable. For example, in a given sampling location on a given night, multiple katydids of multiple species had the same plant in their stomach (Table S3), perhaps reflecting a nearby feeding opportunity that attracted multiple species of katydid.

Transient feeding opportunities on particular plants would be consistent with what is known about the palatability and phenology of many tropical plants. Even though leaves may persist for several years, 25–70% of leaf damage occurs in the weeks when leaves are expanding [50,51]. In response to herbivore pressure, tropical plants have evolved a variety of strategies to minimize their window of vulnerability to herbivores [27]. Herbivore evasion strategies can include exceptionally rapid expansion of leaves, delayed greening, and synchronous flushing, strategies that minimize exposure to herbivory by compressing the window when leaves are maximally palatable [52,53].

The possibility of short-term feeding windows on specific plants is also supported by anecdotal field observations during this study, where katydids of multiple species were observed aggregated on a tree a few days before the tree produced substantial and obvious new growth (L. Symes, personal observation).

If katydids do aggregate to exploit transient feeding opportunities, co-localization on food sources might still facilitate mate finding, even for diet generalists. There are several avenues of investigation that could provide information on whether katydids aggregate to feed on plants during leafout. One strategy is to bait traps with plant volatiles such as benzyl nitrile, phenyl acetaldehyde, and/or 2-phenylethanol [54,55] to determine whether these chemical compounds are attractive to katydids, suggesting targeted feeding on vulnerable plants. A second strategy is to deploy long-term acoustic recorders and test for periods of time when a single location on the landscape has an elevated number of katydid calls, reflecting aggregation or one or more katydid species in an area for a short period of time. Understanding whether katydids aggregate on plants that are producing new leaves is important for understanding herbivore pressures on the forest vegetation, patterns of food availability for insect predators, and impacts of habitat patch size on insect populations and foraging effectiveness.

The results of DNA barcoding, when applied to Neotropical forest katydids, helps to define previously unseen connections between plants and herbivores, including connections that occur out of sight in the forest canopy. The generality of the katydid diet is more or less consistent with observations on other species in other locations, but underscores our lack of knowledge of how animals with rare short-range signals find each other in tropical forests. Our observations also suggest several avenues for future research.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/d14020152/s1, Table S1: Primers utilized for DNA barcode amplification; Table S2: List of accessions of DNA barcodes for BCI trees/shrubs/lianas used in BLAST searches; Table S3: List of katydid samples, plant BLAST result, and collection metadata. Figure S1: The phylogenetic distribution of plant species in the diets of katydids on BCI.

**Author Contributions:** Conceptualization, C.M.P. and L.B.S.; methodology, C.M.P. and L.B.S.; formal analysis, C.M.P. and L.B.S.; investigation, C.M.P., N.L.W., S.J.M. and L.B.S.; resources, C.M.P., H.M.t.H., W.J.K. and L.B.S.; data curation, C.M.P. and L.B.S.; writing—original draft preparation, C.M.P. and L.B.S.; writing—review and editing, C.M.P., H.M.t.H., W.J.K. and L.B.S.; visualization, C.M.P. and L.B.S.; supervision, C.M.P. and L.B.S.; project administration, C.M.P. and L.B.S.; funding acquisition, C.M.P., H.M.t.H. and L.B.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** C.M.P. and N.L.W. were funded by Castleton University and the Vermont Biomedical Research Network (VBRN). Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health, grant number P20GM103449. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. L.B.S. was supported by the Neukom Institute of Dartmouth College. L.B.S., S.J.M. and H.M.t.H. were supported by funding from the Smithsonian Tropical Research Institute, Dartmouth College, and an Artificial Intelligence for Earth Innovation grant from Microsoft/National Geographic, grant number NG5-57246T-18. W.J.K. was supported by the Smithsonian Institution.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Sequence data for chloroplast DNA amplified from gut contents is available in the Supplementary Materials. Reference plant barcodes (accessions found in Table S2) can be found at https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 10 August 2021).

**Acknowledgments:** The authors would like to thank Steier, J., Lehman, K., and Wright, J. for their help in generating the barcode sequence data for shrubs and lianas on Barro Colorado Island.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


**2. Biochemical modifications in cotton plant under water stress**

In many plant activities, water is vital for the transport of nutrients, chemical and enzymatic reactions, cell growth, cell division, and transpiration [43]. Drought stress reduces plant development due to disruptions in the plant's main biochemical and physiological systems [44]. Drought stress affects root penetration, and stem elongation and increases water use efficiency. The leaf water potential, rate of transpiration, and leaf temperature are important traits that affect the growth of plants under water-deficit conditions. Although all stages of cotton development are affected by water stress, however, the reproductive phase, that is, flowering and boll development, are generally accepted as the most sensitive stages [45]. A very close relationship between nutrient uptake and water use efficiency has been observed in many crops. Therefore, nutrient uptake was reduced during drought stress, particularly N and K in cotton. Similarly, K contents in plant tissue also decrease due to drought stress and an overall reduction in nutrient uptake and there utilization [43].

*Enhancing Water Use Efficiency by Using Potassium-Efficient Cotton Cultivars Based… DOI: http://dx.doi.org/10.5772/intechopen.112606*

Root growth is important for plant development as water and nutrients are mainly taken up via roots, whereas root elongation is affected by drought stress which ultimately limits crop yield [20]. Under severe drought stress, the elongation of plants may be inhibited by interrupted water flow from the xylem to the surrounding cells, which impairs mitosis and cell expansion, resulting in a reduction of root growth, nutrient uptake, plant height, and leaf area as well as plant growth [46]. In cotton, under water stress conditions chlorophyll contents decreased which reduced photosynthesis rate, sugar production, and pigments including chlorophyll *a*, chlorophyll *b,* and carotenoids [47].

There is a root-to-leaf transduction of chemical signals caused by water-deficit stress, through the production of abscisic acid (ABA), which results in the closure of stomata. Under drought stress, ABA promotes stomata closure to reduce the transpiration rate [48]. When water potential is low in plants, ABA also stimulates root growth and inhibits shoot growth. Once the soil water availability is reduced, the amount of ABA in the xylem increases, and consequently, ABA concentration in different parts of the leaf is increased. As ABA is directly associated with stomatal conductance, it reduces net CO2 intake and decreased photosynthesis [48]. Existing cotton cultivars vary in tolerance to abiotic stresses, such as drought; therefore, maintenance of optimum plant cell turgor is an indication of the drought tolerance potential of a cultivar [49]. Similarly, a change in carbohydrate metabolism with an increase in glucose concentration in leaves and sucrose concentration in pistils of white flowers of cotton was observed under drought stress [50].

Ascorbate peroxidase activity is reported to increase whenever plants undergo in drought stress as reported in cotton grown under drought stress but glutathione metabolism levels were not changed [51]. However, the investigation of antioxidant activity in cotton plants exposed to drought stress is still controversial and is not well understood and superoxide radicals (SOD) or catalase (CAT) activities remained unchanged under drought stress [52]. It has been shown that CAT activity is not affected by drought stress, while, the Ascorbate peroxidase (APX) and SOD activity was increased [53]. In most plants, osmoregulation reduces the water potential of cells, thus increasing the gradient for water flow in the cell to maintain cell growth [54]. The maintenance of cell turgor contributes to ongoing physiological processes such as stomatal conductance and photosynthesis, as reported by Ref. [21, 55]; however, information on osmotic regulation in modern cotton varieties under drought stress is still lacking.

Yield is essentially the precise integration of the many systems of physiology. Drought stress negatively affects most of these physiological systems. The negative effects of shortage of water on yield depend mainly on the intensity of stress and the growth stage of plant life. In key crops, significant drought stress losses have been observed. The low moisture caused by parenthesis lowered the time to anthesis and reduced the time to fill the grain with cereal by anthesis [56]. Exposure of plants to drought stress can lead to total sterility of pearl millet (*Pennisetum glaucum* L.), typically owing to disrupted mobility in the assimilated ear [57]. The drought may be caused by many causes such as decreasing photosynthetic rate [58], disturbing assimilate partitioning [44], or inadequate growth of a prominent leaf [59]. Maize was significantly reduced in returns when exposed to drought conditions at the tasseling stage [14]. In the same way, the production and abortion of the developed bolls in cotton under drought circumstances were significantly reduced, which eventually affected the lint yield [60]. In drought environments, there was also a substantial drop in barley grain production (*Hordeum vulgare* L.), largely due to lower viable

grain tillers and grain with less than 1000 seeds [61]. Drought stress in the blooming stage produced more than a 50% decrease in the seed output by the exposition of pigeon peas (*Cajanus cajan* L.) [62].

Drought has a primary influence on the plants, namely poor germination and impaired planting. Several studies have demonstrated detrimental effects on germination and seedling development of drought stress [44, 63]. In major crops of fields, including pea (*Pisum sativum* L.), alfalfa (*Medicago sativa* L.), and rice (*Oryza sativa* L.) under drought stress, drought stress was observed to reduce germination, early sowing growth, root and shoot dry weight, hypocotyl ate length, and vegetative development [64–66]. Plant growth is achieved largely through the division of cells, expansion, and differentiation. Drought affects mitosis and cell elongation leading to poor growth [39]. Drought restricts the cell development process, largely owing to turgor loss [67]. Water limitation leads to cell elongation, largely because of the inadequate passage of water from the Xylem to the next cells [68]. Drought also reduces the number of leaves and the size of each leaf. The leaf growth usually depends on the turgor and the availability of assimilates. Reduced turgor and slower photosynthesis rate in circumstances of drought stress particularly restrict the growth of the leaf [59]. Fresh and dry weight in the water limitation conditions are likewise reduced significantly [69]. Plant height, leaf size, and, stem diameter were diminished significantly in maize under water-limiting conditions [70]. Ref. [71] found in another investigation that the bioaccumulation of maize has decreased considerably under dry conditions at different stages of growth.

Certain variables, such as the leaf water, leaf and canopy temperature, transpiration rates, and stomatal conductivity, impact water relations. Drought stress disrupts all of these processes in plants, but stomatal conductance is particularly impacted [44]. Drought conditions, which eventually raised the leaf and canopy temperature, showed a substantial decrease in the leaf water potential and transpiration rates [72]. The efficiency of the dry matter ratio accumulated with the water used is another essential characteristic of plant physiology control. Efficient wheat crops are more efficient at using water during drought conditions [73]. This improvement in the efficiency of water usage is large because the dry matter accumulates by absorbing less water due to the closure of the stomata and less transpiration. When subjected to an early season water scarcity, a decreased water efficiency in potatoes (*Solanum tuberosum* L.) was found, and eventually, biomass buildup and output were low [74].


*3.1. Growth and Development of Tomato Seedlings as A*ff*ected by Supplemental Lighting Treatment in the Glasshouse*

After cultivation in a glasshouse for 10 d, growth and development parameters of the grafted tomato seedlings were measured. Results of the measurements showed that the characteristics of the seedlings' morphologies were significantly influenced by light intensities. The two cultivars of grafted tomato cultivated in *100* and those in *150* showed the greatest scion length and root fresh weight. No significant differences were discovered in scion stem diameter and leaf length between supplementary lighting of *50*, *100,* or *150*. Leaf area and width, number of leaves, and root length of 'SS' were significantly greater in supplementary lighting of *100* and *150* than in supplementary lighting *50*. The greatest root length was observed in light intensity of *150* in 'SD'. In general, there were little differences between supplementary lighting of *100* and *150*. Compared with supplementary lighting of *50*, both of these two treatments significantly improved quality of the two types of grafted tomato seedlings (Table 1).

Dry weight of shoot and dry weight of root are shown in Figure 1. Considering these two weights, there were no significant differences between light intensity of *100* and light intensity of *150*. However, compared to the treatment of light intensity of *50*, these two treatments significantly improved dry weight of the two types of tomato cultivars.

**Figure 1.** Influence of supplementary lighting intensity on dry weight of shoots and dry weight of roots of two cultivars of tomato 'Super Sunload' ('SS') (**A**,**B**) and 'Super Dotaerang' ('SD') (**C**,**D**) grafted onto the 'B-Blocking' rootstock. Error bars represent SEs of nine biological replicates (*n* = 9). According to the Duncan test, lowercase letters at *p* ≤ 0.05 indicate significant differences between different treatments.

**Table 1.** Influence of supplemental light intensity on growth and development of grafted tomato seedlings grown for

 10 d.

Growing compact and sturdy seedlings is an objective of high-quality seedling production. Dry weight to height ratio of scion (WHR) was conducted to indicate the quality of seedlings. For 'SS', supplementary lighting of *100* and *150* significantly improved WHR as compared to supplementary lighting of *50*. There were no significant differences in WHRs between supplementary lighting of *100* and *150*. For 'SD', the greatest WHRs showed in supplementary light of *150*. For both tomato cultivars, the specific leaf weight (a leaf fresh weight per unit leaf area) was significantly improved in light intensities of *100* and *150* as compared to light intensity of *50*. There were no significant differences in the specific leaf weight between light intensity of *100* and 150 (Figure 2).

**Figure 2.** Influence of supplementary lighting intensity on dry weight to height ratio of scion and specific leaf weight of two cultivars of tomato 'SS' (**A**,**B**) and 'SD' (**C**,**D**) grafted onto 'B-Blocking' rootstock. The error bars represent SEs of biological replicates (*n* = 9). According to the Duncan test, lowercase letters at *p* ≤ 0.05 indicate significant differences between different treatments.

Moreover, we determined the effect of lighting intensity on stomatal conductance of the two tomato cultivars. Results showed, compared with light intensity of *50*, light intensity of *100* increased stomatal conductance of the two cultivars. Interestingly, stomatal conductance in light intensity of *150* was similar to that in light intensity of *50* treatment (Figure 3).

**Figure 3.** Influence of supplementary lighting intensity on stomatal conductance of two cultivars of tomato 'SS' (**A**) and 'SD' (**B**) grafted onto the 'B-Blocking' rootstock. The error bars represent SEs of three biological replicates (*n* = 3). According to the Duncan test, lowercase letters at *p* ≤ 0.05 indicate significant differences between treatments.

Figure 4 illustrated seedling morphologies of 'SS' and 'SD' after treating for 10 d by using supplementary lighting with a different light intensity. It showed that tomato seedlings grown in supplementary lighting of *100* and *150* had more biomass and were sturdier.

**Figure 4.** Influence of supplementary light intensity on morphology of 'SS' (**A**) and 'SD' (**B**) after 10 d of cultivation.

#### *3.2. Expressions of Photosynthesis-Related Genes*

Light intensity significantly influenced *PsaA* and *PsbA* genes (Figure 5). Light intensity of *100* enhanced expressions of *PsaA* and *PsbA*. Relative expressions of *PsaA* in 'SS' and 'SD' were 1.37- and 2.27-fold, while that of *PsbA* was 4.01- and 10.29-fold, as compared to that in the light intensity of *50*. However, a dramatic decrease in expression was observed in light intensity of *150* in 'SS' and 'SD'. Particularly, the relative expression of *PsaA* in 'SS' was only 0.44-fold compared to the expression level in *50*.

**Figure 5.** Relative expressions of *PsaA* and *PsbA* genes in 'SS' (**A**,**B**) and 'SD' (**C**,**D**) grafted onto the 'B-Blocking' rootstock. The bars among treatments represent the mean ± SEs of technological replicates (*n* = 3). According to the Duncan test, lowercase letters at *p* ≤ 0.05 indicate significant differences between different treatments.

### *3.3. PsaA and PsbA Immunoblots*

The *PsaA* gene is located in chloroplast genome (cpDNA). It encodes a core protein of a protein-pigment complex in PSI [17] and encodes D1 protein, which is a core component of PSII [18]. Figure 6 shows the protein expressions of PsaA and PsbA in 'SD' and 'SS' cultivated in supplementary lighting with different light intensities (*50*, *100*, or *150*). For both tomato cultivars, expression levels of PsaA and PsbA in light intensities of *100* and *150* increased as compared to those in light intensity of *50*. The highest expression levels of the PsaA and PsbA proteins appeared in light intensity of *100*.

**Figure 6.** Expressions of PsaA and PsbA protein in 'SD' and 'SS' grafted onto the 'B-Blocking' rootstock. Equal loading was verified by the Rubisco large subunit (RbcL) level.

#### **4. Discussion**

Among regulatory factors for light condition, light intensity as an essential element is involved in regulation of growth, yield, and nutritional quality of vegetables [19]. Previous studies have proven that increased light intensity within a certain range may promote growth in lettuce [20,21]. Light condition is the foundation and guarantees healthy growth and yield of tomatoes [22]. Recent studies have shown that tomato seedlings under low-light conditions showed elongated internodes and petioles as well as a larger leaf area, which were caused by a shade-avoidance response [23,24]. Low-light conditions also affect the differentiation time and quality of flower buds in tomatoes [25]. For tomato cultivars 'Matador' and 'Blizzard', plant height, leaf width, number of leaves, and dry weight of aerial plant parts significantly improved due to increased levels of supplementary light (30, 60, and 90 μmol·m−2·s−1 PPFD) [26]. However, these findings do not mean the higher light intensities are always better. Tomato seedlings treated with 150 μmol·m−2·s−1 PPFD using white and blue LEDs showed continuous light-induced injuries, and the degree of the injury was more serious under white and blue LEDs than under orange and red LEDs of the same intensity [27]. This study showed that, compared with stem diameter of grafted tomato seedlings in supplementary lighting of *50*, that of grafted tomato seedlings increased in supplementary lighting of *100* and *150*. Leaf width and leaf thickness were also greater with treatments of light intensity of *100* and *150* (Table 1). Higher over-ground biomass and activeness of antioxidant enzymes were discovered in treatments with increased light intensity (100, 200, and 400 μmol·m−2·s−1 PPFD) in lettuce [28]. For *Nicotiana tabacum*, seedling biomass, soluble sugar concentrations, and primary root growth increased dramatically with light intensity of 300 μmol·m−2·s−1 PPFD as compared to 60 μmol·m−2·s−1 PPFD [14]. It had been proven that low light intensity decreased above- and below- ground biomass in radish [29]. The results also showed, compared with light intensity of *100* and *150*, dry weight of scion and dry weight of root were much lower in light intensity of *50* (Figure 1). Additionally, light intensity influences the development of stomata as well. According to the study of Lee [30], the stomatal conductance and the number of stomata per unit area of leaf increased with light intensity, increasing up to 60 μmol·m−2·s−1 PPFD in *Withania somnifera*. However, width and length of stomata declined in light intensity of 90 μmol·m−2·s−1 PPFD. These findings were consistent with our results in Figure 3. The *100* promoted stomatal conductance of both tomato cultivars, while *150* affected it adversely.

The aim of supplementary lighting in greenhouses are as follows: to increase the light intensity in greenhouse and enable crops to receive proper light, to provide crops with light with specific

wavelengths, to alter the time of flowering and fruiting, and to increase time for photosynthesis via changing the lighted period [31,32]. Advantages of light-emitting diodes (LEDs) include their small volume, longer service life, energy savings, and use of a single wavelength, which are efficient to encourage plant growth by producing the type of light with the specific wavelength needed by plants [33]. Studies have shown that red and blue lights absorbed by plants account for 90% of total light absorption [34], which are involved in regulating gene expression in photomorphogenesis [35]. Our previous study showed that compared with high pressure sodium (HPS), far-red (FR), metal halide (MH), and white LEDs (W), mixed LEDs (W1R2B1) as a supplementary light source had the best effect to improve the quality of grafted tomato seedlings [15]. The application of monochromatic light may cause physiological defects of plants such as disturbance of photosynthetic mechanisms [36], damage to the granule and thylakoid [37], and restrained synthesis of chlorophyll [38], which can be overcome or relieved by the mixture of red and blue light.

Photosynthesis is one of the supreme chemical reactions in plants. Photoreaction takes place in the chloroplast thylakoid membrane. Two light energy-driven systems, PSI and PSII, synergistically function in primary energy conversion reactions. Driven by light, plants and other photoautotrophs are able to synthesize carbon compounds directly from CO2 and H2O and release O2. Processes of consumption and formation in photosynthesis are related to photosynthetic phosphorylation coupled with electron transport. Electrons generated from splitting of H2O by the oxygen-evolving complex (OEC) are used to reduce NADP+ to NADPH. At the same time, synthesis of ATP is driven by a transmembrane electrochemical proton gradient [39,40]. The PSII, a multi-subunit protein-pigment complex, is associated with the photolysis of water and synthesis of ATP [41]. Within the core of the complex, PsbC and PsbB are bond to the photosynthetic pigments and transfer the excitation energy to reaction center proteins D1 (Qb, PsbA) and D2 (Qa, PsbD) [42]. D1 dimerize with D2 as a heterodimer. Studies have suggested that synthesis of D1 is coordinated with linear photosynthetic electron transport as well as the relative activities of PSI and PSII [43]. Light has an important role in the translation of Dl protein [44]. The PSI complex contains reaction center P700, as well as five electron transfer centers (A0, A1, FX, FA, and FB) bound to PsaA and PsaB proteins [45]; it mediates electron transfer and is involved in light-driven conversion of NADP+ to NADPH [46]. The two largest polypeptides, PsaA and PsaB, carrying P700 are believed to form the center Fx through dimerization [47]. In higher plants, the genes *PsaA* and *PsaB* encode the two polypeptides respectively and are located adjacent to each other in the plastid genome [48].

A previous study proved that expression of the *PsaA* gene product during rice plastid development was light-regulated on the translational or post-translational level [49]. In *Synechococcus*, blue light photoreceptor regulates expressions of *PsbA* genes [50]. Moreover, in *Synechocystis* the transcription levels of promoters of *Psba* gene changed in response to dark and light conditions [51].

Expression levels of *PsaA* and *PsbA* genes in light intensities of *100* and *150* increased as compared to those in light intensity of *50* in both cultivars. Compared with those in light intensity of *100*, lower expression levels of genes appeared in light intensity of *150* (Figure 5). Western blotting showed the same tendency of variation in protein expression as shown in Figure 6. Previous studies pointed that high light intensities caused degradation and phosphorylation of PSII core protein and damaged the photosynthetic machinery [52]. It could be speculated that the photosynthetic apparatuses suffered slight damage in light intensity of *150*.

Although light is the basic energy for photosynthesis, light that is too strong will cause light stress for plants, which has side effects for photosynthesis, especially under low temperatures, drought, or other unfavorable conditions [53]. In 1956, Kok firstly proposed the concept of photoinhibition and defined it as 'the photochemical inactivation of complete pigment complexes or photosynthetic units' [54]. Powles (1984) pointed that excessive light energy absorbed by the photosynthetic apparatus caused photoinhibition with the characteristic of reduced photosynthetic capacity [55]. PSII is very sensitive to strong light, and it is the main occurrence site of photoinhibition [40]. There are two mechanisms of photoinhibition in PSII, acceptor-side photoinhibition and donor-side

photoinhibition [56]. In the former situation, the hindered assimilation of CO2 will cause the reduction of the plastoquinone pool. Consequently, the double-reduced form of QA– accumulates and promotes the formation of the triplet state of the primary donor (3P680). The 1O2 generated from the reaction between 3P680 and O2 will cause damage to proteins and pigments, especially the amino acid residue sites in the D1 protein. In another mechanism, electron transport is impaired due to the hindered oxidation of water and prolonged half-life period of P680+ resulting in high oxidation potential on the donor side. Adjacent proteins and pigments are damaged as a consequence [57].

In 1994, Terashima firstly reported photoinhibition in PSI [58]. High-light intensity [59,60], low temperatures [61], and fluctuating light conditions [62,63] will cause PSI photoinhibition. Studies have shown that changes of reducing state and production of ROS (reactive oxygen species) and hydroxyl peroxide on the acceptor side are the main causes of PSI photoinhibition [64,65].

Plants have developed various mechanisms to prevent photoinhibition under high-light intensities because of their long-term evolution and development. The main mechanisms of photoprotection are as follows: to reduce the light absorption via movements of leaf and chloroplast [66]; to increase photosynthetic efficiency via increased levels of the photosynthetic electron transport carriers and contents of the photosynthetic key enzymes [67,68]; to protect the photosynthetic apparatus by photochemical and non-photochemical pathway such as the xanthophyll cycle, photorespiration, and the Mehler reaction [69–71]; to strengthen the active oxygen eliminating enzyme system [72,73]; and to distribute excitation energy and heat dissipation equally via state transition between PSII and PSI [74,75].

Usually, sun and shade plants possess different morphological, physiological, and biochemical characteristics to adapt to different light conditions. Chloroplasts with distinctive characteristics will contribute to levels of photosynthesis. As compared to sun plants, shade plants have higher thylakoid stack areas, fewer grana stacks, and smaller widths of grana stacks to inhibit degradation of the D1 protein [76]. The levels of photosynthetic electron transport carriers and activities of photosynthesis-related enzymes, especially Rubisco, are higher in sun plants [77,78]. When light intensity is changed, the activities of RuBP and Rubisco do respond to the fluctuating environment [79]. Studies in mulberry showed that the leaves, which were at the daily maximum photosynthetic state, showed higher reversibility of photosynthetic quantum conversion, confronting the environment with changing light intensity [80]. After being transferred to an intercropping shaded environment, the lodging-resistant soybean genotypes showed elongated main stems and internodes as well as decreased biomass to the length of the stem ratio. The reasons may be due to downregulated gene expressions related to biosynthesis of structural carbohydrates [81]. Additionally, shade conditions influenced the photosynthetic rate by altering the chlorophyll fluorescence performance in soybean [82] and *Athyrium pachyphlebium* [83]. Shade treatment changed concentrations of chlorophyll and chlorophyll a/b ratios in soybeans [84] and wheat [85]. These studies suggested that light capture and utilization, PSII activity, and the transfer of electron and energy between PSII and PSI may involve the regulation of photosynthetic capacity in shade and light conditions. Phenotypic plasticity of plants refers to the ability to conform their morphology and physiology to the environment [86]. Even though studies have shown that the phenotypic plasticity of some shade plant species is lower [87,88], the mechanism of optimizing light energy capture should be lucubrated. To illustrate that, the mechanism of photosynthesis in natural growth condition with fluctuating light can help plants to adapt to sun and shade conditions and increase yields as a consequence [86]. Additionally, more attention should be paid to the relationship between genotype plasticity and productivity as well as the intraspecific variation in plasticity to fluctuating light [89].

Light required by plants is related to different species, cultivars, growth stages, developmental stages, and environmental factors as well as target of production [19]. Although light has a vital function in photosynthesis, excess light may depress photosynthetic efficiency, which is referred to as photoinhibition [55]. Therefore, to maximize economic benefits of grafted seedlings with high quality and quantity, detailed studies on optimizing light intensity are in urgent need.


*3.1. Distributed Fiber Raman Amplifiers*

In a distributed FRA, pump at the optical frequency ω*P* and signal at the frequency ωs = ω*P* − ων are jointly launched into the same ordinary optical fiber. Raman optical amplification takes place when their frequency difference Ω = ω*P* − ωs lies within the bandwidth of the Raman-gain spectrum. The distributed FRA guides light at both the signal and pump wavelengths and it is normally single mode to ensure the best overlap of all traveling waves. A distributed FRA unit is a pumping unit at 14xx nm without gain medium and it, usually, consists of pump lasers, optical couplers to combine them, isolators and a few monitor taps, as well as the WDM coupler for signal and pump. Depolarizers are necessary when the pump is polarized, in order to suppress the polarization dependence of Raman amplification.

Figure 3 shows a configuration in which pumping light propagates bi-directionally in distributed FRA. Moreover, the system is provided with band pass filter (BPF) and arrayed waveguide grating (AWG) devices which act as demultiplexing unit in the receiving side.

As mentioned before, in a distributed FRA both forward and backward pumping schemes are possible. In the forward pumping, the signal is amplified near its fiber-input end, where the signal power is relatively high. Thus, this scheme corresponds to a booster amplifier. In the backward pumping, the optical signal is amplified near its fiber-output end before photodetection, where the signal power may be so weak as to be comparable to the noise light power. Since this scheme is used to improve detectivity or to reduce the minimum detectable signal power, it can be considered as a detection preamplifier [50,51].

We note that the process of Raman amplification takes place so rapidly that, unless the intensity noise of the forward pumping light is sufficiently small, the pumping light noise will be transferred to the signal light resulting in increasing transmission bit error rates. Since signals propagating in the opposite direction to the pump will average over the fluctuations, in order to mitigate unwanted

crosstalk between the pump and the signal, in many cases only backward pumping is used [52,53]. Typically, high-powered counter propagating Raman pumps are deployed in conjunction with discrete amplifiers, such as EDFAs.

**Figure 3.** Schematic representation of multiplexing/demultiplexing based distributed optical Raman amplifier.

Distributed FRAs offer several advantages: simplicity, because they provide direct optical amplification of signal light in the transmission fiber; flexibility in the use of signal wavelengths, because the Raman gain peak is dependent on the pump wavelength and not the emission cross section of a dopant; broad gain bandwidth, which allows to employ multiple pumps. However, one of the major benefits, only obtainable through the use of distributed amplification, is that signal gain may be pushed into a transmission span preventing the signal from decaying as much as it otherwise would have if no amplification was provided within the span. As a consequence, the signal-to-noise ratio does not drop as much as it would have in a system based on transmission through a passive fiber followed by a discrete amplifier (see Figure 4). Thus, lower signal powers can be used, nonlinear penalty are reduced and higher loss can be tolerated. This improved noise performance may be used in different ways. One way is to extend the reach between repeaters, another is to extend the total reach of a transmission system, and a third one is to improve the transmission capacity.

**Figure 4.** Ideal distributed amplifier: the loss is counterbalanced at every point along the span, leading to an improvement of the signal to noise ratio (SNR) respect to (ideal) discrete amplification.

Using distributed FRAs, the transmission fiber becomes an amplifier, giving an important advantage but, at the same time, the optimization of the fiber design with respect to amplifier performance becomes more difficult due to the need to optimize the fiber for signal transmission, too. Several parameters have to be taken into account to evaluate the performance of a distributed FRAs, such as linear noise accumulation, pulse distortion due to group velocity dispersion, effects due to Rayleigh scattering, nonlinear interactions between pump and signal channels, and pump–signal crosstalk. Considering that in distributed amplifiers the gain is gathered over tens of kilometers and that the Raman effect is a very fast process (response time in the order of femtoseconds), these noise sources become significant.

In Ref. [54] a 25.6 Tb/s experiment was demonstrated over a 240 km repeated span by employing 160 WDM channels with 50 GHz channel spacing in the C+L bands. The authors used backward Raman pumping to increase the received optical signal to noise ratio (OSNR) and simplify the optical repeaters, while an optical equalizer was used in the receiver to minimize channel distortion caused by narrow optical filtering.

#### *3.2. Discrete Fiber Raman Amplifiers*

Lumped or discrete FRAs use a fiber medium that is localized before or after transmission to fully or partially compensate for the transmission loss. Unlike a distributed FRA, all of the pump power is confined to the lumped element (see Figure 5). In this particular case, counterpropagating pump power is confined within the unit by the use of isolators surrounding the amplifier. Compared with Figure 3, no pump power enters the transmission line.

**Figure 5.** Discrete fiber Raman amplifier (FRA) scheme with pump power confined to the lumped element.

In discrete FRAs, the used fiber is a lot shorter (order tens instead of hundreds of km) compared to distributed FRAs. Since gains accumulate with fiber length, Raman amplification better fits in the distribuited FRAs class. Therefore, discrete FRAs are primarily used to increase the capacity of fiber-optic networks, opening up new wavelength windows, which are inaccessible by EDFAs. In FRAs, the signal is amplified at a wavelegth given by the frequency difference between the pump and the Stokes frequencies, thus by choosing the pump wavelength, gain at any wavelength can be obtained. Moreover, by combining multiple pump wavelengths, the shaping of the gain spectrum can be achieved, whereas by combining several pumps centered at different wavelengths, a flat gain in an ultrawide bandwidth is allowed.

The latter issue, i.e., a flat gain, is highly desirable in WDM systems in order to equally amplify all the channels. For this reason, flat gain of broad-band Raman amplifier, is considered one of the most important goal for the optimization of fiber communications. This task can be obtained by simultaneously using pumps at different wavelegths that generate Raman gain lightly shifted from each other so as to partly overlap each other leading to a composite gain (see Figure 6). The final Raman gain can be very flat over a wide range of wavelegth by accurately designing each pump wavelength and power. The gain-band expansion that can be achieved by multi-wavelength pumping is determined by the magnitude of the Stokes shift. A large Stokes shift is therefore desirable for a wideband FRAs using multi-wavelength pumping [55]. The maximum amplification bandwidth of a FRA is about 100 nm for silica fiber in the 1550-nm band, so the composite gain could cover a nearly 100-nm signal band [56]. They are particularly useful for amplifying (dense) WDM systems.

**Figure 6.** Multi-wavelength pumping technique for wideband Raman amplifiers.

Using a broadband pumping approach, amplifiers with gain bandwidths greater than 100 nm have been demonstrated [57] with average net gain of 2 dB gain for SMF, while for dispersion-shifted fiber and reverse dispersion fiber the net gain was of 6.5 dB. A 12- wavelength-channel WDM LD (high-power laser diode (LD) pumping unit), with a range of wavelength from 1405 to 1510 nm and combined by an Il-MZI-integrated, using a planar lightwave circuit technology, were used to enlarge the gain bandwidth of Raman amplifiers [57] However, due to a strong interaction between pumps, these broadband FRAs typically need of more power at the shortest wavelengths pumps which amplify the longer wavelegth pumps, leading to at an increase of the noise properties.

Dispersion-compensating Raman amplifiers integrate two crucial tasks, dispersion compensation and discrete Raman amplification, into a single component [58–60]. Dispersion compensating fiber (DCF) devices, based on typical dispersion for the propagating mode, are usually in the order of several kilometers and, as a result, show important insertion loss. Hansen et al. demonstrated that Raman gain produced in the DCF was as a simple and attractive method to achieve lossless DCF modules, without affect system performance [58]. Due to a small mode-field diameter, a very low pump powers are required, like those available from semiconductor laser diodes. The DCF shows a net gain higher than loss, resulting in a wider system margin and the possibility to introduce other elements such as optical add-drop multiplexers into the system. In Ref. [61] a polarization-combined four channel WDM 14xx nm laser diode unit was used as pumping light source to demonstrate a broadband Raman amplification in a DCF. A fully compensation of the losses in the DCF were obtained by means of the Raman gain over 50 nm bandwidth within +/− 0.5 dB variation.

#### **4. Fiber Raman Lasers (FRLs)**

FRLs are similar to ordinary lasers. They basically involve three key components: A low-effective-area and high-nonlinearity optical fiber for providing Raman gain, mirrors for feedback and components for isolation between the pump laser (usually a rare-earth-doped fiber laser) and the Raman resonator. A first analogy is that, in FRLs, lasing occurs when the Raman-active gain medium is placed inside a cavity, for example, between mirrors reflecting the first Stokes wavelength. A second

analogy is that, in FRLs, the threshold power is obtained when Raman amplification during a round trip is as large as to compensate the cavity losses. However, there are also some important differences between FRLs and traditional lasers. A first one is that an amplifier medium based on Raman gain is used rather than on stimulated emission from excited atoms or ions. A second difference is that the required wavelength for pumping Raman laser does not depend on the electronic structure of the medium, so it can be chosen to minimize absorption. In addition, it is worth highlighting the unique property of Raman laser, i.e., they can be operated at several wavelengths simultaneously. Higher-order Stokes wavelengths can be generated inside the active medium at high pump powers and they can be properly dispersed spatially in association with separate mirrors for each Stokes beam [62].

The first continuous-wave Raman laser in fiber was demonstrated in 1976 [63,64]. Pumping at the watts level, a distributed Raman gain, higher than the linear attenuation plus losses at splice/connector points, was induced in a single-mode glass fiber, placed inside a Fabry-Perot cavity. SRS process occurred in fiber resulting in an intense output, due to wavelength-selective feedback for the Stokes light of the Fabry-Perot cavity, which was formed by two partially reflecting mirrors. Higher-order Stokes wavelengths were generated inside the fiber at high pump powers, too. The spatial dispersion of various Stokes wavelengths through an intracavity prism allowed tuning of the laser wavelength.

A single wavelength shift FRL is a fiber resonator at the Stokes wavelength, in which SRS shifts the spectrum of the propagating pump radiation through an optical fiber towards lower frequency Stokes components. Raman lasing is obtainable in conventional single-mode telecom fibers (e.g., SMF-28) as well as other passive fibers, by trapping Stokes components by reflectors and by pumping the laser by a high-power rare-earth-doped fiber laser (commonly ytterbium). In an all-fiber configuration, enough positive feedback for lasing at the first Stokes wavelength is easily achieved. Directionality to the laser emission is provided by a high-reflecting fiber Bragg grating (reflectivity > 99%) on the input side and by a low-reflectivity output coupler (at Stokes wavelength) on the output side. Fiber Bragg gratings (FBGs) are narrow-band reflectors, acting as the mirrors of the laser cavity and inscribed directly into the core of the optical fiber used as the gain medium. However, we note that in Raman laser, when Raman gain of the active medium is very large, the laser can build up from noise to a substantial signal without any feedback. In addition, a single wavelength shift FRL, with a broadband flat Raman gain profile can be obtained using multiple pump wavelengths. Figure 7 shows a schematic diagram of a specific Raman fiber laser with one wavelength shift. In this particular case, the laser uses a 1117 nm ytterbium-doped fiber laser and converts it to 1178 nm.

**Figure 7.** Schematic representation of a Raman fiber laser with one wavelength shift. (HR—high-reflectivity fiber Bragg grating, OC—output coupler—a low-reflectivity fiber Bragg grating).

A multiple wavelength shifts FRL takes advantage of SRS cascading. The pump light gives rise to the "first-order" laser light in a single frequency-shifting step, which remains trapped in the laser resonator. Afterwards, the "first-order" laser light can be pushed to very high power levels becoming itself the pump for the generation of "second-order" laser light, which is shifted by the same vibrational frequency of first order. Using this technique, conversion of the pump light (typically around 1060 nm) to an "arbitrary" desired output wavelength through several discrete steps can be performed by using a single laser resonator. In Figure 8 is schematically reported the wavelength conversion over two or more Stokes shifts obtained by using a cascaded Raman resonator with fiber Bragg gratings incorporated in it, in order to realize nested cavities at each of the intermediate wavelengths. These intermediate wavelengths are chosen close to the peak of the Raman gain of the respective preceding wavelength; finally, a low-reflectivity output coupler terminates the wavelength conversion. The resulting output is at the designed final wavelength while only small fractions are at the intermediate wavelengths.

**Figure 8.** Schematic representation of a Raman fiber laser with multiple wavelength shifts by using a cascaded Raman resonator (RIG—Raman input grating set, ROG—Raman output grating set).

Tunability is an important property for lasers. The first tunable FRL, using a linear cavity configuration, was reported by Jain et al. [65]. A tuning range of 8 nm with an efficiency of 9% were demonstrated. However, the bulk optics (prisms and rotating mirrors), placed inside the laser cavity for tuning, introduced mechanical instabilities as well as coupling losses, which gave rise to high power thresholds and low efficiencies. In order to eliminate losses and to make a compact device, the approach based on bulk optics was replaced by an all-fiber design. In such a design, cavity mirrors were integrated within the fiber by the development of tunable fiber Bragg gratings [66]. In 1988, the first FRL based on fiber Bragg gratings was reported [67].

Since the Raman gain value of germanosilicate fiber is about eight times higher than for silicate fiber, these fibers are extensively used in FRLs. Due to their peak Raman shift of 440–490 cm−1, in an FRL with a 1064 nm pump source, the third and the sixth Stokes orders produce outputs at 1239 nm and 1484 nm, respectively. So, in order to make a FRL at 1484 nm, one would need six cascaded cavities. In Ref. [68], a GeO–doped high-power and widely tunable all-fiber Raman laser was demonstrated. The FRL, using a linear cavity configuration and a purely axial compression of the FBGs, was continuously tuned over 60 nm, from 1075 to 1135 nm. A Stokes output power up to 5 W, for 6.5 W of launched pump power was provided. A high efficiency over the whole tuning range, from 76.1 to 93.1% and laser thresholds varying from 0.78 to 2.59W were obtained, respectively.

It is worth noting that the peak Raman shift of low-loss phosphosilicate (P2O5-SiO2) fiber is 1330 cm−1, so when they are pumped by a 1064 nm pump source, the first Stokes (S1) and the second Stokes (S2) orders occur at 1239 nm and 1484 nm, respectively. This means that to make a FRL at 1484 nm, only two cascaded cavities would be required, thereby greatly increasing the FRL efficiency. In Ref. [69], a CW high-power Raman flber laser was reported, which was formed by a CW 8.4 W/ 1064 nm Yb-doped double-clad fiber laser as a pump, a 700 m phosphosilicate fiber and cascaded cavities with two pairs of FBG mirrors for the first and the second Stokes orders. A maximum output power of 2.24 W and slope efficiency of 32.8% at 1484 nm were demonstrated. In Ref. [70], Babin et al. demonstrated a simple all-fiber widely tunable phosphosilicate FRL of high efficiency. The laser had more than 50 nm tuning range and generated up to 3.2 W of output power with 72% maximum slope efficiency. The output power was almost constant in the range 1258–1303 nm.

Typically, the operating wavelengths of Raman laser are in the 1–1.5 μm region of the spectrum and have been created using silica glass as the base material with the desired application directed toward telecommunications. However, there are a large number of applications requiring high power laser with wavelengths longer than 2 μm. As a result of the versatility offered by FRLs, these types of sources are attractive for this purpose [71]. Chalcogenide glass fibers have a Raman gain coefficients approximately two orders of magnitude greater than the gain coefficients of silica. For wavelengths longer than 1.8 μm, the development of Raman fiber lasers based on chalcogenide glasses has now become technically achievable [72]. In Ref. [73] a Raman fiber laser, using a nonoxide glass as the gain medium and a 2 μm fiber laser pump source was demonstrated. Due to the low optical losses and high nonlinear index of commercially available glassy As2Se3 fiber, a combined output of 1 W at the first and second Stokes wavelengths of 2062 and 2074 nm, were generated respectively.

Single-frequency laser sources have a great number of applications, but are difficult to implement, suffer from poor robustness, poor quality (linewidth and stability) and are expensive. In recent years, there has been much interest in rare-earth doped distributed feedback (DFB) fiber lasers, due to their very low noise and high efficiency [74]. The oscillating wavelength can be accurately defined by a Bragg grating written directly into the fiber and inserting a π phase-shift (PS) at, or close, to the center of the Bragg grating structure. Of course, the operation of π-PS-DFB fiber lasers has been mostly limited to the spectral bands of rare-earth dopants [75].

A Raman gain based DFB fiber laser would have a number of potential advantages. First, the possibility of generating narrow linewidth low-noise oscillation in wavelength bands outside the one of rare-earth doped materials. Second, Raman based fiber laser systems do not suffer from issues associated with high-concentration rare-earth doped fibers, which limit their efficiency due to thermal effects. Using Raman gain in DFB fiber lasers was proposed for uniform FBGs by Perlin and Winful [76] for the first time, while a Raman DFB fiber laser based on a relatively short (<20cm), π-shift fiber Bragg grating were proposed by Hu et al. [77]. Westbrook et al. have realized the first functional DFB using the Raman gain in fiber, but with a very high threshold (40 W) was observed at 1584 nm. The authors showed that watt-level thresholds can be achieved by using a small-core high-numerical aperture (NA) fiber [78]. Shi et al. demonstrated that a polarized pump and an ultralong FBG (30 cm) could yield sub-watt threshold at a lasing wavelength of 1120 nm [79].

In reference [80] highly efficient Raman DFB fiber lasers with CW output power up to 1.6 W was demonstrated, for the first time. The DFB Bragg gratings were written directly into two types of commercially available passive germano-silica fibers. Two lasers of 30 cm length were pumped with CW power at 1068 nm up to 15 W. The threshold power was ~2 W for a Raman-DFB laser written in standard low-NA fiber, and only ~1 W for a laser written in a high-NA fiber, both of which oscillate in a narrow linewidth at ~1117 nm and ~1109 nm, respectively. The slope efficiencies were ~74% and ~93% with respect to absorbed pump power in the low-NA fiber and high-NA fiber respectively. Recently a Raman-DFB with lower thresholds at 1120 nm and 1178 nm was demonstrated [81]. Despite these developments, the performance in terms of slope efficiency has remained poor (1–7% for single-ended output) compared to the expected simulated efficiencies (40–50%).

By exploiting the multiple scattering of photons in a disordered gain medium, a random laser can be obtained allowing a coherent light source without a traditional cavity [82]. For different scientific and medical applications, ultrafast Raman fiber lasers are an interesting option. An efficient way to obtain a high power ultrafast Raman fiber laser is pulse pumping, but it requires a real-time synchronization between the pump pulses and laser cavity. In order to overcome this limitation, recently, random fiber lasers with distributed Raman gain and Rayleigh feedback in standard telecommunication optical fibers were demonstrated [83,84]. Their advantages are a simple and flexible design, quasi-CW operation, narrow spectrum generation, high beam quality and pump energy conversion efficiencies comparable to the ones of conventional cavity lasers [85]. In recent years, many works have been reported on the mechanism [84,85], the improvement of performance including power scaling [86,87], wavelength

tuning [88,89], and Q-switching [90,91]. Nowadays, random fiber is a unique laser technology with various advantages in the fields of fiber sensing and optical communications [92,93].

A promising approach for the further development of devices based on SRS is represented by Photonic crystal fiber (PCF). The most important advantage of this technology is the reduction of the fiber length and the power levels required. The first preliminary results in photonic crystal fiber for Raman amplification and for an all optical Raman modulator was presented by Yusoff et al. [94]. An analysis of the Raman properties in triangular photonic crystal fiber was reported by Fuochi et al. [95], which investigated the influence of presence of the germanium doped core on the Raman properties. A model of PCF amplifiers was developed by Bottaccini et al. [96], which demonstrated that germanium doped PCF can enhance the gain for the same pump power. Finally, SRS in ethanol core PCF was demonstrated by Yiou et al. [97]. The approach of PCF with liquid could open new perspectives for SRS nonlinear properties with applications in optical sensing.