3.3 Receiver coil design

In practical applications, it is almost always beneficial to reduce the size of an implantable device, whereas the WPT system requires a match of the resonant frequencies of the primary and secondary coils. We have designed a new structure of the implantable coil with a miniaturized size while having enough turns to match the resonant frequency of the primary coil. Figure 20 illustrates our design in which

Figure 16.

Z-component of the magnetic field distribution with different separations between the evaluation plane and the transmitter consisting of seven coils. (a) 3.25 cm, (b) 7.25 cm, (c) 10.25 cm, and (d) 20.05 cm.

Wireless Power Transfer for Miniature Implantable Biomedical Devices DOI: http://dx.doi.org/10.5772/intechopen.89120

Figure 17. HFSS simulation. (a) 3D model of the transmitter mat; (b) Dimensions of each PSC.

Figure 18.

respectively, above the power mat (i.e., the X-Y plane). Color indicates the magnitude of the magnetic field in the z-direction. It can be seen that, at z = 8 cm (Figure 18a), the magnitude of the magnetic field was the highest (peak) at the center of each coil, and the lowest (valley) at the junction of three coils. When the distance to the HPT mat increased to 20 cm, a smoother magnetic field distribution

(Figure 18b). In order to evaluate the evenness of distribution quantitatively, the coefficient of variation (COV) was utilized which was defined as the standard deviation of the field values divided by the mean. Thus, a smaller value of the COV indicates a more uniform distribution. Figure 19 shows the COVs of the magnetic field in the z-direction above the HPT mat at distances from 5 to 40 cm. It can be observed that the COV achieves a value <10% when the distance is larger than the

In practical applications, it is almost always beneficial to reduce the size of an implantable device, whereas the WPT system requires a match of the resonant frequencies of the primary and secondary coils. We have designed a new structure of the implantable coil with a miniaturized size while having enough turns to match the resonant frequency of the primary coil. Figure 20 illustrates our design in which

Z-component of the magnetic field distribution with different separations between the evaluation plane and the

transmitter consisting of seven coils. (a) 3.25 cm, (b) 7.25 cm, (c) 10.25 cm, and (d) 20.05 cm.

was observed, but approaching to the field generated by a large spiral coil

size of the transmitter coil.

Recent Wireless Power Transfer Technologies

3.3 Receiver coil design

Figure 16.

84

Distribution of the z-component of the magnetic field in a plane at (a) 8 cm and (b) 20 cm above the HPT mat at the resonant frequency of 85.2 MHz.

Figure 19.

Variation in coefficient of variation (COV) of vertical field distribution as a function of distance above the HPT mat at the resonant frequency of 85.2 MHz.

#### Figure 20.

(a) Three sub-coils winded in proper directions are combined and connected to form a single coil shown in (b).

coils serve as both implant exterior housing and power receiving elements. It consists of two planar sub-coils and one helical sub-coil. The sub-coils are combined into a single coil within a shallow box assembly. By choosing different geometric designs for the sub-coils, different shaped boxes can be obtained. In practical implementation, the exterior of the box must be covered by a biocompatible material for biological safety.

By measuring the induced voltage in the small receiver coil, the variation of the

∂t ¼ �N ∂BzS

When the measuring coil is sufficiently small and the system is driven by sinusoidal input, the induced voltage V in the measuring coil is proportional to the local Bz. As our objective function cancels out the constants relating these two values, we can directly evaluate the cost function using V instead of Bz and compare with the calculation results. As shown in Figure 22, these measurement locations were chosen because the magnetic field distribution at any interior PSC in a regular

mat can be approximated by the central PSC in each single seven-coil cell

In order to compare the measured voltage with the calculated Bz, we need to normalize both to the same scale, as they are proportional to each other. Figure 23 shows that the measured data matches the calculated ones very well, except for that the measured data tends to be larger than the calculated ones, which is because the receiver can capture a small portion of horizontal field in additional to the field in the vertical direction. When the measured data are normalized to the center value where the horizontal component is almost zero, the other positions will have larger

In our WPT system design (Figure 13), the separation between the primary and secondary coils includes a distance between the mat and the floor. This distance can be adjusted to achieve both a high WPT performance and an acceptably uniform magnetic field distribution. At different separations, we measured the peak-to-peak values of induced RF voltages in the load coil when the system was powered by a sinusoidal wave at the resonant frequency (approximately 26.6 MHz). Our experiments show that, for our particular system design, the separations of approximately 10 cm and 8 cm between the primary and secondary coils provide a good compromise between performance and magnetic field distribution. In order to visualize this distribution, we interpolated the 21 measured values and plotted the results in Figure 24. At a separation of 10 cm, the measured 21 voltage values were in the range between 1.12 and 1.64 V, whereas the mean and standard deviation were

∂t ∝Bz (16)

V ¼ � ∂φ

Wireless Power Transfer for Miniature Implantable Biomedical Devices

DOI: http://dx.doi.org/10.5772/intechopen.89120

vertical magnetic field is given by

Sketch of a seven-resonator mat and the test points.

(Figure 15).

87

Figure 22.

field than the expected one.

## 3.4 Experiment tests

In order to study the performance of the mat-based WPT system experimentally, we constructed a prototype mat-based system shown in Figure 21, where a transmitter consists of seven circular spiral coils arranged in a hexagonal form. Each PSC was 13.2 cm in diameter, 2.9 mm in trace width, and 1.6 mm in trace spacing. On the reverse side of each PSC, several conductor strips were utilized to form distributed capacitances with respect to the coil on the front side. By changing the numbers of these strips, the resonant frequencies of the PSCs became adjustable. The frequencies were adjusted to 29.453 0.072 MHz with a Q-factor of approximately 100. The prototype of a receiver coil includes three coils and is 25 mm in diameter, 7 mm in height, and 3.39 g in weight. The resonant frequency and Q factor were measured to be 29.075 MHz and 61, respectively.

Figure 21. Experimental platform for measuring magnetic field distribution.

Wireless Power Transfer for Miniature Implantable Biomedical Devices DOI: http://dx.doi.org/10.5772/intechopen.89120

Figure 22. Sketch of a seven-resonator mat and the test points.

coils serve as both implant exterior housing and power receiving elements. It consists of two planar sub-coils and one helical sub-coil. The sub-coils are combined into a single coil within a shallow box assembly. By choosing different geometric designs for the sub-coils, different shaped boxes can be obtained. In practical implementation, the exterior of the box must be covered by a biocompatible mate-

(a) Three sub-coils winded in proper directions are combined and connected to form a single coil shown in (b).

In order to study the performance of the mat-based WPT system experimentally, we constructed a prototype mat-based system shown in Figure 21, where a transmitter consists of seven circular spiral coils arranged in a hexagonal form. Each PSC was 13.2 cm in diameter, 2.9 mm in trace width, and 1.6 mm in trace spacing. On the reverse side of each PSC, several conductor strips were utilized to form distributed capacitances with respect to the coil on the front side. By changing the numbers of these strips, the resonant frequencies of the PSCs became adjustable. The frequencies were adjusted to 29.453 0.072 MHz with a Q-factor of approximately 100. The prototype of a receiver coil includes three coils and is 25 mm in diameter, 7 mm in height, and 3.39 g in weight. The resonant frequency and Q

factor were measured to be 29.075 MHz and 61, respectively.

Experimental platform for measuring magnetic field distribution.

rial for biological safety.

Recent Wireless Power Transfer Technologies

Figure 20.

Figure 21.

86

3.4 Experiment tests

By measuring the induced voltage in the small receiver coil, the variation of the vertical magnetic field is given by

$$V = -\frac{\partial \rho}{\partial t} = -N \frac{\partial \overline{B}\_x S}{\partial t} \propto B\_x \tag{16}$$

When the measuring coil is sufficiently small and the system is driven by sinusoidal input, the induced voltage V in the measuring coil is proportional to the local Bz. As our objective function cancels out the constants relating these two values, we can directly evaluate the cost function using V instead of Bz and compare with the calculation results. As shown in Figure 22, these measurement locations were chosen because the magnetic field distribution at any interior PSC in a regular mat can be approximated by the central PSC in each single seven-coil cell (Figure 15).

In order to compare the measured voltage with the calculated Bz, we need to normalize both to the same scale, as they are proportional to each other. Figure 23 shows that the measured data matches the calculated ones very well, except for that the measured data tends to be larger than the calculated ones, which is because the receiver can capture a small portion of horizontal field in additional to the field in the vertical direction. When the measured data are normalized to the center value where the horizontal component is almost zero, the other positions will have larger field than the expected one.

In our WPT system design (Figure 13), the separation between the primary and secondary coils includes a distance between the mat and the floor. This distance can be adjusted to achieve both a high WPT performance and an acceptably uniform magnetic field distribution. At different separations, we measured the peak-to-peak values of induced RF voltages in the load coil when the system was powered by a sinusoidal wave at the resonant frequency (approximately 26.6 MHz). Our experiments show that, for our particular system design, the separations of approximately 10 cm and 8 cm between the primary and secondary coils provide a good compromise between performance and magnetic field distribution. In order to visualize this distribution, we interpolated the 21 measured values and plotted the results in Figure 24. At a separation of 10 cm, the measured 21 voltage values were in the range between 1.12 and 1.64 V, whereas the mean and standard deviation were

higher coupling factor than the conventional solenoid coil when a lateral or angular misalignment exists. In addition, the DH coil achieves the largest coupling factor and energy transfer efficiency when the axis of the DH coil is in parallel with the plane of the planar spiral transmitter coil. Our computer simulation and experiments under lateral and angular misalignments have been conducted and their

Wireless Power Transfer for Miniature Implantable Biomedical Devices

In order to support biomedical studies using the animal model, we have designed a new power mat, enabling wireless power delivery to miniaturized moving targets. The power mat contains a single or multiple transmitter cells and each cell consists of seven hexagonally packed PSCs. We have conducted theoretical, computational and experimental studies on the special WPT system to meet the challenge of distributing the electromagnetic field evenly over the power mat. We have analyzed the HPT cell using the CMT. Formulas have been derived relating the received power to the inputs and system parameters. Then, we utilize computer simulation to study the evenness of the magnetic field distribution over the power mat at different distances between the power mat and the floor of the animal cage. Finally, we constructed a prototype system, measured its magnetic field distribution and verified that our design has met the challenge. We have also presented a new design of the receiver coil consisting of three serially connected sub-coils. This new design of the receiver coil allows it to capture the most magnetic flux produced by the transmitter, facilitates a match of resonant frequencies of the transmitter and

, Wenyan Jia3

1 School of Artificial Intelligence and Automation, Huazhong University of Science

2 Department of Electrical Engineering, Shanghai Jiao Tong University, Shanghai,

3 Department of Electrical and Computer Engineering, University of Pittsburgh,

4 Department of Neurosurgery, University of Pittsburgh, Pittsburgh, USA

5 Department of Bioengineering, University of Pittsburgh, Pittsburgh, USA

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Zhi-Hong Mao3,5

results have verified our analytical results.

DOI: http://dx.doi.org/10.5772/intechopen.89120

receiver, and reduces the volume of the implant.

Qi Xu1,4, Tianfeng Wang2,4, Shitong Mao3

\*Address all correspondence to<EMAIL_ADDRESS>
provided the original work is properly cited.

and Technology, Wuhan, China

Author details

China

89

Pittsburgh, USA

and Mingui Sun3,4,5\*

Figure 23. Comparison of measured and calculated vertical magnetic field over the prototype mat.

Figure 24.

Voltage distribution computed by interpolating the 21 measured voltage values across the load coil at a constant primary and secondary coil separation of (a) 8 cm and (b) 10 cm.

1.23 and 0.11 V, respectively. The relatively small standard deviation indicates a nearly uniform magnetic field distribution, as observed in Figure 24. The results indicate that a flat magnetic field can be achieved by our power mat design and that this design is effective for WPT to biomedical implants in freely moving animals.


**3.3 Effect of EMF on uterine tubes**

The uterine tubes act as the site of fertilization and have a critical role in the conduction of zygote to the uterine cavity. Any changes in the structure and or function of uterine tubes would result in tubal pregnancies. The aim of the present study is evaluating the effect of EMF on uterine tubes by examining histological and morphological features using light and electron microscopy and immunohistochemical techniques.

#### **3.3.1 Light microscopic studies**

Light microscopic studies of uterine tubes from control group showed that: uterine tubes are lined with ciliated simple columnar epithelium and their cilia formed a smooth and orderly arranged ribbon at luminal face. The nuclei of epithelial cells were light and euchromatic. The epithelium was rested on a highly vascularized loose connective tissue (Figure 19). In the EMF-exposed group, epithelial cells were low columnar and mostly had lost their cilia. The nuclei of the epithelial cells were condensed and heterochromatic (Figure 20). Morphometric studies, based on final magnification, showed that the height of epithelial cells were decreased after EMF exposure, it was 2.77±0.19 mm VS 2.83±0.46 mm (Figure 21), the difference between two groups is significant (p<0.05).

Environmental Electromagnetic Field and Female Fertility 73

Fig. 21. A diagram showing the height of epithelial cells in uterine tubes (as mm) in control

Electron microscopy showed clearly the ciliated and secretory cells in the epithelial lining of the uterine tubes. In the control group, the ciliated cells had a basal or central ellipsoid nuclei with a prominent nucleolus. The cytoplasm contained poorly developed organells. The apical cell surface bears numerous cilia. The secretory or non ciliated cells had a central elongated nuclei with a prominent nucleolus. The organells were well developed. Membrane bound granules were located at apical cytoplasm. The epithelial cells were held

Fig. 22. Electron micrograph from uterine tube epithelium in control rat. Secretory cell (SC),

together by junctional complex and rested on a basal lamia (Figure 22).

and EMF-exposed groups.

ciliated cell(CC). 3000X.

**3.3.2 Electron microscopic studies**

Fig. 19. Photomicrograph of uterine tube from control rat. Note epithelium with a ribbonlike cilia at luminal face (arrow). H&E staining. 600X.

Fig. 20. Photomicrograph of a section of uterine tube from EMF-exposed rat. Note loss of cilia in epithelial cells, scattered cilia are left (arrow head). H&E staining. 600X.

Fig. 19. Photomicrograph of uterine tube from control rat. Note epithelium with a ribbon-

Fig. 20. Photomicrograph of a section of uterine tube from EMF-exposed rat. Note loss of

cilia in epithelial cells, scattered cilia are left (arrow head). H&E staining. 600X.

like cilia at luminal face (arrow). H&E staining. 600X.

Fig. 21. A diagram showing the height of epithelial cells in uterine tubes (as mm) in control and EMF-exposed groups.

#### **3.3.2 Electron microscopic studies**

Electron microscopy showed clearly the ciliated and secretory cells in the epithelial lining of the uterine tubes. In the control group, the ciliated cells had a basal or central ellipsoid nuclei with a prominent nucleolus. The cytoplasm contained poorly developed organells. The apical cell surface bears numerous cilia. The secretory or non ciliated cells had a central elongated nuclei with a prominent nucleolus. The organells were well developed. Membrane bound granules were located at apical cytoplasm. The epithelial cells were held together by junctional complex and rested on a basal lamia (Figure 22).

Fig. 22. Electron micrograph from uterine tube epithelium in control rat. Secretory cell (SC), ciliated cell(CC). 3000X.

Environmental Electromagnetic Field and Female Fertility 75

Immunohistochemical studies were restricted to TUNEL technique for detection of apoptotic cells. In the control group, with TUNEL assay, apoptotic cells was neither observed in the lining epithelium nor in the subepithelial layer (Figure 24). In the EMFexposed group, there were numerous apoptotic cells in the lining epithelium. Apoptosis was observed both in secretory and ciliated cell types. Very few apoptotic cells was also present

Fig. 24. Photomicrograph of a section from uterine tube in control rat. TUNEL method,

counterstained with toluidine blue. 360X.

**3.3.3 Immunohistochemical studies**

in the subepithelial layer (Figure 25).

In the EMF-exposed group, the nuclei were condensed, the apical cilia were apparently reduced and organells were sparsely distributed through the cytoplasm. Slight pyknosis, increased peripheral chromatin condensation and some degree of cytoplasmic condensation was observable. The most remarkable feature of the uterine tubes in this group was the presence of cells with preapoptotic characteristics i.e. flattened and cystic endoplasmic reticulum and nuclear condensation (Figure 23).

Fig. 23. Electron micrograph from uterine tube in EMF-exposed rat. Note decreased ciliar number and presence of intercellular spaces and cystic rER. 3500X.

#### **3.3.3 Immunohistochemical studies**

74 From Preconception to Postpartum

In the EMF-exposed group, the nuclei were condensed, the apical cilia were apparently reduced and organells were sparsely distributed through the cytoplasm. Slight pyknosis, increased peripheral chromatin condensation and some degree of cytoplasmic condensation was observable. The most remarkable feature of the uterine tubes in this group was the presence of cells with preapoptotic characteristics i.e. flattened and cystic endoplasmic

Fig. 23. Electron micrograph from uterine tube in EMF-exposed rat. Note decreased ciliar

number and presence of intercellular spaces and cystic rER. 3500X.

reticulum and nuclear condensation (Figure 23).

Immunohistochemical studies were restricted to TUNEL technique for detection of apoptotic cells. In the control group, with TUNEL assay, apoptotic cells was neither observed in the lining epithelium nor in the subepithelial layer (Figure 24). In the EMFexposed group, there were numerous apoptotic cells in the lining epithelium. Apoptosis was observed both in secretory and ciliated cell types. Very few apoptotic cells was also present in the subepithelial layer (Figure 25).

Fig. 24. Photomicrograph of a section from uterine tube in control rat. TUNEL method, counterstained with toluidine blue. 360X.

Environmental Electromagnetic Field and Female Fertility 77

Comparison of oocyte degeneration with apoptosis since Wyllie et al. (Wyllie 1980) first described the morphological characteristics of physiological cell death (apoptosis) few studies have described the ultrastructure of the atretic oocyte and none have examined this in pubertal or adult animals. Most reviews of ovarian follicular atresia focused on changes in granulosa cells or equate the entire process with apoptosis (Tilly 1998; Kaipia and Hsueh 1996). Biochemical analysis of atretic follicles that have measured DNA integrity (188-189)or increases in cell death – related mRNA levels, including bax and Fas, fasL (Hsuhe et al. 1994; Mori et al. 1997), have confirmed that apoptosis is occurring in antral ovarian follicles. Because the oocyts is a very small component of these large follicles, such measurements most probably reflect the status of granulosa cells. Due to these restrictions, microscopic examination is required to study the process of atresia in oocytes in situ. Because ultrastructural characterization is a reliable method for the classification of cell death as

Alterations in oocytes from EMF-exposed ovaries mainly in rat atretic f'ollicles included: loss of both granulosa cell and oocyte microvilli from the zona pellucida, changes in cytoplasmic organelles such as lamellar condensation and shirinkage of oocytes. Loss of microvilli and cytoplasmic condensation do resemble apoptosis, but other events differed from those associated with traditional apoptosis. For example, the mitochondria do not maintain their characteristic appearance during, early stages of atresia, as normally occur in apoptosis. While, cytoplasmic condensation, which is reflected by an increase in electron density at the ultrastructural level, observed in degenerating oocytes. In support of the findings of the present study, condensed chromatin was never observed in oocytes of atretic follicles by other investigators (Devine 2000). These comparisons suggest that there are more differences than similarities between physiological oocyte cell death and apoptosis. Other reports attempting to identify the mechanism of oocyte death have not discussed the possibility of alternative, nonapoptotic, types of physiological death (Perez et al. 1999). Early ultrastructural studies occurred before apoptosis was characterized (Vazquez and Sotello 1967; Franchi and Mandl 1962). More recent studies using ovulated oocytes failed to prove definitively that apoptosis was the mechanism of oocyte death (Perez et al. 1999; Van et al. 1998; Phillips et al. 1992). Therefore, it seems likely that oocytes in postnatal rats have unique cell-death triggers, signal transduction pathways, and clearance mechanisms as compared with other cell types. Such flexibility has not been described for traditional

events

The unique nature of the oocyte relative to other cell types may be the cause for its unusual manner of cell death. Oocytes can remain arrested in meiosis for years, are surrounded by an acellular zona pellucida, are nonproliferating, and are known to rely on surrounding granulosa cells for survival (Hirshfield 1991). Apoptosis is an active process thought to protect the rest of an organism from an aberrant cell. Meiotic oocyte may not be required to undergo apoptosis, because they pose no threat of excessive proliferation and tumor formation. Overall, the results presented here support that oocyte loss in atretic follicles of postnatal rats can be morphologically distinguished from the two more widely described mechanisms of cell death, necrosis, and apoptosis. While it is generally accepted that granulosa cells are lost by apoptosis, the ability of the oocyte to undergo apoptosis is still in

apoptosis (Payne et al. 1995).

apoptosis.

**4.2 The unique aspects of oocytes**

Fig. 25. Photomicrograph from uterine tube in EMF-exposed rat. Note apoptotic cells with golden brown color at luminal face (arrow). TUNEL method, counterstained with toluidine blue. 360X.


**4. Discussion**

*4.1. Thermal Comfort*

The thermal comfort evaluation of the historical residential buildings of Stone Town based on subjective and objective surveys is summarized in Table 5.

**Table 5.** Summary of subjective and objective survey results.

The air temperature obtained from both the subjective and objective surveys was above 26 ◦C, which is higher than the comfort zone for summer conditions (23–26 ◦C). Similarly, the relative humidity, which was around 63% as obtained from the questionnaires and measurements, is also higher than the value in the comfort zone (50–60%). Referring to the results obtained for the PMV and PPD values in the field measurements, neither of the case study buildings are controlled within the specified acceptable thermal comfort range based on the ASHRAE Standard 55, which is [−0.5 < PMV < +0.5] with a PPD < 10%. Indeed, numerous thermal comfort studies carried out worldwide have revealed that the PMV-PPD index cannot evaluate thermal comfort under non-air-conditioned space without modification due to the ignorance of subjects' adaptations. Although at least 20% of occupants are not satisfied with the current thermal environment, the TSV reflecting real thermal sensations indicates that the majority (73.57%) of the respondents' thermal sensation votes are within the central three categories of the 7-point scale. It once again proves that the PMV-PPD index is not applicable to assess thermal comfort in naturally ventilated buildings. Therefore, in Table 5, we add TSV with the purpose of reflecting the actual thermal satisfaction of occupants.

#### *4.2. Passive Cooling Strategies*

Based on the questionnaire and field survey results, passive cooling strategies implemented in the historical residential buildings in Stone Town include the north-south orientation (HRB1), natural ventilation (HRB1 and HRB2), pitched roof (HRB1), light color finishing (HRB1 and HRB2), and various types of shading. The majority (73.57%) of respondents' TSV results are within the central three categories, which indicates that these passive techniques are beneficial for indoor thermal comfort in hot areas.

Similar results have been found in previous research in recent decades. Lapisa et al., and Ozarisoy indicated that the main façade the buildings with large area windows should avoid exposure to direct solar radiation [35,36]. In this study, bedroom 3 in HRB2 is facing south, while the living room is exposed to the worst orientation (west) to solar radiation. This is one of the possible reasons why the indoor temperature in bedroom 3 is much lower than that in the living room. Window shading is an optimal way to compensate for the non-ideal orientation in retrofitting historical buildings. Lapisa et al. confirmed that slope roofs can improve the thermal comfort effect of buildings, while high-slope roofs increase costs and cause potential safety problems in earthquakes [35]. Therefore, the application of pitched roofs to historical buildings in developing countries such as Zanzibar in this study should take the cost, potential safety risk, and thermal comfort into consideration. Previous research found that solar radiation can be effectively reflected by using cool roofs and walls, thereby decreasing the heat gain in the interior [35,37]. The exterior walls of the two case study buildings in this research are painted in white or light yellow, which contributes to thermal comfort inside the buildings. This technique can be further implemented on roofs to improve thermal performance.  This technique is also implemented in the historical residential buildings in Stone Town, where a large number of openings and cross-ventilation can be found. However, large areas of openings will increase the potential energy demand for heating, so the decision making of the window areas should depend on the heating/cooling demand.

In addition, numerous studies have focused on the energy demand of residential buildings by using passive cooling strategies. Fernandez-Antolin et al. found that northsouth orientation and light color finishing contribute to low cooling energy demand for residential buildings in Spain. This is consistent with the thermal comfort evaluation results obtained in this research, which implemented the same strategies [40]. Pero et al. Proposed four main processes for passive building design, including site planning, building's shape and envelope, window design, and renewable energy sources [41]. These strategies are effective for new buildings. However, many are not applicable to the renovation of historical buildings, such as the orientation and building shape. In some cases, window openings and shadings are also restricted due to the local culture. Therefore, design strategies

for improving the thermal comfort of historical residential buildings are proposed in the following subsections.

#### *4.3. Design Strategies for Historical Residential Buildings*

Three factors should be considered for the improvement of thermal comfort in historical residential buildings: (1) improve the thermal comfort conditions; (2) respect the local culture and history; and (3) reduce energy consumption.

Sun radiation is the main source of heat gain inside buildings; therefore, one of the best means by which to improve the thermal comfort of buildings is to create a quality building envelope using construction materials that help reduce the sun's heat. For historical buildings, traditional building materials, such as lime plaster and lime wash, are suitable for solving the problem.

#### 4.3.1. Lime Plaster

Lime plaster is generally made by heating lime rock (rock made of calcium carbonate, such as limestone or marble). Pure lime is white, lighter in weight than the original rock, and reacts violently with water. The lime plaster has the benefits of protecting masonry against weathering conditions, improving the thermal and acoustical performance.

Historical buildings in Stone Town were built hundreds of years ago, and damage is commonly detected nowadays. Lime plaster can improve the appearance of walls by hiding the imperfections of rough work, giving it an attractive texture compatible with the local environment.

The climate in Stone Town is hot and humid. Excessive humidity may enter the masonry in different ways and forms, such as the penetration of rainwater, fog, rising damp, and condensation, thereby leading to damage to the building's structure. Thus, lime plaster has an important role in allowing the walls of the property to breathe by letting out excess humidity.

#### 4.3.2. Wash Lime

White and light colors are beneficial in reducing cooling energy [35,37]. Although the two case study buildings already have light-colored paint on the walls, as presented in Section 3.2.1, the indoor temperatures and thermal comfort are still under the requirements indicated by the ASHRAE; thus, wash lime is proposed. Wash lime is mostly applied to stone and brick walls and adobe. It is widely used due to its availability and low cost, making it a good choice for developing countries. In addition, wash lime can brighten up the exterior walls and form calcite crystals, which reflect the sun rays striking on the walls. A light color is preferred for wash lime, as indicated earlier.

#### **5. Conclusions**

A thermal comfort study of historical residential buildings in a tropical hot–humid climate was conducted in Zanzibar. A subjective survey was carried out among 159 occupants living in the historical site of Stone Town to investigate the passive cooling techniques used in historical residential buildings and the occupants' subjective evaluations of indoor environmental parameters. The objective indoor environmental parameters were collected by field measurements from two case study buildings over six continuous days to evaluate the thermal comfort in the historical buildings.

Even with the use of passive cooling technologies, such as the use of sun shading devices and more openings to create better ventilation, thermal comfort was still not acceptable, according to international ASHRAE Standard 55. In the questionnaire survey, 73.57% of the participants voted within the three central categories of the thermal sensation vote, and 68.55% of the occupants stated that they would prefer to be cooler. This indicates that their thermal environment is not acceptable. In the field survey, the indoor temperatures and relative humidity, as well as the PMV–PPD values, of the two case study buildings were found to be higher than the acceptable limits for thermal comfort zones, which indicates

the necessity of improving the thermal environment in these historical residential buildings that already take passive cooling into consideration.

Based on the analysis of passive cooling technologies (north-south orientation, natural ventilation, pitched roof, light color finishing, and window shading) and their positive and negative effects on the thermal environment in historical residential buildings, design strategies are proposed to improve the local thermal environment. The design strategies include the application of lime plaster and wash lime, which have the potential to reduce energy consumption, improve thermal comfort, and respect the local culture and history.

This research provides guidelines to assist architects in designing energy-efficient residential buildings while also taking into account cultural heritage and thermal comfort. The retrofitting of historical buildings should avoid the large destruction of historical buildings, construct as much as possible with local materials, and preserve coordination with the local environment. Further studies concerning thermal comfort should be performed in suburban areas over a longer period, or more historical residential buildings, in order to obtain a broader picture of thermal comfort phenomena in areas with different cultural and behavioral patterns.

**Author Contributions:** Conceptualization, H.X. and H.M.A.; methodology, H.X. and H.M.A.; writing—original draft preparation, C.L. and H.M.A.; writing—review and editing, C.L., H.X. and J.L.; supervision, H.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Science and Technology Research Program of Chongqing Municipal Education Commission, grant number KJQN201900113.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.


**3. Macrophytic algae**

#### **3.1 Macrophytic algae are well-adapted to coral reefs**

Macrophytic algae are major benthic contributors to the living intertidal and subtidal biomasses of most temperate and subpolar coastal regions. In contrast to kelp forests dominating nutrient-rich colder waters, the presence of macrophytic algae appears secondary to that of invertebrates in coral reefs that naturally develop in oligotrophic conditions. However the three macroalgal lineages (red, brown and green) have many representatives that are well adapted to the numerous communities found in all reef types and zones, as witnessed by their extremely varied growth forms. In addition, the rhodophyte (red) and the chlorophyte (green) algae have calcifying forms that actively contribute to substrate-forming calcification (by calcareous green algae) and to the cementation of loose aggregates (by encrusting coralline red algae). If stony corals are usually regarded as the framework architects, algae certainly play a role in the building and consolidation of reef assemblages, along with other biomineralizers such as sponges, tube worms and foraminifera.

#### **3.1.1 Calcareous chlorophytes and coralline rhodophytes are essential components of reef ecosystems**

Calcification, mostly in the form of calcium carbonate, has arisen independently in the three algal lineages (though to a much lesser extent in the brown algae), reflecting different growth strategies to those of non-calcifying forms. On coral reefs, heavily calcifying algae can adopt two forms: (i) geniculate, i.e. made up of calcified segments separated by flexible joints called genicula, and (ii) crustose , i.e. encrusting forms that lack genicula and grow as thin encrusting patches on hard substratum. The articulated chlorophyte *Halimeda* provides a major calcium carbonate contribution to the substratum of reef flats, i.e. the sand, and to sea grass beds which host many juvenile forms of fish and invertebrates. Crustose coralline red algae are arguably the most abundant organism (plant or animal) to occupy hard substrata within the world's marine photic zone. Unattached ball-like rhodoliths and maerl are responsible for reef formations called algal ridges in tropical wave-exposed environments (Steneck & Martone, 2007), while encrusting forms are responsible for the cementation of rubble and mineral debris into larger structures onto which other benthos can attach. Coralline algae provide food to sea urchins, parrot fish, limpets and chitons, and together with scleractinian corals and sponges they provide the framework for the development of complex communities of invertebrates. Furthermore, crustose coralline algae (CCA) are known to play a crucial role in the settlement and metamorphosis of larvae of sea urchins (Huggett et al., 2006), starfish (Johnson & Sutton, 1994), mollusks (Williams et al., 2008), corals (Negri et al., 2001; Webster et al., 2004), and possibly

biota. When mangroves are removed, the nutrient cycling efficiency needed to maintain biodiversity along a seaward gradient is overproductive for heterotrophic microorganisms and underproductive for eukaryotic consumers. Biodiversity loss in coastal waters is the result of ever-increasing activity of sea farming, fishing and tourism in tropical countries. Poor household waste management and the use of fertilizers cause oxygen depletion of seawater with suffocation of reef invertebrates and fish. In the longer term, nutrient enrichment, by favoring algal growth, will cause a strong imbalance between trophic

Macrophytic algae are major benthic contributors to the living intertidal and subtidal biomasses of most temperate and subpolar coastal regions. In contrast to kelp forests dominating nutrient-rich colder waters, the presence of macrophytic algae appears secondary to that of invertebrates in coral reefs that naturally develop in oligotrophic conditions. However the three macroalgal lineages (red, brown and green) have many representatives that are well adapted to the numerous communities found in all reef types and zones, as witnessed by their extremely varied growth forms. In addition, the rhodophyte (red) and the chlorophyte (green) algae have calcifying forms that actively contribute to substrate-forming calcification (by calcareous green algae) and to the cementation of loose aggregates (by encrusting coralline red algae). If stony corals are usually regarded as the framework architects, algae certainly play a role in the building and consolidation of reef assemblages, along with other biomineralizers such as sponges, tube

**3.1.1 Calcareous chlorophytes and coralline rhodophytes are essential components**

Calcification, mostly in the form of calcium carbonate, has arisen independently in the three algal lineages (though to a much lesser extent in the brown algae), reflecting different growth strategies to those of non-calcifying forms. On coral reefs, heavily calcifying algae can adopt two forms: (i) geniculate, i.e. made up of calcified segments separated by flexible joints called genicula, and (ii) crustose , i.e. encrusting forms that lack genicula and grow as thin encrusting patches on hard substratum. The articulated chlorophyte *Halimeda* provides a major calcium carbonate contribution to the substratum of reef flats, i.e. the sand, and to sea grass beds which host many juvenile forms of fish and invertebrates. Crustose coralline red algae are arguably the most abundant organism (plant or animal) to occupy hard substrata within the world's marine photic zone. Unattached ball-like rhodoliths and maerl are responsible for reef formations called algal ridges in tropical wave-exposed environments (Steneck & Martone, 2007), while encrusting forms are responsible for the cementation of rubble and mineral debris into larger structures onto which other benthos can attach. Coralline algae provide food to sea urchins, parrot fish, limpets and chitons, and together with scleractinian corals and sponges they provide the framework for the development of complex communities of invertebrates. Furthermore, crustose coralline algae (CCA) are known to play a crucial role in the settlement and metamorphosis of larvae of sea urchins (Huggett et al., 2006), starfish (Johnson & Sutton, 1994), mollusks (Williams et al., 2008), corals (Negri et al., 2001; Webster et al., 2004), and possibly

compartments of coral reefs, to the detriment of coral survival.

**3.1 Macrophytic algae are well-adapted to coral reefs**

**3. Macrophytic algae**

worms and foraminifera.

**of reef ecosystems**

sponges (Carballo & Avilla, 2004). Several CCA metabolites have been identified as potential inducers, e.g. 11-deoxyfistularin-3, a bromotyrosine derivative which stimulates settlement of coral planulae in the presence of algal carotenoids (Kitamura et al., 2007), aminovaleric acid and other salts which induce competent abalone larvae (Stewart et al., 2008), and dibromomethane which induces sea-urchins and the invasive slipper limpet *Crepidula fornicata* (Taris et al., 2010). Bacterial consortia which form specific biofilm-like CCA-associated assemblages, and/or their products have also been shown to act as inducers (Johnson et al., 1991) in the settlement and metamorphosis of larvae (reviewed by Hadfield, 2011). Coralline algae do not grow in sediment-rich coastal waters. Organisms with larvae that specialize in settling on CCA are naturally excluded from zones that are unsuitable for the growth of their host (Fabricius & De'ath 2001). This is a possible explanation for the lower species abundance and community composition of CCAs in coastal environments receiving sediments from rivers or from heavy rainfall washing unstabilized top soil (e.g. mining sites), in addition to insufficient bioavailability of calcium for calcification. Increasing metabolic cost associated with global climate change may offset the advantages of calcification, and recent increases in disease may indicate that oceans are becoming a more stressful environment for calcified algae. Dessication, temperature and light are capable of inducing 50% pigment loss within 24 minutes of emersion in the model species *Calliarthron tuberculosum*, and predictionoriented models based on the combined effects of these parameters are being developed to evaluate the effects of progressive climatic changes (Martone et al., 2010). Taking *Neogoniolithon fosliei* (a primary reef-builder) as a case study, Webster et al. (2010a) demonstrated a strong correlation between elevated sea water temperature (32°C) and (i) de-pigmentation, (ii) a large shift in the structure of microbial communities associated with CCAs, (iii) development of chlorophytic endophytes, and (iv) a dramatic decrease in the ability to induce metamorphosis of coral planulae, i.e. loss of bacterial surface flora. Physiological experiments have recently shown that temperature stress induced bleaching of the coralline alga *Corallina officinalis* was the result of pigment loss following an oxidative burst, i.e. an increase in production of H2O2 and other reactive oxygen species and decrease in quenching capacity of the haloperoxidase system (Latham, 2008).

#### **3.2 The phycosphere and its associated microbiome**

#### **3.2.1 Algae are phylogenetically ancient and have coevolved in association with microbes**

Macrophytic algae belong to three distinct lineages that diverged early in the history of eukaryotic evolution. Red and green algae (including plants) arose from a common ancestor (Keeling, 2010) through primary endosymbiosis, i.e. engulfing of a cyanobacterium by an aerobic eukaryote, the former becoming the original plastid. Fossils as old as 1 to 1.2 billion years (mid-Proterozoic) indicate the existence of filamentous forms of both red and green algae at the root of present day plants (Javaux et al., 2004). Brown algae (stramenopiles) evolved much later as a consequence of secondary endosymbiosis (engulfing of a unicellular red alga by an aerobic eukaryote). Analysis of the genome of the filamentous brown alga *Ectocarpus siliculosus* (Cock et al, 2010) has provided evidence of the independent evolution of multicellularity within the stramenopile lineage which also includes diatoms. All three lineages have since developed into thousands of different forms with sizes ranging from microscopic (e.g. endophytes) to gigantic (e.g. brown kelp *Macrocystis*) and adaptation to most known aquatic ecosystems. Their capacity to survive eons of biotic and abiotic stresses

Coral Reef Biodiversity in the Face of Climatic Changes 83

and green algae. Compounds with have antibacterial activities are now inspiring ecological alternatives to classical antifouling paints and coatings based on genotoxic heavy metal

Certain parasitic endophytes (fungi and minute filamentous algae) are capable of penetrating the outer cuticle of red and brown algae and causing modifications of the growth of the host (e.g. Gauna et al., 2009). Some bacteria have the enzymatic machinery capable of breaking down cell wall polymers into absorbable energy sources, as well as being resistant to the defense compounds elicited following their intrusion. Their biotechnological potential for the production of low MW bioactive components from bulk tissue is being investigated (e.g. Kim et al., 2009; Colin et al., 2006). Bacterial epidemics are occasionally reported, leading to the complete destruction of local natural populations (e.g. *Laminaria hyperborea* in western Brittany), or of seaweed farms of brown (e.g. *Laminaria japonica*, Wang et al., 2008), or of tropical red algae (e.g. carrageenophytes, Largo et al., 1999).

**3.2.4 Bacteria are also essential to the development, fitness and defense of algae**  The life cycle of Ulvales (Chlorophyta) is clearly dependent upon the presence or the association of adequate bacterial strains. Production by a bacterium of thallusin, a Ncontaining carboxylated diterpene, induces the morphogenesis of foliose thalli of the green ulvophyte *Monostroma oxyspermum* from filamentous cell aggregates (Nishizawa et al., 2007), and more generally from *Enteromorpha*-like filamentous forms into *Ulva*-like foliose forms (Matsuo et al., 2005). Swimming *Ulva* tetrazoospores are strongly influenced by specific quorum-sensing signals liberated from bacterial biofilms (review by Joint et al., 2007) resulting in settlement and metamorphosis in their vicinity. *Ulva australis* favors colonization of its surfaces by *Roseobacter gallaeciensis* and by *Pseudoalteromonas tunicata* which coexist as segregated microcolonies, and modulate the settlement of other competing strains essentially by (i) production of the antibacterial protein AIpP by *P. tunicata* and (ii) biofilm invasion and dispersion by *R. gallaeciensis* (Rao et al., 2006).  The *Roseobacter* clade is now regarded as an essential functional component of the microbiome of phytoplankton (both intra- and extracellularly), assisting in both primary and secondary roles (Geng & Belas, 2010), as well as seemingly in a number of macrophyte associations of symbiotic nature. Brown algae shelter bacterial communities that may vary according to their location on the thallus, partly due to differences in surface chemistries (q.v. review by Goecke et al., 2010), and to the mode of colonization, i.e. by planktonic bacteria on distal surfaces and by surface contamination of the holdfast by epibionts and complex microbial biofilms. Among the numerous and unidentified non-cultivatable strains,

it can be assumed that some of have neutral or beneficial interactions with their host.

Lam & Harder (2007) have described how macroalgae can selectively control their immediate surroundings by diffusing waterborne chemicals that prevent settlement by potentially fouling microbiota and epibiota. The emission of cocktails of volatile halogenated C1-C3 compounds has been extensively reported in red (e.g. Kladi et al., 2004) and in brown (e.g. La Barre et al., 2010) algae, with impacts beyond the proximal underwater effects on atmospheric chemistry. Green algae are responsible for massive

**3.3.1 Thalli control their immediate hydrosphere…and the atmosphere**

**3.3 Algae and their chemical language**

formulations.

and their adaptability to colonize extreme environments has emerged through finely tuned interactions and co-evolution with microbial organisms (La Barre & Haras, 2007).

#### **3.2.2 Macrophytic algae control surface microbial colonization and biofilm formation**

A recent paper by Goecke et al. (2010) reviews the various ways in which extant marine macroalgae and bacteria interact positively and negatively via chemical mediators. Adult macroalgae are strongly susceptible to surface colonization, as they provide potential surfaces for settlement of epibionts. As well as increasing the weight of the thallus and making it mechanically fragile, epibiosis significantly reduces the surface area available for photosynthesis. On inert substrata, macrofouling is generally facilitated by the presence of bacterial biofilms, as demonstrated by numerous studies (Fusetani, 2011). Marine algae produce photosynthates that serve as prime carbon sources for bacteria, as witnessed by the surprisingly large microbial biodiversity found on their apparently "clean" surfaces, but the surfactant nature of these carbohydrates strongly discourages adhesion. Biofilm formation is also discouraged by molecules that (1) interfere with bacterial quorum-sensing activation, e.g. hypobromous acid in the brown kelp *Laminaria digitata* (Borchardt et al., 2001) or halogenated furanones produced by the rhodophyte *Delisea pulchra*, and (2) act as decoy analogs of bacterial acyl-homoserine lactones which induce quorum sensing (Manefield et al., 2002). Surface compounds produced by the phaeophyte *Fucus vesiculosus* are capable of modulating both epibiotic development and bacterial biofilm production (Lachnit et al., 2010). Flow-cell experiments (this author, unpubl. results) have shown that the thickness and 3-D architecture of monospecific biofilms of model bacteria isolated from inert substrata and from *Laminaria digitata* blades are differentially affected by exposure to exudates and surface compounds extracted from this kelp. Antifouling programs are now integrating biofilm denaturation studies into their strategies, an ecologically sound alternative to the use of toxic ingredients.

#### **3.2.3 Microbial pathogenic infection results in coordinated immune responses from infected algae**

Bacteria interact with algae mostly in a non-invasive manner until older tissues can no longer resist saprophytic degradation by bacteria or bacterial consortia that have the appropriate lytic enzymes. However, fortuitous bacterial intrusion following mechanical damage or exposure to specific signal substances (elicitors) may trigger an oxidative burst followed by intracellular responses comparable to classical inflammation, as part of an innate immune mechanism (Weinberger, 2007). In brown algae, microbial infection is strongly inhibited by the emission of halogenated compounds that may have bactericidal or bacteriostatic activities in the immediate hydrosphere of the thallus, due to an efficient apoplastic enzymatic machinery (Butler & Carter-Franklin, 2004) that can be activated in a non-systemic fashion. Indeed, red and brown algae are reported to use both animal‐like (eicosanoid) and higher plant‐like (octadecanoid) oxylipins in the regulation of defense metabolism for protection against pathogens and grazers or in response to elicitors of defense responses (Cosse et al., 2009). Tetracyclic brominated diterpenes active against multi-resistant strains of the nosocomial bacteria *Staphylococcus aureus* have recently been isolated from the red alga *Sphaeorococcus* (Smyrniotopoulos et al 2010), suggesting efficient antibiotic responses against potentially infective strains. Kornprobst (2010a) provides a comprehensive review of bioactive metabolites of various chemical classes from red, brown

and their adaptability to colonize extreme environments has emerged through finely tuned

**3.2.2 Macrophytic algae control surface microbial colonization and biofilm formation**  A recent paper by Goecke et al. (2010) reviews the various ways in which extant marine macroalgae and bacteria interact positively and negatively via chemical mediators. Adult macroalgae are strongly susceptible to surface colonization, as they provide potential surfaces for settlement of epibionts. As well as increasing the weight of the thallus and making it mechanically fragile, epibiosis significantly reduces the surface area available for photosynthesis. On inert substrata, macrofouling is generally facilitated by the presence of bacterial biofilms, as demonstrated by numerous studies (Fusetani, 2011). Marine algae produce photosynthates that serve as prime carbon sources for bacteria, as witnessed by the surprisingly large microbial biodiversity found on their apparently "clean" surfaces, but the surfactant nature of these carbohydrates strongly discourages adhesion. Biofilm formation is also discouraged by molecules that (1) interfere with bacterial quorum-sensing activation, e.g. hypobromous acid in the brown kelp *Laminaria digitata* (Borchardt et al., 2001) or halogenated furanones produced by the rhodophyte *Delisea pulchra*, and (2) act as decoy analogs of bacterial acyl-homoserine lactones which induce quorum sensing (Manefield et al., 2002). Surface compounds produced by the phaeophyte *Fucus vesiculosus* are capable of modulating both epibiotic development and bacterial biofilm production (Lachnit et al., 2010). Flow-cell experiments (this author, unpubl. results) have shown that the thickness and 3-D architecture of monospecific biofilms of model bacteria isolated from inert substrata and from *Laminaria digitata* blades are differentially affected by exposure to exudates and surface compounds extracted from this kelp. Antifouling programs are now integrating biofilm denaturation studies into their strategies, an ecologically sound alternative to the use

**3.2.3 Microbial pathogenic infection results in coordinated immune responses from**

Bacteria interact with algae mostly in a non-invasive manner until older tissues can no longer resist saprophytic degradation by bacteria or bacterial consortia that have the appropriate lytic enzymes. However, fortuitous bacterial intrusion following mechanical damage or exposure to specific signal substances (elicitors) may trigger an oxidative burst followed by intracellular responses comparable to classical inflammation, as part of an innate immune mechanism (Weinberger, 2007). In brown algae, microbial infection is strongly inhibited by the emission of halogenated compounds that may have bactericidal or bacteriostatic activities in the immediate hydrosphere of the thallus, due to an efficient apoplastic enzymatic machinery (Butler & Carter-Franklin, 2004) that can be activated in a non-systemic fashion. Indeed, red and brown algae are reported to use both animal‐like (eicosanoid) and higher plant‐like (octadecanoid) oxylipins in the regulation of defense metabolism for protection against pathogens and grazers or in response to elicitors of defense responses (Cosse et al., 2009). Tetracyclic brominated diterpenes active against multi-resistant strains of the nosocomial bacteria *Staphylococcus aureus* have recently been isolated from the red alga *Sphaeorococcus* (Smyrniotopoulos et al 2010), suggesting efficient antibiotic responses against potentially infective strains. Kornprobst (2010a) provides a comprehensive review of bioactive metabolites of various chemical classes from red, brown

interactions and co-evolution with microbial organisms (La Barre & Haras, 2007).

of toxic ingredients.

**infected algae**

and green algae. Compounds with have antibacterial activities are now inspiring ecological alternatives to classical antifouling paints and coatings based on genotoxic heavy metal formulations.

Certain parasitic endophytes (fungi and minute filamentous algae) are capable of penetrating the outer cuticle of red and brown algae and causing modifications of the growth of the host (e.g. Gauna et al., 2009). Some bacteria have the enzymatic machinery capable of breaking down cell wall polymers into absorbable energy sources, as well as being resistant to the defense compounds elicited following their intrusion. Their biotechnological potential for the production of low MW bioactive components from bulk tissue is being investigated (e.g. Kim et al., 2009; Colin et al., 2006). Bacterial epidemics are occasionally reported, leading to the complete destruction of local natural populations (e.g. *Laminaria hyperborea* in western Brittany), or of seaweed farms of brown (e.g. *Laminaria japonica*, Wang et al., 2008), or of tropical red algae (e.g. carrageenophytes, Largo et al., 1999).

#### **3.2.4 Bacteria are also essential to the development, fitness and defense of algae**

The life cycle of Ulvales (Chlorophyta) is clearly dependent upon the presence or the association of adequate bacterial strains. Production by a bacterium of thallusin, a Ncontaining carboxylated diterpene, induces the morphogenesis of foliose thalli of the green ulvophyte *Monostroma oxyspermum* from filamentous cell aggregates (Nishizawa et al., 2007), and more generally from *Enteromorpha*-like filamentous forms into *Ulva*-like foliose forms (Matsuo et al., 2005). Swimming *Ulva* tetrazoospores are strongly influenced by specific quorum-sensing signals liberated from bacterial biofilms (review by Joint et al., 2007) resulting in settlement and metamorphosis in their vicinity. *Ulva australis* favors colonization of its surfaces by *Roseobacter gallaeciensis* and by *Pseudoalteromonas tunicata* which coexist as segregated microcolonies, and modulate the settlement of other competing strains essentially by (i) production of the antibacterial protein AIpP by *P. tunicata* and (ii) biofilm invasion and dispersion by *R. gallaeciensis* (Rao et al., 2006).  The *Roseobacter* clade is now regarded as an essential functional component of the microbiome of phytoplankton (both intra- and extracellularly), assisting in both primary and secondary roles (Geng & Belas, 2010), as well as seemingly in a number of macrophyte associations of symbiotic nature. Brown algae shelter bacterial communities that may vary according to their location on the thallus, partly due to differences in surface chemistries (q.v. review by Goecke et al., 2010), and to the mode of colonization, i.e. by planktonic bacteria on distal surfaces and by surface contamination of the holdfast by epibionts and complex microbial biofilms. Among the numerous and unidentified non-cultivatable strains, it can be assumed that some of have neutral or beneficial interactions with their host.

#### **3.3 Algae and their chemical language**

#### **3.3.1 Thalli control their immediate hydrosphere…and the atmosphere**

Lam & Harder (2007) have described how macroalgae can selectively control their immediate surroundings by diffusing waterborne chemicals that prevent settlement by potentially fouling microbiota and epibiota. The emission of cocktails of volatile halogenated C1-C3 compounds has been extensively reported in red (e.g. Kladi et al., 2004) and in brown (e.g. La Barre et al., 2010) algae, with impacts beyond the proximal underwater effects on atmospheric chemistry. Green algae are responsible for massive

Coral Reef Biodiversity in the Face of Climatic Changes 85

Overfishing, besides causing mechanical damage to coral biota (wading, use of explosives and use of dragnets) modulates the predation pressure on benthic algae in many reefs worldwide, with consequences for corals and their associated biodiversity. Near extinction of reef sharks and other carnivorous fish and of grazing herbivores has a dramatically positive incidence on algal biomass and average size in overexploited reefs worldwide (Hay, 1997 , Sotka & Hay, 2009). The resulting increase in the production of photosynthates feeds a bacterial population that is potentially pathogenic to corals. Disrupting the coral–microbe relationship by organic carbon loading (dissolved organic carbon (DOC), i.e. mainly carbohydrates) can directly cause coral mortality by over-stimulating growth of coral mucus-associated microbes (Kuntz et al., 2005). An analysis of the gradual decline of Jamaican coral reefs by the marine microbiologist Forest Rohwer (2010) led him to define this self-feeding loop as the DDAM model (DOC>disease>algae>microbes), which can only be broken by top down herbivory that reduces the algal biomass to levels compatible with the development of a functional scleractinian microbiome. Future management policies should ensure that key algae consumers be identified and protected in reef areas susceptible

**3.4.2 Overfishing is linked to algal and microbial development and to coral decline**  The Philippines and Indonesia include the western half of the Coral Triangle of marine biodiversity, which extends eastwards to New Guinea and the Solomon islands. One quarter or more of the human populations of these islands live in coastal areas and derive their

revenues from coral reef production, on a non-sustainable basis.

to recurrent blooming of coral damaging species (Rasher & Hay, 2010b).

**3.5 Exotic algae may adapt to new environments as climate changes**

may be an aggravate bacterial infections.

**3.5.1 Invasive species**

**3.4.3 Farming of macroalgae in tropical regions also suffers from climatic changes**  Farming of macrophytic algae for the food and the pharmacological industries provides an alternative activity to fishing and tourism for entire communities in tropical islands (Indonesia, Philippines, Madagascar and the Caribbean islands). However, algal monoculture can be risky. The carrageenophytes *Eucheuma* and *Kappaphycus*, that have the capacity to adjust to hyper and hypo salinity changes encountered in shallow tropical waters (q.v. Teo et al., 2009), are prone to bacterial plague disease ("ice-ice"). Such epidemics have caused occasional eradication of entire populations (Largo et al., 1999), with total loss of the livelihood of farmers. Seasonal infestations by filamentous endophytes of Malaysian *Kappaphycus/Eucheuma* farms which are attributed to seasonal changes (Vairappan, 2006)

The large-scale introduction of non-indigenous species and homogenization of the world's biota has long been considered among the greatest threats to species diversity (Carlton & Geller, 1993). Before global warming became the central issue in the coral reef biodiversity literature, the necessity for proper management of reef resources had already been highlighted (Maragos et al., 1996), including the issue of species of commercial interest imported into developing countries due to the low cost of local labor. Introductions may result in competition with native biota, eventually affecting human livelihood. Whether deliberately imported (introduced) or accidentally established (invasive), alien species, like endangered species, are now identified on periodically upgraded lists. For example, the International Coral Reef Initiative (ICRI) devotes a whole section of its website (http://www.icriforum.org/) to this topic. Alien seaweed, fish, mollusks and even corals

releases of dimethylsulfoproprionate (DMSP) and diffusion of their breakdown products (acrylate and dimethylsulfide) into the atmosphere, which may create conditions favorable for the dispersal of their gametes (Welsh et al., 1999). This is reminiscent of cloud-forming emissions of iodinated compounds above kelp beds (Ball et al., 2010). Thus benthic algae and phytoplankton actively participate in the cycling of iodine, bromine, chlorine and sulfur at the water-air interface, while responding to requirements at both cellular and population levels. Biomass breakdown following massive blooms of DMSP algae and Prymnesophytes may cause severe anoxia and hydrogen sulphide intoxications to local fauna and seashell farming, pointing out the necessity for proper control of nutrient enrichment of coastal waters.

#### **3.3.2 Algal metabolites alter the fitness and growth of their benthic neighbors**

In addition to volatile compounds, macroalgae owe their competitiveness to the production of whole arrays of metabolites that are bioactive (i) by contact interactions with adjacent alien tissues, using lipid-soluble compounds (Rasher & Hay, 2010a), (ii) by diffusing waterborne chemicals, or (iii) by altering the functional microbial flora of their invertebrate neighbors thereby encouraging the development of pathogenic strains. Damage to the scleractinian cover on the outer reef slopes in various localities of the Central and South Western Pacific by blooms of *Asparagopsis taxiformis* may be the result of one or more of these modes of action. Toxic volatile or diffusible halogenated compounds, like haloforms, methanes, ketones, acetates and acrylates, were described for *A. taxiformis* and its sibling species *A. armata (*Mc Connell & Fenical, 1977; Woolard et al., 1979; Kladi et al., 2004). Thick mats can overrun live scleractinian colonies and diffuse a range of volatile halocarbons that are considered toxic in addition to having various antimicrobial activities (Genovese et al., 2009). Hypoxia and tissue disruption of polyps at coral-algal tuft and coral-macroalgae interfaces led Barott et al. (2009) to the conclusion that erect (i.e. non crustose) algae were a constant cause of stress to adjacent coral colonies in contact or close vicinity in pairwise experimental associations.

#### **3.4 Algae as a crucial ecological link between corals and microbes 3.4.1 Control of biomass of algae by grazers is essential to coral reef diversity**

Algae are regarded as superior space competitors on hard substrata. However, predation is an important pressure on non-calcifying algae (Hay, 1997). The epilithic algal community is grazed by herbivorous fish, echinoderms (sea urchins), mollusks, crustaceans and worms, themselves serving as food to carnivores in a bottom-up succession of predators. Thus, small turf-like species, sporelings of macrophytes, and the unicellular forms that are associated with surface slime on sand and rubble, e.g. protein-generating cyanobacteria, represent an essential primary trophic component of the reef, generating more than half of the edible biomass of the whole food chain (Hay, 1997). A number of larger algae produce chemicals (terpenes, polyphenols and halogenated compounds) that have an inhibitory effect on grazers (see recent review by Paul et al., 2011). Fish being the largest consumers of algae and being selective in their food source, the biodiversity of seaweeds on the reef is a reflection of both the diversity of grazing modes (Burkepile & Hay, 2010) and of the chemodiversity of the defence compounds they produce. To complete the picture, aggressive fish such as the common Pomacentrid damselfish tend to fend intruders off their territory, including foraging herbivores, thus promoting spatial and taxonomic diversity in the distribution of reef algae (Brawley & Adey, 1977).

releases of dimethylsulfoproprionate (DMSP) and diffusion of their breakdown products (acrylate and dimethylsulfide) into the atmosphere, which may create conditions favorable for the dispersal of their gametes (Welsh et al., 1999). This is reminiscent of cloud-forming emissions of iodinated compounds above kelp beds (Ball et al., 2010). Thus benthic algae and phytoplankton actively participate in the cycling of iodine, bromine, chlorine and sulfur at the water-air interface, while responding to requirements at both cellular and population levels. Biomass breakdown following massive blooms of DMSP algae and Prymnesophytes may cause severe anoxia and hydrogen sulphide intoxications to local fauna and seashell farming, pointing out the necessity for proper control of nutrient enrichment of coastal

**3.3.2 Algal metabolites alter the fitness and growth of their benthic neighbors**

**3.4 Algae as a crucial ecological link between corals and microbes**

**3.4.1 Control of biomass of algae by grazers is essential to coral reef diversity**

Algae are regarded as superior space competitors on hard substrata. However, predation is an important pressure on non-calcifying algae (Hay, 1997). The epilithic algal community is grazed by herbivorous fish, echinoderms (sea urchins), mollusks, crustaceans and worms, themselves serving as food to carnivores in a bottom-up succession of predators. Thus, small turf-like species, sporelings of macrophytes, and the unicellular forms that are associated with surface slime on sand and rubble, e.g. protein-generating cyanobacteria, represent an essential primary trophic component of the reef, generating more than half of the edible biomass of the whole food chain (Hay, 1997). A number of larger algae produce chemicals (terpenes, polyphenols and halogenated compounds) that have an inhibitory effect on grazers (see recent review by Paul et al., 2011). Fish being the largest consumers of algae and being selective in their food source, the biodiversity of seaweeds on the reef is a reflection of both the diversity of grazing modes (Burkepile & Hay, 2010) and of the chemodiversity of the defence compounds they produce. To complete the picture, aggressive fish such as the common Pomacentrid damselfish tend to fend intruders off their territory, including foraging herbivores, thus promoting spatial and taxonomic diversity in the distribution of

In addition to volatile compounds, macroalgae owe their competitiveness to the production of whole arrays of metabolites that are bioactive (i) by contact interactions with adjacent alien tissues, using lipid-soluble compounds (Rasher & Hay, 2010a), (ii) by diffusing waterborne chemicals, or (iii) by altering the functional microbial flora of their invertebrate neighbors thereby encouraging the development of pathogenic strains. Damage to the scleractinian cover on the outer reef slopes in various localities of the Central and South Western Pacific by blooms of *Asparagopsis taxiformis* may be the result of one or more of these modes of action. Toxic volatile or diffusible halogenated compounds, like haloforms, methanes, ketones, acetates and acrylates, were described for *A. taxiformis* and its sibling species *A. armata (*Mc Connell & Fenical, 1977; Woolard et al., 1979; Kladi et al., 2004). Thick mats can overrun live scleractinian colonies and diffuse a range of volatile halocarbons that are considered toxic in addition to having various antimicrobial activities (Genovese et al., 2009). Hypoxia and tissue disruption of polyps at coral-algal tuft and coral-macroalgae interfaces led Barott et al. (2009) to the conclusion that erect (i.e. non crustose) algae were a constant cause of stress to adjacent coral colonies in contact or close vicinity in pairwise

waters.

experimental associations.

reef algae (Brawley & Adey, 1977).

#### **3.4.2 Overfishing is linked to algal and microbial development and to coral decline**

The Philippines and Indonesia include the western half of the Coral Triangle of marine biodiversity, which extends eastwards to New Guinea and the Solomon islands. One quarter or more of the human populations of these islands live in coastal areas and derive their revenues from coral reef production, on a non-sustainable basis.

Overfishing, besides causing mechanical damage to coral biota (wading, use of explosives and use of dragnets) modulates the predation pressure on benthic algae in many reefs worldwide, with consequences for corals and their associated biodiversity. Near extinction of reef sharks and other carnivorous fish and of grazing herbivores has a dramatically positive incidence on algal biomass and average size in overexploited reefs worldwide (Hay, 1997 , Sotka & Hay, 2009). The resulting increase in the production of photosynthates feeds a bacterial population that is potentially pathogenic to corals. Disrupting the coral–microbe relationship by organic carbon loading (dissolved organic carbon (DOC), i.e. mainly carbohydrates) can directly cause coral mortality by over-stimulating growth of coral mucus-associated microbes (Kuntz et al., 2005). An analysis of the gradual decline of Jamaican coral reefs by the marine microbiologist Forest Rohwer (2010) led him to define this self-feeding loop as the DDAM model (DOC>disease>algae>microbes), which can only be broken by top down herbivory that reduces the algal biomass to levels compatible with the development of a functional scleractinian microbiome. Future management policies should ensure that key algae consumers be identified and protected in reef areas susceptible to recurrent blooming of coral damaging species (Rasher & Hay, 2010b).

#### **3.4.3 Farming of macroalgae in tropical regions also suffers from climatic changes**

Farming of macrophytic algae for the food and the pharmacological industries provides an alternative activity to fishing and tourism for entire communities in tropical islands (Indonesia, Philippines, Madagascar and the Caribbean islands). However, algal monoculture can be risky. The carrageenophytes *Eucheuma* and *Kappaphycus*, that have the capacity to adjust to hyper and hypo salinity changes encountered in shallow tropical waters (q.v. Teo et al., 2009), are prone to bacterial plague disease ("ice-ice"). Such epidemics have caused occasional eradication of entire populations (Largo et al., 1999), with total loss of the livelihood of farmers. Seasonal infestations by filamentous endophytes of Malaysian *Kappaphycus/Eucheuma* farms which are attributed to seasonal changes (Vairappan, 2006) may be an aggravate bacterial infections.

#### **3.5 Exotic algae may adapt to new environments as climate changes 3.5.1 Invasive species**

The large-scale introduction of non-indigenous species and homogenization of the world's biota has long been considered among the greatest threats to species diversity (Carlton & Geller, 1993). Before global warming became the central issue in the coral reef biodiversity literature, the necessity for proper management of reef resources had already been highlighted (Maragos et al., 1996), including the issue of species of commercial interest imported into developing countries due to the low cost of local labor. Introductions may result in competition with native biota, eventually affecting human livelihood. Whether deliberately imported (introduced) or accidentally established (invasive), alien species, like endangered species, are now identified on periodically upgraded lists. For example, the International Coral Reef Initiative (ICRI) devotes a whole section of its website (http://www.icriforum.org/) to this topic. Alien seaweed, fish, mollusks and even corals

Coral Reef Biodiversity in the Face of Climatic Changes 87

summer. Though considered efficient at absorbing excess nutrient enrichment, if not collected they can themselves become a source of chemical pollution (hydrogen sulfide in particular) through massive bacterial degradation of the decaying biomass. In tropical environments, developing economies associated with fishing and farming of marine resources generate massive effluxes of untreated urban sewerage leading to localized blooming of chlorophytic algae. In the vicinity of reefs, bacterial enrichment by resident algae may be detrimental to coral polyps by promoting the growth of pathogenic strains at

In association with spongin (proteinaceous) fibres, marine sponges typically biomineralize non-aragonitic calcium and magnesium carbonates (subphylum Calcispongia) or silica (subphylum Silicispongia). One of the most ancient eumetazoan lineages, sponges have adopted different modes of biomineralization and reef-forming capacities through geological time, as seawater chemistry and the bioavailability of dissolved salt species has changed periodically due to tectonic events (Stanley & Hardie, 1998). Most reef-forming sponges have disappeared since the Phanerozoic, and present-day sponges are mostly siliceous Demosponges which are ubiquitous worldwide in their distribution (see Hooper &

The position of sponges at the root of metazoan evolution is still a hotly debated topic (see eg. Maldonaldo, 2004). Choanoflagellates, with which sponge choanocytes and metazoan lineages share genomic similarities (King et al., 2008), have emerged as model organisms for studies of early metazoan evolution. For example, the choanoflagellate genome carries the markers of three types of molecules that cells use to achieve phospho-tyrosine signaling proteins, involved in important processes (cell-cell communication, immune system responses, hormonal stimulation, etc.) in metazoans. These molecules are tyrosine kinases (TyrK), protein tyrosine phosphatases (PTP) and Src Homolgy 2 (SH2) molecules that operate as a tandem system to achieve signal recognition (Manning et al., 2008). Recent studies have also demonstrated the role of associated bacteria in the colony-forming behavior of otherwise freeliving choanoflagellates (e.g. a glycosphingolipid produced by the bacterium *Algoriphagus* that affects the choanoflagellate *Salpingoeca rosetta* (Alegado et al., 2010). Other studies mention the role bacteria may have had in the transfer of genes between unicellular eukaryotes. Nedelcu et al. (2008) provide an example of bacterially mediated lateral transfer of four stress-related genes of algal origin to a choanoflagellate host, supposedly providing the recipient cell the

Going one evolutionary step further, Srivstava et al. (2010) have shown that the genome of the demosponge *Amphimedon queenslandica* contains the set of genes that correlates with critical aspects of more evolved metazoans (body plan, cell cycle control and growth, development somatic and germ-cell differentiation, cell adhesion innate immunity and allorecognition). Adaptive responses to environmental changes are known to be more rapid in microbial symbionts than in sponge cells, and bacteria may act as environmental sentinels to marine holobionts as they are particularly sensitive to environmental stressors such as heavy metal pollution (Webster et al., 2001), elevated seawater temperature, sedimentation

the expense of the regular associated bacterial microflora (Rohwer, 2010).

**4. Sponges**

Van Soest, 2002).

**4.1 Sponges and reef structuration**

**4.2 Why are sponges so unique?**

capacity to adapt to stress under environmental changes.

are listed as invasive species competing with native species for resources and being potential causes of new diseases of resident scleractinian corals.

The primary cause of invasion of new territories by alien algae is not always easy to determine, and a combination of factors usually contribute to the success of their expansion in new territory. Regular de-ballasting of huge amounts of seawater by container ships is often held responsible for the spread of exotic algae and microalgae along major maritime trade routes, eventually leading to endemic settlement points in regions where acclimation is possible (Carlton, 1996). On the other hand, massive invasions along the Mediterranean shores of the toxic Australian green alga *Caulerpa taxifolia* (and later of *Caulerpa racemosa*) are examples of accidental contaminations starting with a few individuals (Klein & Verlaque, 2008). The displacement by these two *Caulerpa* species of resident halophyte meadows (e.g. *Posidonia*) which are home to juveniles of a number of important demersal fish is considered a threat to local biodiversity. Rhodophytes (red algae) include a number of "cosmopolitan" species that have become established in tropical environments. An example is the territorial expansion of *Asparagopsis taxiformis* in the South Pacific that is currently considered a serious threat to corals in New Caledonia and in French Polynesia, like its sibling species *A. armata* in other parts of the world.

#### **3.5.2 Cataclysmic events may favor supremacy of algal communities**

In tropical seas, hurricanes are becoming more frequent and more severe with global warming, resulting in near-total destruction of the coral cover in the most exposed localities, with negative impacts on larval recruitment (Crabbe et al., 2008) and hence on biodiversity. Initially, only the most resilient and hardy scleractinian species are likely to reestablish and create the replacement coral cover. Also, colonies killed by sudden and massive rainfall in shallow lagoons, or broken into fragments by brutal wave action, will provide clean substrate for fast-growing opportunists, initially mostly algal species, usually via some mediation by microbial biofilms. Once established, and provided adequate nutrients are available, algae tend to replace lost coral cover. A "side effect" of cyclone damage is the temperature-associated blooming (Chateau-Degat et al., 2005) of the neurotoxic dinoflagellate *Gambierdiscus toxicus* growing on algal turf that colonizes newly available substrate. With the observed trend of the increase in frequency and in severity of tropical cyclones associated with global warming, important edible fish species may increasingly become unfit for human consumption in impacted areas, a tragic situation in remote islands with low revenue populations with little choice for food substitutes.

#### **3.6 Nutrient enrichment favors algal development to the detriment of corals**

Non-calcifying green algae and in particular the ulvaceans, a ubiquitous group of noncalcifying chlorophytic algae, represent the most visible sign of pollution due to organic enrichment. They are distributed worldwide, from cold temperate waters to warm tropical latitudes, and from fresh or brackish to saline coastal environments, as they are both opportunistic and resilient to environmental stresses. Their taxonomy may prove difficult (Loughlane et al., 2008) with occasional reassessments made necessary owing to their phenotypic flexibility (e.g. Kang & Lee, 2002). In temperate regions, green algae tend to be seasonal, their development dependant not only on the amount of sunlight, but also on nutrient availability and on microbial consortia associated to particular developmental stages. Spectacular biomass explosions attributed to nutrient enrichment generated by agricultural and farming practices and long daylight exposure occur during spring and summer. Though considered efficient at absorbing excess nutrient enrichment, if not collected they can themselves become a source of chemical pollution (hydrogen sulfide in particular) through massive bacterial degradation of the decaying biomass. In tropical environments, developing economies associated with fishing and farming of marine resources generate massive effluxes of untreated urban sewerage leading to localized blooming of chlorophytic algae. In the vicinity of reefs, bacterial enrichment by resident algae may be detrimental to coral polyps by promoting the growth of pathogenic strains at the expense of the regular associated bacterial microflora (Rohwer, 2010).

### **4. Sponges**

86 Biodiversity Loss in a Changing Planet

are listed as invasive species competing with native species for resources and being

The primary cause of invasion of new territories by alien algae is not always easy to determine, and a combination of factors usually contribute to the success of their expansion in new territory. Regular de-ballasting of huge amounts of seawater by container ships is often held responsible for the spread of exotic algae and microalgae along major maritime trade routes, eventually leading to endemic settlement points in regions where acclimation is possible (Carlton, 1996). On the other hand, massive invasions along the Mediterranean shores of the toxic Australian green alga *Caulerpa taxifolia* (and later of *Caulerpa racemosa*) are examples of accidental contaminations starting with a few individuals (Klein & Verlaque, 2008). The displacement by these two *Caulerpa* species of resident halophyte meadows (e.g. *Posidonia*) which are home to juveniles of a number of important demersal fish is considered a threat to local biodiversity. Rhodophytes (red algae) include a number of "cosmopolitan" species that have become established in tropical environments. An example is the territorial expansion of *Asparagopsis taxiformis* in the South Pacific that is currently considered a serious threat to corals in New Caledonia and in French Polynesia, like its sibling species *A. armata*

In tropical seas, hurricanes are becoming more frequent and more severe with global warming, resulting in near-total destruction of the coral cover in the most exposed localities, with negative impacts on larval recruitment (Crabbe et al., 2008) and hence on biodiversity. Initially, only the most resilient and hardy scleractinian species are likely to reestablish and create the replacement coral cover. Also, colonies killed by sudden and massive rainfall in shallow lagoons, or broken into fragments by brutal wave action, will provide clean substrate for fast-growing opportunists, initially mostly algal species, usually via some mediation by microbial biofilms. Once established, and provided adequate nutrients are available, algae tend to replace lost coral cover. A "side effect" of cyclone damage is the temperature-associated blooming (Chateau-Degat et al., 2005) of the neurotoxic dinoflagellate *Gambierdiscus toxicus* growing on algal turf that colonizes newly available substrate. With the observed trend of the increase in frequency and in severity of tropical cyclones associated with global warming, important edible fish species may increasingly become unfit for human consumption in impacted areas, a tragic situation in remote islands

potential causes of new diseases of resident scleractinian corals.

**3.5.2 Cataclysmic events may favor supremacy of algal communities**

with low revenue populations with little choice for food substitutes.

**3.6 Nutrient enrichment favors algal development to the detriment of corals**

Non-calcifying green algae and in particular the ulvaceans, a ubiquitous group of noncalcifying chlorophytic algae, represent the most visible sign of pollution due to organic enrichment. They are distributed worldwide, from cold temperate waters to warm tropical latitudes, and from fresh or brackish to saline coastal environments, as they are both opportunistic and resilient to environmental stresses. Their taxonomy may prove difficult (Loughlane et al., 2008) with occasional reassessments made necessary owing to their phenotypic flexibility (e.g. Kang & Lee, 2002). In temperate regions, green algae tend to be seasonal, their development dependant not only on the amount of sunlight, but also on nutrient availability and on microbial consortia associated to particular developmental stages. Spectacular biomass explosions attributed to nutrient enrichment generated by agricultural and farming practices and long daylight exposure occur during spring and

in other parts of the world.

#### **4.1 Sponges and reef structuration**

In association with spongin (proteinaceous) fibres, marine sponges typically biomineralize non-aragonitic calcium and magnesium carbonates (subphylum Calcispongia) or silica (subphylum Silicispongia). One of the most ancient eumetazoan lineages, sponges have adopted different modes of biomineralization and reef-forming capacities through geological time, as seawater chemistry and the bioavailability of dissolved salt species has changed periodically due to tectonic events (Stanley & Hardie, 1998). Most reef-forming sponges have disappeared since the Phanerozoic, and present-day sponges are mostly siliceous Demosponges which are ubiquitous worldwide in their distribution (see Hooper & Van Soest, 2002).

#### **4.2 Why are sponges so unique?**

The position of sponges at the root of metazoan evolution is still a hotly debated topic (see eg. Maldonaldo, 2004). Choanoflagellates, with which sponge choanocytes and metazoan lineages share genomic similarities (King et al., 2008), have emerged as model organisms for studies of early metazoan evolution. For example, the choanoflagellate genome carries the markers of three types of molecules that cells use to achieve phospho-tyrosine signaling proteins, involved in important processes (cell-cell communication, immune system responses, hormonal stimulation, etc.) in metazoans. These molecules are tyrosine kinases (TyrK), protein tyrosine phosphatases (PTP) and Src Homolgy 2 (SH2) molecules that operate as a tandem system to achieve signal recognition (Manning et al., 2008). Recent studies have also demonstrated the role of associated bacteria in the colony-forming behavior of otherwise freeliving choanoflagellates (e.g. a glycosphingolipid produced by the bacterium *Algoriphagus* that affects the choanoflagellate *Salpingoeca rosetta* (Alegado et al., 2010). Other studies mention the role bacteria may have had in the transfer of genes between unicellular eukaryotes. Nedelcu et al. (2008) provide an example of bacterially mediated lateral transfer of four stress-related genes of algal origin to a choanoflagellate host, supposedly providing the recipient cell the capacity to adapt to stress under environmental changes.

Going one evolutionary step further, Srivstava et al. (2010) have shown that the genome of the demosponge *Amphimedon queenslandica* contains the set of genes that correlates with critical aspects of more evolved metazoans (body plan, cell cycle control and growth, development somatic and germ-cell differentiation, cell adhesion innate immunity and allorecognition). Adaptive responses to environmental changes are known to be more rapid in microbial symbionts than in sponge cells, and bacteria may act as environmental sentinels to marine holobionts as they are particularly sensitive to environmental stressors such as heavy metal pollution (Webster et al., 2001), elevated seawater temperature, sedimentation

Coral Reef Biodiversity in the Face of Climatic Changes 89

core-consortia of bacteria which are found in distant conspecific sponge populations and all year around indicates a high degree of functional specificity and species selectivity. Vertical transmission of "essential" bacteria from parent to offspring via the eggs or the larvae in, respectively, oviparous and ovoviviparous species have been investigated by Webster et al. (2010b) in major poriferan clades: Demospongiae, Homoscleromorpha, Calcarea and

Archaea are found in a wide variety of sponges, and their populations have been characterized in several taxa, e.g. *Axinella*, either as clade-specific mutualists or not, with some sponges lacking them altogether (Holmes & Blanch, 2007). This understudied component is primarily involved in ammonia oxidation, but the co-production of bioactive

In addition, sponges are host to microscopic algae, viruses, yeast and fungi, which add to the biodiversity and, at least for the fungi, to the chemodiversity of the holobiome (e.g. König et al., 2006). Sponges are host to a strain of *Aspergillus sydowii*, a fungus which has been identified as a causative agent of epidemics that affect gorgonian corals. The authors of the study (Ein-Gil et al., 2009) postulate that sponges may act as reservoirs of potential marine pathogens, in the same way as the bacterial populations associated with turf and macroalgae are considered as potential sources of infection to scleractinian corals which are

Polychaetes, prosobranchs, ophiuroids and crustaceans are also commonly found in association with sponges, and the disappearance of the latter would automatically reduce

The sponge microbiome is a prime example of natural chemodiversity, occupying an extensive range of functions in primary (C and N cycling – see e.g Li, 2009) and secondary metabolism (thousands of original molecules of various classes and modes of action) which

The biochemical nature of sponge-microbe symbioses is largely unknown, and ideally requires investigations at single strain microniche consortium and whole microbiome levels (Kamke et al., 2010) for a given host to obtain a better insight into the functional dynamics of the holobiont. Several basic (primary metabolism) sponge-associated microbial processes

i. nitrification, the oxidation of ammonia (NH3) to nitrite (NO2-) and subsequently to nitrate (NO3-) for energy purposes, both steps being carried out by two different bacterial groups: AOB or ammonia oxidizing bacteria and archaea, and NOB or nitrite

ii. nitrogen fixation which appears important in nutrient poor reef environments (Mohamed et al., 2008). Recently Hoffman et al. (2009) described the complex nitrogen cycle of the sponge *Geodia barretti* and speculated upon the possible role of marine

iii. photosynthesis with cyanobacteria (Arillo et al., 1993; Li., 2009) and even zooxanthellae that appear to resist elevated temperatures (but not light) better than coral

Hexactinellida (Ereskovsky, 2011), also including cyanobacteria (Usher et al., 2001).

**4.3.4 Other components of the sponge holobiont**

host to specific microbiomes (Barott et al., 2009).

the specialized associated invertebrate biodiversity.

**4.4 The sponge holobiont – Functional aspects**

**4.4.1 Functional "primary" aspects of symbiosis**

oxidizing bacteria (Bayer et al., 2008),

have been described, including:

sponges as nitrogen sinks,

has been studied worldwide by natural product chemists.

metabolites is not excluded.

and disease (Webster et al., 2011), and tolerance to eutrophication (Turque et al., 2010). Garderes et al. (2011) showed that specific bacterial quorum sensing signals can be recognized by sponge cells, triggering phagocytosis, a response proposed as part of a symbiont population-regulating mechanism.