**Meet the editor**

Ibrahim Al-Bahadly received a B.Sc. (Eng.) degree from the Baghdad University of Technology in 1987, followed by an M.Sc. and Ph.D. from Nottingham University, in 1990 and 1994, respectively, all in electrical and electronic engineering. From 1994 to 1996, he was a research associate with the electric drives and machines group at the University of Newcastle upon Tyne, UK. Since 1996,

he has been with the Massey University, where he is currently Associate Professor in electrical and electronic engineering. His research interests include power electronic applications, variable speed machines and drives, energy conversion and renewable energy system, instrumentation and automation. Ibrahim is a senior member of the Institute of Electrical and Electronics Engineers (IEEE).

Contents

**Preface VII**

Choon

**Energy System 1**

Chapter 3 **Solar Cooling Technologies 39**

Anthony Paul Roskilly

**Photovoltaic Systems 21**

Salman Ajib and Ali Alahmer

**and Utilization in Industry 55**

Giulia Massaglia and Marzia Quaglio

Chapter 6 **The Bioenergy Potentials of Lignocelluloses 93**

Chapter 7 **Free-Piston Stirling Engine Generators 105** Songgang Qiu and Laura Solomon

Chapter 1 **The Solutions of DC-DC Converters for Renewable**

Chapter 2 **Electrical Rating—Long-Term Performance Potential of**

Chapter 4 **State-of-the-Art Technologies on Low-Grade Heat Recovery**

Chapter 5 **The Role of Material Selection and Microfluidics for Optimized Energy Conversion in Microbial Fuel Cells 75**

Olatunde Samuel Dahunsi and Munachi Enyinnaya

Muhammad Burhan, Muhammad Wakil Shahzad and Ng Kim

Janie Ling-Chin, Huashan Bao, Zhiwei Ma, Wendy Taylor and

Nguyen The Vinh and Vo Thanh Vinh


*Article* **Parrot Ownership and Capture in Coastal Ecuador: Developing a Trapping Pressure Index**

**Rebecca Biddle 1,\*, Ivette Solis-Ponce 1, Martin Jones 2, Mark Pilgrim 1 and Stuart Marsden 2**

**Abstract:** We located rural communities with pet parrots and used these locations to predict the probability of illegal parrot ownership across coastal Ecuador, using variables related to demand for pets, parrot availability, and trapping accessibility. In 12 pet keeping communities, we carried out in-depth interviews with 106 people, to quantify ownership, trapping, and interviewees' attitudes towards these behaviours. We combined these data to calculate a trapping pressure index for four key roosting, feeding and nesting sites for the Critically Endangered Lilacine or Ecuadorian Amazon Parrot *Amazona lilacina*. We found that 66% of all communities had pet parrots and 31% had pet Lilacines. Our predictive models showed that pet parrot ownership occurs throughout coastal Ecuador, but ownership of Lilacines by rural communities, is more likely to occur within the natural distribution of the species. The number of people per community who had owned Lilacines in the last three years varied from 0–50%, as did the number of people who had trapped them—from 0–26%. We interviewed 10 people who had captured the species in the last three years who reported motives of either to sell or keep birds as pets. Attitudes towards pet keeping and trapping differed among the 12 communities: 20–52% believed it was acceptable to keep pet parrots, and for 32–74%, it was acceptable to catch parrots to sell. This being said, most people believed that wild parrots were important for nature and that local people had a responsibility to protect them. We conclude that trapping pressure is greatest in the southern part of the Lilacine's range, and urgent conservation measures such as nest and roost protection, and local community engagement are needed.

**Keywords:** *Amazona lilacina*; poaching; conservation threats; mangrove; dry forest; local knowledge; attitudes; Lilacine Amazon

### **1. Introduction**

Parrots (Psittaciformes) are among the most endangered and rapidly declining bird groups, with 28% of their species classified as threatened [1]. Globally, over a third of parrot species are caught to fulfil the demand of the international wildlife trade [1–3]. In the Neotropics, over half of the studied parrot populations are in decline [4], and one reason for this is the high demand for the pet trade [5]. Neotropical species are particularly favoured as pets [2,6], and it is suggested that trapping is a stronger threat to their conservation than habitat loss [7]. Amazon parrots and macaws are preferred due to their attractiveness and ability to mimic the human voice [8]; this is illustrated in Costa Rica, where nearly 20% of households have a pet parrot and half of these are *Amazona* species [9]. Consequently, the rate at which Amazon parrots and macaws are trapped is much higher than expected considering their availability in the wild [10].

Trapping risk is highest where parrots are abundant in the wild, where demand is high and where parrots are relatively easy to catch and sell [11]; therefore, trapping pressure may differ across a species' range and also between species. Additional factors found to drive hunting and trapping include overlap with human population [11,12] and proximity to

**Citation:** Biddle, R.; Solis-Ponce, I.; Jones, M.; Pilgrim, M.; Marsden, S. Parrot Ownership and Capture in Coastal Ecuador: Developing a Trapping Pressure Index. *Diversity* **2021**, *13*, 15. https://doi.org/ 10.3390/d13010015

Received: 25 November 2020 Accepted: 21 December 2020 Published: 5 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

infrastructure or towns [13]. Attitudes and subjective norms are also factors that influence decision making [14], and are therefore likely to affect the level of pet keeping and capture in different areas. In Ecuador, wild bird keeping is illegal [15], and whilst ownership appears to be declining in major cities [16] demand is still high in rural areas, where over half of coastal communities still keep pet parrots [17]. The most frequently reported confiscated bird species in the country are those with wild distributions exclusive to this coastal region [16,18]. Moreover, this region is one of the most densely populated and impoverished [19] parts of Ecuador, the habitats here have been drastically reduced [20] and are greatly underrepresented in the country's national protected areas system [21].

The Critically Endangered Lilacine or Ecuadorian Amazon *Amazona lilacina*, a species recently split from the *A. autumnalis* group, is found exclusively within the coastal region of Ecuador [22]. CITES reported thousands of individuals of this species being trapped and exported in the early 1980s [23] and although frequency of trapping is likely to have reduced significantly in recent years, there are still multiple reports of capture and petkeeping within rural communities [17]. An average of 392 wild-caught parrots, including 30 *A. autumnalis*, were confiscated annually in Ecuador between 2003 and 2016 [16]. Although some of these may be older birds, and they may be either *A. lilacina* or *A. a. salvini*, this figure suggests that some level of trapping is still occurring to fulfil the demand for pets. The goal of this study was to understand the risk of trapping in rural communities and formulate a strategy for conservation support. Specific objectives were:

### **2. Materials and Methods**

### *2.1. Surveys to Locate Communities with Pet Parrots*

In order to locate rural communities with pet parrots, we conducted surveys between January and July 2017. The study area encompassed the extent of occurrence of the Lilacine Amazon *Amazona lilacina* and communities close (<10 km) to forest patches, where wild parrots may occur were selected. Participants were asked to confirm if they knew of pet parrots in their community, and if possible to identify the species. Prior verbal consent was obtained from each participant and full ethical approval of survey content and methods was gained from The North of England Zoological Ethical Review Committee. We aimed to survey at least four households per community; however, some communities were made up of just a few houses, so this was not always possible. We recorded the geographic coordinates of communities with all pets, pet parrots and pet Lilacines, and calculated how many communities each species was recorded in. We used IUCN Red List range maps provided by BirdLife International [24] in order to determine if species were native to the study area. Range maps are frequently updated so we report the year of update for each range map in the results. ArcGIS (version 10.8.1) [25] was used, clipping the distribution shape files, to calculate the size of each species range within our study area.

It is illegal to keep native bird species as pets in Ecuador [15], yet in our experience, people speak openly about their parrots and are proud to show them off. However, it was important that participants did not feel threatened or that we were collecting information to inform the authorities. Therefore, surveys were conducted by a local Ecuadorian researcher, in Spanish, with only the researcher and interviewee present, and it was made clear that all information given was anonymous, and only to be used for scientific research.

Although we refer to *A. lilacina* as the Lilacine or Ecuadorian Amazon Parrot, neither of these common names have Spanish translations that are used in Ecuador. Most local

communities refer to "loro frentirrojo" (Red-lored Parrot), which in English describes the *A. autumnalis* group and includes *A. a. salvini* in northern Ecuador. To avoid confusion, we use *A. lilacina* in our communication with local communities and use photographs to confirm identity, but refer to the Lilacine Amazon in this manuscript.

### *2.2. Distribution Models to Predict Parrot Ownership*

From our surveys we created two groups of geographic coordinates to represent (1) communities with pet parrots, and (2) communities with pet Lilacines. The MaxEnt package in R (version 4.0.3) [26,27] was used to build distribution models based on these coordinates combined with random background points within 30 km buffers of community locations, to predict the distribution of pet parrots, and the distribution of pet Lilacines. Variables were extracted to match each corresponding location and were chosen due to their influence over parrot ownership and trapping [11]: opportunity (presence of parrots and their desirability); demand (presence of people and the infrastructure for trade); and accessibility (into the forest).

For each location, we calculated a "species value" to represent parrot trapping opportunity. For the pet model, this was calculated based on the presence of wild parrot species at that location using species range maps [24], combined with the frequency of the species being reported as a pet; 0.1 was allocated for each species present in that area, and an additional 0.1 was added if that species was reported in a single community, 0.2 if in two communities, etc. This value was used just for comparative purposes within the study and we gave equal weighting to wild species presence and popularity in captivity, as we had no evidence that either was more important than the other. For the pet Lilacine model, this value was replaced with the predicted occupancy area from our distribution models created using observations of the wild population [28]. For both models, we also used the estimated human population [29], the Euclidean distance to the nearest town and nearest road calculated in ArcGIS using OpenStreetMap [30] data, and the mean annual Normalised Difference Vegetation Index (NDVI) from the monthly MODIS product over 2010–2015 as a proxy of vegetation cover.

For each group of points, spatial autocorrelation was controlled for by limiting them to one per 1 km using the R package spThin [31]. Predictors were checked for pairwise correlation across random points within the study area, using pair plot for collinearity [32]. Model evaluation was performed with five-fold cross validation and the mean AUC +/− SD are presented to demonstrate the predictive ability. An AUC of 0.7 means there is a 70% chance that the fitted model will be able to correctly distinguish between presence and absence [33]. All data were included in the final models. We present the permutation importance (%) of variables, with a high value indicating that the final model depends heavily on that variable [27].

### *2.3. Interviews to Quantify Parrot Ownership, Trapping and Attitudes*

We selected 12 communities where pet parrots were present to conduct interviews with community members about their experiences and attitudes towards parrot ownership and capture. These communities ranged in size from 50 to 300 people. The reason these sites were chosen was because our focus was on understanding risk to Lilacine Amazons, so the selected communities fell within the species extent of occurrence and were <15 km away from key roosting, nesting and feeding grounds [17]. These 12 communities were grouped into four clusters (Figure 1). We interviewed at least six participants from different households in each community. Participants were outdoor workers (i.e., agriculturalists, fishers and crab fishers) selected for their familiarity with parrots in their local area. The same methods regarding informed consent and data anonymity as described in Section 2.1 were followed. Due to low literacy levels amongst participants, all questions were read out aloud and the answer provided was recorded by the researcher. Age and gender of each participant was recorded. The interview consisted of eight questions and seven attitude statements arranged on a five point symmetric Likert scale (Table 1). The Likert

package [34] in R (version 4.0.3) [26] was used to visualise attitude statements. Responses were grouped into positive, neutral or negative and a non-parametric test (Kruskal–Wallis) was used to determine significant differences in responses between the four community clusters.

**Table 1.** Interviews about parrot ownership and capture asking eight questions and seven attitude statements, which were read out aloud by the researcher in Spanish. Interviews were anonymous and participants could decline to answer any questions.

**Figure 1.** Interviews about parrot ownership and capture were conducted in 12 communities, grouped into four clusters (A, B, C, and D) near key Lilacine Amazon roost sites. Each cluster contains three communities <10 km apart.

### *2.4. Trapping Pressure Index*

To prioritise areas for conservation support, we calculated a trapping pressure value for each of the four community clusters, to represent the level of risk to the wild Lilacine Amazon population from capture and local desire for pet keeping. This risk value was calculated by combining the following six factors: (1) the mean model value for pet parrot keeping (which represents a probability that pets occur at that location); (2) the mean model value for pet Lilacine keeping; (3) the percentage of people who have owned a pet Lilacine in the last three years; (4) the percentage who have trapped Lilacines in the last three years; (5) the percentage of people who think it is OK to keep a parrot as a pet; and (6) the percentage who think it is OK to catch wild parrots to sell.

### **3. Results**

### *3.1. Locations and Species of Pet Parrots*

Surveys were carried out in 65 communities (mean = 6 interviewed people per community; range 3–20). In 43 (66%), pet parrots were confirmed, and in 20 (31%), pet Lilacines were confirmed. Of the 19 wild parrot species, nine were reported in at least one community, with the most frequently reported being Lilacine Amazons (Figure 2) and Grey-cheeked Parakeets *Brotogeris pyrrhoptera*. The mean range size within the study area of parrots found as pets was 27,370 km2, compared to 8677 km2 for those not kept as pets (Table 2).

**Figure 2.** Examples of pet Lilacine Amazons in four rural communities in coastal Ecuador. Parrots were kept in a variety of situations; indoors or outdoors, caged or with clipped wings. In some cases pet parrots that were housed in gardens were not initially considered to be captive by the owner, but for the purposes of this study any parrot living in the locality of people was classed as a pet.

**Table 2.** The number of communities in which each of the 19 species was reported as a pet and the range size within the study area according to BirdLife International [24].

### *3.2. Predicted Distribution of Pet Parrots*

The locations of the 43 communities with pet parrots and the 20 communities with pet Lilacines were reduced to 42 and 19, respectively, after limiting each group of locations to one per 1 km. A total of 3803 background points were randomly allocated. The mean AUC of resulting models was 0.69 ± 0.06 (sd) for pet parrots and 0.62 ± 0.20 (sd) for pet Lilacines. The most important variables predicting the presence of pet parrots were distance to nearest road (permutation importance, PI = 40%) and distance to nearest town (PI = 28%); the key factors for the presence of pet Lilacines were the mean annual NDVI (PI = 33%) and species value, representing the native distribution (PI = 27%) (Table 3). Predictions show that pet parrots are likely to be widespread throughout the study area, whereas pet Lilacines seem to be more likely within the species range. Both models show a high probability of occurrence of pets to the west of Guayaquil and out towards the coast (Figure 3).

**Table 3.** Permutation importance values for variables used to create models predicting the distribution of pet parrots and pet Lilacines in coastal Ecuador.

**Figure 3.** Model predictions showing the distribution of pet parrots (**a**) and pet Lilacines (**b**).

### *3.3. Incidence of Parrot Ownership and Trapping*

Within 12 selected communities where pet parrots occur, 106 (96 men/10 women) participants (min 6, max 13, mean 8.8 per community) took part in interviews. All participants worked outdoors as farmers (57), fishers (25), crab fishers (18), bee keepers (3) or wildlife guides (3). Participants were familiar with *A. lilacina*, the majority seeing them daily (68%), weekly (19%), or monthly (8%), with the remaining 5% just a few times per year. Of all participants, 66% (70) had owned a pet parrot either previously or currently, and 36% (38) a pet Lilacine. The majority (74%, 28) of Lilacine pets had been caught by the owner themselves, with the remainder received as gifts (16%, 6), bought (2%, 6), or found (2%, 6). In the last three years, 15 people have owned a total of 24 Lilacines. In total, 34 people (32%) confirmed that they had previously captured Lilacines, the majority (76%, 26) to keep as a pet themselves, the others to sell (9%, 26) or for undisclosed reasons (15%, 5). Pet ownership and trapping varied between community clusters, with the highest rates of historic and current ownership and trapping of Lilacine Amazons occurring in the crab fishing communities (D) in the southern part of the range (Table 4).

**Table 4.** The number, age and occupations of people interviewed from each community cluster and the number who reported owning parrots or catching parrots, either previously or in the last three years.

In the last three years, 10 interviewees reported that they had caught Lilacines, with at least 16 birds among them, to either keep the bird as a pet (7), to sell it (1), or for an undisclosed reason (2). All had either no or primary level schooling, and were men 23–72 years old. They reported seeing wild Lilacines daily (9) or weekly (1), and all but one believed the wild population was stable or increasing. In cases where the capture location was given, this always corresponded to the person's occupation, i.e., farmers reported catching parrots in the forest, fishers and crab fishers reported trapping parrots in mangroves (Table 5). Seven of the 10 people who had caught Lilacines in the last three years reported that multiple other people within their community also catch Lilacines, and all 10 knew of multiple pet Lilacines in their community (mean 5.2 Lilacines).

**Table 5.** The age, gender, schooling, and occupation of all interviewees who reported catching Lilacines in the last three years. We report the trapping location, reason for capture and how many were caught.

### *3.4. Attitudes towards Parrot Ownership and Trapping*

Across all communities, responses to attitude statements show a strong feeling that wild parrots are important for nature and participants indicated that local people have a responsibility to protect the environment. This is mirrored by a strong feeling of discomfort with outsiders coming to catch parrots and with parrots disappearing from their area. On the contrary, 46% of all participants believe it is OK to catch wild parrots to sell and 32% that it is acceptable to keep a pet parrot. Furthermore, 17% of people did not believe that catching wild parrots could make them become extinct in the local area (Figure 4). There were no significant differences between communities in the distribution of positive, neutral and negative responses to all attitude statements apart from one: "I think it is OK to keep a parrot as a pet". For this, there was a significant difference between mean responses of the community groups (H = 6.613, *p* = 0.022), with 52% of community cluster D believing this is acceptable, and just 20% of cluster A believing so.

**Figure 4.** Responses to attitude statements are reported on a five point Likert scale (1 = strongly disagree, 2 = disagree, 3 = neutral, 4 = agree, 5 = strongly agree).

### *3.5. Trapping Pressure Index*

When combining our results into a trapping pressure index, we can see variation between the four community clusters, with higher values suggesting a higher risk to the wild Lilacine Amazon population in that area (Table 6). Wild Lilacines occurring around community cluster D are at greatest risk, due to the high level of reported parrot ownership and capture, and a strong local attitude that this is acceptable. Those occurring around community cluster C are also at high risk, as model predictions here show a high probability of pet Lilacine occurrence (0.78), which suggests a high probability of trapping as pet owners mostly report catching their pet themselves. The wild population occurring around community cluster A appears to be at the lowest risk from trapping, as there were no reports here of current Lilacine ownership or capture; however, this is the smallest remaining subpopulation of the species within its range, which could also explain the low prevalence of pets. When considered against participants' responses to their perceived status of the wild population locally, we see more negative responses from the southern community clusters, with the most frequent response in cluster C being 'decreasing' (76%), in cluster D 'stable' (39%), whilst 'increasing' in cluster B (83%) and A (42%).

**Table 6.** Trapping pressure index for each community cluster, calculated by adding together factors of predicted or reported level of pet ownership and trapping, and attitudes towards pet keeping, capture and trade. Predicted probabilities were converted into percentages for this calculation.

### **4. Discussion**

This study found that 66% of rural coastal communities in Ecuador have pet parrots and 31% have pet Lilacine Amazons *A. lilacina*. Within these communities, 66% of people had owed a pet parrot during their lifetime, and 14% currently owned Lilacines. This is similar to Costa Rica, where 18% of households owned a pet parrot in 2001 [9]. Our current ownership questions focused on just one species, so we expect the level of current ownership of all parrot species to be much higher and similar to Colombia where 58% of all people had pet parrots [10]. Current ownership and reports of Lilacine trapping in the last three years varied between communities, with 0% to 50% and 0% to 26%, respectively. Ten participants confirmed that they had taken Lilacines from the wild in the last three years to keep birds as pets, or to sell them, so we suggest that, similarly to Yellow-shouldered Amazon *Amazona barbadensis* harvesting in Venezuela, there are at least two categories of trappers—"poacher-keepers" and "poacher-sellers" [35], with only the latter having the contacts and logistics to sell birds. People in occupations with an established and frequent trade link—for example, fishers or crab fishers—may have more opportunity to transport trapped birds to other towns to sell. We also found that capture location corresponded to the occupation of the poacher, which may explain the variation between rural communities as occupation depends heavily on location, i.e., in-land or coastal. Our trapping pressure index identified that the southern distribution of the species is likely to be at greatest risk, which agrees with earlier work showing a vast population decline in this area [17] and provides further evidence that this area should be prioritised for conservation support.

Understanding whether taking parrots from the wild is opportunistic or selective is important because selective capture can lead to the extinction of species through overharvesting [10]. Our results suggest that trapping is selective given the differences in the popularity of species, with some kept in 20 communities and some in none. The two most frequently reported pet parrot species differed greatly in body mass, which, in general, is linked to longevity in captivity [36], suggesting that variation in popularity is not a side effect of survival rates in captivity. Similarly to previous research, we have noted a preference for Amazon parrots, with all three wild occurring species and two non-native species being kept as pets [8]. However, parrot ownership and capture, at least within

rural communities, may also be opportunistic. Most parrot owners had caught their bird locally, within areas they visit during a normal days' work, and our predictions showed that pet Lilacines were more likely within the species' wild distribution. Moreover, parrot species kept as pets had a larger average wild range size than those that are not. This all suggests that ownership and capture are driven in part by parrot availability and accessibility [11], but more research including a true measure of wild parrot abundance, and surveys and interviews in larger towns and cities are needed. According to anecdotal reports in some rural communities, orders are placed by outsiders from cities such as Guayaquil or Quito, which fits the typical multi-level chain involving trappers, middlemen and markets described by Pires [37] and needs investigation.

Our interviews revealed that Lilacine Amazons were trapped both in mangroves, where they roost, and dry forests, where they feed and nest [38,39], suggesting that both adults and chicks are being taken from the wild. Anecdotal reports from communities suggest past events of outsiders casting nets over mangrove islands to remove an entire roost of Lilacines at a time. Research has shown that the removal of adults from a population can have more drastic consequences on population size and growth rate, than removal of chicks [40]. In a study of illegal wildlife trade markets in Bolivia, contrary to the idea that most parrots come from nest poaching, 70% of parrots were adults [41]. Our results also suggest that 60% of Lilacines caught in the last three years were from mangroves, so are likely to be adult or juvenile birds. A number of studies have shown that anti-poaching efforts, in the form of additional human presence, can benefit bird populations [42–44] and that recruitment of young people (who may be facilitators in parrot trapping) from the local community to act as nest monitors, can significantly decrease poaching rate [45]. In some cases, nest protection implemented at the correct time of year can have a significant effect [42], but we suggest that year-round protection is needed to safeguard both vulnerable roosting and nesting sites for this species.

The lack of environmental education in Ecuadorian schools is a barrier to reducing parrot ownership and capture [16]. The main purpose of any environmental education strategy is to change people's knowledge and attitudes, and ultimately behaviours [46]. Alone, or in combination with other conservation interventions, environmental education projects can result in a decrease in the persecution of parrots and consequently an increase in population size [43,47]. Most people in our study believed that wild parrots were important for nature and that they themselves had a responsibility to protect parrots. Local people do not want parrots to disappear and are strongly opposed to outsiders coming in to their community to catch them. Contrastingly, up to 74% per community agreed that it was OK to take parrots from the wild to sell, and up to 52% believed that it was OK to keep them as a pet. Furthermore, up to 30% disagreed that catching parrots could make them locally extinct. We found similarity between attitudes and reported behaviours. In areas with more pet Lilacines and reports of parrot trapping, there was also a stronger belief that this was acceptable, compared to areas with fewer pets and trapping. This suggests that changing these attitudes could have an impact on future behaviour, and that the implementation of a targeted behaviour change education project could have conservation benefits to the Lilacine Amazon. We suggest following the practices of the successful PRIDE campaigns [48] which inspire people to take pride in the species and habitats that make their communities so unique, whilst introducing viable alternatives to environmentally destructive practices.

We therefore recommend that a combination of environmental education to change attitudes towards parrot ownership and trapping, and increased protection of wild birds through nest and roost guarding, particularly in the southern part of its range, are conservation priorities for the Lilacine Amazon.

**Author Contributions:** Conceptualization, R.B., M.J., M.P. and S.M.; Data curation, R.B. and I.S.-P.; Formal analysis, R.B.; Funding acquisition, R.B. and M.P.; Investigation, R.B. and I.S.-P.; Methodology, R.B., M.J. and S.M.; Project administration, R.B.; Resources, R.B. and M.P.; Supervision, M.J. and S.M.; Visualization, R.B.; Writing—original draft, R.B.; Writing—review and editing, R.B., M.J. and S.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the North of England Zoological Society.

**Institutional Review Board Statement:** The study was approved virtually by The North of England Zoological Society Ethical Review Committee, on Friday 17 November 2017.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to anonymity and sensitive content.

**Acknowledgments:** We would like to extend our thanks to the Amazona Lilacina Education Project volunteers who assisted us with community surveys and interviews. This study would not have been possible without the support of local communities and we thank every person who volunteered their time, valuable knowledge and personal experiences to this research. Thank you to Paolo Escobar for providing one of the pet parrot photographs and to three reviewers for their constructive comments.

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

### **References**


*Review* **Selective Autophagy by Close Encounters of the Ubiquitin Kind**

**Anna Vainshtein 1 and Paolo Grumati 2,\***

Received: 4 September 2020; Accepted: 21 October 2020; Published: 24 October 2020

**Abstract:** Autophagy, a bulk degradation process within eukaryotic cells, is responsible for cellular turnover and nutrient liberation during starvation. Increasing evidence indicate that this process can be extremely discerning. Selective autophagy segregates and eliminates protein aggregates, damaged organelles, and invading organisms. The specificity of this process is largely mediated by post-translational modifications (PTMs), which are recognized by autophagy receptors. These receptors grant autophagy surgical precision in cargo selection, where only tagged substrates are engulfed within autophagosomes and delivered to the lysosome for proteolytic breakdown. A growing number of selective autophagy receptors have emerged including p62, NBR1, OPTN, NDP52, TAX1BP1, TOLLIP, and more continue to be uncovered. The most well-documented PTM is ubiquitination and selective autophagy receptors are equipped with a ubiquitin binding domain and an LC3 interacting region which allows them to physically bridge cargo to autophagosomes. Here, we review the role of ubiquitin and ubiquitin-like post-translational modifications in various types of selective autophagy.

**Keywords:** selective autophagy; ubiquitin; mitophagy; aggrephagy; lysophagy; xenophagy; lipophagy; nucleophagy; ER-phagy; cargo receptors

#### **1. Introduction**

The cellular life cycle is complex, having to contend with ever-changing and at times competing internal and environmental demands, can be stressful. Fail-safe degradation mechanisms are therefore required for the effective disposal of potentially toxic and harmful components and their recycling into building blocks needed for biosynthesis. These degradation systems are vital for the survival and continuity of both long-lived and dividing cells. Several cellular degradation processes have evolved to fill this need and their importance is illustrated through their conservation across evolution, and the pathology that ensues with their perturbance [1,2].

The ubiquitin-proteasome and autophagy-lysosome are the two major cellular degradation systems found in eukaryotic cells and organisms. These processes have remained conserved among species and failure of either one can result in the accumulation of toxic or damaged proteins and organelles, culminating in a number of severe pathologies including cancer, failure to thrive, degenerative diseases, and premature death [1,2]. The ubiquitin-proteasome pathway mainly relies on the 26S proteasome for the final degradation of its substrates [3]. Considered to be the more selective of the two degradation systems, proteasomal substrates are largely composed of individual proteins, requiring large complexes to be disassembled before degradation can take place [4]. The autophagy-lysosome pathway utilizes double membraned vesicles, termed autophagosomes, for the encapsulation and delivery of components to the lysosome for breakdown [5]. Autophagy substrates tend to be larger, including protein aggregates, organelles- either in their entirety or select portions, and invading

pathogens. Although autophagy was historically considered to be a bulk degradation pathway, it is now universally accepted that it can be quite selective, with a wide range of substrates under its jurisdiction. Despite the two degradation mechanisms being fairly distinct, they both appear to utilize ubiquitin modification for substrate recognition [6]. It is intriguing that two seemingly independent degradation pathways, which have evolved largely different components and substrates, converge on the same PTM for cargo recognition. This suggests some crosstalk and redundancy between these pathways, with ubiquitin acting as a universal degradation signal.

#### **2. Ubiquitin**

Ubiquitin (Ub) was originally discovered for its role in tagging proteins for proteasomal degradation. Indeed, Aaron Ciechanover, Avram Hershko, and Irwin Rose received the 2004 Nobel Prize in Chemistry for their discovery and important contributions in unravelling the mechanisms of ubiquitin-mediated proteasomal degradation and the involvement of this pathway in cellular physiology. They demonstrated that ubiquitin was critical for the degradation of proteins involved in cellular regulation, notably cyclins, which play a role in determining cellular replication and mitosis.

Much like its name implies ubiquitin is ubiquitous, it is expressed in all eukaryotic cells and tissues and is evolutionary conserved across species as diverse as plants, yeast, and mammals. A small protein, composed of only 76 amino acids, it is a powerful molecular modifier that governs the fate of proteins and organelles. The ligation of ubiquitin moieties onto a substrate requires a cascade of three distinct enzymes E1, E2, and E3 [3]. E1 is a ubiquitin-activating enzyme that must first prime ubiquitin in an ATP-dependent process. The activated ubiquitin is then transferred to E2, a conjugating enzyme, and finally covalently bonded to a specific substrate with the help of E3, a ubiquitin ligating enzyme. E3 enzymes catalyze the rate-limiting step in Ub conjugation, with over 600 distinct E3 ligases identified so far; this process is extremely substrate-specific and bestows Ub with its selectivity [7]. The ubiquitin tag is then identified by downstream receptors containing a ubiquitin-binding domain (UBD) [8]. Ubiquitin can also be removed from a substrate by deubiquitinating (DUB) enzymes, which antagonize the cell's ubiquitination machinery [9]. By mediating the degradation of cellular regulators ubiquitin controls a variety of cellular processes including cell cycle, transcription, cellular signaling, metabolism, and more. This molecule is central to cellular decision making and is involved in immune response, development, cell death, and growth [3]. Deficiencies in this process can result in a number of pathologies including cancer and neurodegeneration.

In addition to its central role in proteasomal degradation, ubiquitin has been implicated in autophagy regulation and cargo selection [10]. Substrates can be either monoubiquitinated, through the addition of a single ubiquitin moiety, poly monoubiquitinated through the addition of several single ubiquitin moieties, or polyubiquitinated through the formation of polymeric ubiquitin chains [11]. Although there are multiple possible Ub linkage types, while Lysine 48 (K48) and Lysine 63 (K63)-linked chains are among the most abundant ones [11]. Where K48-linked polyubiquitination more readily targets proteins for proteasomal degradation, K63-linked polyubiquitination appears to be more strongly associated with molecular signaling including autophagy [12]. K63 ubiquitination also regulates protein function, interactions, and localization. Although ubiquitin is itself soluble and remarkably stable, in vitro studies, using uncleavable polyubiquitin moieties, showed that ubiquitin chains, the longer they are, the less stable and prone to aggregation they become [13]. This results in the formation of ubiquitin fibrils, which signal for autophagic clearance. The failure to remove these fibrils results in disease. Evidence suggests that polyubiquitination protects cells from actions by proteins that are destined for degradation, by arresting them in solid aggregates and signaling for their autophagic removal [13]. In this scenario, aberrant autophagy owing to lack of essential autophagy components such as Atg5 or Atg7 results in the accumulation of various types of ubiquitin chain topologies suggesting that autophagy is not dependent on any one specific type of ubiquitination [10].

Thus, one important function of ubiquitin is to deliver the "kiss of death" to proteins, organelles, and invading pathogens. It does so by acting as a molecular beacon for the arrest, aggregation, and removal of substrates by cellular proteolytic systems. For a long time, Ubiquitin was thought to be a lone worrier, however, several functionally and structurally related molecules, collectively termed ubiquitin-like (UBL) proteins have been identified. Among them, LC3s/GABARAPs and ATG12 are the most well-studied in the autophagy field [14,15]. Other UBLs include SUMO, FAT10, ISG15, Hub1/Ubl5, NEDD8, UFM1, and URM1. UBLs share many similarities with Ub, including the three-step conjugation process composed of E1, E2, and E3 enzymes and much like Ub, they are also involved in a growing number of cellular processes, including autophagy [16–19].

#### **3. Autophagy**

Macroautophagy, herein on termed Autophagy, is a cellular recycling mechanism responsible for cellular housekeeping and turnover during steady-state conditions and nutrient scavenging during starvation. This process involves the formation of double membraned vesicles, known as autophagosomes, around a substrate or a cytoplasmic domain, and the subsequent delivery of these vesicles to the lysosome for proteolytic degradation (Figure 1). Autophagy depends on core autophagy proteins which contribute to the initiation of autophagosome formation, the lipidation of the autophagosomal membrane protein Atg8/LC3/GABARAP, the fusion of the autophagosome to the lysosome, and finally, the degradation of the autophagosome with its cargo within the lysosomal lumen [20]. In 2016 Yoshinori Ohsumi received the Nobel prize in Physiology and Medicine for his discovery of the mechanisms of autophagy in yeast. Ohsumi systematically uncovered autophagy-related genes, classified as ATGs, and his discoveries resulted in an autophagy renaissance.

**Figure 1.** Autophagy Induction: first a pre-autophagosome (phagophore) is formed by the lipidation of LC3. Cargo Selection: Ubiquitinated cargo is tagged for degradation by autophagy receptors equipped with an LC3 binding motif and a ubiquitin binding domain. The Autophagosome is then elongated around the cargo to be degraded. Fusion: the mature autophagosome fuses with the lysosome. Degradation and efflux: The contents of the autophagosome are degraded by proteolytic enzymes within the lysosomal lumen and nutrients are released.

Once considered an indiscriminate bulk degradation process, autophagy is now well recognized for its selectivity. With the aid of various receptors and signaling molecules, autophagy can discern between healthy cellular components and toxic organelles, proteins, as well as invading organisms. Indeed, constitutive autophagy can be extremely selective and is responsible for the removal of cellular components, like misfolded protein aggregates or exhausted organelles, to ensure a proper physiological turnover [21]. Moreover, autophagy induced by a specific trigger can also be very selective; for instance, xenophagy induced by a pathogen infection triggers a selective autophagic response against the invading organism. Likewise, when mitochondria are chemically depolarized, mitophagy ensues and targets strictly depolarized mitochondria [22–24]. Contrarily, autophagy induced by severe nutrient depletion may not be overly selective, as its sole goal is nutrient liberation under conditions of cellular stress. With this in mind, various types of substrates are eliminated through selective autophagy and this number is continuously on the rise. These include protein aggregates (aggrephagy/proteophagy), mitochondria (mitophagy), endoplasmic reticulum (reticulophagy/ER-phagy), peroxisome (pexophagy), nucleus (nucleophagy), pathogens (xenophagy), lipids (lipophagy), and even lysosomes themselves

(lysophagy) (Figure 2) [23,25]. During selective autophagy, dysfunctional or obsolete proteins and organelles are targeted for degradation by various receptors, which, in turn, entice the arrival of the autophagosome. The selectivity in this process is largely mediated by PTMs such as phosphorylation and ubiquitination, with UFMylation, ISGylation, and SUMOylation recently arising as potential PTMs, similar to Ubiquitination, that modulate autophagy [17].

**Figure 2.** Mammalian Ub-mediated Organellophagy. Damaged, dysfunctional or otherwise superfluous organelles are tagged for degradation by ubiquitin (Ub) or ubiquitin-like molecular modifiers (SUMO, FAT10, ISG15). These post translational modifications (PTMs) are then recognized by autophagy cargo receptors (p62, NBR1, OPTN, NDP52, TAX1BP1, NUFIP1) which bind both the ubiquitinated cargo and the microtubule-associated protein 1B light chain 3B (LC3B). The autophagosome engulfs the receptor cargo complex and delivers it to the lysosome for breakdown. *ER-Phagy*/*Reticulophagy***:** ER stress sensor IRE1α, is activated upon ER stress. IRE1α interacts with p62, OPTN, and NBR1 to promote reticulophagy, but whether Ub is involved in this process is unclear. Self-ubiquitination of the E3 ligase TRIM13 also recruits p62 to damaged ER. This results in the sequestration of the ER within autophagosomes and its delivery to the lysosome for elimination. *Pexophagy***:** PEX2 ubiquitinates the peroxisomal import receptor PEX5 which is then recognized by autophagy receptors p62 and NBR1. PEX3 is also involved in this process and enhances peroxisomal ubiquitination, while peroxisomal acyl-CoA binding domain containing 5 (ACBD5) regulates the pexophagy receptor protein complex. OPTN was also shown to be involved in this process. *Mitophagy*: Mitochondrial damage results in the stabilization of PINK1 on the mitochondrial outer membrane, which entices the arrival of E3 ligases Parkin and GP78. These ligases, in turn, ubiquitinate a variety of substrates on the mitochondrial membrane which recruits autophagy receptors p62, NDP52, OPTN, NBR1, and TAX1BP1. *Aggrephagy*/*Proteophagy:* Protein aggregates are

recognized by selective autophagy receptors p62, NBR1, OPTN, and TOLLIP, with autophagy-linked FYVE protein (ALFY) acting as a scaffold protein promoting the assembly of p62 bodies. Cue5 the yeast homolog of mammalian TOLLIP binds ubiquitinated proteasomes targeting them for proteophagy. Ubiquitin and ubiquitin like PTMs (UBLs SUMO, FAT10, and ISG15) also play a role in aggrephagy. *Xenophagy:* The E3 ligases Parkin and Lrsm1 ubiquitinate invading pathogens which are then detected by p62, NBR1, NDP52, and OPTN. Other UBLs have also been demonstrated to promote xenophagy including FAT10 and ISG15. With FAT10 interacting directly with p62. Recognition by cargo receptors then promotes the incarceration of pathogens within autophagosomes and their delivery to lysosomes for elimination. *Ribophagy:* The large ribosomal subunit is targeted for autophagosomal degradation by the cargo receptor NUFIP1, however, the involvement of Ub or other UBLs in this process has not been determined. *Nucleophagy:* The selective autophagy receptor p62 and autophagic adaptor ALFY, have been documented to shuttle between the nucleus and cytoplasm, however whether Ub is involved in this process is not known. SUMOylation, was documented to drive nucleophagy where DNA damage promotes the accumulation of SUMO E2 ligase UBC9, resulting in the SUMOylation of lamin A/C. *Lipophagy:* Ancient Ubiquitous Protein 1 (AUP1) localizes to LDs and recruits the E2 ubiquitin-conjugating enzyme G2, which ubiquitinates targets on the lipid droplet, thus inviting autophagy receptor p62. *Lysophagy:* Glycans are exposed following lysosomal membrane rapture and are identified by galectins-3 and -8 (Gal3/8) or by the SCF ubiquitin ligase FBXO27. Gal3 then stimulates ubiquitination by TRIM16 and recruits the autophagy receptor p62, while Gal8 engages NDP52. The E2 ubiquitin-conjugating enzyme UBE2QL1 was also found to participate in this process, as were autophagy receptors TAX1BP1 and OPTN. The AAA-ATPase VCP97 is another essential player in the elimination of injured lysosomes.

Moreover, there are two ubiquitin-like (UBL) protein conjugation systems that act to promote the formation of the autophagosome precursor, the phagophore [26]. These are involved in the conjugation of Atg12, and ultimately, microtubule-associated protein light chain 3 (LC3) and its close relatives GABARAP and GATE16 [27–29]. One pathway involves the covalent conjugation of Atg12 to Atg5. This occurs with the help of Atg7 and Atg10, E1- and E2-like enzymes, respectively. The Atg12–Atg5 heteromer then interacts with Atg16-like 1 (Atg16L1) [30]. This complex acts as a *de facto* E3-like ligase to promote the second conjugation reaction, involving LC3. The conjugation of LC3 to phosphatidylethanolamine (PE) is essential for phagophore expansion and sealing. LC3 exists in its inactive form free in the cytosol and must first be cleaved by the protease Atg4 giving rise to LC3-I [31]. The E1-like enzyme Atg7 and the E2-like enzyme Atg3 are then tasked with priming LC3-I for its final conjugation to PE, by the E3-like complex of Atg12-Atg5. Lipid conjugation converts LC3-I, to the LC3-II form that is attached to either side of the growing phagophore membrane.

#### **4. Selective Autophagy**

The benefits of selective autophagy are vast, as it allows for the surgical removal of targeted substrates. For instance, the removal of mitochondria during hypoxia, organelle damage, or even when the organelle becomes obsolete, such as during erythrocyte maturation, all occur selectively through mitophagy [32–34]. In bulk autophagy, the ubiquitination of autophagic components often acts as a regulatory signal. However, when it comes to selective autophagy ubiquitination also acts as a signal for cargo recognition, process initiation, and rate of autophagosome formation. For instance, the ubiquitination of components of the PI3K-Beclin1 complex contributes to the regulation of autophagic induction and autophagosome formation [35,36]. Selective autophagy relies on core autophagy components but is also aided by specific selective autophagy receptors. The cargo selection step of this process is highly dependent on these receptors (Table 1).

Generally, during selective autophagy, protein aggregates, damaged organelles or portions thereof are identified and tagged for degradation by E3 Ub-ligases. Once cargos are tagged, ubiquitin acts as an "eat me" signal for autophagy receptors, which further flag targets for degradation. Autophagy receptors are also implicated in segregating and coalescing materials destined for autophagic degradation, effectively preparing them for the arrival of the phagophore. Indeed, autophagy receptors harbor a

UBL domain to sense Ub molecules, and a LIR domain to bind the LC3s/GABARAPs, which are present on autophagosomal membranes. By binding to mATG8s, cargo receptors promote the recruitment of autophagosomal membranes around the Ub-tagged materials, eventually encapsulating them completely. Mature autophagosomes are then delivered to the lysosome for degradation [37,38].

Considering that potentially all cytosolic components could be selectively degraded via lysosomes, it is plausible that selective autophagy plays a role in a number of diverse physiological processes and their associated diseases. The ability to specifically target the elimination of desired cellular constituents presents great therapeutic potential for the treatment of many diseases characterized by the accumulation of toxic materials including neurodegenerative diseases like Huntington's and Alzheimer's, lysosomal storage diseases, and mitochondrial DNA disorders [39,40]. Moreover, selective autophagy has also been implicated in chronic obstructive pulmonary disease (COPD) [41] as well as other pulmonary disorders [42]. Of note, one of the most well-studied functions of selective autophagy in human health is its role in infections [43]. Once bacteria invade the host cell, they are immediately labeled with ubiquitin chains and selectively degraded via xenophagy [44]. The removal of viral pathogens by selective autophagy is also termed virophagy [45,46]. Of note, host cells counteract some types of viral infections, like flaviviruses and Ebola virus, by directly eliminating their replicative niche in the endoplasmic reticulum, thus activating another form of selective autophagy named ER-phagy [47,48].

#### **5. Cargo Receptors**

Most autophagy cargo receptors are characterized by the presence of a ubiquitin-binding domain (UBD), enabling the detection of marked cargo, and an LC3-interacting region (LIR) allowing for interaction with LC3/GABARAP/GATE16 proteins on the autophagosomal membrane [8]. These properties permit receptors to act as middlemen, effectively bridging tagged cargos with autophagosomes. Indeed, receptors accompany their targets to the lysosome and are degraded alongside them. The UBD, composed of either a UBA, UBAN (ubiquitin-binding in ABIN and NEMO), or ubiquitin-binding zinc finger motif, can recognize various flavors of Ub chain linkages, and its affinity for ubiquitinated substrates can be further enhanced through phosphorylation [49–51]. The core of the LIR motif consists of a minimal consensus sequence W/F/Y-x-x-L/V/I, which is common to most cargo receptors [52], and the phosphorylation of this domain was also demonstrated to augment binding affinity to LC3 and related proteins [53]. Of note, some cargo receptors like NDP52 and TAX1BP1 present an unconventional LIR domain (LVV) that is missing the consensus aromatic amino acid. This non-canonical LIR is important for the function of NDP52 and TAX1BP1 in xenophagy and seems to confer the receptor preferential binding to LC3C, at least in the case of NDP52 [54,55].

Although LIR was the first domain recognized to mediate the binding of ATG8s to autophagy adaptors and receptors, another domain with a similar function has been recently characterized. Several ATG8 interactors exploit a ubiquitin-interacting motif (UIM)-like sequence that confers them with a high affinity for autophagy modifiers [56]. However, the presence of a LIR or UIM motif does not "a priori" confer cargo receptors ATG8-binding abilities. A real interaction should always be experimentally confirmed. Nevertheless, the characterization of the UIM domain greatly expands the number of cellular proteins that could serve as autophagy receptors and adaptors.

The first cargo receptor to be identified was p62 and it is undoubtedly the most versatile and well-studied receptor to date. p62 is ubiquitously expressed and mediates the removal of a wide range of ubiquitinated proteins, protein aggregates, and organelles [57]. Due to its common role in sequestering ubiquitin positive protein aggregates, it earned the name sequestosome-1 (SQSTM1) and was demonstrated to selectively bind to ubiquitinated protein aggregates such as neurofibrillary tangles, Lewy bodies, tau and α-synuclein and alike [58]. p62 contains both a UBA and an LIR domain, thus mediating the association between cargos and autophagosomes [59]. p62 itself is degraded alongside its cargo, as it accompanies the targeted components to their final destination. Indeed, conditions that are characterized by defective autophagy often display large accumulations of p62 along

with ubiquitinated proteins. Interestingly, in some cases elimination of p62 in autophagy-deficient models results in protein aggregate clearance [10], suggesting a role for p62 in the coalescing of materials for autophagic degradation. In addition to p62, many other cargo receptors have been described to participate in various types of selective autophagy including NBR1 (neighbor of BRCA1 gene 1), NDP52 (nuclear dot protein 52 kDa), Optineurin (OPTN), TAX1BP1 (Tax1-binding protein 1), and TOLLIP (Toll-interacting protein) (Table 1), with the number of receptors continuing to grow as more types of selective autophagy are identified.

A screen for Atg8 interactors identified NBR1 as an autophagy receptor containing both LIR and a UBA ubiquitin-binding domain. NBR1 is recruited to Ub-positive protein aggregates and is degraded by autophagy, a process that requires a functional LIR domain [49,60–62]. NBR1 and p62 both act as selective autophagy receptors but appear to function independently as NBR1-positive aggregates are cleared by autophagosomes even in the absence of p62. The depletion of NBR1 abolishes the formation of Ub-positive p62 bodies upon the puromycin treatment of cells [61].

Similar to p62 and NBR1, Optineurin (OPTN) is a selective autophagy receptor equipped with a UBAN-type UBD and a LIR [50,53]. OPTN is present in protein inclusions observed in various neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Huntington's, Alzheimer's, Parkinson's, Creutzfeld-Jacob's, and Pick's disease. Its depletion significantly increases protein aggregation in HeLa cells and morpholino-silencing of the OPTN ortholog in zebrafish causes a motor axonopathy phenotype similar to ALS [63]. Mutations in OPTN's UBAN and LIR domains abolish its ability to bind Ub and LC3/GABARAP modifiers, respectively; these mutations do not appear to compromise its ability to colocalize with protein aggregates, suggesting that OPTN interacts with protein aggregates in a manner that is ubiquitin and LC3 -independent [53]. Indeed, OPTN recognizes various protein aggregates via its C-terminal coiled-coil domain in a ubiquitin-independent manner. Moreover, TANK1 binding kinase 1 (TBK1) phosphorylates OPTN, which regulates its ability to interact with autophagy modifiers and clear protein aggregates [53,63]. OPTN can itself be ubiquitinated by the E3 ligase HACE1 (HECT domain and ankyrin repeat-containing E3 Ub protein ligase 1) which promotes its complexing with p62 and enhances autophagy flux [64]. The HACE1-OPTN interaction synergistically suppresses the growth and tumorigenicity of lung cancer cells [64].

Thus, selective autophagy relies on receptors for the targeting and coalescing of materials into autophagosomes for degradation. Numerous such receptors have been identified, each with its own repertoire of autophagy targets.

#### **6. Mitophagy**

Mitochondria, the powerhouse of the cell, provide energy in the form of ATP to fuel most cellular activities. However, these powerful organelles also produce toxic reactive oxygen species (ROS) as a by-product of oxidative phosphorylation. This leaves them vulnerable to ROS-induced damage and dysfunction. Recycling damaged mitochondria is therefore vital for cellular health and bioenergetics. The wholesale removal of mitochondria occurs through a selective process termed mitophagy. Mitophagy is ever-ongoing at low levels and accounts for routine mitochondrial turnover which occurs approximately every 7–14 days, but may be accelerated during energetic imbalance or oxidative stress [65]. Mitophagy also participates in the pruning of healthy mitochondria during various physiological processes such as erythrocyte maturation and the elimination of paternal mitochondria during oocyte fertilization [34,66–68]. Interestingly, the stimulation of mitochondrial oxidative phosphorylation has also been demonstrated to enhance mitophagy through the small GTPase Rheb [69]. This acts as a pre-emptive mechanism to facilitate mitochondrial renewal in response to enhanced metabolic demands. Here, we provide evidence to the role of ubiquitin in targeting mitochondria for autophagic degradation, mechanisms of mitophagy have been extensively reviewed elsewhere and are beyond the scope of this review [22,24].

When mitochondria withstand irreversible damage and all other quality control mechanisms have failed to restore proper mitochondrial function, mitophagy is triggered. The loss of mitochondrial membrane potential and integrity results in the stabilization of mitochondrial health sensor PINK1 (PTEN-induced putative kinase protein 1) on the organelle's outer membrane. PINK1 serves as a docking station for the arrival of the cytosolic E3 ubiquitin ligase Parkin to damaged mitochondria. The phosphorylation of Parkin and ubiquitin chains by PINK1 ensures Parkin is retained on the surface of depolarized mitochondria effectively tagging damaged mitochondria for degradation [70–73]. To keep mitochondria stationary, PINK1 phosphorylates Miro, a component of the motor adaptor complex. Miro is responsible for anchoring the motor protein kinesin to the mitochondrial surface and facilitating mitochondrial movement along microtubule tracks. Miro phosphorylation predisposes it for degradation in a Parkin-dependent manner [74] thus, keeping mitochondria at a standstill. Moreover, Parkin also mediates the ubiquitination and degradation of mitochondrial fusion effectors, Mitofusins 1 and 2 (MFN1 and MFN2). This ensures the isolation of the damaged organelle. Parkin has also been documented to ubiquitinate a variety of other outer mitochondrial membrane proteins such as the voltage-dependent anion channels (VDACs), translocases of the outer membrane (TOMs), as well as many others [73]. The ER-associated E3 ligase Glycoprotein 78 (Gp78) was also found to ubiquitinate mitochondrial outer membrane proteins such as MFN1 and induce mitophagy of depolarized mitochondria independently of Parkin [75]. On the other hand, deubiquitinase USP30 (ubiquitin carboxyl-terminal hydrolase 30) acts in opposition to Parkin, removing Ub from depolarized mitochondria thus blocking mitophagy [76]. Defective mitophagy, resulting from deficiencies in Parkin or PINK1 can be rescued by the knockdown of USP30 [76]. The presence of ubiquitinated proteins on the mitochondrial outer membrane, act as signals for the arrival of autophagy receptors NBR1, p62, OPTN, NDP52, and TAX1BP1 [61,77–79]. However, some evidence suggests that p62 may not be required for mitochondrial degradation by autophagy [22,79,80]. Indeed, a comprehensive study demonstrated NDP52 and OPTN to be the main receptors responsible for PINK1- and Parkin-mediated mitophagy, where they also activate and recruit core autophagic machinery [79].

#### **7. Aggrephagy**/**Proteophagy**

Busy making proteins to sustain life, cells make mistakes in protein folding, proteins get damaged and oxidized by toxic metabolic byproducts, and certain proteins, such as polyglutamine (polyQ), are inherently prone to aggregation. Aggrephagy refers to the autophagic degradation of protein aggregates, which aberrantly accumulate due to processing mistakes, damage, or proteasomal dysfunction [81]. Protein aggregates are common in proteinopathies such as neurodegenerative diseases and aging [21]. The usual aggregate prone suspects, including amyloid-β [82], huntingtin [83,84], and alpha synuclein [85], can all be degraded through this process. Ubiquitination is vital for the autophagic removal of protein aggregates in proteinopathies [86–89], with p62, NBR1, and OPTN all participating as cargo receptors in mammals [23]. Scaffolding proteins such as ALFY also appear to play a role in this process by promoting the assembly of p62 bodies and their degradation by autophagy [90,91]. The most recently identified aggrephagy receptor, in yeast, is Cue5 (coupling of ubiquitin to ER degradation-5). Cue5 promotes aggrephagy by facilitating the interaction between Ub and Atg8 on protein aggregates. Cue5 also acts as an autophagy receptor for proteasomal subunit degradation, known as proteophagy, by binding to ubiquitinated proteasomes and promoting their removal by autophagy [92–94]. The overexpression of TOLLIP, the human homolog of Cue5 has been demonstrated to mediate the degradation of polyQ proteins in human cell lines [95].

Moreover, other UBLs including SUMOylation (the post-translational modification of proteins with small ubiquitin-like modifiers), FAT10 (the addition of the cytokine-inducible HLA-F adjacent transcript 10, also known as ubiquitin D, onto proteins) and ISGylation (the covalent attachment of Interferon-stimulated gene 15 (ISG15) to substrates) have been documented to play a role in aggrephagy. SUMOylation, like ubiquitination, involves lysine residue modifications of proteins through a three-step process involving distinct enzymes: SUMO E1 (the heterodimer SAE1 and SAE2), SUMO E2-conjugating enzyme (UBE2I/UBC9) and several E3 ligases [21,22]. SUMO-1 was found to accelerate the accumulation of autophagic vacuoles in neurons, thus increasing the production

of amyloid-β (Aβ) [96]. FAT10 was found to regulate the solubility of polyQ proteins, such as huntingtin [97], and ensure the delivery of aggregated or damaged proteins to the aggresome during proteasome dysfunction by interacting with histone deacetylase 6 (HDAC6) [98]. Similarly, ISGylation marks protein aggregates for autophagic disposal and also interacts with p62 and HDAC6 to enhance aggrephagy [99].

#### **8. ER-Phagy**/**Reticulophagy**

The endoplasmic reticulum is a jack of many trades, involved in proper protein folding, processing and secretion, as well as calcium homeostasis, and lipid synthesis. The ER has also been documented to contribute membranes to growing autophagosomes [100–102]. The ER is a continuous membrane structure composed of an extensive network of sheets and tubules [103]. The accumulation of unfolded proteins induces an ER stress response known as the unfolded protein response (UPR). This program activates the following ER stress sensors: inositol-requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6) [104], which bring protein synthesis to a halt, enhance the degradation of misfolded proteins, and increase the expression of molecular chaperones. If these actions do not resolve ER stress, the selective autophagy of the ER or ER-phagy will ensue in a final attempt to avoid the induction of apoptosis [105,106]. Through this process, the ER undergoes significant remodeling and is targeted for degradation by the following cargo receptors: FAM134B, SEC62, RTN3, CCPG1, ATL3, and TEX264 [107–113]. Thus far, the role of ubiquitin in ER-phagy, and its relationship with cargo receptors involved in this process, remains largely unknown. However, some recent data suggest that ubiquitination does indeed play a role in this process. In this scenario, the N-degron pathway mediates ER-phagy through the auto-ubiquitination of the transmembrane E3 ligase TRIM13. Once ubiquitinated, TRIM13 signals for the arrival of cargo receptor p62 thus recruiting the phagophore to the damaged organelle. This is dependent on an N-degron destabilizing residue on the N-terminal arginine (Nt-Arg) [114]. p62, in turn, facilitates the encapsulation of ER into autophagosomes and their turnover in the lysosome, by binding to ER transmembrane protein IRE1α [115,116]. IRE1α has also been demonstrated to bind other Ub binding receptors such as OPTN and NBR1 [115].

#### **9. Ribophagy**

Ribosomes are the cellular protein manufacturing facilities and can be found free-floating in the cytosol or attached to the ER. These tiny factories sustain life through continuous protein biosynthesis. During starvation, ribosomes are selectively eliminated via ribophagy to generate nucleotides/nucleosides required for cellular survival [117].

In yeast selective degradation of mature ribosomes is mediated by the Ubp3p ubiquitin protease complex composed of Ubp3p, Bre5, Cdc48, and Ufd3 [117,118]. This complex deubiquitinates Rpl25 on the large ribosomal subunit, which results in its degradation. This process is antagonized by Ltn1-mediated ubiquitination of the same residue on Rpl25, creating a dynamic and selective ribophagy signal. Therefore, ubiquitin-mediated regulation of ribophagy, in yeast, is counterintuitive to other types of selective autophagy. Where, in most other types of selective autophagy ubiquitination functions as a marker for cargo recognition, in ribophagy ubiquitination appears to be protective, and must be removed to trigger autophagic degradation, at least in yeast. Interestingly this interplay only controls the turnover of the large ribosomal subunit. This suggests ribophagy is even more selective as independent mechanisms are responsible for the degradation of the different ribosomal subunits [117].

Ribophagy has also been demonstrated to occur in human cells in response to starvation, mTOR inhibition, and treatment with cellular stressors such as sodium arsenite and reversine [119]. However, the molecular mechanisms that regulate ribophagy are not clear and some controversial theories co-exist. NUFIP1 (nuclear fragile X mental retardation–interacting protein 1) was initially characterized as a selective ribophagy receptor, responsible for mediating ribosomal degradation by

directly interacting with LC3B and ribosomes during starvation [120]. Moreover, ribophagy appeared to be particularly important during nutrient depletion as it represents a source of nucleosides for starving cells [120]. Further studies on ribophagy revealed that the delivery of ribosomes to lysosomes could follow one of three paths: (i) random engulfment of ribosomes into autophagosomes during bulk autophagy; (ii) by-stander flux following ER-phagy induction and (iii) receptor-mediated selective autophagy [113,119]. Moreover, recent proteomic studies revealed that during acute nutrient stress, ribosomal protein degradation occurs mainly via a non-autophagic pathway. No obvious differences in ribosomal protein levels were detected between wild-type and *ATG7* or *RB1CC1* deficient cells following treatment with Torin 1. Moreover, similar results were obtained in NUFIP1 deficient cells, bringing the role of this protein in ribophagy into question [121].

Although the involvement of ubiquitination in ribophagy has not been determined yet, ribosomal protein ubiquitination has been demonstrated to occur under stressful conditions such as translation stalling, oxidative stress, and UPR [122–125]. Indeed, the E3 ligases HUWE1 (HECT, UBA, and WWE Domain Containing Protein 1) and UBE2O (Ubiquitin Conjugating Enzyme E2 O) have been implicated in the ubiquitination and proteasomal degradation of ribosomal proteins [126,127].

#### **10. Pexophagy**

Peroxisomes are small organelles responsible for lipid metabolism and redox regulation in most eukaryotic cells. As both producers and scavengers of reactive oxygen species (ROS), these organelles play a multipurpose role in cellular signaling and metabolism. The peroxisomal lifecycle is quite dynamic, with a half-life of only about two days. The elimination of peroxisomes takes place through a selective autophagy process termed pexophagy. Like other selective autophagy substrates, peroxisomes must be ubiquitinated and decorated by cargo receptors to be targeted for elimination.

PEX5 is an import receptor that shuttles between the peroxisomal membrane and the cytosol. Phosphorylation of PEX5 by ataxia-telangiectasia mutated (ATM) predisposes it to mono-ubiquitination, which blocks its export and signals for peroxisomal degradation by autophagy [128,129]. The E3 ubiquitin ligase Peroxin 2 (PEX2) has been implicated in the ubiquitination of PEX5, and PMP70 during starvation [130]. Moreover, PEX14, a peroxisomal membrane protein that acts as PEX5 s docking partner can directly interact with LC3B-II during starvation. An additional peroxisomal import factor, PEX3, has also been documented to play a role in pexophagy, where the overexpression of PEX3 induces ubiquitination and elimination of peroxisomes by autophagy [131].

Selective autophagy receptors NBR1 [62] and p62 [132] were both shown to participate in this process. Indeed, NBR1 was demonstrated to be both sufficient and required to drive peroxisomal elimination by pexophagy, a role that requires its amphipathic α-helical J domain, ubiquitin-associated (UBA) domain, LC3-interacting region, and coiled-coil domain. p62 binding to NBR1 does not appear required for its function but does enhance pexophagy [62]. Moreover, some emerging evidence is suggesting that the peroxisomal protein ACBD5 (Acyl-CoA-binding domain containing protein 5), may be important for regulating the pexophagy receptor protein complex [133]. However, pexophagy research is still in its infancy and more studies are required to further elucidate the molecular mechanisms regulating this process.

#### **11. Lysophagy**

Lysosomes are acidic membrane-bound organelles, which contain a wide variety of hydrolytic enzymes required for the degradation of various substrates including proteins, lipids, carbohydrates, nucleic acids, and invading pathogens. Lysosomes play a key role in cellular homeostasis by controlling both cellular clearance, and energy production in response to environmental cues [134]. These organelles contain a slew of hydrolases (e.g., proteases, nucleases, esterases, polysaccharidases, and glycosidases) capable of degrading a wide spectrum of components. Therefore, upon injury the spilling of lysosomal contents poses a threat to cellular health and can culminate in inflammation and cell death [135,136]. It is not surprising then that damaged lysosomes themselves undergo a selective form of autophagy, termed lysophagy.

Several cellular events can render the lysosomal membrane prone to permeabilization including excessive cholesterol, toxic aggregates such as α-synuclein, mutant huntingtin, and Aβ or tau fibrils, as well as alterations in lipid composition and ROS, just to name a few. Physical damage to lysosomes induced by silica, lysosomotropic agents, and urate crystals have all been documented to induce lysophagy [137]. Upon lysosomal membrane rupture and failing any repair efforts, glycans that normally reside within the lysosomal lumen are exposed. These glycans are then identified by galectins-3 and -8 or by the SCF ubiquitin ligase, FBXO27, leading to the ubiquitination of lysosomal components [138,139]. Indeed, Galectin-3 (Gal3) stimulates the ubiquitination of lysosomal proteins by TRIM16 [140,141], thus recruiting the autophagy receptor p62, while Gal8 engages NDP52 [142].

Moreover, it appears that both K63- and K48-linked ubiquitination are involved in lysophagy. The E2 ubiquitin-conjugating enzyme UBE2QL1 ubiquitinates lysosomal membrane proteins with K48-linked Ub chains [4,143]. While p97 (Valosin-containing protein) translocates to the lysosome to form a complex with UBXD1, PLAA, and YOD1. This complex acts downstream of K63 ubiquitin chains and removes K48-linked ubiquitin conjugates from damaged lysosomes to promote the recruitment of autophagic membranes [4].

In this process ubiquitinated lysosomes recruit autophagy receptors which promote their engulfment within autophagosomes. They are subsequently cannibalized by healthy lysosomes [144]. Interestingly, multiple cargo receptors have been demonstrated to play a role in this process including p62 [4,145], NDP52 [142], TAX1BP1 [143], and OPTN [146]. These are essential for forming a docking platform for ULK1, which activates the core autophagic machinery and induces autophagosome formation regulated by TBK1 [146].

This fits well within the autophagy wheelhouse and can be particularly useful for host-defense. Indeed, one-way pathogens are able to establish an infection, is by breaking vacuolar membranes and escaping into the cytoplasm [147]. This has been documented during Shigella infection, where host cell-membrane remnants attached to a bacterium, activate an autophagic response resulting in pathogen removal [148].

#### **12. Xenophagy**

Xenophagy refers to the selective removal of invading pathogens and constitutes an important faction of innate and acquired immune responses. Autophagy plays a role in combating a number of pathogens including bacteria such as *Streptococcus pyogenes*, *Mycobacterium tuberculosis*, *Listeria monocytogenes, Shigella flexneri, Salmonella enterica,* and *Toxoplasma gondii.* Similarly, viruses such as Sindbis are also targeted by autophagy [149]. Like other forms of selective autophagy, pathogens are first tagged for degradation by Ub [148,150–152] and are then recognized by cargo receptors. This process also requires functional core autophagy machinery. Ubiquitin is vital for xenophagy and is responsible for linking bacteria-containing endosomes with autophagic machinery, such as Atg16L1 [145]. Some examples of E3 ubiquitin ligases that function as xenophagy regulators are: ARIH1/HHARI (ariadne homologue 1) and LRSAM1 (leucine rich repeat and sterile alpha motif containing 1) that ubiquitinate cytosolic *Salmonella Typhimurium* [153,154]. Linear ubiquitin chains have also been detected on the surface of Salmonella; therefore, the E3 complex LUBAC (linear ubiquitin chain assembly complex) and its specific DUB OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) play an active role in xenophagy [155,156]. Moreover, Parkin was shown to ubiquitinate invading bacteria *Mycobacterium tuberculosis* [157]. The cargo receptors p62 [158,159], NBR1 [61], NDP52 [158,160], and OPTN [53] have all been demonstrated to facilitate the sequestration of invading bacteria in autophagic vesicles.

Interestingly, other PTMs such as interferon-α,β inducible ubiquitin-like modifiers FAT10 and ISG15 have also been implicated in xenophagy. Where FAT10 was found to decorate autophagy-targeted *Salmonella* and recruit p62, thus contributing to resistance against *Salmonella* infection in mice, through the induction of xenophagy [18]. Similarly, ISG15 was found to be essential for optimal mycobacterial immunity, by mediating the release of IFN-γ-inducing secreted molecule [161,162]. Indeed, enhanced ISGylation in cells and animals results in greater basal and infection-induced autophagy. This is largely mediated by temporary metabolic reprogramming induced by the ISGylation of mTOR, WIPI2, and AMBRA1 following infection [16].

#### **13. Lipophagy**

Autophagic degradation of lipid droplets, the organelles responsible for cellular lipid storage, is termed lipophagy. Although it was first described over a decade ago, the molecular mechanisms involved in this process remain largely elusive [163]. Lipophagy is particularly important in the liver, where it is responsible for lipid mobilization in response to a multitude of cellular stressors. Indeed, lipid droplets were found to co-localize with autophagy marker LC3B in mouse liver [163]. Later studies uncovered that Huntingtin, the protein mutated in Huntington's disease, is involved in stress-induced lipophagy and appears to act by facilitating the binding between LC3B and p62 [164]. Another protein, Ancient Ubiquitous Protein 1 (AUP1), has been shown to localize to LDs and act as a recruiter for the E2 ubiquitin conjugase G2 (Ube2g2). These findings were the first to suggest that the ubiquitination machinery localizes to LDs and promotes their degradation by autophagy [165]. Moreover, ethanol-induced lipophagy occurs through the increased presence of Ub and p62 on LDs [166]. It appears that ubiquitin may play a role in this selective autophagy process, but solid evidence to this effect, as well as the molecular mechanisms involved, remain to be illuminated.

#### **14. Nucleophagy**

The nucleus is the "brain" of the cell, entrusted with safeguarding the genome and dictating cellular activities by controlling gene expression. The health and integrity of this organelle is therefore the cell's priority. Nuclear recycling by autophagy, known as nucleophagy, is an evolutionarily conserved process among eukaryotes and is responsible for the degradation of nuclear components. Although in some multinucleated eukaryotic cells the nucleus is degraded wholesale by autophagy [167], in most eukaryotes, nucleophagy is thought to occur piecemeal, and require a series of events [168]. This process begins by first sensing and flagging damaged components which are then exported from the nucleus, encapsulated within the autophagosome, and eventually degraded by the lysosome. The precise molecular mechanisms, and whether protein ubiquitination is involved, remain largely unexplored.

In yeast nuclear recycling was documented to occur piecemeal through micronucleophagy [169,170], or by macronucleophagy with the help of the autophagy receptor Atg39 [171], under physiological and nutrient stress conditions, respectively. Nucleophagy has also been described to occur in differentiating murine and human keratinocyte, where perinuclear vesicles containing histone interacting protein and heterochromatin protein 1α, were shown to localize near Lamin A and B1 [172]. Although nucleophagy is not well understood in mammals, dysfunction in this process appears to contribute to pathologies such as cancer and neurodegeneration and is also implicated in cellular senescence and aging [168]. It is unclear, however, whether nucleophagy is an ongoing physiological process that is further triggered by pathology, as a maladaptation or protective mechanism, or rather strictly arising due to pathology. It is also unclear if Ub plays a role in this process, and thus far no autophagy receptors have been identified to participate in mammalian nucleophagy. Several autophagy-related proteins, including the selective autophagy receptor p62 and autophagic adaptor ALFY, have been documented to shuttle between the nucleus and cytoplasm [90,168,172]; however, there is no clear-cut evidence for their role in nucleophagy yet.

Interestingly, recent studies identified SUMOylation to play an important role in cellular activities including nucleophagy. DNA damage was shown to facilitate the accumulation of SUMO E2 ligase UBC9, and SUMOylation of lamin A/C [173]. This modification mediated the interaction between lamin A/C and LC3B, thus promoting nucleophagy. The knockdown of UBC9 prevented SUMOylation of lamin A/C and its interaction with LC3B, which attenuated nucleophagy and resulted in the degradation of nuclear components and leakage of nuclear DNA. SUMOylation has been previously implicated in the autophagic degradation of the polyglutamine (polyQ) protein ataxin-3, thus suggesting a role for SUMOylation in aggrephagy [19].

#### **15. Conclusions**

Ubiquitin is a universal degradation signal used to tag proteins, organelles, and pathogens for disposal by the ubiquitin-proteasome and autophagy-lysosome systems. A large number of selective organelle-phagies are regulated, at least in part, by ubiquitination or other ubiquitin-like modifications. Ubiquitin acts as a beacon for the arrival of selective cargo receptors, and although the various selective types of autophagy rely on specific E3 ligases, of which there are upwards of 600, only a handful of Ub-dependent selective autophagy receptors have been identified thus far. Moreover, selective autophagy relies on common core autophagy machinery for the induction and formation of autophagosomes. This suggests common elements amongst the various types of selective autophagy which have likely evolved from bulk autophagy.

Despite the remarkable growth of knowledge in the field, spearheaded by talented "autophagians", there is still much work to be done. The continuous identification of new selective types of autophagy must be followed up by a detailed characterization of the molecular mechanisms governing them. Likewise, novel cargo receptors involved in the various types of selective autophagy remain to be identified and characterized. Unraveling the molecular mechanisms that regulate the spatio-temporal ubiquitin signaling that controls selective autophagy, will be of great importance, to better understand its role in physiological and pathological processes.

**Author Contributions:** A.V. and P.G. conceived and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Telethon Foundation (TMPGCBX16TT; TMPGGLU16TT), Roche Foundation (Roche per la ricerca 2019), AFM Telethon (Trampoline Grant 2020).

**Acknowledgments:** We kindly acknowledge Alessio Reggio for critical comments.

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


5.1.3. Ecological Restrictions

Table 10 shows the ecological restrictions. Given the planning studies of the Semnan province [74], the location of environmental protected areas, urban and rural areas, and water bodies and rivers were identified, and buffer zones were specified according to the mentioned references in Table 10.

**Table 10.** Ecological restrictions.

Figure 10 exhibits the suitable and unsuitable ecological areas according to the buffer zones of Table 10. According to the ecological restrictions, the red and green areas are unsuitable and suitable zones. Based on the calculations made according to Table 10, 18,052.24 km2 (18.51%) of the province's total area is among the unsuitable areas.

**Figure 10.** Suitable and unsuitable areas according to ecological restrictions.

Figure 11 shows Semnan province by applying all the restrictions and buffer areas. The areas marked in red and green are unsuitable and suitable, respectively. The total unsuitable areas are equal to 35,094.042329 km2, which is 36.2% of the total province area.

**Figure 11.** All buffer areas of Semnan province.


**3. River flow measurements in Amazon North Channel**

In the Amazon River (North Channel) two up to date measurements of net discharges were made. The measuring process, consists of: 1) performing a series of flow measures over a minimum period of 12.30 h, using ADCP with an average of 12 experimental measurements;

anomalies, setting the north / south basins of the tropical Atlantic Ocean. The dipole negative phase of the configuration is essentially opposed. Several observational studies showed that the phase of the dipole directly interferes with north-south migration of the Intertropical Convergence Zone (ITCZ). The ITCZ is the main inducer of the rain weather system in the eastern Amazon, especially in the states of Amapá and Pará, at its southernmost position defines climatologically the quality of the rainy season in these states (Souza & Cunha, 2010). The behavior of the climate is important because it significantly influences the hydrological cycle and, therefore, the hydrodynamic and mixing processes in

According to Van Maren & Hoesktra (2004) the mechanisms of intra-tidal mixing depend strongly on seasonally varying discharge (climate) and therefore hydrodynamics. In this case, during the dry season, there is a breakdown of stratification during the tidal flood that occurs in combination with the movements of tides and advective processes. Intra-tidal mixing is probably greater in semi-diurnal than in diurnal tides, because the semi-diurnal flow velocity presents a non-linear relationship with the mixture generated in the river bed

A second, Hu & Geng (2011), studying water quality in the Pearl River Delta (PRD) in China, found that coupling models of physical transport and sediments could be used to study the mass balance of water bodies. Thus, most of the flows of water and sediment occur in wet season, with approximately 74% of rainfall, 94% water flow and 87% of suspended sediment flow. Moreover, although water flow and sediment transport are governed primarily by river flow, tidal cycle is also an important factor, especially in the regulation of seasonal structures of deposits in river networks (deposition during the wet season and erosion in the dry season). As well as net discharge there are several types of physical forces involved in these processes, including: monsoon winds, tides, coastal currents and movements associated with gravitational density gradients. Together these forces seem to jointly influence the control of water flow and sediment transport of that

A third example, according to Guennec & Strasser (2009), hydrodynamic modeling along a stretch of the Manacapuru-Óbidos river in the upper Amazon a stretch of the *Manacapuru-Óbidos* river in the upper Amazon revealed that the ratio of liquid flow that passes through the floodway changes from 100% during the low water period to 76% (on average) during the high water period. Expressed in volume, this means that about 88% of the total volume available during a hydrological cycle moves through the floodway of the river, and only 12% moves through the mid portion. The volume that reaches the fringe of the flood plain is

Based on the climatic characteristics of the State of Amapá, one of the main challenges for both hydrological and hydrodynamic studies is to integrate meteorological information from the Amazon Basin and include these forces when evaluating the responses of aquatic ecosystems in the Lower Amazon River estuary (Brito, 2010; Bastos, 2010; Cunha et al., 2006; Rickey et al., (1986), Rosman (2007), Gallo (2004), ANA (2008) and Nickiema et al., (2007).

In the Amazon River (North Channel) two up to date measurements of net discharges were made. The measuring process, consists of: 1) performing a series of flow measures over a minimum period of 12.30 h, using ADCP with an average of 12 experimental measurements;

approximately 4% and appears to be temporarily stored.

**3. River flow measurements in Amazon North Channel**

the water.

estuary.

and the mean velocity.

2) interpolate the temporal evolution of flow and velocity from these measurements; 3) integrate the values with the tidal cycle to obtain the average flow rate (or velocity); 4) analyze the maximum and minimum flow, and the relationship between flow/velocity and level, as described by ANA (2008), Cunha et al. (2006) and Silva & Kosuth (2001).

Fig. 1 shows the location of Transect T2 (blue line) of the North Channel and Matapi River, both studied by Brito (2010) and Cunha (2008) nearly to city of *Macapá*, respectively .The requirement for local knowledge of the river bathymetry is demonstrated by the geometric complexity of the channels and variations in the average depths of the channel. Cunha (2008) observed depths ranging from 3 m (minimum) to approximately 77 m (maximum) in the section indicated.

Brito (2010) has studied the water quality sampled water quality and participated in the quantification of the measurements of liquid discharge in the North Channel. The width of the North Channel is approximately 12.0 kilometres (30/11/2010). The width of the South Channel was approximately 13.0 kilometres (12/02/2010).

Fig. 1. Features in river sections close to Transect T2 located in the North Channel of the Amazon River – Amapá State (S0 03 32.2 W51 03 47.7)


Chapter 4

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Compressive Strength of Concrete