The Culprit behind the Mass Death of Mangroves: Egrets or Rats ( Rattus losea )?

: Mangroves play a crucial role in maintaining coastal ecological balance. This study focused on the impact of branch-breaking behavior on the mortality of Rhizophora stylosa in the Guangxi Shankou Mangrove Reserve. However, we found mangrove mortality in areas devoid of egret habitation, prompting a reevaluation of our research hypothesis. Further investigation suggested that nesting behavior was the primary cause of mangrove mortality. A comparison of the data from areas with egrets ( Egretta garzetta , Ardea intermedia ) and lesser rice-field rats ( Rattus losea ) activity indicated significant mechanical damage caused by rats to mangroves as the main cause of mortality. Additionally, we found that the biological characteristics of R. stylosa , particularly its stunted growth and recovery abilities after branch breaking, were key factors affecting its survival. These findings imply that rat-induced mortality may not occur in other less susceptible mangrove species. The results contradict assumptions regarding the impact of egret behavior and highlight the importance of the biological characteristics of R. stylosa . This offers fresh insights into mangrove conservation and management, emphasizing the need for ongoing observation and hypotheses verification. Future studies should explore the influence of lesser rice-field rats’ activity and the intrinsic characteristics of R. stylosa on the ecosystem’s long-term stability.


Introduction
Mangroves are key components of coastal ecosystems and play an important role in maintaining ecological balance [1].In the Guangxi Shankou Mangrove Reserve, particularly in the Ma'anshan Bay area of Yingluo Port, the mangrove plant Rhizophora stylosa serves as an ecological symbol of this region and has faced unprecedented ecological challenges in recent years, with over twenty acres lost to mass mortality.This phenomenon not only threatens the survival of R. stylosa but also jeopardizes the entire mangrove ecosystem.The primary goal of this study was to reveal the causes and mechanisms behind the mortality of R. stylosa, aiming to provide a scientific foundation for the protection and restoration of mangroves.Mangroves are one of the most threatened ecosystems in coastal areas due to increasing urban and industrial development [2][3][4].In addition, extreme weather [5][6][7][8], pathogens [8,9], insects [8], and wood-borers [10,11] can cause tree dieback.The mortality of mangrove forests in the Beihai city is mainly caused by the wood-borer Sphaeroma [12] and sea reclamation [13].
Mangroves serve as nesting sites, roosting trees, and foraging habitats for egrets [14][15][16].In the initial phase of our research, we hypothesized that the large-scale death of R. stylosa in Yingluo Port could potentially be related to large gatherings of egrets, considering that egret feces would contribute to soil pollution and eutrophication [17][18][19].Egret habitation can alleviate P deficiency by increasing soil P supply capacity, but may accelerate P leaching and cause soil nutrient imbalance [20].Subsequently, our focus shifted to the branch-breaking nesting behavior of egrets, which would severely impact the branches of R. stylosa [21].Egrets mostly picked twigs from the ground, but sometimes they broke living and dead twigs off trees and bushes [22].Egrets roost in mangroves for long periods of time and may have a negative impact on this forest.When we were investigating the nests, we found a different kind of nest-rat's nest, which is different from the bird's nest.However, as the study progressed, we discovered the widespread nesting behavior of Rattus losea, a lesser rice-field rat, in areas without egret gatherings, along with the observed mass mortality of mangroves.This observation led us to reevaluate the causes of mangrove mortality, ultimately determining that lesser rice-field rats were the main contributors [23][24][25][26].
This study examines the various factors contributing to the mortality of R. stylosa while also exploring the ongoing evolution of hypotheses in scientific inquiry.Furthermore, it provides new insights into mangrove conservation and management, emphasizing the importance of ongoing observation and hypothesis testing in environmental protection endeavors.This comprehensive analysis offers valuable insights for future environmental conservation efforts.

Field Surveys and Nesting Animals
Systematic field surveys were conducted in the mangrove conservation areas of Shankou in Yingluo Village, Guangxi Province, China (21 • 28 ′ N, 109 • 43 ′ E), and in egret habitats in Yanlou Village, Dangjiang Town, and Hepu County (21 1).Due to the distribution of mangrove mortality areas in the center of Shankou mangrove forests, away from the shore, there is little direct impact from humans.There is freshwater from the Nanliu River flowing into the Yanlou mangrove forest, but no freshwater flow to Shankou mangrove, so we determined soil interstitial water salinity in two plots with salinity meters (HYSYA2-2, Tianjin, China) in November 2023.We studied nest-site plant species, height, and location of shoot growth points by randomly measuring 30 plants in two plots.We made visits twice a month from November 2023 to January 2024 and once a month from February to May 2024 (no egrets were seen) with drones (FEIMA E2000, Shenzhen, China and DJI Mavic 3, Shenzhen, China) to determine the egrets' species in the Shankou Yingluo mangrove.We made visits to Yanlou's egrets' habitat twice in November 2023 as a control site (no rats).Drones and field surveys were mainly used to investigate the rat species, due to the dense foliage and the rats' ability to hide themselves.The rats built their nests among the mangrove forests of Shankou Mangrove Reserve using the foliage and short shoots of mangrove trees.Random sampling was used to collect nest materials in the Shankou plot, and bring them back to count the number of branches and leaves in the nest.

Mangrove Forest Damage Condition
Drones were employed for aerial photography and field surveys to investigate the extensivity of damaged mangrove plants in the egret nesting range.The aerial survey of mangrove forests in severely degraded areas started in January 2023, with a total of five acquisitions, obtaining more than 100 GB of 0.5 m aerial orthophotos, and using Pix4Dmapper (4.5.6) or DJI Zhitu software (3.6.8) for DOM orthophoto generation, according to the different models of drones used to collect the data.The human-computer interactive interpretation method was adopted to visually interpret the mangrove forests and severely degraded areas in the study area on the ArcGIS 10.4 software platform, and screen vectorization was carried out, combined with use of network RTK (iRTK5X, Guangzhou, China) for root point placement and fragmentation point measurement.Finally, we determined the area of mangrove mortality.Google Earth (7.1.5.1557) and ArcGIS was used to analyze changes in egrets' habitats and mangrove damage.We collected high-definition images (0.5 m resolution) of the area from 2018 to 2022 through Google Earth, and manually interpreted the area of mangrove deaths and egret colonies over the years, then plotted it as a map with use of ArcGIS.

Mangrove Leaf Index
Fifty mature and intact leaves were randomly collected from the four mangrove plants (Rhizophora stylosa, Avicennia marina, Aegiceras corniculatum, Kandelia obovata) and their leaf area was determined using leaf area meter (Yaxin-1241, Beijing, China) in December 2023.They were then dried in an oven (WGL-230B, Tianjin, China) at 75 • C until constant weight, and the dry weights were measured on a 1 in 10,000 balance and calculator specific leaf area (Leaf area/dry weight).We recorded leaf drop monthly by labeling 20 newborn leaves of four adult plants, and broke 50 cm branches of four adult plants to observe branch sprouting monthly from January 2023 to May 2024.

Relationship between Egret Aggregations and Mangrove Mortality
Studies in the mangrove conservation areas of the Shankou Yingluo and Yanlou villages have revealed significant egret populations (Figure 2).Despite the high number and density of egrets in these regions, negative effects have not consistently been observed.In Yingluo Village, up to 1974 egrets (Egretta garzetta, Ardea intermedia) were predominantly found inhabiting R. stylosa from November 2023 to January 2024, but no egret roosted on the site from February to May 2024.Furthermore, in the Beihai Yanlou mangroves, 1508 egrets (Egretta garzetta, Ardea intermedia) were observed nesting in Avicennia marina, Aegiceras corniculatum, and Kandelia obovate (Table 1) in November 2023.A comparative analysis of these two areas (Table 1) showed that although the Shankou Yingluo mangrove conservation area contained more egrets, only one species R. stylosa grow in this area due to the lack of river freshwater replenishment and high soil interstitial water salinity (28 psu).We investigated and found 20 acres of dead mangrove forest in the Shankou Yingluo mangrove.Additionally, branch-breaking nesting behavior was relatively common in the Shankou Yingluo mangrove.Conversely, in the Beihai Yanlou mangrove, despite the significant egret population, the impact on plants was relatively minor owing to river freshwater replenishment and lower soil interstitial water salinity (15 psu).Only isolated plant deaths were observed, along with less frequent instances of branch-breaking nesting behavior (Table 1).A comparative analysis of these two areas (Table 1) showed that although the Shankou Yingluo mangrove conservation area contained more egrets, only one species R. stylosa grow in this area due to the lack of river freshwater replenishment and high soil interstitial water salinity (28 psu).We investigated and found 20 acres of dead mangrove forest in the Shankou Yingluo mangrove.Additionally, branch-breaking nesting behavior was relatively common in the Shankou Yingluo mangrove.Conversely, in the Beihai Yanlou mangrove, despite the significant egret population, the impact on plants was relatively minor owing to river freshwater replenishment and lower soil interstitial water salinity (15 psu).Only isolated plant deaths were observed, along with less frequent instances of branch-breaking nesting behavior (Table 1).

Non-Correlation between Egret Aggregations and R. stylosa Mortality
Since 2018, egret aggregation areas (indicated in white in Figure 3) have maintained a relatively fixed location.Historical images show that the first mangrove deaths (blue areas in Figure 3) occurred in 2019 to the west of the egret habitat, but not in the egret habitat itself.Mangrove mortality in the egret habitat (cyan areas in Figure 3) was only observed in September 2022.Moreover, the mortality area continued to expand toward the egret habitat until the end of 2023.It showed that the initial appearance of the R. stylosa mortality area was in a separate location from the egret aggregation area.Subsequent remote-sensing monitoring indicated that the R. stylosa mortality area gradually expanded without egret aggregation (Figure 3).

Internal Factors Influencing Mangrove Mortality
Comparing the ecological characteristics of the four mangrove plants, namely R. stylosa, A. marina, A. corniculatum, and K. obovate, revealed noteworthy differences in the number of branches, leaf size, leaf lifespan, and other ecological traits between the species (Table 2).
R. stylosa is typically characterized by a lower number of branches, a larger mature leaf area (2855 mm 2 ), and a higher specific leaf area (3687.26mm 2 /g) (Table 2).These traits

Internal Factors Influencing Mangrove Mortality
Comparing the ecological characteristics of the four mangrove plants, namely R. stylosa, A. marina, A. corniculatum, and K. obovate, revealed noteworthy differences in the number of branches, leaf size, leaf lifespan, and other ecological traits between the species (Table 2).R. stylosa is typically characterized by a lower number of branches, a larger mature leaf area (2855 mm 2 ), and a higher specific leaf area (3687.26mm 2 /g) (Table 2).These traits suggest a strong K-strategy for R. stylosa, but they also indicate a relatively poor ability to recover after damage.The lifespan of R. stylosa leaves extends up to 18 months.However, this species lacks the ability to regenerate new branches and leaves from the base of the trunk, as its growth points are primarily located at the terminal buds.
Conversely, A. marina and A. corniculatum exhibit different ecological traits.They contain a higher number of branches, with 10-20 leaves per twig, smaller leaf areas (702 mm 2 and 1280 mm 2 respectively), and higher specific leaf areas (4084.97mm 2 /g and 5350.52 mm 2 /g, respectively).These species are also distinguished by shorter leaf lifespans (13 months and 15 months), employ an r-strategy, and possess faster recovery ability after damage (Table 2).Moreover, A. marina and A. corniculatum were capable of regenerating new branches and leaves from the base of the trunk, with growth points distributed at the nodes of the branches (Figure 4).

The Role of R. losea in Mangrove Mortality
In the drone-captured images of the mangrove (Figure 5), the center of the mortality zone features a distinct green patch surrounded by a ring of bare mangrove.This green patch was identified as the sole nest of lesser rice-field rats and was characterized by a mix of fresh and dried branches and leaves.These branches and leaves were directly broken off from the adjacent mangrove, a phenomenon not observed in other trees in the region.K. obovata comprises a mixture of these traits, with a mature leaf area of 1546 mm 2 and a specific leaf area of 5594.42 mm 2 /g, as well as a weak K-strategy (Table 2).The leaf lifespan of K. obovata was 16 months, with growth points located at the terminal buds, a height of 2-3 m, and no ability to regenerate new branches or leaves from the base of the trunk (Table 2).

The Role of R. losea in Mangrove Mortality
In the drone-captured images of the mangrove (Figure 5), the center of the mortality zone features a distinct green patch surrounded by a ring of bare mangrove.This green patch was identified as the sole nest of lesser rice-field rats and was characterized by a mix of fresh and dried branches and leaves.These branches and leaves were directly broken off from the adjacent mangrove, a phenomenon not observed in other trees in the region.

The Role of R. losea in Mangrove Mortality
In the drone-captured images of the mangrove (Figure 5), the center of the mortality zone features a distinct green patch surrounded by a ring of bare mangrove.This green patch was identified as the sole nest of lesser rice-field rats and was characterized by a mix of fresh and dried branches and leaves.These branches and leaves were directly broken off from the adjacent mangrove, a phenomenon not observed in other trees in the region.

Extensive Harvesting of Fresh R. stylosa Branches for Nest Building
Lesser rice-field rats tended to build nests in the higher canopy of R. stylosa, which required the breaking of numerous small branches to enable nest construction (Figure 6).On average, approximately 57 small branches (length ~11 cm, fresh branch ratio ~19%) were required to build one rat's nest, and lesser rice-field rats need fresh branches to reinforce the nest, reflecting the rat's intensive use of R. stylosa branches.However, 125 branches (length ~25 cm, all dead branches) were required to build one egret's nest.
Lesser rice-field rats tended to build nests in the higher canopy of R. stylosa, which required the breaking of numerous small branches to enable nest construction (Figure 6).On average, approximately 57 small branches (length ~11 cm, fresh branch ratio ~19%) were required to build one rat's nest, and lesser rice-field rats need fresh branches to reinforce the nest, reflecting the rat's intensive use of R. stylosa branches.However, 125 branches (length ~25 cm, all dead branches) were required to build one egret's nest.

Discussion
The comparable numbers of egrets at the two study sites but the vastly different mangrove mortality (Table 1) may be related to tree species or other factors, not necessarily egrets causing mangrove mortality.R. stylosa is a tall tree characterized by a low number of leafy branches (approximately 161 twigs per tree) and, resultantly, relatively few buds.Leaf renewal occurs in the terminal bud only (Table 1).R. stylosa suffered significant damage due to the frequent breaking of its branches and leaves during interactions with lesser rice-field rats (Figure 6).Moreover, broken branches and leaves may become pathways for pathogen invasion, increasing the risk of plant infection and increasing the susceptibility of damaged tissues to fungi, bacteria, and other pathogens.Compared to other mangrove plants, such as Avicennia and Aegiceras, R. stylosa has weaker recovery capabilities, reducing its ability to recover rapidly from mechanical damage.Importantly, this damage directly affects the growth and development of R. stylosa.Broken branches and leaves cannot continue to grow normally, leading to stunted growth in the affected areas.This growth restriction impacts both the appearance and structure of the plant and produces long-term negative impacts on its overall growth potential and reproductive capacity.Therefore, the recovery and regenerative abilities of R. stylosa are severely challenged and require the implementation of effective protection measures to mitigate the effects of lesser

Discussion
The comparable numbers of egrets at the two study sites but the vastly different mangrove mortality (Table 1) may be related to tree species or other factors, not necessarily egrets causing mangrove mortality.R. stylosa is a tall tree characterized by a low number of leafy branches (approximately 161 twigs per tree) and, resultantly, relatively few buds.Leaf renewal occurs in the terminal bud only (Table 1).R. stylosa suffered significant damage due to the frequent breaking of its branches and leaves during interactions with lesser rice-field rats (Figure 6).Moreover, broken branches and leaves may become pathways for pathogen invasion, increasing the risk of plant infection and increasing the susceptibility of damaged tissues to fungi, bacteria, and other pathogens.Compared to other mangrove plants, such as Avicennia and Aegiceras, R. stylosa has weaker recovery capabilities, reducing its ability to recover rapidly from mechanical damage.Importantly, this damage directly affects the growth and development of R. stylosa.Broken branches and leaves cannot continue to grow normally, leading to stunted growth in the affected areas.This growth restriction impacts both the appearance and structure of the plant and produces long-term negative impacts on its overall growth potential and reproductive capacity.Therefore, the recovery and regenerative abilities of R. stylosa are severely challenged and require the implementation of effective protection measures to mitigate the effects of lesser rice-field rats' activity.It was determined that the nest-building behavior of lesser rice-field rats significantly impacted the health and growth capabilities of R. stylosa.The collection of withered and fresh buds, as well as the number of dead and new leaves from the nest, clearly demonstrated the detrimental effects of lesser rice-field rats on the growth of R. stylosa.The loss of branches and leaves not only reduces the photosynthetic potential of the tree but also decreases the surface area available for effective transpiration, leading to an imbalance in water regulation.In particular, the frequent removal of fresh branches may damage tree growth points that are crucial for plant recovery and growth.These characteristics (Table 2) make R. stylosa more susceptible to phenomena such as leaf herbivory, which results in the death of branches [27].In this study, we showed that R. stylosa mortality is closely linked to its biological characteristics as well as external environmental stresses [27].Increased branch breakage impacts the growth of R. stylosa, ultimately leading to tree mortality.The relationship between mangrove mortality trends and egret roosting sites in the Shankou Yingluo mangrove (Figure 3) implies that egrets may, to some extent, impede the spread of the R. stylosa mortality zone toward the egret aggregation area.In addition, this phenomenon also refutes the previous hypothesis that bird fecal pollution leads to mangrove death, as the area with the highest quantity of bird feces within the egret aggregation zone did not exhibit widespread R. stylosa mortality.This trend further supports a non-correlation between egret aggregation and R. stylosa mortality.
No signs of egret activity in Figure 5, such as nesting or other characteristics, were identified in the images.Lesser rice-field rats were the only observable disturbance factor within this area.It can, therefore, be reasonably inferred that the pattern of tree mortality centered around the nest was caused by the activity of lesser rice-field rats.The presence of fresh branches and leaves in the nest indicated that it was an active nest, with lesser rice-field rats likely continuing to gather branches from the surrounding R. stylosa trees during the study period.Rats, being omnivorous, have highly variable diets, likely in response to available prey.Their diet includes plant materials [28,29], as well as small animals like grasshoppers, spiders, cockroaches, and flies [30,31].Additionally, rats also consume seabird eggs and sea turtle hatchlings as a food source [29,32].In coastal areas, rats are widely distributed in mangrove forests [33,34].Lesser rice-field rats prey on small birds [24], Sparsparia alterniflora [35], mangrove seedlings [36], and propagules [37][38][39].In some instances, lesser rice-field rats may feed on food remnants discarded by egrets during rearing.This behavior could increase the survival and reproductive capacity of lesser rice-field rats in mangroves, thereby exacerbating their destructive effects on R. stylosa.These complex food chain relationships and ecological interactions may have indirect but significant effects on the growth and survival of R. stylosa.While this study primarily focused on the effect of lesser rice-field rats on the mortality of R. stylosa in the Guangxi Shankou Mangrove Reserve, it may prove valuable to explore the potential interactions between egrets and lesser rice-field rats.The growth and recovery abilities of R. stylosa are relatively weak, making it more susceptible to frequent branch-breaking nest-building behaviors by lesser rice-field rats.
This study conducted a comparative analysis of the effects of lesser rice-field rats on mangrove forests in Shankou Yingluo and Dangjiang Yanlou, to investigate the processes and mechanisms behind the mortality of R. stylosa in the Guangxi Shankou Mangrove Reserve due to the branch-breaking nest-building behavior of lesser rice-field rats.A core finding of this study was that the primary cause of R. stylosa mortality was the activity of lesser rice-field rats rather than the previously assumed egret behavior (Figures 3 and 5).
Notably, the effect of lesser rice-field rats' nesting behavior on R. stylosa far exceeded the capacity of the ecosystem to adapt, particularly in areas without egret activity.Thus, the death of mangroves is a result of the combined effects of the biological characteristics of tree species, and lesser rice-field rats' activities.
These findings hold significant theoretical and practical implications for future studies.Firstly, research on the biological characteristics of R. stylosa has unveiled its vulnerability to external disturbances, offering crucial insights for selecting suitable species for ecological restoration and protection efforts.Additionally, the direct impact of lesser rice-field rats on R. stylosa emphasizes the profound effects of biological activity on ecosystems, necessitating scientific management to protect the ecological balance.
However, the interactions between egrets and lesser rice-field rats warrant further investigation.These interactions may play a significant role in mangrove ecosystems and are crucial for understanding and managing the ecological health and viability of R. stylosa.Future research should aim to determine the impacts of the relationship between egrets and R. losea on the overall health and stability of mangrove forests.
Certain limitations were noted during this study.Laboratory studies on the growth and recovery abilities of R. stylosa may not fully simulate complex natural environments.Ultimately, more in-depth research will enhance our comprehension of the dynamic changes occurring in mangrove ecosystems, thus furnishing a scientific foundation for formulating effective protection and management strategies.

Conclusions
In this study, we investigated the increased mortality of R. stylosa observed in the Shankou Mangrove Reserve in Guangxi, China, under extreme environmental conditions.Egret nesting behavior was initially suspected to be the cause; however, our findings indicated that the biological characteristics of the trees, such as low branch numbers and a reduced ability to recover after damage, resulted in an increased vulnerability to external disturbances.
A critical external factor contributing to the mortality of R. stylosa revealed in this study was the nesting behavior of lesser rice-field rats [6].This mangrove rate species frequently collects branches from R. stylosa for nest building, directly leading to obstructed photosynthesis and a disrupted water balance in the trees.Although high-salinity conditions and a lack of freshwater influx into rivers pose certain threats to the survival of R. stylosa, they are not the main drivers of its death.
The results of this study have significant implications for the protection and management of mangrove ecosystems.Our findings highlight the need to consider climate change, the biological characteristics of the species, and the impact of biological activities when devising mangrove conservation measures.To mitigate the adverse effects of lesser rice-field rats on R. stylosa, comprehensive ecological management measures are required, including enhanced habitat protection (conduct of anti-rodent operations), increased biodiversity (planting a variety of mangroves in dead zones), and the scientific management of animal populations within mangroves.
Future research should explore how ecological engineering and the restoration of natural ecosystems can enhance the adaptability and resilience of R. stylosa and other mangrove plants.Additionally, research should focus on balancing the survival needs of animals with ecological conservation objectives while protecting mangrove ecosystems.

Forests 2024 , 11 Figure 3 .
Figure 3. Relative positions of the contiguous mangrove mortality areas from 2019-2022, along with the perennial gathering area of egrets in Shankou Yingluo.

Figure 3 .
Figure 3. Relative positions of the contiguous mangrove mortality areas from 2019-2022, along with the perennial gathering area of egrets in Shankou Yingluo.

ForestsFigure 4 .
Figure 4. Regeneration of new branches and leaves sprouting from the trunk of Aegiceras corniculatum (A) and Avicennia marina (B).

Figure 4 .
Figure 4. Regeneration of new branches and leaves sprouting from the trunk of Aegiceras corniculatum (A) and Avicennia marina (B).

Figure 4 .
Figure 4. Regeneration of new branches and leaves sprouting from the trunk of Aegiceras corniculatum (A) and Avicennia marina (B).

Table 1 .
Comparative Analysis of Egret Populations in the Mangroves of Shankou Yingluo and Dangjiang Yanlou.Comparison Item Shankou Yingluo Egret Forest Dangjiang Yanlou Egret Forest

Table 1 .
Comparative Analysis of Egret Populations in the Mangroves of Shankou Yingluo and Dangjiang Yanlou.

Table 2 .
Comparative Ecological Characteristics of Four Mangrove Plants.