The Influence of Environmental Factors on the Behavior and Mortality Risk of Polypedates megacephalus Tadpoles

Jakub PACOŃ ,Wojciech KRUSZYŃSKI and Jarosław PACOŃ

1 Department of Genetics,Wrocław University of Environmental and Life Sciences,Kożuchowska 751-631 Wrocław,Wrocław 51631,Poland

2 Department of Internal Medicine and Clinic of Diseases of Horses,Dogs and Cats,Wrocław University of Environmental and Life Sciences,Pl.Grunwaldzki 4750-366 Wrocław,Wrocław 50366,Poland

Abstract Since tadpoles change their behavior when facing adverse environmental conditions,observing tadpoles in a tank can help detect problems in their maintenance.Detecting such abnormal behavior early,an amphibian breeder can reactquickly,by changing the conditions.If quick and appropriate,such a reaction can save unnecessary deaths.The paper presents an experimental study on the behavior of tadpoles of Polypedates megacephalus in various adverse environmental conditions.In addition to a control 25-liter tank,5-liter tanks with 10 tadpoles each were studied,each having various improper conditions.Observations included the degree of tank exploration by the tadpoles,their mortality,anomalies in swimming behavior,and eating behavior.

Keywords Polypedates megacephalus,tadpole,mortality,the spot-legged tree frog

1.Introduction

One of the most difficult moments amphibian breeders face is the transformation of a tadpole into an adult.To make this process possible,tadpoles need appropriate temperature and water parameters,but also food that meets their needs.Constant and appropriate environmental conditions are sometimes difficult to impose,and their fluctuations often give negative effects—even an increase in mortality.

Tadpole abnormal behavior can mean that their welfare is affected,quite often leading to an increased tadpole mortality.Thus,breeders should observe tadpoles,paying particular attention to any atypical behavior,which can signal environmental anomalies in the tank.Noticing such changes,the breeder should quickly react,by checking the conditions in the tank andmaking appropriate changes,if water parameters changed in an undesirable way.

Tree frogs constitute one of the most popular amphibian families in amateur breeding,this popularity resulting from a wide range of their colors,from intensely green (

Hyla cinerea

,Lamb and Avise,2006) to emerald (

Litoria caerulea

,Manzano

et al.

,2008).Widespread,tree frogs occur on all the continents except for Antarctica.Their characteristic feature is that pads at the tips of their toes enable them to move on vertical and flat surfaces (such as glass).The spot-legged tree frog (Figure 1) (

Polypedates megacephalus

)is a tree frog living,among others,in India (Ao

et al.

,2013) and Thailand (Ningombam and Bordoloi,2013).Temperatures in both these regions can be very high,and to survive in such extreme conditions,anuras developed so-called metabolic compensation,a specific physiological strategy they employ to minimize the influence of water temperature on their internal physiological processes (Wu

et al.

,2007).Thanks to this process,this species also occurs in Taiwan,where it is classified as invasive (Lee

et al.

,2019),and in the Guam island,located in Micronesia in the western Pacific Ocean,where it is considered an alien species,as it did not live in this area naturally (Christy

et al.

,2007).The current literature has focused on determining how environmental pollution affects the behavior and mortality of various amphibian species,such as

Rana clamitans

(Boone and Bridges,2003) and

Litoria citropa

(Broomhall,2002).Knowledge on tadpole behavior in abnormal breeding conditions,however,is lacking,even though it could help amphibian breeders,with atypical behavior being used as an indicator of adverse breeding conditions,thereby offering them clues for what they could do to improve the conditions.

The present research aims to link atypical tadpole behavior to abnormal environmental conditions that increase the mortality of tadpoles in breeding aquariums.

2.Material and Methods

2.1.Tadpoles

The material studied constituted 90 tadpoles of the spot-legged tree frog (

Polypedates megacephalus

).All the specimens were full siblings and originated from the same foam nest,with their parents having been about four years old when the cocoon was laid.The tadpoles hatched in July 2017,and the study was conducted from July to the end of September 2017.

2.2.Experimental conditions

Eighty tadpoles were introduced into eight cubic containers (so-called boxes) with a capacity of 5 liters,10 specimens per box,each having different environmental parameters (temperature,type of water,and type of food).Ten specimens introduced into a 25-liter cubic aquarium (box 9) with standard conditions (in accordance with the instructions by the caretakers of the Terrarium of the Wroclaw Zoological Garden) constituted a control.The aquarium and the boxes were placed in a room devoted to this experiment;no other animals,including tadpoles of other species,were kept there.The room had two windows and artificial light,turned on during the day and turned off during the night,so a 12-hour photoperiod was maintained during the experiment.The aquarium was made of glass,and the other boxes were made of plastic dedicated for keeping animals.

Figure 1 Polypedates megacephalus during mating embrace.(Property of the authors)

The different environments were intended to provoke abnormal behavior.We recognized abnormal behavior as behavior that deviated from the behavior we had previously observed in healthy tadpoles and from the norms set by breeders and caretakers from the Wroclaw Zoological Garden.Likewise,we understood correct environmental conditions as amphibian breeders and caretakers do:A container should be adapted to the number of individuals,and it should have a permanent filter and a neutral pH of water (Table 1). Table 2 shows the conditions in the individual boxes.

The specimens from the 5-liter boxes were observed three times a day (every day):between 7 and 8 a.m.,about noon(which observation was combined with feeding),and about 2 p.m.Each observation lasted at least five minutes per container.The observations were made by two people,directly in the experimental room.During the observations,the behavior of tadpoles from the experimental boxes was assessed and compared with that of the individuals from the control aquarium.

The tadpoles from the protein-fed and mixed-fed boxes were fed with two feeding sticks (per box) for the first month;then they grew up,and so were fed with three feeding sticks per box.Those from the mixed-fed and Ketapang-fed boxes were fed with one leave per box;it was slightly moistened,so that it was not too hard to eat.Feeding took place three times a week:onMondays,Wednesdays,and Fridays.Water was changed three times a week and when a specimen died,using the following procedure.The tadpoles were fished out using a net,and each was placed in a separate container (a plastic cup) filled with the same type of water that was in the container.Then the container was emptied,cleaned using osmotic water,and then refilled with fresh water.The tadpoles were let in by pouring the contents of the cups from a small height,so that they would not be accidentally injured.

Table 1 Mineral ingredients of the water used in the experiment.

Table 2 Environmental conditions in the nine boxes in which the tadpoles of the spot-legged tree frog (Polypedates megacephalus) were kept.

The following parameters were analyzed:

Mortality

Expressed in percentages per box,it was determined as the ratio of the number of tadpoles that died during the experiment to all the tadpoles kept in the box.

Eating behavior

Expressed in percentages per box,it represents the interest of the specimens in the food they received;so,eating behavior was represented by a share of individuals interested in food (that is,coming straight to food immediately after feeding),relative to all the individuals in the box.The observations were carried out immediately after feeding (after introducing the food into the container).In the case of mixed food (box 2),the parameter was determined as the mean value for both types of food:protein and plant food.

The degree of tank exploration

This parameter represents the behavior of all the specimens in the given box.The behavior was scored on a scale of 1 to 5,where

1—no exploration:specimens almost do not move and so do not explore the container at all or almost at all (90%-100% of the individuals do not move during the entire observation time);

2—slight exploration:specimens seldom move and slightly explore the box (60%-80% of the individuals do not move during the observation;those which move do it very slowly,stay motionless for 80% of the observation time,and do not explore more than half of the box);

3—moderate exploration:specimens move often but slowly,and they do not explore the whole box (40%-50% of the individuals do not move during the observation;those which move do it slowly,stay motionless for 60%-70% of the observation time,and explore over half of the box—but not the whole of it) ;

4—slow exploration:specimens explore the whole box,moving often but slowly (0%-30% of the individuals do not move;those which move do it fastand explore the whole box,though staying motionless for 20%-40% of the observation time);

5—intensive exploration:specimens move quickly and explore the whole box (all the individuals move;they stay motionless for 0%-10% of the observation time and explore whole box many times).

A given level of exploration was noted for analysis if it lasted for the entire observation of tank.If the individual began to exhibit behaviors assigned to another level,then the observation was extended for another minute and the noticed level that was the longest was noted.

Anomalies in swimming behavior

Expressed as the number of specimens behaving abnormally per box,it determines whether any anomalies occur in movement direction,like swimming only in one plane (for example,front-back or updown) or even no swimming (meaning no exploration of the box).Such a phenomenon was considered an anomaly when it was occurring continuously for at least twenty consecutive observations,when it lasted until the end of the experiment,or if the specimen showing this abnormal behavior died.

Statistical analysis

The relation between mortality and the degree of tank exploration was analyzed using Spearman’s correlation (because the variables were not normally distributed) while that between mortality and eating behavior using Pearson’s correlation.The analyses were performed in Statistica v.13.3.

3.Results and Discussion

The degree of tank exploration significantly negatively correlated with mortality (Spearman’s correlation of

r

=-0.92) (Table 3):The higher the degree of tank exploration,the lower the mortality in the tank.Mortality,however,was not significantly related with eating behavior (Pearson’s correlation of

r

=0.30).This phenomenon was well represented by box 6,which had full mortality despite the optimal eating behavior (all the specimens showed immediate interest in the food).In fact,all deaths (in all the boxes) took place during the first month of the experiment.Figures 2-10 show changes in behavior and mortality.None of the tadpoles in the control tank died,and all showed interest in exploring the tank (showing the highest degree of tank exploration),a phenomenon suggesting that the degree of tank exploration can be used to decide whether the environmental conditions in a tank are appropriate or not.A lower exploration degree can result from the intoxication of a tadpole’s organism.Fioramonti

et al

.(1997) and Lefcort

et al

.(1998) reported similar results for tadpoles of several species of the genus

Rana

.In both cases,unfavorable water parameters—variable pH in Fioramonti

et al.

’s study and contamination with heavy metals in Lefcort

et al.

’s—were associated with an increase in tadpole mortality.In addition,Lefcort

et al.

noted that heavy metals could impair tadpoles’ motor reactions associated with danger.Temperature fluctuations do not have to strongly influence tadpole survival,as demonstrated by Broomhall (2002).In her study on

Litoria citropa

tadpoles,a variable temperature cycle increased the tadpoles’ mortality only when they were exposed to sublethal endosulfan concentrations.When not exposed to this compound,tadpoles exposed to a variable temperature cycle had similar mortality as those under stable temperature.In their research on

Polypedates megacephalus

,Wu

et al.

(2007)showed that tadpoles of this species had high metabolic compensation,a process enabling them to grow properly in varying temperature,reaching up to 30°C.To the best of the authors’ knowledge,no one has studied so far whether feeding frequency affects tadpole behavior.Anholt

et al.

’s (2013) study on the genus Rana,however,showed that the availability of food affected the amount of time tadpoles spent exploring the tank.In their research,the increased amount of food decreased both the time the tadpoles spent on exploration and their swimming speed.

Anomalies in swimming behavior were so rare—only two tadpoles showed them—that they might be considered an atypical phenomenon even in such unfavorable environmental conditions (Table 2).Thus,they were not considered in the statistical analyses.Since these two instances occurred in tanks with high mortality,one might suppose that the two phenomena could be correlated,both resulting from stressful environmental conditions.To confirm this observation,however,a study is needed in which such anomalies would be more frequent than in this experiment,where they were too rare to be used for such interpretation.

As is possible in any biological experiment,some tadpoles might have died not due to the environmental factors,but due to their overall worse conditions—especially because tadpoles are characterized by rather large inter-individual variability.

Table 3 Observations of the tadpoles of the spot-legged tree frog (Polypedates megacephalus) made in nine different conditions.

Figure 2 Changes in behavior and mortality in box 1.

Figure 3 Changes in behavior and mortality in box 2.

Figure 4 Changes in behavior and mortality in box 3.

Figure 5 Changes in behavior and mortality in box 4.

Figure 6 Changes in behavior and mortality in box 5.

Figure 7 Changes in behavior and mortality in box 6.

Figure 8 Changes in behavior and mortality in box 7.

Figure 9 Changes in behavior and mortality in box 8.

Figure 10 Changes in behavior and mortality in box 9.

Such high variability results from the breeding strategy of this species,aiming at the quantity rather than the quality of offspring.In Karraker and Dudgeon’s (2014) study,females of

P.megacephalus

deposited foam nests consisting of as many as 245 to 461 eggs.The authors,however,did not know the parental history of the specimens studied,which hindered interpretation and,to some extent,blurred the conclusions.

Unfavorable environmental conditions in a tank can increase mortality of the tadpoles living in it.In the most severe situations,mortality can reach 100%,as it did in four out of eight experimental tanks in our study.It is unlikely,however,that tadpoles would not change their behaviorbefore dying.They can,for example,start to move slowly,change their swimming behavior,decrease or even cease eating,or limit their exploration of the tank.In other words,poor conditions in a tank can take away the tadpoles’ vitality and interest in anything around,including food.

4.Conclusions

Observing tadpole behavior offers a simple method for a breeder to notice that something can be going wrong in the tank.But to correctly interpret such misbehavior,however,he or she has to be equipped with sufficient knowledge.This study is likely the first to offer such knowledge,and more like this are needed to extend it.

Having noticed abnormal tadpole behavior due to adverse environmental conditions,a breeder can react by improving them.Well-chosen and quick enough,such a reaction can make it possible to undo the damage—or at least part of it.Even mere observation of tadpoles’ exploratory behavior can help detect possible problems with the conditions in the tank.It is possible that observing swimming anomalies can also be useful,but during the study they were observed only for two specimens from different tanks,in both of which mortality was very high.This aspect thus needs further research.

What we have learned from this research is that poor environmental conditions in a tank make tadpoles of the spot-legged tree frog change their behavior,in various ways.Future research should focus on the recovery after noticing tadpole misbehavior and reacting to it.For example,how quickly should the breeder correct the conditions in order to avoid increased mortality? Are there kinds of tank pollution that cannot be corrected? Such wide knowledge will allow for creating useful recommendations for amphibian breeders.

Since similar studies have not been conducted on other amphibian species in amateur breeding,we have no grounds to generalize the conclusions—here drawn for the spot-legged tree frog—to other species.In order to do so,more studies dealing with tadpoles of different species are needed,especially those popular in amateur amphibian breeding,such as poison dart frogs.