Effect of Fungi Presence on Wing Morphometry and Flight Behaviour in the Common Pipistrelle Bats ( Pipistrellus pipistrellus )

Bats play an important role in ecosystem function, serving as a biocontrol agent of insects and pollination in plants, hence the need to understand the factors affecting their health and performance. Infestation of fungi has been identified as a potential threat to populations of mammals, especially bats, yet their specific effects on wing morphology and flight behavior remain understudied. In this study, swabs of Pipistrellus pipistrellus bat wings were taken from bat carers around the United Kingdom. For identification, samples were exposed to conventional PCR using fungal-specific primers to amplify the ribosomal DNA's internal transcribed spacer (ITS). The wind morphometric parameters such as wing angles (maximum, minimum, mean, and amplitude) and wing frequency were determined for both wings with fungi infestation and without fungi infestation. A rain cloud distribution plot with a box plot was used to determine the distribution of flight parameters of the wings. A principal component analysis biplot was used to determine the relationship between the flight parameters in terms of wings with infestation by fungi and without infestation. The findings in this study are important to bat biology, ecology, and conservation and give insights into the need for wildlife management and conservation, ultimately contributing to the ongoing efforts to preserve bat populations and support ecological balance in diverse ecosystems.


Introduction
Bats are mammals belonging to the order Chiroptera and comprise about 20% of mammalian diversity [1- 3] .Bats play a crucial role in the ecosystem such as seed dispersion, pollination, and control of insect populations [4][5][6][7] .Bats also help to maintain ecosystem stability [7] .
In the realm of mammals, bats stand out as the sole creatures endowed with the ability of powered flight [2-3]   .This distinctive trait merits exploration in its own right and serves as the foundation for further discussion.Regarding flying behaviour, bats usually fly at night to forage for food [7] .Regarding flight membranes in bats, which are called patagia, they comprise 85% of the body surface area of a bat [8] .
The membranes are made up of 4 sections; the first section is called the protopatagium, which spreads from the bat's shoulder to the thumb; the second section is called chiropatagium (or dactylopatagium), which is the skin between fingers 1 and 5; the third section is called the plagiopatagium, which is the skin between the fifth fingers and the body trunk, [9][10][11] .The fourth section involves a tail membrane between the hind limbs, found in various species of bat and called the uropatagium [10,11] .The bat wings have an important role in critical for powering flight, as all those sections have a function in bat flight [2,12] .However, the large and thin wing membranes make them particularly susceptible to injuries, holes, and tears [8, 13]   .
As with many animals, bats face some threats during their lives; those threats could be related to anthropogenic activities such as light pollution, noise, collisions with man-made structures, and habitat loss [7,[14][15][16] .On the other hand, bats could face other natural threats, such as weather, fungal infections, and cat predation [17][18][19][20][21] .Some of those threats affect the bat wing, thus affecting bats' flight behaviour.
One of the critical fungal infections in bats is caused by a fungus called keratin-digesting fungus (Pseudogymnoascus destructans), which infects the bat's skin and causes a disease called White-Nose Syndrome (WNS) [18][19][20][21][22][23] .That fungus grows quickly through the winter during the hibernation season for bats [24] .Through the hibernation season, bats experience some factors that permit the fungus to grow and spread commonly among the bats' populations, such as deficiency of food and water, a reduction in metabolism, immune function, and body temperature, so bats commonly avoid losing moisture by find humid or heavily populated areas for hibernation [18][19][20]22] .
This infection by WNS kills millions of bats and threatens some bat species in the USA and Canada [22-23]   .Moreover, the WNS-affected Little Brown Bats (Myotis lucifugus) during the winter, the wings of the infestation bats seemed crumpled due to losing their elasticity and they were no longer strong so they would tear easily [19,24] .Additionally, the microscopic analysis of the affected wings by P.
destructans infestation, exposed damage to some glands as a result of the skin corrosion as the fungus digests the wing skin, then which would influence all structures in the wing such as connective tissues and blood vessels [19,24] .The P. destructans not only infects the skin, but it could interrupt the physiological homeostasis which causes bat mortality [19] .
The P. destructans fungus has been identified in European hibernating bats [25,26] .Moreover, it was identified by molecular analysis in several European countries, including the United Kingdom [26] .
In this study, we aim to 1) investigate the fungal presence in the wing of Common Pipistrelle bats (Pipistrellus pipistrellus) in the United Kingdom and detect the fungal species; 2) identify the effect of fungal presence on bat flying behavior in those bats.

Collected samples:
Swabs of P. pipistrellus bat wings were collected from bat carers around the United Kingdom.The total number of samples collected was 72.All the collected swabs were stored at -20C before genetic analysis.

Fungal DNA
The internal transcribed spacer (ITS) region of ribosomal DNA from fungi was amplified by standard PCR on the materials using primers specific to fungi [27,28] .In a total volume of 20 μL, the following reagents are used in the DNA amplification reactions: 0.5 μM ITS1 forward primer (5'-CTTGGTCATTTAGAGGAAGTAA-3') (Invitrogen, Thermo Fisher Scientific), 0.

Agarose gel electrophoresis
The amplified PCR products were performed on a 1% agarose gel in 1x TBE to evaluate the PCR results.The PCR results were placed onto the gel in an aliquot of 10 μL along with 5 μL of a DNA size ladder (HyperLadder TM 100 bp, Bionline, UK).Gels were made in accordance with the procedure outlined in section 5.2.4.

Sanger sequencing
Sanger

Phylogenetic tree
To find the relationship between the fungal DNA sequences, a phylogenetic tree was created using MEGA7 software (version 7.0.26)(https://www.megasoftware.net) and aligning sequences using the Maximum Likelihood method [30]   .

Flight behaviour analysis
From all the bat wing swabs that were received for genetic analysis, 12 P. pipistrellus bats have been filmed over 3 summers around the United Kingdom.
The bats were in good condition without any wing injuries.From all specimens, 7 bats did not reveal any fungal infection, while five bats obtained fungal infection.The filming protocol that was used was mentioned in Khayat et al. (2020) [31] .
The wing angles were measured in degrees to

Fungal analysis
The bat swab samples were analyzed and showed the presence of fungus in 24 samples out of all samples (33.33% of cases).The gel results exposed PCR products in 24 samples ranging from 265bp to 297bp (Fi.1), which is expected for the ITS1 and ITS2 regions [32] .The positive control also exposed a PCR product at 450 bp, also within the expected size range of S. cerevisiae [32,33] .Moreover, the sequencing results validate S. cerevisiae spp., with a 90% similarity match.The negative control was pure, which represented no contamination.
Figure 1: Photo shows the agarose gel electrophoresis of the PRC products of the amplified fungal DNA.The numbers above each lane refer to the number of bat swabs, +ve is the positive control, and -ve is the negative control.
DNA sequencing of these samples showed the presence of 12 different fungal species (Fig. 2, 3), which are usually found in the environment, like water, soil, air, and plants.The most common species in a quarter of the samples was Cladosporium (Fig. 3).The phylogenetic tree shows the presence of 8 different families, which are Sydowia, Cladosporium, Penicillium, Trichoderma, Aureobasidium, Coniochaeta, Talaromyces, Septoria (Fi.3).Bat Wing Morphometry Fig. 4 is the distribution and rain cloud plot for bat wing morphometry for bat wings with fungi and wings with no presence of fungi for the left (Fig. 4a) and right (Fig. 4b) wings.In this plot, the max.angle morphometry is more distributed in both the left and right bat wings with no fungi infestation.However, higher distributions were recorded at the final count for both max. the angle of the left and right wings.The wing without fungi infestation still recorded more distribution in terms of the max.angle.Additionally, the initial counts for both the right and left wings in terms of no fungi and fungi infestation were sparse, while the max.The angle distribution count was higher in the right-wing than in the left (Fig. 4b).The morphometry of the wing was highly magnified in terms of the distribution following almost normal distribution of the count for the mean angle of both the left and right wings, irrespective of whether they had been infected with fungi (Figs.6a and b).However, the distribution count was higher in the mean angle of the wing without fungi infestation for both left and right.It is important to note that for the mean angle of the left wing, even though the count was higher in the wing with no infestation, sparse distribution was recorded at the highest count of mean angle morphometry.For the right-wing mean angle, the distribution was normal in the wing without fungi infestation than what was observed in the wing with fungi infestation, even though a negligible sparse distribution was observed in the right mean angle of the wing without fungi infestation (Fig. 6b).The RMS results revealed the wing's morphometry to be highly magnified in terms of the distribution, with the count for left-wing RMS for the wing with fungi infestation following an almost normal distribution (Fig. s 7a and b).For the RMS of the left wing, the distribution count was higher in the wing without fungi infestation, although the distribution for both the wings with infestation and not was sparse at the initial count of the RMS (Fig. 7a).In addition, for the right wing, even though RMS count was higher in the wing without fungi infestation, the count for the RMS was almost normally distributed than the count in the wing without fungi infestation (Fig. 7b) Frequency as one of the variables used for the wing's morphometry was highly magnified in terms of the distribution, with the count for the left and right wings revealing the same patterns of distribution (Figs.8a  and b).However, a more normally distributed count was recorded for wings with no fungi infestation, while the highest peak count in terms of single value was recorded for wings with fungi infestation (Figs.8a and b).

Relationship Between Morphometric Parameters of Bat Wing
The group Principal Component Analysis (PCA) biplot for the relationship between Bat wing morphometric parameters in terms of the presence and absence of fungi infestation revealed components 1 and 2 accounted for a total variation of 49.9 % of the total variation (Fig. 9).In this result, a strong positive relationship exists between the min angle of the left and right-wing, the mean angle of the left and right-wing, and the RMS of the left and right-wing, max.angle of left and right wings, all in terms of the presence and absence of fungi infestation.The results from the plot revealed a strong association between the two groups as an influence on the relationship between the morphometric parameters (Fig. 9).As depicted in this study, the analysis of bat wing morphometry provides valuable insights into the impact of fungi infestation on the distribution of the maximum angle measurements for both left and right wings.Notably, the rain cloud plot reveals distinct patterns in the morphometric distributions under different conditions.The distribution and rain cloud plot for bat wing morphometry for wings with fungi and wings with no presence of fungi are presented for the left and right wings.Surprisingly, even in the presence of a fungi infestation, the maximum angle morphometry is more widely distributed in both left and right wings when there is no fungi infestation.This observation suggests that the absence of fungi may contribute to a broader range of wing morphological variations [36] .
However, it is noteworthy that higher distributions were recorded at the final count for both left and right wings, indicating potential changes in wing morphology over time [37] .
Interestingly, the wing without fungi infestation consistently exhibited a more extensive distribution in terms of the maximum angle.This finding aligns with a study, which demonstrated that fungal infestation in bat wings can lead to alterations in wing morphology [19] .Moreover, it was observed reduced wing flexibility and increased wing damage in bats with fungal infestation [19,24,38] .This supports our observation that wings without fungi exhibit a broader range of maximum angle measurements.
Furthermore, the initial counts for both right and left wings were sparse, categorized by the presence or absence of fungi infestation.This suggests there might be limited variability in wing morphometry at the outset.However, as the study progressed, the final count showed a higher distribution of maximum angle measurements, particularly in the right wing.This asymmetry in distribution between the right and left wings is consistent, where asymmetrical wing morphometry was observed in bat populations [39] .
The observed patterns in the distribution of minimum angle measurements for both left and right wings are in line with recent findings in the literature.as it has been reported that, the bats with fungal infestation exhibited restricted wing flexibility, leading to altered wing angles [40] .Our results align with this, as we observed a more widespread distribution of minimum angles in wings without fungal infestation, indicating greater morphological variability.
Intriguingly, from this study, higher counts were recorded at the initial count for both left and right minimum angles, and as the count increased, the distribution became more sparse.This temporal pattern suggests that there might be initial uniformity or limited variability in minimum angle measurements, and as the study progressed, a divergence in wing morphometry became evident.
Also, it has been documented that the dynamic changes in bat wing morphometry over time, support the idea that wing angles may undergo variations during specific phases of the study [41] .It is crucial to acknowledge that the observed patterns in minimum .The observed pattern may be attributed to the harmful effects of fungal infestation on wing flexibility and overall wing morphology, as reported by these studies [43] .
An interesting finding emerged when analyzing the mean angle of the left wing, where, despite the higher count in the wing without infestation, a sparse distribution was recorded at the highest count of mean angle morphometry.This suggests that certain angles may be less prevalent or exhibit higher variability even in the absence of fungal infestation.
This finding adds complexity to our understanding of mean wing angles and highlights potential asymmetries in the distribution of morphometric measurements [43] .For the right-wing mean angle, the distribution was more normal in wings without fungi infestation compared to those with infestation.
This aligns with the notion that fungal infestation may lead to asymmetrical changes in wing morphology, as reported by recent studies such as Zepeda Mendoza et al. (2018) [44] .Although a negligible sparse distribution was observed in the right mean angle of wings without fungi infestation, this further emphasizes the potential impact of fungal infestation on the overall distribution of mean wing angles.
The RMS results reveal a highly magnified distribution for the left wing, with the count for the wing with fungi infestation following an almost normal distribution (Fig. 7a and b).However, the distribution count for the left-wing RMS was higher significant changes in wing surface characteristics in bats with fungal infestation [45,46] .The sparse distribution at the initial count of RMS for both wings suggests that certain morphometric features may be less prevalent initially, regardless of the presence of fungi.The higher RMS count for rightwing in this study in wings without fungi infestation, and the count distribution was almost normally distributed compared to the wing with fungi infestation, even in the absence of fungal infestation, can be due to a typical distribution of morphological features across the wing surface.This finding is in line with the work of Bure and Moore (2019), who observed asymmetrical changes in wing surface morphology in bats with fungal infestation [47] .
As a key variable in bat wing morphometrics determined in this study, frequency exhibited highly magnified distributions in both left and right wings, suggesting bilateral symmetry in response to considered variables [48] .Notably, wings without fungi infestation displayed a more normally distributed frequency count, while those with fungi infestation exhibited a pronounced deviation from normal distribution, marked by the highest peak count.This indicates a potential wing morphology disruption under the influence of fungi [19] .The findings underscore the importance of considering external stressors in bat wing studies, with implications for understanding adaptive mechanisms and vulnerabilities, as it was found the potential wing morphology change affects the wing movement during flight [31] .
The correlation that exists between the minimum angle, mean angle, and RMS of both left and right wings, as well as the maximum angle of both wings, which also identified strong associations among different morphometric parameters in bat wings [49,50] .They emphasized the interdependence of wing angles, supporting our observation of a positive relationship between minimum, mean, and maximum angles [49,50] .Elsewhere, Hedenström (2023) highlighted the influence of external factors, such as fungal infestation, on multiple morphometric parameters [51] , corroborating our findings of an association between morphometric parameters and fungi infestation.
The PCA biplot indicates a cohesive influence of fungi infestation on the relationship between morphometric parameters, portraying the two groups distinctly. it has been demonstrated how fungi and external factors, such as environmental stressors, can impact the overall wing morphology in bats [52] .Our results underscore the importance of considering fungi infestation as contributing to the integrated nature of bat wing morphometry.

Conclusion
In calculate the flight parameters, which are maximum angle (Max.angle), minimum angle (Min.angle), mean angle (Mean.angle), and the Root Mean Square Amplitude (RMS).Also, the wing beat per second was determined in Hertz to calculate the wing frequency (FREQ).The raw data were subjected to Leven's test of homogeneity of variance and the Shapiro-Wilk test for normality before other analyses.A rain cloud distribution plot with a box plot was used to determine the distribution of flight parameters of the wings.A principal component analysis biplot was used to determine the relationship between the flight parameters in terms of wings with infestation by fungi and without infestation.R statistics software for Windows v.4.0.3 and SPSS v.23 were used for the analysis.

Figure 2 :
Figure 2: The 12 different fungal species found on P. pipistrellus bat wing.

Figure 3 :
Figure 3: Molecular Phylogenetic Tree analysis by the Maximum Likelihood method

Fig. 4
Fig. 4 Distribution of max.angle a) Left b) Right as a morphometric for bats' wings with fungi and wings with no presence of fungi.

Fig. 5
Fig. 5 Distribution of min.angle a) Left b) Right as a morphometric for bat wings with fungi and wings with no presence of fungi.

Fig. 6
Fig. 6 Distribution of mean.angle a) Left b) Right as a morphometric for bats' wings with fungi and wings with no presence of fungi.

Fig. 7
Fig. 7 Distribution of RMS a) Left b) Right as a morphometric for bat wings with fungi and wings with no presence of fungi.

Fig. 8
Fig. 8 Distribution of frequency a) Left b) Right as a morphometric for bat wings with fungi and wings with no presence of fungi.

Fig. 9
Fig. 9 Relationship between Bat wing morphometry in terms of presence and absence of fungi infestation angle distribution are complex and may be influenced by various factors, including environmental conditions and individual bat behaviour.The comprehensive assessment of wing morphometry, considering fungi infestation, adds nuance to our understanding of bat wing adaptations.123Thehigher count distribution in the mean angle of wings without fungi infestation aligns with recent studies by Vanderwolf (2022), which demonstrated that bats with fungal infestation exhibited wing structure alterations, leading to wing angle changes[42] in wings without fungi infestation, indicating potential alterations in the wing surface structure due to fungal infestation.This observation aligns with recent studies by Federici et al. (2022) and Hathaway et al. (2023), which demonstrated irrespective of fungi infestation, underscores the intricate nature of bat wing morphology.It suggests that alterations in one morphometric parameter may be mirrored in others.These findings align with Voigt (2013) and Forsythe et al. (2022) research, conclusion, our comprehensive investigation into both fungal infestation and bat wing morphometry has provided valuable insights into the intricate relationships governing bat adaptations.Fungal analysis revealed a high prevalence of fungi in bat swab samples, with Cladosporium being the most common species.The identification of 12 different fungal species, as confirmed by DNA sequencing, and the diverse phylogenetic tree underscore the environmental ubiquity of these fungi.The evaluation of bat wing morphometry using diverse parameters revealed nuanced responses to fungi infestation.Rain cloud plots effectively depicted the distribution patterns of wing angles, exposing heightened variability in wings unaffected by fungal infestation, a trend consistent with recent research on the impact of fungi on bat wing morphology.Furthermore, the RMS results underscored an amplified distribution of wing morphometry in reaction to fungal presence, emphasizing the intricate nature of these adaptations and suggesting potential implications for the flight behavior of the bats.The Principal Component Analysis (PCA) biplot explained a strong positive relationship among morphometric parameters, irrespective of fungi infestation.Components 1 and 2 collectively explained a substantial portion of the total variation, emphasizing the interconnectedness of wing features.These findings contribute to the broader understanding of the dynamic interplay between fungi infestation and bat wing morphometry, providing a foundation for further ecological studies and conservation efforts.The observed associations underscore the need for a holistic approach when examining the ecological implications of fungal infections on bat populations.