Sternal Keel Affects Swimming Speed in Giant Water Scavenger Beetles (Coleoptera: Hydrophilidae: Hydrophilini)

Canadian Journal of Zoology

Evidence of morphological adaptation to life underwater: Sternal keel affects swimming speed in giant water scavenger beetles (Coleoptera: Hydrophilidae: Hydrophilini)

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2020-0247.R1

Manuscript Type: Article

Date Submitted by the 23-Dec-2020 Author:

Complete List of Authors: Matsushima, Ryosuke; University of Tsukuba

Is your manuscript invited for consideration in a Special Not applicableDraft (regular submission) Issue?:

Aquatic beetles, gas gill, motion analysis, oscillatory movement, Keyword: submergence, water scavenger beetles, Hydrophilus acuminatus

Evidence of morphological adaptation to life underwater: Sternal keel affects swimming speed in giant

water scavenger beetles (Coleoptera: Hydrophilidae: Hydrophilini)

Ryosuke MATSUSHIMA1

1Laboratory of Conservation Ecology, Graduate School of Life and Environmental Sciences, University of

Tsukuba, Tsukuba, Ibaraki, Japan Draft

Correspondence: Ryosuke Matsushima, Laboratory of Conservation Ecology, Graduate School of Life and

Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan

E-mail: [email protected]

ORCID: https://orcid.org/0000-0001-5131-4147

Abstract

Fundamentally, insects evolved on land and secondarily inhabited aquatic environments multiple times. To live underwater, aquatic insects have acquired enormously variable morphological, developmental, physiological, and ecological traits, such as gas exchange systems and swimming-related characteristics. Giant water scavenger beetles of the tribe Hydrophilini (Coleoptera: Hydrophilidae) are characterised by the presence of sternal keel, which often extends posteriorly. Despite being a conspicuous morphological trait, its function remains unclear.

Here, I verified two hypotheses: keel affects (1) submergence time following air replacement as well as (2) speed and oscillatory movement during forward swimmingDraft in Hydrophilus acuminatus Motschulsky, 1854.

Submergence time was affected by body mass rather than keel removal; in other words, larger individuals replaced their gas gills more frequently. Keel removal reduced swimming speed by 12.5%. These observations support hypothesis (2) and are also consistent with previous speculations that sternal keel is a key adaptation to swim, but the results showed that the degree of oscillation was closely related to body mass but not keel removal. Further studies are warranted to elucidate precise factors through which the presence of keel increases swimming speed. Such studies would provide clues into understanding the associations amongst body size, swimming methods, and morphological traits.

Keywords: Aquatic beetles; gas gill; Hydrophilus acuminatus; motion analysis; oscillatory movement; submergence; water scavenger beetles

INTRODUCTION

Fundamentally, insects evolved on land, and some orders, including Diptera, Hemiptera, and Coleoptera,

secondarily inhabited aquatic environments multiple times (e.g. Andersen 1995; Jäch and Balke 2008). To live

underwater, these insects have acquired enormously variable morphological, developmental, physiological, and

ecological traits (Lancaster and Downes 2013; Bilton et al. 2019). One of the most specialised traits in aquatic

insects is gas exchange system. For instance, most adult diving beetles (Dytiscidae) and water boatmen

(Corixidae) use gas gills: they hold air bubblesDraft under elytra and hemielytra or trap them using a layer of

hydrophobic hair (Rahn and Paganelli 1968). The water bug Aphelocheirus sp. (Aphelochiridae) possess plastron

gills, which can fully satisfy body’s oxygen demand by diffusion of oxygen from water into the gas store

(Thorpe 1950; Flynn and Bush 2008; Seymour et al. 2015). Moreover, swimming-related traits of aquatic

insects, such as body shape and limb structure, are evidently modified in various ways. For instance, adult

dytiscids and gyrinids show dorso-ventrally flattened or streamlined bodies and oar-like legs possessing

swimming hair. These specialised morphological traits help energy-efficient swimming by minimising drag as

well as increasing stability and manoeuvrability (Nachtigall 1961; Ribera and Foster 1997).

Water scavenger beetles (Coleoptera: Hydrophilidae) comprise over 3000 described species that show nearly

global distribution (Short and Fikáček 2013). Many of these species are well-known as aquatic beetles inhabiting

various environments, including small ponds, stream margins, and wetlands. Some species inhabit both semiaquatic and terrestrial environments. Aquatic hydrophilids hold air bubbles on their ventral thorax and/or abdomen in addition to under wings, namely, gas gill breathers. Under air deficiency, these insects extend their antennae above water and send fresh atmospheric air to the gas store (Lancaster and Downes 2013). However, the air bubbles on the ventral side would make it difficult for them to maintain posture in the water. Indeed, several groups with smaller body sizes (e.g., Amphiops) move through the water with their ventral side above

(Angus 1966). As for their swimming method, they follow alternate-leg swimming (legs on either side are paddled alternately), producing a distinct side-to-sideDraft body movement, similar to the members of Haliplidae and

Curculionidae (Hughes 1958; Barr and Smith 1980). Majority of hydrophilids bear swimming hair on the tibiae and/or tarsi (Hughes 1958; Short and Fikáček 2013). Overall, the gas exchange system and swimming-related traits of hydrophilids play pivotal roles in enabling life underwater.

The tribe Hydrophilini contains some of the largest aquatic insects in the world, with some species exceeding a body length of 50 mm (Short 2010; Short and Fikáček 2013). Importantly, all members of this tribe are characterised by the presence of sternal keel, resulting from the fusion of meso- and metaventral elevations, which often extends posteriorly over the abdominal ventrites as a sharp spine (Hansen 1991; Short and Fikáček

2013). Interestingly, it is similar to the structure at the bottom of a ship—also called the keel. Although the genus Hydrophilomima (Hydrophilidae: Laccobiini) also has the sternal keel, it is not extend to the same extent

as in most Hydrophilini, indicating the sternal keel is unique to this tribe of aquatic beetles. However, despite

being a conspicuous morphological trait, its function remains unclear.

Typically, larger animals show higher oxygen requirements, and increase in oxygen storage capacity allows

them to submerge longer (Zeuthen 1953; Verberk et al. 2020). Therefore, members of Hydrophilini, which are

amongst the largest aquatic beetles, may exhibit specific traits to meet high oxygen demand. Additionally, since

they store air bubbles ventrally, where the keel is located, this structure may affect the ability to hold air bubbles

(Watanabe 1982; Dettner 2019). Previously, Barr and Smith (1980) hypothesized that sternal keel damps out

wobble, providing a more stable forward trajectory.Draft In the present study, to clarify the functions of sternal keel in

Hydrophilini, I examined the effects of keel removal on (1) submergence time following air replacement as well

as (2) speed and oscillatory movement during forward swimming.

MATERIAL AND METHODS

Study animal

Hydrophilius acuminatus Motschulsky, 1854 (body length, 33–40 mm) belongs to the family Hydrophilidae and

is distributed in China, Japan, Russia, Korea, Myanmar, Indonesia, and Taiwan (Hansen 1999). This species

usually inhabits lentic water systems, such as ponds and paddy fields (Satô and Yoshitomi 2018). From late July

to early August 2020, six males and six females of adult H. acuminatus were collected from paddy fields in

Tsukuba, Ibaraki Prefecture, Japan (36°11′33″N, 140°06′42″E, ca. 52 m above sea level). The individuals were not fed and maintained in plastic containers (31 cm × 16 cm basal width × 22 cm height) with moistened filter paper in the laboratory at approximately 25 °C. Within 12 h of collection, the keel of three males and three females was cut and filed using a nail clipper and a nail file. The ridge on the head side and the spine were cut and filed, and the raised section between the middle and hind legs was filed (Fig. 1a). The experiments were conducted within at least 48 h of collection, after confirming that their activity levels had not decreased.

Draft Experiment 1. Submergence time following air replacement

One individual with or without keel was placed into a plastic container (19.5 cm × 17.5 cm basal width × 10 cm height) filled with water up to 2.5 cm and containing a plastic net (13 cm × 10 cm) on the bottom (Fig. 1b).

Air-replacing behaviour was observed, and submergence time, defined as is the duration from when the insects extended their antennae above water surface and replaced gas gills to when they did so again, was recorded after every air replacement event. Ten consecutive observations were recorded for twelve individuals (with keel: three males and three females, without keel: three males and three females, n = 60 per treatment).

Experiment 2. Swimming speed and oscillatory movement

After experiment 1, the insects were transferred to a large plastic container (40.7 cm × 28.5 cm basal width ×

18.5 cm height) filled with water up to 12 cm. The insects were gently released at the water surface and allowed

to swim freely. This set was performed ten times for all twelve individuals (with keel: three male and three

female, without keel: three male and three female) and recorded using a digital video camera (HDR-XR500V,

Sony Corporation, Japan). From the videos of each individual, the three most linear swimming trajectories were

selected for motion analysis (n = 18 per treatment). The motion capture software Kinovea ver. 0.8.15 was used

to measure the speed and oscillatory movement during swimming (Video S11). First, the tip of the elytra at the

start of the swim was used as the start point (closed symbol in Fig. 1c) and that at the end of the swim was used

as the goal point (open symbol in Fig. 1c). SwimmingDraft speed was calculated based on linear distance between the

two points and the time required to move between them. Second, by tracking the tip of the elytra, the length of

its undulating trajectory was measured (Fig. 1c). In the present study, the difference between the linear distance

and trajectory of the tip of the elytra per unit distance was defined as the degree of oscillation. Wet weight (body

mass) was measured using an electronic balance (A & D Company, Limited, Japan) following experiment 2.

Statistical analyses

Data were analysed using R ver. 4.0.2 (R Core Team 2020). Generalised linear mixed models (GLMMs) with a

gamma error distribution were developed with a log link function using the ‘lme4’ (Bates et al. 2015) package in

1 Video S1 A movie of the swimming of Hydrophilius acuminatus in experiment 2 as 0.30× speed. The green

line shows the linear distance and the blue line shows the trajectory drawn by the tip of the elytra.

R to test the effects of keel removal, sex, and body mass on submergence time after air replacement, swimming speed, and oscillatory movement during swimming. These response variables were continuous and most closely fit a gamma distribution. All analyses were controlled for pseudo-replication by including individual identity as a categorical random variable.

RESULTS

Experiment 1. Submergence time following air replacement

Submergence time after air replacement was 10.57Draft ± 6.40 min (mean ± SD), reaching the maximum value of

35.97 min. Submergence time after air replacement was significantly associated with body mass; as such, larger individuals replaced their gas gills more frequently; however, this trait was not associated with keel removal and sex (Table 1 and Fig. 2).

Experiment 2. Swimming speed and oscillatory movement

With or without keel, the beetles swam with most of their bodies underwater and only part of their backs out of the water, with the ventral side facing downward in the water. Results of swimming speed are shown in Table 2a and Fig. 3. Swimming speeds significantly varied between individuals with or without keels. The swimming speed (mean ± SD) of individuals with and without keel was 13.39 ± 1.45 and 11.72 ± 1.57 cm·s-1, respectively

(Table 2a and Fig. 3). In other words, keel removal reduced mean swimming speed by 12.5%. However, there

were no significant effects of body mass and sex on swimming speed (Table 2a).

Regarding oscillatory movement during swimming, there were no significant effects of keel removal and sex

(Table 2b and Fig. 4); however, the degree of oscillation was significantly lower in larger individuals (Table 2b).

DISCUSSION

Results of experiment 1 did not support hypothesis (1). Submergence time after air replacement was affected by

body mass rather than keel removal; as such, largerDraft individuals more frequently replaced their gas gills (Fig. 2).

Typically, metabolic rates are positively correlated with body mass of insects. In other words, larger insects tend

to show more oxygen uptake (Addo-Bediako et al. 2002; Niven and Scharlemann 2005). Thus, the association

between submergence time and body mass may be explained by differences in the metabolic rates of individual

insects. In aquatic beetles, submergence time varies across species, activity, and water temperature (Madsen

1967; Calosi et al. 2007; Kehl and Dettner 2009). In my experiment, submergence time slightly varied within

individuals, even though water temperature was constant (approximately 25 °C). This variability may be

explained by differences in activities, such as swimming and walking, during submersion within individuals.

In experiment 2, the presence of keel increased swimming speed (Fig. 3). This result supported hypothesis (2),

which is consistent with the hypothesis put forth by Barr and Smith (1980) that the presence of keel is associated

with swimming. These authors also proposed that the development of a prominent keel is a key adaptation to damp out wobble (yaw). However, my results showed that the degree of oscillation was closely associated with body mass, but not with the presence or absence of keel (Fig. 4). Therefore, the increase in swimming speed due to the presence of keel may be explained by other factors that are unrelated to the reduction of oscillation.

Because the keel is hollow and its centre of gravity is close to the body axis, it is also unlikely that the keel plays a role in reducing the roll (rotation of the body axis). One possibility is that the keel may help them to maintain their body posture in the water by dividing the air bubbles to the left and right of the ventral side of the thorax.

The control of the posture would affect the swimmingDraft speed. To clarify the fluid dynamics affecting the difference in swimming speeds, additional studies, such as water tunnel testing or computational fluid dynamics, would be necessary. Moreover, such studies would provide clues into the evolution of keel in Hydrophilini. Of the 3000 described species of Hydrophilidae, only about 100 exceed the body length of 15 mm, and all of them belong to the tribe Hydrophilini (Short 2010). In addition to alternate-leg swimming in Hydrophilidae, which produces lateral instability, gigantism in Hydrophilini may also have influenced the acquisition of keel. In this light, the results of the present study may help for understanding the associations amongst body size, swimming methods, and morphological traits.

In the present study, the function of sternal keel, which is characteristic to giant water scavenger beetles of the tribe Hydrophilini, was experimentally investigated for the first time. By removing the keel of H. acuminatus,

two hypotheses were tested: the keel affects (1) submergence time after air replacement as well as (2) speed and

oscillatory movement during forward swimming. My results suggest that sternal keel is associated with

swimming speed. The presence of keel would reduce energetic costs by improving swimming efficiency. Not

only that, but faster swimming may help beetles escape predation by birds and frogs. Dettner (2019) have

suggested that the keel serve as defensive structure. Further study is needed to examine whether it also functions

as a direct defense against predators.

Acknowledgements Draft

I gratefully acknowledge the thoughtful and constructive comments of the reviewers. I am grateful to Dr. Martin

Fikáček (National Museum and Charles University, Prague) and Mr. Fang-Shuo Hu (National Chung Hsing

University, Taiwan) for providing valuable information regarding swimming of Hydrophilidae. I also thank Dr.

Masateru Maeda (Royal Veterinary College) for technical comments on fluid dynamics. I would like to thank

Dr. Tomoyuki Yokoi (University of Tsukuba) for renting me the digital video camera and electronic balance for

experiments. I would like to acknowledge Mr. Yuta Nagano (University of Tsukuba) and Mr. Yuya Suzuki

(University of Tsukuba) for their thoughtful and constructive comments. I would also like to thank Editage

(www.editage.com) for English language editing.

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Figure captions

Figure 1 (a) Lateral view of giant water scavenger beetles with (top) and without (bottom) keel. (b) A submerged Hydrophilius acuminatus in experiment 1. (c) Overview of methods for measuring swimming distance and oscillatory movement in experiment 2. Closed and open symbols indicate the start and goal points, respectively. The linear distance between these two points is shown as a solid line, and the length of the trajectory drawn by the tip of the elytra is shown as a dashed line.

Figure 2 Relationship between submergence timeDraft after air replacement and body mass in Hydrophilus acuminatus. The solid line represents the estimated association and the shaded range indicates the 95% confidence intervals. Closed symbols indicate individuals with keel, and open symbols indicate individuals without keel.

Figure 3 Boxplot of relationship between the presence of keel and swimming speed in Hydrophilius acuminatus.

Figure 4 Boxplot of relationship between the presence of keel and degree of oscillation in Hydrophilius acuminatus.

Table 1 Generalised linear mixed model for submergence time after air replacement in Hydrophilus

acuminatus.

Submergence time

Explanatory variable Estimate SE z value Pr(<|z|)

Keel (removal) 0.086 0.203 0.422 0.673

Sex (male) −0.278 0.205 −1.355 0.175

Body mass −0.887 0.231 −3.849Draft< 0.001

Table 2 Generalised linear mixed models for (a) swimming speed and (b) degree of oscillation in

Hydrophilus acuminatus.

(a) Swimming speed (b) Degree of oscillation

Explanatory variable Estimate SE z value Pr(<|z|) Estimate SE z value Pr(<|z|)

Keel (removal) −0.138 0.065 −2.124 0.034 0.102 0.248 0.411 0.681

Sex (male) 0.043 0.065 0.661 0.509 −0.253 0.250 −1.013 0.311

Body mass −0.091 0.074Draft−1.232 0.218 −0.612 0.287 −2.132 0.033

Draft

Figure 2 Relationship between submergence time after air replacement and body mass in Hydrophilus acuminatus. The solid line represents the estimated association and the shaded range indicates the 95% confidence intervals. Closed symbols indicate individuals with keel, and open symbols indicate individuals without keel.

88x88mm (300 x 300 DPI)

Draft

Figure 3 Boxplot of relationship between the presence of keel and swimming speed in Hydrophilius acuminatus.

88x88mm (300 x 300 DPI)

Draft

Figure 4 Boxplot of relationship between the presence of keel and degree of oscillation in Hydrophilius acuminatus.

88x88mm (300 x 300 DPI)