Life history of Laccophilus dikinohaseus Kamite, Hikida, and Satô, 2005 (Coleoptera: Dytiscidae) and its preferences for oviposition substrates

Abstract We describe the life history of Laccophilus dikinohaseus Kamite, Hikida, and Satô, 2005 using laboratory rearing-based methods to contribute to future ex situ and in situ conservation. At 26 °C, each developmental stage had the following duration: egg, 6–7 days; first-instar, 3–5 days; second-instar, 3–5 days; third-instar in water, 7–18 days; landing to leaving the pupal chamber, 9–11 days; and total duration of the immature stages, 30–44 days. The total larval period was significantly longer than those of L. yoshitomii Watanabe and Kamite, 2018, L. kobensis Sharp, 1873, and L. hebusuensis Watanabe and Kamite, 2020. The maximum complete lifespan (egg to adult death) and adult lifespan (exiting the pupal chamber to adult death) were 792 and 762 days, respectively, indicating that L. dikinohaseus can live for more than two years after the adults exit the pupal chamber. Laccophilus dikinohaseus laid eggs not only in aquatic plants but also in driftwood and withered plants, suggesting a diverse oviposition substrate availability. This is the first study on the immature stages of L. dikinohaseus and lifespan of Laccophilus. We propose in situ conservation measures for L. dikinohaseus based on its findings.


Introduction
Dytiscidae is the largest aquatic Coleoptera family, with 4,615 known species worldwide (Nilsson and Hájek 2023).In Japan, 137 species are known (Watanabe and Biström 2022;Watanabe and Yoshitomi 2022), approximately 40% of which have been listed on the 2019 Red List (Hayashi, Nakajima, Ishida, Kitano, and Yoshitomi 2020).Of these, the genus Laccophilus Leach, 1815 clearly shows significant decline, as five species, which represent approximately 42% of the 12 species recorded at the time, have been listed on the Red List (Watanabe 2022).
Laccophilus dikinohaseus Kamite, Hikida, and Satô, 2005 (Coleoptera: Dytiscidae: Laccophilinae) (Figure 1a) is endemic to Japan (Nakajima, Hayashi, Ishida, Kitano, and Yoshitomi 2020; Watanabe and Yoshitomi 2022) and belongs to the L. kobensis species group (Kamite, Hikida, and Satô 2005;Nilsson and Hájek 2023).It is not listed in the Red List; however, it is rare and its habitats are limited (Nakajima et al. 2020).Since there are only a few of these habitats left, this species can be evaluated as an endangered species.It is thought to be dependent on the wetlands associated with large riverbeds in the plains; however, its suitable habitats and life history are unknown.Information on the life history is essential for planning conservation strategies for L. dikinohaseus.Recently, rearing methods for Laccophilus have been introduced (Watanabe 2021a).The life histories of some species can be clarified using this method (e.g., Watanabe 2021bWatanabe , 2022)).Describing the details of the process from egg to adult for the target species in the laboratory will contribute to the realisation of future ex situ and in situ conservation methods (e.g., Watanabe, Suda, and Fukutomi 2017;Watanabe 2022).
Laccophilus species are known to lay eggs in plant tissues (e.g., Watanabe 2020, 2021a), and many species are found in habitats with abundant aquatic plants (Nakajima et al. 2020).Studies have shown that L. hebusuensis Watanabe and Kamite,  Watanabe and Kamite, 2018 are found in shady ponds and wetlands where aquatic plants are present and fallen leaves accumulate (Hirasawa, Mitamura, Yoshii, and Watanabe 2021).This suggests that Laccophilus species may use not only live aquatic plants, but also withered plants, as oviposition substrates.

and L. yoshitomii
The purpose of this study was to describe the life history, lifespan, and preferred oviposition substrates of L. dikinohaseus in the laboratory.

Material and rearing methods
Laccophilus dikinohaseus adults were collected in Tochigi Prefecture, Japan, in June-July 2020.All individuals used in the rearing experiments were offsprings obtained from these adults.They were brought to the rearing room, which was maintained at 26 °C with a 9 h light (08:15-17:15, JST) and 15 h dark (9 L:15D) photoperiod, at the Ishikawa Insect Museum on October 6, 2020.The rearing methods for adults, larvae, and pupae followed those described in Watanabe (2021a), and Pogostemon sp.'Dassen' (Lamiaceae) was used as the oviposition plant.After 24 h of rearing, the adults were removed, and the eggs were retained.
We reared 21 of the 41 hatched larvae individually, and observations regarding egg-laying, hatching, moulting, transition to soil, and the emergence dates of adults from the pupal chamber to the soil surface were recorded every 24 h.

Comparison of larval durations of six Laccophilus species
The total larval period of L. dikinohaseus was compared with those of L. lewisioides Brancucci, 1983(Watanabe 2022), L. vagelineatus Zimmermann, 1922(Watanabe 2020), L. yoshitomii (Watanabe 2021a), L. kobensis Sharp, 1873(Watanabe 2021b), and L. hebusuensis (Watanabe and Kamite 2020), which were obtained under the same rearing conditions as larvae in this study.The total larval period of the six species was analysed using the Kruskal-Wallis test, and pairwise comparisons were made using Wilcoxon rank-sum test with continuity correction (P-value adjustment using Holm's method).All analyses were conducted using R version 4.0.4(R Core Team 2020).

Lifespan
To determine lifespan, we transferred seven newly emerged adults reared using the above described method to separate plastic cups (8 cm diameter, 4 cm height, 2 cm water depth).The rearing method followed that described by Watanabe and Ohba (2021), wherein Java moss (Hypnaceae) was placed in each plastic cup to provide for hiding and resting sites, and the rearing room was maintained at 26 °C with a 9 L:15D photoperiod.The adults were fed frozen chironomid larvae every 2-3 days.
We checked for death every 24 h, and the rearing experiment continued until all the adults died.We counted the days from when the egg was laid and from when the adult exited the pupal chamber until its death.
Prior to the experiment, adults were bred in the laboratory of the University of Tsukuba from September to November 2021, and four males and eight females were obtained.After emergence, the sexes were separated and reared under 26 °C conditions until January 2022, and then under 10 °C conditions until May 2022.From June 2022, mixed sexes were reared under 26 °C conditions and randomly mated.
Mated females and the eight substrates were set in eight plastic cups (10 cm diameter, 10 cm height, 8 cm water depth); there was one female per substrate.Substrate was replaced with another type of substrate every 3 days.The experiment was conducted for three days × 12 terms, for a total of 36 days, so that each female experienced eight different substrates once or twice.To prevent sperm depletion in the females, we randomly mated the females again at the end of each of the four terms for nine days in the first and 10 days in the second.The substrate with the laid eggs was divided into separate plastic cups, and the number of hatched larvae was counted.All of these were conducted at 26 °C with 16 L:8D.
For determining the preference for oviposition substrates under laboratory conditions, the number of hatched larvae for the eight oviposition substrates were analysed using the Kruskal-Wallis test, and pairwise comparisons were made using the Wilcoxon rank-sum test with continuity correction (P-value adjustment using Holm's method).All analyses were conducted using R version 4.0.4(R Core Team 2020).
After transitioning to land, the larvae dug into the soil or stayed on the soil surface to construct a pupal chamber.The duration from landing to leave the pupal chamber was 9-11 days (9.7 ± 0.6 days, n = 17) (Table S1).The total duration of the immature stages was 30-44 days (33.5 ± 3.3 days, n = 17).

Life history
This is the first study on the immature stages of L. dikinohaseus and lifespan of Laccophilus.The life history during the immature stages of L. dikinohaseus was similar to that of previously reported Laccophilus species (Watanabe 2019(Watanabe , 2020(Watanabe , 2021a(Watanabe , 2021b;;Watanabe and Kamite 2020).
Previous studies have shown that the third-instar larvae of the L. kobensis species group, L. hebusuensis, L. kobensis, L. nakajimai, L. vagelineatus, and L. yoshitomii, were dark brown to yellowish brown and changed to green several days before landing (Watanabe 2019(Watanabe , 2020(Watanabe , 2021a(Watanabe , 2021b;;Watanabe and Kamite 2020;Watanabe and Okada 2023).This study revealed that L. dikinohaseus larvae underwent similar changes in colouration as that observed in the L. kobensis species group as they grew.
Comparison of the larval periods of six species showed that L. dikinohaseus had the second longest larval period after L. lewisioides (Table 1).Among the L. kobensis species group, L. dikinohaseus and L. vagelineatus had the longest larval periods.Laccophilus dikinohaseus, similar to L. lewisioides and L. vagelineatus, inhabits ponds (Kamite et al. 2005;Nakajima et al. 2020).These results support the suggestion by Watanabe (2022) that differences in larval periods are related to stability of the inhabited water bodies.
The habitat where adults were collected for this study had no water on April 7, 2020, but was flooded from April 23 to July 21, 2020, and new teneral adults were collected in this habitat on July 21, 2020 (R. Uchiyama, personal observation).The total duration of the immature stages was 30-44 days; thus, it is considered that reproduction starts after the end of April in the habitat.Future field surveys are necessary to determine the larval appearance period and whether the new adults that emerge begin to reproduce during the same year.
Our results showed that the maximum whole and adult lifespans of L. dikinohaseus were 792 (egg to adult death) and 762 days (exiting the pupal chamber to adult death), respectively.The current experiment was conducted under the same conditions as those described by Watanabe and Ohba (2021): (a) no reproduction, (b) no overwintering (year-round activity), and (c) separate rearing of individuals.Although the experiment was conducted under laboratory conditions, which may differ from field conditions, the results showed that L. dikinohaseus could potentially live for more than two years after the adults exit the pupal chamber.Therefore, assuming that adults start reproducing in the next year of emergence, L. dikinohaseus could live for up to two reproductive seasons.In contrast, the lifespan of L. biguttatus Kirby, 1837(as L. strohmi Thomson, 1874in Hagenlund and Nilsson (1985)) was estimated to be approximately one year based on the results of field surveys (Hagenlund and Nilsson 1985); however, Hagenlund and Nilsson (1985) mentioned that longer-lived L. biguttatus individuals may occur.Although the research methods of the present study were different, the results show that Laccophilus species can be more long-lived than previously considered.However, the potential for interspecies differences in lifespan cannot be excluded, and lifespan of more species should be confirmed using the same method in the future.

Egg-laying behaviour and comparison of preference for oviposition substrates
Female adults of Laccophilus were known to lay eggs into the aquatic plant tissues (e.g., Watanabe and Kamite 2020;Watanabe 2021a), but the present study revealed that they use more diverse oviposition substrates.It is especially remarkable that they laid eggs into driftwood and withered plants and that those eggs hatched (Figures 3, 4).
Among Dytiscidae that lay eggs in aquatic plant tissues, Cybister Curtis, 1827 and Dytiscus Linnaeus, 1758 have been studied for their preference of oviposition substrates (Ichikawa 2002;Inoda 2011a, b).Cybister prefers plants with sponge-like tissue as oviposition substrates (Tsuzuki, Taniwaki, and Inoda 2000;Ichikawa 2002), while Dytiscus prefers plants with a hollow centre (Inoda 2011a).This difference in preference is considered to be due to their egg-laying behaviour.Cybister creates an oviposition pore by biting the oviposition substrates with their mandibles before egg-laying.This pore provides an exit for the larvae (Inoda 2011b).Dytiscus instead use their ovipositor to create crevices in the substrates without biting, laying their eggs directly (Inoda 2011a).The egg-laying behaviour of L. dikinohaseus was similar to that of Dytiscus as they inserted their ovipositor into the substrate and laid the eggs directly.In contrast, L. dikinohaseus uses not only aquatic plants as oviposition substrates but also emergent plants, water-soaked terrestrial plants, and driftwood, suggesting a more diverse substrate availability than Cybister and Dytiscus.The similar morphology of Laccophilus ovipositors and the fact that similar egg-laying behaviour has been observed in some species to date (e.g., Watanabe and Kamite 2020; Watanabe 2021a) suggest that many species of Laccophilus can utilise a variety of oviposition substrates.
Several species of Laccophilus often inhabit temporary water (e.g., Larson, Alarie, and Roughley 2000).The capacity to lay eggs on a variety of oviposition substrates, whether living or withered, may contribute to increasing the reproductive success in various types of aquatic habitats.Laccophilus has major faunas in every biogeographic region and is the second most diverse genus in the family Dytiscidae (Miller and Bergsten 2016).The great diversity of Laccophilus may be related to the availability of a wide range of oviposition substrates.

In situ conservation
Laccophilus dikinohaseus is known to inhabit ponds (Kamite et al. 2005) but is also considered a wetland-dependent species that thrives in habitats with fluctuating water levels (Nakajima et al. 2020).These environments have been reduced by intensive agriculture and development factors, and the only place in recent years where this species has been found is around Watarase Yusuichi, which is registered under the Ramsar Convention.The current status of the species suggests that there is an urgent need to consider the conservation of L. dikinohaseus, including its addition to the Red List.For in situ conservation, we propose two conservation measures: (1) maintaining plants (aquatic plants, emergent plants, and water-soaked terrestrial plants) and driftwood as oviposition substrates for this species: excessive removal of dead plants and driftwood around water bodies can lead to loss of oviposition substrates.Notably, S. chinensis, Persicaria sp., M. sacchariflorus, M. sacchariflorus (withered), and driftwood are present in the natural habitat of L. dikinohaseus and, therefore, available as oviposition substrates; (2) preserving environments where water bodies are maintained for at least 45 days (> 2 months preferred) to allow the eggs to develop into adults.
In this study, the life history of L. dikinohaseus was determined by laboratory rearing.Further studies are required to identify the reproductive season in the field and to find pupal habitats, as in the study by Uchiyama and Watanabe (2021), to appropriately conserve this species.

Figure 2 .
Figure 2. Histogram of lifespan of Laccophilus dikinohaseus Kamite, Hikida, and satô, 2005: (a) the number of days from when the adult exited the pupal chamber until its death (n = 7); (b) the number of days from egg to adult death (n = 7).

Table 1 .
Habitat and total larval period of the Laccophilus species.