Noninvasive sampling of DNA from larval exuvia in diving beetles of the genera Cybister and Dytiscus

Nonlethal DNA sampling is a highly recommendable method in molecular genetic studies of protected and endangered species. To develop a demonstrably nonlethal method of obtaining DNA from larvae of endangered diving beetles (Cybister brevis, C. lewisianus, C. limbatus, C. rugosus, Dytiscus sharpi sharpi and D. sharpi validus), we obtained the larval exuvia (molted skin) of these endangered diving beetles under laboratory conditions. A single exuvia 24 h after molting was sufficient to allow polymerase chain reaction (PCR) detection of a mitochondrial DNA gene, cytochrome‐c oxidase subunit I (COI), and the sequence of the COI gene could be determined directly. Sequences obtained from the exuvial samples were used to further find similarities within DDBJ/EMBL/GenBank. Genomic DNA from the samples was successfully isolated, and we identified the species. This process suggests that exuvia provides a good sample for extracting DNA from endangered diving beetle larvae without killing them.     

Cracks or holes in the stems of oviposition plants provide the only exit for hatched larvae of diving beetles of the genera Dytiscus and Cybister

Diving beetles such as Dytiscus and Cybister species (Coleoptera: Dytiscidae) usually oviposit inside an aquatic plant stem beneath the surface of the water. The hatched larvae need to escape from the stem to intake oxygen from the air. To determine where larvae of these diving beetles hatch in the plant stem, the hatchability and escape rates in larvae of Dytiscus sharpi Wehncke, Cybister chinensis Motshulsky, Cybister lewisianus Sharp, and Cybister brevis Aubé were investigated under laboratory conditions. Hatchability of D. sharpi in the stem of Sagittaria trifolia L. (Alismataceae) was extremely low (8.2%). However, it was high (>90%) when late‐stage eggs (2–3 days before hatching) were isolated from the stem and kept in water. On the other hand, the hatchability of Cybister spp. was high (88–95%) in S. trifolia. Usually, Cybister spp. females bite a hole in the plant stem on oviposition. When the oviposition pore in the stem was plugged with glass wool, no larvae could escape from the stem, indicating that the oviposition pore was the only exit for hatched larvae of Cybister spp. In contrast, females of D. sharpi oviposited directly by making a crack in the stem of Oenanthe javanica (Blume) DC. (Apiaceae) without biting. Eggs grew to a length and diameter equal to the stem crack size 2–3 days before hatching. Dytiscus sharpi eggs isolated from O. javanica were artificially inserted into plant stems of O. javanica or S. trifolia (so‐called inserted egg model), and the hatchability and larval escape rates were determined. Larval escape strongly depended on the stem crack width of both O. javanica and S. trifolia, suggesting that the stem crack was an exit for hatched larvae of D. sharpi.     

Status of Japanese Dytiscus species (Coleoptera: Dytiscidae) based on mitochondrial DNA sequence data

We sequenced 628 base pairs of the mitochondrial cytochrome‐c oxidase subunit 1 (cox1) gene to investigate the genetic differentiation among Japanese Dytiscus diving beetles (Coleoptera: Dytiscidae). Dytiscus beetles are a charismatic part of the fauna of lakes, ponds and swamps. Three species are known from Japan: D. dauricus Gebler, 1832; D. marginalis czerskii Zeitzev * , 1953; and D. sharpi Wehncke, 1875. Adults of D. sharpi collected in Chiba and Ishikawa Prefectures were morphologically highly similar, but here we found that they differed by 20 cox1 base pairs (3.18%). Our results imply that conservation strategies especially for Japanese D. sharpi sharpi and D. sharpi validus Régimbart, 1899 might need to be adjusted to address the presence of two evolutionarily significant units.     

Female dimorphism in Japanese diving beetle Dytiscus marginalis czerskii (Coleoptera: Dytiscidae) evidenced by mitochondrial gene sequence analysis

Three species of the Japanese diving beetle Dytiscus have been identified: D. dauricus Gebler, 1832; D. marginalis czerskii Zeitzev, 1953; and D. sharpi. At present, the latter consists of the subspecies D. sharpi sharpi Wehncke, 1875 and D. sharpi validus Régimbart, 1899 based on the comparative data of mitochondrial DNA cytochrome‐c oxidase I (COI) sequences. Many Dytiscus species have smooth and grooved elytra, which are female dimorphic traits. For many years it has been thought that Japanese D. marginalis czerskii has a single morph, that is, only grooved females, although there were some collecting reports of smooth females occurring at the foot of Mt. Chokaisan in Yamagata and Akita Prefectures. However, the population of smooth females (smooth population) has not yet been identified by DNA markers. To understand the species status of the smooth population, we sequenced 769 bp of COI of a male derived from a smooth mother insect and compared it with the sequence from a known grooved female. The sequences of 769 bp of the COI gene in the smooth population were identical to that in the grooved female, indicating that Japanese D. marginalis czerskii has female dimorphic traits.     

Mass breeding larvae of the critically endangered diving beetles Dytiscus sharpi sharpi and Dytiscus sharpi validus (Coleoptera: Dytiscidae)

For conservation purposes, and to supply critically endangered insects for laboratory use, a system for artificial breeding is crucial. However, in the case of carnivorous insects such as diving beetles, the larvae must be isolated because they are cannibalistic. We developed a method for mass breeding the larvae of two diving beetles, Dytiscus sharpi sharpi (Wehncke) and Dytiscus sharpi validus (Régimbart) (Coleoptera: Dytiscidae), which are designated critically endangered species in Japan. Ten to twenty larvae were raised in a small tank (35 cm × 25 cm × 10 cm; water depth 7 cm) with Rana ornativentris (Werner) tadpoles as prey. At low prey density, ~80 % of the larvae were cannibalized. At moderate prey density, 50–60 % were cannibalized. However, at high prey density, <3 % were cannibalized. Well-fed mass-bred adults were larger than individually bred and field-collected adults. This mass breeding method can be used for the conservation and breeding of these rare diving beetles in a manageable number of aquaria.     

Effects of temperature on life histories of three endangered Japanese diving beetle species

To elucidate population-increasing factors in the diving beetle Cybister tripunctatus lateralis (Fabricius) (Coleoptera: Dytiscidae) in Japan in recent years, life histories and oviposition patterns were compared among three endangered diving beetle species, Cybister brevis Aubé (qualified by the Japanese Red Data List as ‘near threatened’), Cybister chinensis Motschulsky (vulnerable), and C. tripunctatus lateralis (vulnerable). Oviposition in C. brevis, C. chinensis, and C. tripunctatus lateralis was observed from late April to mid-June, from late April to early July, and from late May to mid-August, respectively, under semi-outdoor conditions. There were no interspecies differences in total hatchling production during the reproductive season. In rearing experiments at various temperatures (20, 23, 25, 28, and 30 °C), the mortality of C. tripunctatus lateralis larvae was higher at 20 °C, and gradually lower with increasing temperature up to 30 °C. Adult body size of females in C. tripunctatus lateralis is larger than that of males but there were no significant differences among temperatures (25–30 °C). Cybister brevis had a higher emergence rate at 23–28 °C than at 20 and 30 °C. In C. brevis, the body size of adults reared at 25 or 28 °C was significantly larger than at other temperatures. Cybister chinensis did not differ in emergence rate and adult body size among the five temperature conditions. The developmental zero (i.e., the lower developmental threshold) from the first instar to adult emergence was 11.1 °C for C. brevis, 8.7 °C for C. chinensis, and 16.8 °C for C. tripunctatus lateralis. We speculate how the influence of global warming may have a positive impact on the growth and survival of C. tripunctatus lateralis.     

Field observation of predation on a horsehair worm (Gordioida: Chordodidae) by a diving beetle larva Cybister brevis Aubé (Coleoptera: Dytiscidae)

Diving beetles (Coleoptera: Dytiscidae) are carnivorous in both the larval and adult stages; larvae are exclusively predatory, whereas adults also scavenge for food. They are known to prey on zooplankton, insects, gastropods, fish, amphibians and reptiles. However, there have been no previous reports detailing direct predation on adult Gordioida by Dytiscidae in the field. This study represents the first observation of a diving beetle larva, Cybister brevis Aubé (Coleoptera: Dytiscidae), predating on an adult horsehair worm (Gordioida: Chordodidae). This might be the first report of predation on horsehair worms by insects.     

Specific detection of the floodwater mosquitoes Aedes sticticus and Aedes vexans DNA in predatory diving beetles

Floodwater mosquitoes (Diptera: Culicidae) are associated with periodically flooded wet meadows, marshes, and swamps in floodplains of major rivers worldwide, and their larvae are abundant in the shallow parts of flooded areas. The nuisance caused by the blood‐seeking adult female mosquitoes motivates mosquito control. Larviciding with Bacillus thuringiensis israelensis is considered the most environmentally safe method. However, some concern has been raised whether aquatic predatory insects could be indirectly affected by this reduction in a potential vital prey. Top predators in the temporary wetlands in the River Dalälven floodplains are diving beetles (Coleoptera: Dytiscidae), and Aedes sticticus and Ae. vexans are the target species for mosquito control. For detailed studies on this aquatic predator–prey system, we developed a polymerase chain reaction (PCR) assay for detection of mosquito DNA in the guts of medium‐sized diving beetles. Primers were designed for amplifying short mitochondrial DNA fragments of the cytochrome C oxidase subunit I (COI) gene in Ae. sticticus and Ae. vexans, respectively. Primer specificity was confirmed and half‐life detectability of Ae. sticticus DNA in diving beetle guts was derived from a feeding and digestion experiment. The Ae. sticticus DNA within diving beetle guts was detected up to 12 h postfeeding, and half‐life detectability was estimated to 5.6 h. In addition, field caught diving beetles were screened for Ae. sticticus and Ae. vexans DNA and in 14% of the diving beetles one or both mosquito species were detected, showing that these mosquito species are utilized as food by the diving beetles.     

Temperature-dependent regulation of reproduction in the diving beetle Dytiscus sharpi (Coleoptera: Dytiscidae)

The effects of temperature on the mating behavior, gonad development, germ cell maturation, and egg spawning of the predaceous diving beetle Dytiscus sharpi (Coleoptera; Dytiscidae), were investigated. By field observations, we found that mating behavior started in October and occurred more frequently from November to December. Under our laboratory breeding conditions, we observed almost the same seasonal variation in mating behavior. We found that temperatures lower than 20 degrees C were required to trigger mating behavior. We also found the same temperature threshold triggered gonadogenesis as well as spermatogenesis. Furthermore, for females, exposure to lower temperatures (<8 degrees C) during the winter was required for egg maturation and spawning in spring; that is, there was a second threshold for successful female reproduction. We conclude that the termination of summer reproductive diapause of D. sharpi is regulated in a temperature-dependent manner, thus effecting the adaptation of D. sharpi to southern warm habitats.     

Biology of the Eucalyptus leaf beetle Paropsisterna selmani (Chrysomelidae: Paropsini): a new pest of Eucalyptus species (Myrtaceae) in Ireland

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Effects of diet and host access on fecundity and lifespan in two fruit fly species with different life‐history patterns

The reproductive ability of female tephritids can be limited and prevented by denying access to host plants and restricting the dietary precursors of vitellogenesis. The mechanisms underlying the delayed egg production in each case are initiated by different physiological processes that are anticipated to have dissimilar effects on lifespan and reproductive ability later in life. The egg‐laying abilities of laboratory‐reared females of the Mediterranean fruit fly (Ceratitis capitata Wiedmann) and melon fly (Bactrocera cucurbitae Coquillett) from Hawaii are delayed or suppressed by limiting access to host fruits and dietary protein. In each case, this is expected to prevent the loss of lifespan associated with reproduction until protein or hosts are introduced. Two trends are observed in each species: first, access to protein at eclosion leads to a greater probability of survival and a higher reproductive ability than if it is delayed and, second, delayed host access reduces lifetime reproductive ability without improving life expectancy. When host access and protein availability are delayed, the rate of reproductive senescence is reduced in the medfly, whereas the rate of reproductive senescence is generally increased in the melon fly. Overall, delaying reproduction lowers the fitness of females by constraining their fecundity for the remainder of the lifespan without extending the lifespan. © 2013 The Royal Entomological Society     

Sexual dimorphism and allometry in two seed beetles (Coleoptera: Bruchidae)

Male Callosobruchus chinensis (Coleoptera: Bruchidae) have elaborated, pectinate antennae, which are absent from conspecific females and both sexes of a congener, Callosobruchus maculatus. To begin to unravel the mechanisms producing this striking dimorphism, we examined which morphological traits best explain body size variation in bruchid beetles and quantified sexual dimorphism of antenna size through allometric analyses. Using principal component analyses, we found that elytron length and pronotum width were significantly correlated with the first principal component, which was interpreted as explaining variation in body size. Regressions of log‐transformed body size measures on log‐transformed antenna length revealed that males of both species had longer antennae than conspecific females for any given body size, although most of this effect was attributable to higher intercepts, rather than increased allometry, in males. Comparisons among heterospecific males revealed that C. maculatus males have noticeably longer antennae than C. chinensis males at large body sizes. Callosobruchus chinensis males, thus, appear to have increased the receptive area of their antennae by adding to the width of, rather than further elongating, their antennae. Finally, we found evidence for positive allometry between log‐transformed antenna length and log‐transformed antenna width in C. chinensis males. We discuss our results in the context of evidence supporting the presence of an additional, and potentially unique, sex pheromone in C. chinensis females.     

Larval population density affects female weight and fecundity in the dung beetle Aphodius ater

1. Competition for food at high densities during larval development leads to reduced adult weight in the northern temperate dung beetle Aphodius ater. 2. Analysis of female beetles caught in the field showed that numbers of eggs and total egg load per female were correlated positively with beetle size. 3. Female beetles reared at different population densities during larval development in the laboratory were analysed with regard to their lifetime fecundity and reproductive lifespan. 4. High population densities during development had a negative influence on the number of eggs per female and on reproductive lifespan. Lifetime fecundity was correlated positively with female weight. 5. It was concluded that competition during larval development in the first generation of offspring will result in a lower number of offspring in the second generation in Aphodius ater, and thereby reduce parental fitness.     

Quantifying the effects of host discrimination on egg‐laying decision of the cowpea seed beetle,Callosobruchus maculatus

Many parasitic and endophagous insect species are capable of discriminating among the quality of their hosts. However, there is no appropriate way to quantify their discrimination performance. In this study, we quantified how oviposition of the cowpea seed beetle, Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae), was affected by the relative contributions of both egg number and host size discrimination. The effect of egg density and resource heterogeneity on these discrimination performances was also explored. Egg‐distribution predictions were made by combining time‐dependent available resource fitness (egg discrimination) and host weight factors (size discrimination). The χ² test was then used for goodness‐of‐fit testing. The effects of both egg and size discrimination on oviposition in environments with different levels of resource heterogeneity were compared. It was found that host size, rather than the number of eggs on the host, plays a larger role in the egg‐laying decision for most individual seed beetles, especially when egg density is high. Host size discrimination behavior was reinforced when the beetles experienced increasing resource heterogeneity, but the performance might reach a plateau. This is the first quantitative evaluation of the effect of host discrimination on egg‐laying decisions of seed beetles.     

Balanced intake of protein and carbohydrate maximizes lifetime reproductive success in the mealworm beetle,Tenebrio molitor (Coleoptera: Tenebrionidae)

Recent developments in insect gerontological and nutritional research have suggested that the dietary protein:carbohydrate (P:C) balance is a critical determinant of lifespan and reproduction in many insects. However, most studies investigating this important role of dietary P:C balance have been conducted using dipteran and orthopteran species. In this study, we used the mealworm beetles, Tenebrio molitor L. (Coleoptera: Tenebrionidae), to test the effects of dietary P:C balance on lifespan and reproduction. Regardless of their reproductive status, both male and female beetles had the shortest lifespan at the protein-biased ratio of P:C 5:1. Mean lifespan was the longest at P:C 1:1 for males and at both P:C 1:1 and 1:5 for females. Mating significantly curtailed the lifespan of both males and females, indicating the survival cost of mating. Age-specific egg laying was significantly higher at P:C 1:1 than at the two imbalanced P:C ratios (1:5 or 5:1) at any given age throughout their lives, resulting in the highest lifetime reproductive success at P:C 1:1. When given a choice, beetles actively regulated their intake of protein and carbohydrate to a slightly carbohydrate-biased ratio (P:C 1:1.54–1:1.64 for males and P:C 1:1.3–1:1.36 for females). The self-selected P:C ratio was significantly higher for females than males, reflecting a higher protein requirement for egg production. Collectively, our results add to a growing body of evidence suggesting the key role played by dietary macronutrient balance in shaping lifespan and reproduction in insects.     

Reproductive assessment of the great hornbill (Buceros bicornis) by fecal hormone analysis

The population of great hornbills (Buceros bicornis) in the United States is rapidly aging, and captive breeding efforts have not met population managers' expectations for a sustainable captive group. Little is known about the reproductive physiology of these birds. This study reports the first data on the re‐productive endocrinology of the great hornbill. The hormone profiles of the only pair of these birds that hatched a chick in the 1999–2000 breeding season are compared to the profiles of six other pairs of hornbills, from different institutions in the United States, that did not reproduce successfully that season. The study investigates the estradiol, corticosterone, and testosterone profiles of these seven pairs of birds, establishing a base of knowledge from which endocrine data may be used to improve the success of captive breeding programs. The estradiol profiles from this study indicate a difference in hormonal patterns between laying and non‐laying female great hornbills. Egg‐laying females had significantly higher estradiol concentrations during the breeding season than the non‐laying females (P<0.003). Testosterone concentrations of the males were not significantly different between the mates of egg‐laying and non‐egg‐laying females. The corticosterone concentrations tended to be lower in the females that laid eggs vs. the non‐egg‐laying group. The males of the egg‐laying pairs showed a significantly lower (P<0.036) corticosterone concentration than the non‐egg‐laying male pairs. This, combined with the extremely low corticosterone levels (compared to the other birds in the study) of the pair of hornbills that hatched a chick in the 1999–2000 breeding season, suggests that adrenal activity may play a role in the reproductive failure of some captive great hornbills. Zoo Biol 22:135–145, 2003. © 2003 Wiley‐Liss, Inc.     

Comparing macrophyte herbivory by introduced Louisiana crayfish (Procambarus clarkii) (Crustacea: Cambaridae) and native Dytiscid beetles (Cybister tripunctatus) (Coleoptera: Dytiscidae), in Kenya

The omnivorous Louisiana crayfish, Procambarus clarkii, has caused significant changes to macrophyte communities worldwide and may have similar negative effects in Kenya if used as a biological control agent for snails harbouring human schistosomes. Here we examine how herbivory by the introduced Louisiana crayfish might change macrophyte abundance and species composition relative to herbivory by a prevalent native beetle Cybister tripunctatus. Our observations are thought to be the first on the consumption of live macrophytes by adult dytiscid beetles, which are commonly known as predaceous diving beetles. In a laboratory experiment, we compare herbivory rates and macrophyte species-selectivity between the beetle and the crayfish. Combining the herbivory rates with population estimates of crayfish and beetles in a local pond, we estimate that herbivory in the natural environment is of the same order of magnitude. Preferences among macrophyte species are also similar between herbivores, except that crayfish are likely to have a larger impact than beetles on cultivated rice (Oryza sativa). Results from a laboratory toxicity experiment suggest that the crayfish is not currently a rice pest in Kenya because it is killed by Furadan^® 5G, the commonly applied pesticide.     

Effect of water depth on predation frequency by diving beetles on mosquito larvae prey

Diving behavior and its frequency may differ among species of mosquito larvae because of differences in predation pressure. The present study aimed to investigate the relationship between water depth and predation frequency on two mosquito species, Culex tritaeniorhynchus (wetland breeder) and Aedes albopictus (container breeder), by the diving beetle Eretes griseus. Culex tritaeniorhynchus spends more time at the surface than A. albopictus, which spends more time thrashing underwater. When intact mosquito larvae of both species were present, the diving beetles consumed almost all A. albopictus larvae (98.3%). After all the A. albopictus larvae had been consumed, the diving beetles began to prey on C. tritaeniorhynchus. In order to compare the effect of position on the predation preference of the diving beetles, equal numbers of both species were heat‐killed and allowed to settle on the bottom of the container. When all the dead mosquito larvae had sunk to the bottom of a plastic container, the diving beetles caught both mosquito species at random. These results indicate that mosquito larvae near the surface were eaten less frequently by diving beetles than those at the bottom. The low diving frequency of C. tritaeniorhynchus is regarded as a form of anti‐predatory behavior.     

Terrestrial snails use predator-diet to assess danger

Some aquatic snails are able to use chemical cues (kairomones) to differentiate between predators that have fed on snails and predators that have eaten other prey. However, it is unknown if terrestrial snails are able to differentiate between snail-fed predators and predators that have not recently consumed snails. Here we document diet-based chemical discrimination of a predator, the ground beetle Carabus carabus, by a terrestrial snail Theba pisana. When exposed to the feces of snail-fed beetles, snails initially stopped all movements and then increased climbing speed. The snails also decreased the time to deposition of their egg clutch. The snails did not react to an extract of crushed snails. Snails had only a partial reaction to the feces of beetles that had fed on chicken (Gallus domesticus) livers—they decreased climbing speed but did not alter egg laying times. These responses may be adaptive in that they allow snails to differentiate between individual beetles that may pose an immediate threat and beetles that may not. This is one of only a few studies to examine predator-diet effects on reproductive behavior.