Shun Ichiro Takano

Two Cryptic Species in Brontispa longissima (Coleoptera: Chrysomelidae): Evidence from Mitochondrial DNA Analysis and Crosses between the Two Nominal Species

ABSTRACT The beetle Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae) is a serious invasive pest of the coconut palm, Cocos nucifera I., in Southeast Asia and the Pacific region. Genetic analysis is essential to ecological and evolutionary study of such invasive species. We therefore conducted molecular analyses by using partial sequences (1044 bp) of the mitochondrial cytochrome oxidase subunit I (COI) genes of B. longissima collected from several locations. We found two monophyletic groups: one distributed over a limited area (Australia, Papua New Guinea, Samoa, and Sumba Island; referred to as the Pacific group) and the other collected from a wide area of Asia and the Pacific region (referred to as the Asian group). We compared the biological and morphological traits of the two groups. We found that insects in the Pacific group had a shorter developmental time from hatching to adult emergence, produced fewer eggs, and had a larger adult body size than insects in the Asian group. Elytral color patterns did not differ between the two groups. Our interpopulation crosses produced significantly fewer progeny than intrapopulation crosses, suggesting that B. longissima represents two cryptic species.

Parasitisation of Tetrastichus brontispae (Hymenoptera: Eulophidae), a biological control agent of the coconut hispine beetle Brontispa longissima (Coleoptera: Chrysomelidae)

Abstract The gregarious endoparasitoids Tetrastichus brontispae Ferrière is one of the important natural enemies of the coconut hispine beetle Brontispa longissima (Gestro), a serious invasive pest on coconut palm plants (Cocos nucifera L.) in Southeast Asia. Development at different temperatures, effect of host and female ages, effect of food and oviposition frequency and superparasitism were investigated in the laboratory. Females were allowed only one attack against one host in all experiments. The wasp developed in a host between 19 and 30°C, whilst no wasp completed its immature development at 16 and 31°C. Host and female ages affected parasitisation. Parasitoid emergence was high on day 0 and day 1 pupal hosts, and younger females produced more offspring than older females. The longevity of the female was affected not only by food supply, but also by oviposition frequency. The female survived longer when oviposition frequency was low. However, the total number of hosts parasitised by the female during her lifetime did not differ at different oviposition frequencies. In superparasitism, although the percentage of adult emergence and body size of offspring decreased with an increasing number of attacks per host, a host parasitised by up to four females could produce parasitoid offspring.

Flight orientation behavior of Ooencyrtus nezarae (Hymenoptera : Encyrtidae), an egg parasitoid of phytophagous bugs in soybean

We conducted field experiments to examine flight orientation behavior of Ooencyrtus nezarae Ishii, an egg parasitoid of soybean feeding bugs, using sticky traps with synthetic aggregation pheromones of the host, Riptortus clavatus (Thunberg). Experiments were conducted in plots either containing a soybean field or not containing one. When the traps were suspended at heights of 0.5, 1.5 and 3.0 m above a soybean field in Kobe, O. nezarae females were most frequently caught in the 0.5 m traps. In an open field on the same campus where the vegetation was predominantly tall goldenrod, females were more frequently trapped at 1.5 or 3.0 m. In a soybean field in Kumamoto, females were most frequently trapped in the 0.8 m traps, compared to traps placed at 2.0, 4.0 and 6.0 m. In an oat field in the same area, females were trapped most frequently at heights of 0.8 and 2.0 m. Unlike females, only a small number of males were trapped in the fields in both areas. The result that O. nezarae females were caught in traps at higher positions in non‐host habitats than in soybean fields probably reflects differences in host searching behavior in or movement between the habitats. In host habitats, females mainly walk on the plants or fly within the plant canopy to search for hosts. In non‐host habitats, females may not intensively search for hosts within the plants, and directly respond to host pheromones in the traps at heights equal to or a little higher than the plant canopy.

Unique clade of alphaproteobacterial endosymbionts induces complete cytoplasmic incompatibility in the coconut beetle

Significance Maternally inherited bacterial endosymbionts in arthropods manipulate host reproduction to increase the number of infected females. Cytoplasmic incompatibility (CI) is one such manipulation, in which infected females can produce offspring by mating with both infected and uninfected males, but uninfected females cannot or seldom produce offspring with infected males. Two bacterial endosymbionts, Wolbachia and Cardinium, are known CI inducers. Here we report a third CI inducer that belongs to a unique clade of Alphaproteobacteria. This bacterial clade was found to cause complete CI between two clades of the coconut beetle, a serious invasive pest of coconut palms. We discuss the potential use of this bacterium as a biological control agent and its effects on speciation of the coconut beetle. Maternally inherited bacterial endosymbionts in arthropods manipulate host reproduction to increase the fitness of infected females. Cytoplasmic incompatibility (CI) is one such manipulation, in which uninfected females produce few or no offspring when they mate with infected males. To date, two bacterial endosymbionts, Wolbachia and Cardinium, have been reported as CI inducers. Only Wolbachia induces complete CI, which causes 100% offspring mortality in incompatible crosses. Here we report a third CI inducer that belongs to a unique clade of Alphaproteobacteria detected within the coconut beetle, Brontispa longissima. This beetle comprises two cryptic species, the Asian clade and the Pacific clade, which show incompatibility in hybrid crosses. Different bacterial endosymbionts, a unique clade of Alphaproteobacteria in the Pacific clade and Wolbachia in the Asian clade, induced bidirectional CI between hosts. The former induced complete CI (100% mortality), whereas the latter induced partial CI (70% mortality). Illumina MiSeq sequencing and denaturing gradient gel electrophoresis patterns showed that the predominant bacterium detected in the Pacific clade of B. longissima was this unique clade of Alphaproteobacteria alone, indicating that this endosymbiont was responsible for the complete CI. Sex distortion did not occur in any of the tested crosses. The 1,160 bp of 16S rRNA gene sequence obtained for this endosymbiont had only 89.3% identity with that of Wolbachia, indicating that it can be recognized as a distinct species. We discuss the potential use of this bacterium as a biological control agent.

Artificial diets for rearing the coconut hispine beetle Brontispa longissima and its parasitoid Asecodes hispinarum

We tested artificial diets for rearing the coconut hispine beetle, Brontispa longissima, a serious invasive pest of coconut (Cocos nucifera) in Southeast Asia. We examined three artificial diets that were identical except for their agar content. The survival rate from hatching to adult emergence was 26.0% when beetles were reared on a ‘soft diet’ (20 g/l agar), 16.0% on a ‘hard diet’ (40 g/l agar), and 41.0% on a ‘mixed diet’, in which the hard diet was used for the first instar and the soft diet for later instars. Females raised as larvae on the mixed diet and then as adults on the soft diet laid few eggs, which did not hatch. However, females reared on the mixed diet as larvae and then on fresh leaves as adults consistently laid eggs that hatched. We then examined the suitability of B. longissima larvae reared on the mixed diet as hosts for the larval parasitoid Asecodes hispinarum, a specialist parasitoid of this beetle. Of the oviposited hosts, 75.0% became mummified and 41.7% produced adult wasps. These results indicate that the mixed diet may be useful for rearing B. longissima larvae as hosts for the rearing of A. hispinarum.

Host recognition by the tick parasitoid Ixodiphagus hookeri (Hymenoptera: Encyrtidae)

Abstract We conducted laboratory experiments to determine whether the tick parasitoid Ixodiphagus hookeri Howard uses chemicals of the host tick Amblyomma variegatum F. as host recognition cues. When given a piece of polyethylene sheet containing an air bubble (a dummy host) treated or untreated with hexane, I. hookeri females did not respond to the dummy. However, when females contacted the dummy host treated with hexane extracts from unfed nymphs, engorged nymphs, or unfed adults of the host ticks, they probed the dummy with their ovipositors. When given a choice of dummies treated with hexane extract of engorged nymphs, hexane, or nothing, they did not demonstrate any selective attraction for the dummy with hexane extract from engorged nymphs over the other dummies. A fraction (hexane 9: ether 1) of hexane extract from engorged nymphs strongly stimulated ovipositor probing by females. These results suggest that I. hookeri females use chemicals contacted on host ticks as host recognition cues.

Comparative developmental and reproductive biology of geographical populations from two cryptic species in Brontispa longissima (Coleoptera: Chrysomelidae)

Brontispa longissima is a serious pest of the coconut palm Cocos nucifera, presumed to have originated in Papua New Guinea and Indonesia. It recently invaded Southeast and East Asia, where outbreaks have been reported. Mitochondrial DNA analysis reveals two cryptic species in B. longissima: one is distributed over a wide area including Asia and the Pacific region (the Asian clade) and the other in a limited area in the Pacific region (the Pacific clade). Recent invasions and outbreaks have been reported only from the area where the Asian clade has been found, suggesting that this clade has become a pest in Asia. To infer if the Asian clade has the ability to establish, spread and outbreak in novel habitats more effectively than the Pacific clade, we compared life‐history traits between the two populations of different clades. The net reproduction rate (R0) was 130.0 and 94.0, the mean length of a generation (T) was 57.7 and 54.7 days, and the intrinsic rate of natural increase (r) was 0.084 and 0.083 per day for the population from Ishigaki Island, Japan (ISH) (the Asian clade) and for the population from Papua New Guinea (PNG) (the Pacific clade), respectively. Although the difference in r was little, the simulated population growth showed that the ISH population can be 1.6 times larger than that of the PNG after ten generations. The rapid population growth of the Asian clade would be partly responsible for its establishment, spread and frequent outbreaks in Asia.

Rapid discrimination of two cryptic species within Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae) by PCR–RFLP

Brontispa longissima (Coleoptera: Chrysomelidae) is a serious invasive pest of coconut palm (Cocos nucifera) supposedly originating from Indonesia and New Guinea. It has recently invaded Southeast and East Asia, where it has caused serious damage to coconut plants. Brontispa longissima as currently defined contains two cryptic species: we herein referred to one as the “Asian clade”, which is distributed over a wide area, including Asia and the Pacific region; and we referred to the other one as the “Pacific clade”, which is found in a limited area in the Pacific region. We developed a PCR–RFLP method for differentiating the two clades. Digestion of the PCR product of a 1,014-bp region within the mitochondrial cytochrome oxidase I gene (COI) with BslI, HpyCH4III, or NlaIV resulted in clade-specific patterns as estimated by the sequence data. We applied the method to specimens newly obtained from various locations to investigate the geographical distribution of B. longissima. Although B. longissima collected from Samoa in April 2003 had been placed in the Pacific clade, specimens collected from the same island in April 2010 were placed in the Asian clade, suggesting that the predominant clade may have been changing from the former to the latter. On Timor, specimens included both clades in apparently segregated habitats.

Life history traits and damage potential of an invasive pest Brontispa longissima (Coleoptera: Chrysomelidae) on Satakentia liukiuensis

The coconut hispine beetle Brontispa longissima has been causing serious damage to the coconut palm Cocos nucifera in the Pacific, and Southeast and East Asia. This beetle also attacks Satakentia liukiuensis, an endemic palm on Ishigaki and Iriomote Islands, Japan. To assess the potential impact of B. longissima on S. liukiuensis, we examined the development period, survival rate, egg production and body size of B. longissima on S. liukiuensis at 22–31°C and consumption of S. liukiuensis leaf at 28°C. We also examined these life history traits on C. nucifera at 28°C to compare with those on S. liukiuensis. Brontispa longissima completed their development and reproduced on S. liukiuensis at 22–30°C. Linear regression analysis was used to estimate the effective cumulative temperature (k) as 891.2 degree‐days, and the lower developmental threshold as 13.3°C. On the basis of these values and monthly average temperatures on Ishigaki Island, it was estimated that B. longissima has four generations per year on the island. Satakentia liukiuensis was less suitable for B. longissima than C. nucifera in terms of immature survival, development time, resultant adult size and reproduction. The low quality of S. liukiuensis as a host‐plant may prevent the outbreak of B. longissima, which has not occurred yet on Ishigaki and Iriomote Islands.

Artificial diets for rearing the coconut hispine beetle, Brontispa longissima (Coleoptera: Chrysomelidae), and their suitability to two specialist parasitoids

Abstract The coconut hispine beetle, Brontispa longissima (Gestro), is a serious invasive pest that infests young unopened fronds of coconut palms (Cocos nucifera L.) in Southeast Asia. We previously developed the first artificial diet for rearing B. longissima larvae, which contained a leaf powder of young coconut fronds. Because the fronds are required for healthy growth of coconut palms, it is necessary to reduce their use for rearing the beetles. In this study, we tested two new artificial diets for the beetle larvae, which contained the leaf powders of mature coconut leaves or orchard grass (Dactylis glomerata L.). Brontispa longissima successfully developed from hatching to adulthood on both the mature coconut leaf diet and orchard grass diet. The beetles reared on the mature coconut leaf diet and orchard grass diet developed faster than those reared on the young coconut leaf diet. Fecundity and egg hatchability of beetles did not differ among the three diet treatments. We then examined the suitability of beetle larvae or pupae reared on each diet as hosts for two specialist endoparasitoids, Asecodes hispinarum Boucek and Tetrastichus brontispae Ferriere. The survival rate from oviposition to adult emergence for A. hispinarum was 43.8% in hosts reared on a young coconut leaf diet, 77.1% on a mature coconut leaf diet, and 85.7% on an orchard grass diet. For T. brontispae, the survival rate was 70.0% in hosts reared on the young coconut leaf diet, 38.1% on the mature coconut leaf diet, and 66.7% on the orchard grass diet. Our results indicate these artificial diets can be useful for rearing B. longissima and its two parasitoids, helping to reduce the costs of mass rearing these insects.