Spatial and morphological reorganization of endosymbiosis during metamorphosis accommodates adult metabolic requirements in a weevil.

Bacterial intracellular symbiosis (endosymbiosis) is widespread in nature and impacts many biological processes. In holometabolous symbiotic insects, metamorphosis entails a complete and abrupt internal reorganization that creates a constraint for endosymbiont transmission from larvae to adults. To assess how endosymbiosis copes-and potentially evolves-throughout this major host-tissue reorganization, we used the association between the cereal weevil Sitophilus oryzae and the bacterium Sodalis pierantonius as a model system. S. pierantonius are contained inside specialized host cells, the bacteriocytes, that group into an organ, the bacteriome. Cereal weevils require metabolic inputs from their endosymbiont, particularly during adult cuticle synthesis, when endosymbiont load increases dramatically. By combining dual RNA-sequencing analyses and cell imaging, we show that the larval bacteriome dissociates at the onset of metamorphosis and releases bacteriocytes that undergo endosymbiosis-dependent transcriptomic changes affecting cell motility, cell adhesion, and cytoskeleton organization. Remarkably, bacteriocytes turn into spindle cells and migrate along the midgut epithelium, thereby conveying endosymbionts to midgut sites where future mesenteric caeca will develop. Concomitantly, endosymbiont genes encoding a type III secretion system and a flagellum apparatus are transiently up-regulated while endosymbionts infect putative stem cells and enter their nuclei. Infected cells then turn into new differentiated bacteriocytes and form multiple new bacteriomes in adults. These findings show that endosymbiosis reorganization in a holometabolous insect relies on a synchronized host-symbiont molecular and cellular "choreography" and illustrates an adaptive feature that promotes bacteriome multiplication to match increased metabolic requirements in emerging adults.

Isolation and expression of five genes in the mevalonate pathway of the Chinese white pine beetle, Dendroctonus armandi (Curculionidae: Scolytinae).

The Chinese white pine beetle Dendroctonus armandi (Tsai and Li) is a significant pest of the Qinling and Bashan Mountains pine forests of China. The Chinese white pine beetle can overcome the defences of Chinese white pine Pinus armandi (Franch) through pheromone-assisted aggregation that results in a mass attack of host trees. We isolated five full-length complementary DNAs encoding mevalonate pathway-related enzyme genes from the Chinese white pine beetle (D. armandi), which are acetoacetyl-CoA thiolase (AACT), geranylgeranyl diphosphate synthase (GGPPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MPDC), and phosphomevalonate kinase (PMK). Bioinformatic analyses were performed on the full-length deduced amino acid sequences. Differential expression of these five genes was observed between sexes, and within these significant differences among topically applied juvenile hormone III (JH III), fed on phloem of P. armandi, tissue distribution, and development stage. Mevalonate pathway genes expression were induced by JH III and feeding.

hRNAi-mediated knock-down of Sphenophorus levis V-ATPase E in transgenic sugarcane (Saccharum spp interspecific hybrid) affects the insect growth and survival.

KEY MESSAGE: Transgenic sugarcane expressing V-ATPase subunit E dsRNA affects growth and survival of Sphenophorus levis. Plants being sessile organisms are constantly confronted with several biotic and abiotic stresses. Sugarcane (Saccharum spp) is a major tropical crop widely cultivated for its sugar and other by-products. In Brazil, sugarcane plantations account for significant production losses due to Sphenophorus levis (sugarcane weevil) infestations. With the existing control measures being less effective, there arises a necessity for advanced strategies. Our bioassay injection experiments with V-ATPase E dsRNA in S. levis larvae showed significant mortality and reduction in transcription levels. Furthermore, we down-regulated the V-ATPase E gene of S. levis in transgenic sugarcane using an RNAi approach. The resultant RNAi transgenic lines exhibited reduction in larval growth and survival, without compromising plant performance under controlled environment. Our results illustrate that RNAi-mediated down-regulation of key genes is a promising approach in imparting resistance to sugarcane weevil.

Novel associations in antibiosis stemming from an insect pupal cell.

The pupal soil cell of the pecan weevil, Curculio caryae (Coleoptera: Curculionidae), was reported previously to exhibit antibiosis to an entomopathogenic fungus, Beauveria bassiana. The objectives of this study were to examine 1) if the antimicrobial effect occurs in other insects that form pupal cells, 2) whether the effect extends to plant pathogenic fungi, and 3) identify the source of antibiosis in pupal soil cells of C. caryae. Antibiosis of pupal cells against B. bassiana was confirmed in-vitro in three additional curculionids, Diaprepes abbreviatus, Conotrachelus nenuphar, and Pissodes nemorensis, all of which had fewer fungal colonies relative to controls. Pupal soil cells were found to suppress phytopathogenic fungi in-vitro, including suppression of Alternaria solani by D. abbreviatus pupal cell, and that of Monilinia fructicola by C. caryae. The detection of antibiosis of soil cells formed by surface-sterilized insects using sterile soil implies the antimicrobial effect stemmed from inside the insect. Further, a novel biotic mechanism was identified: a bacterium related to Serratia nematodiphila was isolated from C. caryae pupal soil cells and was found to be associated with antibiosis. The bacterial cultures with or without autoclave had similar effects but were not as potent as pupal soil cells for suppressing B. bassiana. Also, autoclaved soil cells and autoclaved bacterial culture suppressed M. fructicola but were not as inhibitory as non-autoclaved soil cells. This indicates that antibiosis may be due to bacterial metabolites, although other factors may also be involved. Our findings suggest potential to develop the antibiotic compounds as novel bio-fungicides to control plant diseases.

Damage Potential, Effect on Germination, and Development of Sitophilus oryzae (Coleoptera: Curculionidae) on Wheat Grains in Northwestern Himalayas.

The present investigation was carried out to study the biological parameters and orientation of Sitophilus oryzae (L.) toward wheat cultivar HPW-236 and mixed grains of different cultivars (HPW-155, HPW-236, HPW-249, HPW-349, HPW-360, HS-490, and VL-892). The incubation period, larval period, pupal period, and total life cycle period of S. oryzae was longer when fed on mixed grains as compared to HPW-236. Also, the weevils were more oriented toward HPW-236 and lower germination rates were observed from HPW-236 than mixed cultivars when exposed to S. oryzae. We also evaluated quantitative losses caused by S. oryzae in different cultivars of wheat recommended in the northwestern Himalayas under free-choice conditions. The weevil inflicted greater damage and weight loss in grains of HPW-236 while it was negligible in the case of HPW-360 and HPW-249. HPW-236 which is the most cultivated variety of wheat in northwestern Himalayas proved to be highly susceptible to the weevil and provided a more suitable environment for weevil's development. Therefore, this particular cultivar can be avoided for prolong storage and the farmers should prefer cultivars such as HPW-360 and HPW-249, which proved to be least affected the weevil.

Body Size Plasticity of Weevil Larvae (Curculio davidi) (Coleoptera: Curculionidae) and Its Stoichiometric Relationship With Different Hosts.

Parasites obtain energy and nutrients from the host, and their body size is also usually limited by host size. However, the regulatory mechanisms that control the plasticity of parasite body sizes and the stoichiometric relationships with their hosts remain unclear. Here we investigated the concentrations of 14 elements (C, H, O, N, P, S, K, Na, Ca, Mg, Al, Fe, Mn, and Zn) in the acorns of three oak species (Quercus spp.), in their endoparasitic weevil (Curculio davidi Fairmaire) (Coleoptera: Curculionidae) larvae and in the larval feces, and the weight of weevil larvae within different hosts in a warm-temperate zone of China. Our results showed that the three acorn species exhibited significant differences in C, H, O, P, K, Mg, and Mn concentrations. However, in the weevil larvae, only P, Mn, and C:P ratio revealed significant differences. Weevil larvae preferentially absorbed and retained N, Zn, Na, and P, whereas Mn, K, Ca, and O were passively absorbed and transported. The weevil larvae weight was associated with acorn stoichiometry, and positively correlated with acorn size. Weevil larvae P decreased, but Mn and C:P increased with their weight, implying highly variable in somatic stoichiometry are coupled with the plasticity of body size. Interestingly, weevil larvae weight was negatively correlated with acorn infection rate, indicating small-size parasitic insects might have higher fitness level in parasite-host systems than larger-size ones. Our results suggest that variation in P, Mn, and C:P in parasites may play critical roles in shaping their body size and in improving their fitness.

Emergence and Infestation Level of Hypothenemus hampei (Coleoptera: Curculionidae) on Coffee Berries on the Plant or on the Ground During the Post-harvest Period in Brazil.

The coffee berry borer (CBB), Hypothenemus hampei Ferrari (Coleoptera: Curculionidae), is the most important coffee pest in most of the coffee growing countries. CBB females leave old dry berries after harvest and search for dry noninfested berries on the plant or on the ground to lay eggs or to use as refuge until new berries are available on the coffee trees in the following season. The CBB infestation level and emergence from berries on the ground or on the plants were evaluated in two fields post-harvest in the Spring in Brazil over two seasons. Twenty infested or noninfested berries in separate cages (250 ml plastic cups) were placed on the plants or on the ground under the tree canopy, in each field. The number of infested berries and CBB females that emerged from the infested berries were recorded weekly. CBB emergence was higher from berries on the ground than those on the coffee trees in both seasons, whereas CBB infestation was higher on coffee berries on the plants than those on the ground in season I. Insolation (hours of sunlight) and temperature were the main covariates that affected emergence and infestation by this insect. The results are discussed for monitoring CBB during the time of dispersal with implications on integrated management of this pest.

High-temperature shock consequences on the red flour beetle (Tribolium castaneum) and the rice weevil (Sitophilus oryzae).

Temperature shocks have profound effects on biological and physiological functions at all levels of organization. However, the recovery periods from these shocks and their subsequent impacts remain unknown. Herein, our study investigated the effect of short temperature stress on survival, dormancy recovery time, nutritional indices, life traits and development rate for T. castaneum (larvae and adults) and S. oryzae adults. The results showed significant effects on survival rates of T. castaneum (larvae and adults) and S. oryzae adults. When both insects had been exposed to high-temperature shock, survival rates decreased with higher temperatures and longer periods of exposure. Furthermore, recovery times varied between and within the insect species, as prolonged exposure to thermal shocks increased recovery periods. Moreover, dormancy time resulting from the high-temperature shocks significantly affected food deterrence and food intake, regardless of the stage of development, species, exposure periods and temperature-exposure conditions. Subsequently, differences in body growth rates and food consumption rates are an appropriate indicator of differences in food conversion rates under high-temperature shocks, regardless of the species and developmental stages. On the other hand, our results indicated that as high-temperature shocks increased, the total development period increased of T. castaneum. Likewise, the pupal stage increased with increasing high-temperature shocks, and the larval stage decreased with increasing thermal shocks and increasing the periods of exposure. In summary, our study showed the importance of dormancy recovery time and its subsequent effects for improving disinfestation effectiveness of heat treatment, and understanding insect response to high temperatures.

Bacterial chitinase biochemical properties, immobilization on zinc oxide (ZnO) nanoparticle and its effect on Sitophilus zeamais as a potential insecticide.

It has been planned to minimize the yield and quality impairment of the seed corn, which is strategically important in the world, by pests under storage conditions with a biological product produced with a biotechnological approach. In this context, the present study aimed to control the maize weevil Sitophilus zeamais, known as a warehouse pest, using a nanoformulation. In the study, the chitinase enzyme from Lactobacillus coryniformis was purified first using ammonium sulfate precipitation and then by using the HiTrap Capto DEAE column, and the molecular mass of the purified enzyme was determined to be ~ 33 kDa, and the optimum pH and the values as pH 6.0 and 65-75 °C, respectively. Five different doses of nanoformulation (2, 4, 6, 8 and 10 mg/L) were applied to corn grains by the spraying method with three repetitions so that the insect can ingest the formulation through feeding. The effects of the applications on the death rate and mean time of death of Sitophilus zeamais were determined. According to these findings, it was concluded that the best practice was nanoformulation with 6 mg/L, considering both the mortality rate (100%) and the average death time (2.4 days). Chitinase from L. coryniformis is a promising candidate for corn lice control and management.

Evaluation of hydrolytic enzyme activities from digestive fluids of Anthonomus grandis (Coleoptera: Curculionidae).

The cotton boll weevil, Anthonomus grandis, is a major pest of cotton crops in South America. In this work, partial biochemical characterizations of (hemi) cellulases and pectinases activities in the digestive system (head- and gut- extracts) of A. grandis were evaluated. Gut extract section from third instar larvae exhibited endoglucanase, xylanase, ß-glucosidase, and pectinase activities. The endoglucanase and xylanase activities were localized in the foregut, whereas ß-glucosidase activity was mainly detected in the hindgut. In addition, no difference in pectinase activity was observed across the gut sections. Thus, A. grandis digestive system is a potentially interesting reservoir for further lignocellulolytic enzymes research.

Temperature-dependent development and survival of immature stages of the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae).

Although the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae) is the most destructive insect pest of coffee worldwide, there is much to learn about its thermal biology. This study aimed to develop temperature-based models for H. hampei development and to provide the thermal requirements of immature stages in the laboratory. Using a new observation method, larval development and survival were monitored daily on fresh Arabica coffee seeds, under seven constant temperatures in the range 15-35°C, with 80 ± 5% RH and 12:12 L:D photoperiod. Linear and non-linear functions were fitted to the development data plotted against temperature, using Insect Life Cycle Modelling software (ILCYM). Temperature significantly affected the development time of all immature stages. Egg incubation period ranged 4.6-16.8 days, under temperature between 30 and 15°C. No development occurred at 35°C and the larval stage did not develop to pupa at 15°C. The minimum temperature threshold (Tmin) estimated from linear regression was 10.5, 13.0, 15.0 and 13.0°C, for egg, larva, pupa and the total development from egg to adult, respectively. The maximum temperature threshold (Tmax) estimated from the Sharpe and DeMichele function was 32°C for egg to adult development. The thermal constant (k) was estimated at 78.1, 188.7, 36.5 and 312.5 degree days, for egg, larva, pupa and for egg to adult, respectively. Our results will help understand and predict the pest population dynamics and distribution in coffee plantations as impacted by temperature, and as such, will contribute to a more efficient management of the pest.

Low-cost modified-atmosphere hermetic storage structures to reduce storage losses of maize (Zea mays L.) cobs and sorghum (Sorghum bicolor L.) heads.

BACKGROUND: Production and marketing of cereal grains are some of the main activities in developing countries to ensure food security. However, the food gap is complicated further by high postharvest loss of grains during storage. This study aimed to compare low-cost modified-atmosphere hermetic storage structures with traditional practice to minimize quantitative and qualitative losses of grains during storage. The study was conducted in two phases: in the first phase, seven hermetic storage structures with or without smoke infusion were compared, and one selected structure was further validated at scaled-up capacity in the second phase. RESULTS: Grains stored in PVC bag-supported structures (with or without smoke infusion) resulted in low live weevil population, low percentage of damaged grains and reduced weight loss with better retention of crude protein and fat contents. Results from validation study also demonstrated that maize and sorghum stored in improved storage structures experienced, respectively, 9.8% and 10.4% weevil damage as compared with 47.3% and 42.3% when stored in traditional storage structures. The same was true in terms of crude protein and fat contents. CONCLUSIONS: The study demonstrated that storage structures supported with PVC bags are efficient and low-cost structures for reducing storage-related losses and supporting food security efforts as compared to traditional methods. Furthermore, the bags can be made locally and with various storage capacities to store either shelled or unshelled products. © 2019 Society of Chemical Industry.

Noninvasive and Nondestructive Detection of Cowpea Bruchid within Cowpea Seeds with a Hand-Held Raman Spectrometer.

Insect damage to crops is a serious issue, in particular when the pest dwells within its host. The cowpea bruchid ( Callosobruchus maculatus) is an herbivore of legumes including beans and peas. The bruchid lays its eggs on the seeds themselves; after hatching, the larvae burrow into and develop inside the seed, complicating detection and treatment. Left unchecked, two insects could destroy up to 50% of 1 ton of harvest cowpea ( Vigna unguiculata) after several months of storage. In this study, we investigated the possibility of using a hand-held Raman spectrometer to detect the pest during its development within intact cowpeas. Our results show that Raman spectroscopy can detect chemical signatures of bruchid larvae as well as their excrements inside the intact seeds. Additionally, using chemometric methods, we distinguished between healthy and infested seeds as well as among seeds hosting developmentally early or late-stage larvae with high accuracy. This study demonstrates Raman spectroscopy's efficacy in not only detection of pathogens and pests present on the surface of plant leaves and the grain but also inside the seeds. This Raman-based method may prove useful as a rapid means of screening crops for internal pests.

Anthonomus grandis aggregation pheromone induces cotton indirect defence and attracts the parasitic wasp Bracon vulgaris.

Insect-derived volatiles seem to provide reliable chemical cues that plants could employ to defend themselves. Here we investigated the effect of pheromone emission from a closely associated (Anthonomus grandis; boll weevil) and an unassociated (Tibraca limbativentris) herbivore on cotton volatile emission. Exposure to A. grandis aggregation pheromone induced cotton defence response by enhancing the emission of volatiles attractive to the natural enemy of A. grandis, the parasitic wasp Bracon vulgaris, but only when the pheromonal blend was complete (all four components). Individual components of A. grandis aggregation pheromone were not able to induce cotton plants to increase the release of volatiles. On the other hand, T. limbativentris sex pheromone did not induce any change in the cotton constitutive volatile profile. Our results support the hypothesis that plants are able to detect pheromones of tightly co-evolved herbivores. Moreover, A. grandis pheromone exposure induced similar volatile compounds to herbivore-induced cotton, such as linalool, (E)-ocimene, (E)-4,8-dimethylnona-1,3,7-triene (DMNT), and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). We also showed that the larval ectoparasitoid B. vulgaris relies on boll weevil's aggregation pheromone and pheromone-induced plant volatiles as kairomones to locate suitable hosts.

Four new Ophiostoma species associated with conifer- and hardwood-infesting bark and ambrosia beetles from the Czech Republic and Poland.

Fungi under the order Ophiostomatales (Ascomycota) are known to associate with various species of bark beetles (Coleoptera: Curculionidae: Scolytinae). In addition this group of fungi contains many taxa that can impart blue-stain on sapwood and some are important tree pathogens. A recent survey that focussed on the diversity of the Ophiostomatales in the forest ecosystems of the Czech Republic and Poland uncovered four putative new species. Phylogenetic analyses of four gene regions (ITS1-5.8S-ITS2 region, ß-tubulin, calmodulin, and translation elongation factor 1-α) indicated that these four species are members of the genus Ophiostoma. All four newly described species can be distinguished from each other and from closely related species based on DNA sequence comparisons, morphological characters, growth rates, and their insect associations. Based on this study four new taxa can be circumscribed and the following names are provided: Ophiostoma pityokteinis sp. nov., Ophiostoma rufum sp. nov., Ophiostoma solheimii sp. nov., and Ophiostoma taphrorychi sp. nov. O. rufum sp. nov. is a member of the Ophiostoma piceae species complex, while O. pityokteinis sp. nov. resides in a discrete lineage within Ophiostoma s. stricto. O. taphrorychi sp. nov. together with O. distortum formed a well-supported clade in Ophiostoma s. stricto close to O. pityokteinis sp. nov. O. solheimii sp. nov. groups within a currently undefined lineage A, which also includes Ophiostoma grandicarpum and Ophiostoma microsporum. This study highlights the need for more intensive surveys that should include additional countries of Central Europe, insect vectors and host tree species in order to elucidate Ophiostoma species diversity in this region.

Genome-Wide Association Studies (GWAS) for Yield and Weevil Resistance in Sweet potato (Ipomoea batatas (L.) Lam).

KEY MESSAGE: We apply the GWAS to sweet potato genome, and identified the SNPs associated with yield and weevil resistance. The sweet potato (Ipomoea batatas (L.) Lam) is a highly heterozygous, outcrossing, polyploid species, which presents challenges for genetic analysis. Therefore, we considered that genome-wide association studies (GWAS) may be applied to the study of the sweet potato genome. The yield of two sweet potato varieties [Purple Sweet Lord (PSL) and 90IDN-47] was assessed at two locations (Kumamoto and Okinawa prefectures) in Japan in 2013 and the yield scores were used for GWAS. The results showed that there were several single nucleotide polymorphisms (SNP) above the significance thresholds in PSL; two peaks were detected in Kumamoto and Okinawa on the Ib03-3 and Ib01-4 linkage groups of PSL, respectively. As for 90IDN-47, one relatively high peak was detected in Kumamoto on the Ib13-8 linkage group. Interestingly, although high peaks above significance thresholds were detected in Kumamoto and Okinawa in PSL, the peaks were located in different linkage groups. This result suggests that the genetic regions controlling yield may change in response to environmental conditions. Additionally, we investigated the degree of weevil damage to the plants, which is the greatest problem in sweet potato cultivation in Okinawa. In this experiment, no SNPs were identified above the significance thresholds. However, one relatively high peak was found in the 90IDN-47 genotype, which showed resistance to weevils. On the other hand, one relatively high peak was also detected in the PSL genotype, which showed susceptibility to weevils. These results suggest that two regions could affect weevil resistance and may contain the gene(s) controlling weevil resistance.

[Effect of temperature on post-diapause reproductive development in Listronotus maculicollis (Coleoptera: curculionidae)].

The annual bluegrass weevil Listronotus maculicollis requires chilling exposure to terminate reproductive diapause during overwintering, but the effects of temperature on its post-diapause development in spring remain unclear. To explore this effect, overwintering adults were transferred from cold conditions (6°C/4°C, L:D 10:14) to different warm-up temperatures at L:D 12:12. When weevils were transferred to 7, 14 and 21°C in December and late January, the sizes of male and female reproductive organs were significantly smaller at 7°C than at 14 and 21°C. When weevils were transferred to 7, 9, 11, 13 and 15°C in late January, higher temperatures facilitated the post-diapause development. In both sexes, the sizes of reproductive organs and developmental rate increased with temperature. Reproductive organs did not grow significantly at 7°C in males and at 7-9°C in females, at which the percentage of developing weevils remained low. The time required for 50% of individuals to resume development was 44, 18, 13 and 8 days at 9, 11, 13 and 15°C, respectively, in males and 19, 14 and 8 days at 11, 13 and 15°C, respectively, in females. The threshold temperature for post-diapause development was 7.8°C in males, based on which 61.7 degree-days coincided with 50% of individuals developing. Under field conditions, the percentage of male and female maturity and insemination rate were low until early March, but all reached 100% by late March.

Phenology of the sugar beet weevil, Bothynoderes punctiventris Germar (Coleoptera: Curculionidae), in Croatia.

The sugar beet weevil (SBW), Bothynoderes punctiventris Germar, 1824, is a significant pest in most of Eastern Europe. Here, the SBW is described and its seasonal activity characterized, in terms of its different developmental stages in relation to Julian days (JDs), degree-day accumulations (DDAs), and precipitation, as a key to improving monitoring and forecasting of the pest. The phenology and population characteristics of SBW were investigated in sugar beet fields in eastern Croatia over a 4-year period (2012-2015). By using the degree-day model (lower development threshold of 5°C, no upper development threshold, biofix 1 January), the first emergence of overwintering adults was determined as becoming established when the DDA reached 20. The adult emergence was completed when the DDA reached 428. SBW males emerged first, following which the females dominated the adult population. Overwintering adults were present in the field until early July. In August, adults of the offspring generation began to appear. The eggs laid by the overwintering generation required, on average, 10-15 days to develop into larvae; however, eggs were found in soil samples over a period of 102 days (between JDs 112 and 214). Larvae were present in the soil samples over a period of a maximum of 143 days (the first larvae were established on JD 122 and the last one on JD 265), and pupae were established in the soil over a period of 102 days (between JDs 143 and 245). This study provides important data for understanding SBW population dynamics and developing potential population dynamic models for pest forecasting on a regional scale.

Long-term population persistence of flightless weevils (Eurhoptus pyriformis) across old- and second-growth forests patches in southern Appalachia.

BACKGROUND: Southern Appalachian forests are dominated by second-growth vegetation following decades of intensive forestry and agricultural use, although some old-growth patches remain. While it's been shown that second-growth areas may exhibit comparable species richness to old-growth in the area, the extent to which populations of arthropods in second-growth areas have persisted vs. recolonized from other areas remains unexamined. The implications for conservation of both classes of forest are significant. Here we analyze population diversity and relatedness across five old-growth and five second-growth populations of flightless, leaf litter-inhabiting beetles in the genus Eurhoptus (Coleoptera: Curculionidae: Cryptorhynchinae). Our main goal is asking whether second-growth areas show diminished diversity and/or signals of recolonization from old-growth sources. RESULTS: Population genetic and phylogenetic analyses do not reveal any consistent differences in diversity between the old-growth and second-growth populations examined. Some second-growth populations retain substantial genetic diversity, while some old-growth populations appear relatively depauperate. There is no phylogenetic indication that second-growth populations have recolonized from old-growth source populations. CONCLUSIONS: Most populations contain substantial and unique genetic diversity indicating long-term persistence in the majority of sites. The results support substantial resilience in second-growth populations, though the geographic scale of sampling may have hindered detection of recolonization patterns. Broad scale phylogeographic patterns reveal a deep break across the French Broad River basin, as has been reported in several other taxa of limited dispersal abilities. In Eurhoptus this break dates to ~ 2-6 Ma ago, on the older end of the range of previously estimated dates.

Rafting on floating fruit is effective for oceanic dispersal of flightless weevils.

Terrestrial species, especially non-vagile ones (those unable to fly or swim), cannot cross oceans without exploiting other animals or floating objects. However, the colonisation history of flightless Pachyrhynchus weevils, inferred from genetic data, reveals their ability to travel long distances to colonise remote islands. Here, we used captive-bred Pachyrhynchus jitanasaius to analyse (i) the physiological tolerance of weevils (egg, larva and adult stages) to different levels of salinity; (ii) the survival rate of larvae in a simulated ocean environment in the laboratory; and (iii) the survival rate of larvae in a field experiment in the ocean using fruit of the fish poison tree floating on the Kuroshio current in the Pacific Ocean. We found that the survival rate of larvae in seawater was lower than in fresh water, although if the larvae survived 7 days of immersion in seawater, some emerged as adults in the subsequent rearing process. No adults survived for more than 2 days, regardless of salinity level. After floating separately for 6 days in salt water in the laboratory and in the Kuroshio current, two of 18 larvae survived in the fruit. This study provides the first empirical evidence that P. jitanasaius larvae can survive 'rafting' on ocean currents and that the eggs and larvae of these weevils have the highest probability of crossing the oceanic barrier. This ability may facilitate over-the-sea dispersal of these flightless insects and further shape their distribution and speciation pattern in the Western Pacific islands.