Detecting Symbioses in Complex Communities: the Fungal Symbionts of Bark and Ambrosia Beetles Within Asian Pines.

Separating symbioses from incidental associations is a major obstacle in symbiosis research. In this survey of fungi associated with Asian bark and ambrosia beetles, we used quantitative culture and DNA barcode identification to characterize fungal communities associated with co-infesting beetle species in pines (Pinus) of China and Vietnam. To quantitatively discern likely symbioses from coincidental associations, we used multivariate analysis and multilevel pattern analysis (a type of indicator species analysis). Nearly half of the variation in fungal community composition in beetle galleries and on beetle bodies was explained by beetle species. We inferred a spectrum of ecological strategies among beetle-associated fungi: from generalist multispecies associates to highly specialized single-host symbionts that were consistently dominant within the mycangia of their hosts. Statistically significant fungal associates of ambrosia beetles were typically only found with one beetle species. In contrast, bark beetle-associated fungi were often associated with multiple beetle species. Ambrosia beetles and their galleries were frequently colonized by low-prevalence ambrosia fungi, suggesting that facultative ambrosial associations are commonplace, and ecological mechanisms such as specialization and competition may be important in these dynamic associations. The approach used here could effectively delimit symbiotic interactions in any system where symbioses are obscured by frequent incidental associations. It has multiple advantages including (1) powerful statistical tests for non-random associations among potential symbionts, (2) simultaneous evaluation of multiple co-occurring host and symbiont associations, and (3) identifying symbionts that are significantly associated with multiple host species.

Differential Strategies of Two Arbuscular Mycorrhizal Fungi Varieties in the Protection of Lycium ruthenicum under Saline-Alkaline Stress.

To delve into the growth and physiological adaptations exhibited by the economically vital black wolfberry (Lycium ruthenicum) upon inoculation with arbuscular mycorrhizal fungi (AMF) under varying levels of saline-alkaline stress A series of pot experiments were conducted in a gradient saline-alkaline environment (0, 200, 400 mM NaCl: NaHCO3 = 1:1). One-year-old cuttings of black wolfberry, inoculated with two AMF species-Funneliformis mosseae (Fm) and Rhizophagus intraradices (Ri)-served as the experimental material, enabling a comprehensive analysis of seedling biomass, chlorophyll content, antioxidant enzyme activities, and other crucial physiological parameters. This study demonstrated that both Fm and Ri could form a symbiotic relationship with the root of Lycium ruthenicum. Notably, Fm inoculation significantly bolstered the growth of the underground parts, while exhibiting a remarkable capacity to scavenge reactive oxygen species (ROS), thereby effectively mitigating membrane oxidative damage induced by stress. Additionally, Fm promoted the accumulation of abscisic acid (ABA) in both leaves and roots, facilitating the exclusion of excess sodium ions from cells. Ri Inoculation primarily contributed to an enhancement in the chlorophyll b (Chlb) content, vital for sustaining photosynthesis processes. Furthermore, Ri's ability to enhance phosphorus (P) absorption under stressful conditions ensured a steady influx of essential nutrients. These findings point to different strategies employed for Fm and Ri inoculation. To holistically assess the saline-alkaline tolerance of each treatment group, a membership function analysis was employed, ultimately ranking the salt tolerance as Fm > Ri > non-mycorrhizal (NM) control. This finding holds paramount importance for the screening of highly resilient Lycium ruthenicum strains and offers invaluable theoretical underpinnings and technical guidance for the remediation of saline-alkaline soils, fostering sustainable agricultural practices in challenging environments.

Morphology and distribution of sensilla on antennae and mouthparts of the adult bruchid beetles, Bruchidius coreanus (Coleoptera: Bruchidae).

Bruchidius coreanus is a serious pest on Gleditsia sinensis Lam during seed storage, causing significant losses to their yield in southwest China. To gain insight into their behavioral mechanisms, the external morphology, ultrastructure, and distribution of sensilla on antennae, maxillary palps, and labial palps of both male and female B. coreanus were observed using a scanning electron microscope. The results revealed that both male and female adults had serrated antennae comprising a scape, a pedicel, and nine flagellomeres (F1-F9). There were eight types and seven subtypes of antenna sensilla observed in both sexes, including Böhm sensilla (BS), two subtypes of sensilla chaetica (SC1 and SC2), two subtypes of sensilla trichodea (ST1 and ST2), three subtypes of sensilla basiconica (SB1, SB2, and SB3), sensilla auricillica (SA), sensilla styloconicum (SS), capitate pegs (CP), and sensilla cavity (SCa). The average length of BS and ST (ST1 and ST2) showed significant differences between males and females. Furthermore, the number of SC (SC1 and SC2), ST1, and SCa differed significantly between the sexes. Four types of sensilla were found on the maxillary palps and labial palps, with the length of ST on these palps significantly differing between males and females. Additionally, SS on male labial palps was significantly longer than in females. The number of SC significantly differed between the male and female maxillary palps and labial palps, while ST and SS showed significant differences in the maxillary palps. These findings will contribute to further electrophysiological recording and behavioral research. RESEARCH HIGHLIGHTS: The external morphology and distribution of various sensilla on the antennae, maxillary palps, and labial palps of Bruchidius coreanus were described. Eight types and seven subtypes of antenna sensilla were observed on the antennae, while four types of sensilla were observed on the maxillary palps and labial palps. The capitate pegs were found exclusively on the antennae of female B. coreanus.

Spatial Distribution Pattern and Sampling Plans for Two Sympatric Tomicus Species Infesting Pinus yunnanensis during the Shoot-Feeding Phase.

Tomicus minor (Hartig) and Tomicus yunnanensis Kirkendall and Faccoli are two sympatric species that infest Pinus yunnanensis (Franchet) in southwest China, contributing to growth losses. Accurate sampling plans are needed to make informed control decisions for these species. We investigated three pine forests within experimental sites in Yuxi, Yunnan province, China from 2016 to 2018. The spatial distribution patterns of two pine shoot beetles during the shoot-feeding phase were determined using Taylor's power law. The optimum sample sizes and stop lines for precision levels of 0.25 and 0.10 were calculated. The model was validated using an additional 15 and 17 independent field datasets ranging in density from 0.06 to 1.90 beetles per tree. T. minor and T. yunnanensis adults showed aggregated spatial distributions. For T. minor, sample sizes of 41 and 259 trees were adequate for a D of 0.25 and 0.10, respectively, while for T. yunnanensis, a mean density of one individual per tree required sample sizes of 33 plants (D = 0.25) and 208 plants (D = 0.10). The software simulations of this sampling plan showed precision levels close to the desired levels. At a fixed-precision level of 0.25, sampling is easily achievable. This sampling program is useful for the integrated pest management (IPM) of two sympatric Tomicus species.

Transgenerational effects of multiple mating in Spodoptera litura Fabricius (Lepidoptera: Noctuidae).

Polyandrous mating can result in sexual conflict and/or promote the evolution of mating patterns. Does multiple mating by females support the genetic benefits hypothesis and can it be validated as an evolutionary strategy? If we are to decipher the consequences of sexual interactions and understand the interplay of sexual conflict and multiple generational benefits, the transgenerational effects need to be followed over multiple generations. We investigated the effects of three mating patterns, single mating, repeated mating, and multiple mating, on parental Spodoptera litura copulation behavior, and then identified the impact on the development, survival, and fecundity of the F1 and F2 generations. Fecundity was not significantly affected in the F1 generation but was substantially enhanced in the F2 generation. There was a reversal of offspring fitness across the F2 generations from the F1 generations in progeny produced by multiple mating. In addition, the intrinsic rate of increase, finite rate of increase and net reproductive rate in the F1 generation the multiple mating treatment was significantly lower than in the single mating treatment, but there was no apparent effect on the F2 generation. Repeated mating had no significant effects on progeny fitness. We postulate that multiple mating imposes cross-transgenerational effects and may ultimately influence multigenerational fitness in S. litura.

Cold tolerance strategy and cryoprotectants of Megabruchidius dorsalis in different temperature and time stresses.

The honey locusts (genus Gleditsia) are a genus of high-value trees in Asia. Seed beetle, Megabruchidius dorsalis (Fåhraeus) (Col.: Chrysomelidae: Bruchinae), is a Gleditsia oligophagous pest that causes severe yield reduction. To understand the cold tolerance of M. dorsalis adults, this study investigated its cold tolerance strategy and the influence of low temperatures on its physiology and biochemistry. The low-temperature treatments were divided into three groups: long-term temperature acclimation (Group 1; 15°C, or 20°C, or 25°C, or 28°C [control check, CK] for 10 days), short-term low-temperature exposure (Group 2; 0°C or 4°C for 2 h), and long-term low-temperature induction (Group 3; 0°C or 4°C for 1, 3, or 5 d). The supercooling point (SCP; temperature at which spontaneous nucleation and ice lattice growth begin), freezing point (FP; temperature at which insect fluids freeze), low lethal temperature (LLT; temperature at which all individuals are killed), water, lipid, glycerol, and total sugars contents were measured under different temperature stresses. The results showed that M. dorsalis adults were a freeze-avoidant species. The SCP and LLT at 28°C were -10.62°C and -19.48°C, respectively. The SCP and FP of long-term temperature acclimation (15°C, or 20°C, or 25°C) were significantly lower than that of the control group (28°C). The water content of the long-term low temperature induction (0°C) group was significantly lower than that of the control group. The lipid and glycerol content in the acclimated group at 20°C and 25°C were significantly higher than in the control group. M. dorsalis adults may maintain their biofluids in a supercooled state via cryoprotectant accumulation and cryoprotective dehydration to prevent ice nucleation. This study provides a theoretical basis for future research on overwintering and potential distribution and related prediction of M. dorsalis adults.

Two new invasive Ips bark beetles (Coleoptera: Curculionidae) in mainland China and their potential distribution in Asia.

BACKGROUND: Ips is a bark beetle genus of 45 species, many of which are pests of conifer forests and plantations under stress. Twelve Ips species are recorded from China and presumably native there. From 2016 to 2018, specimens suspected to be Ips calligraphus and Ips grandicollis, were collected from traps with ethanol as a sole lure in Zhuhai, Guangdong, China. Both species originate in North America and infest various species of native or introduced pines. Since Ips species are known to cause or exacerbate problems in pine plantations, and a regional survey using traps baited with attractants were implemented in this study to investigate the extent of the introduction. RESULTS: Both I. calligraphus and I. grandicollis have been collected repeatedly from several traps with Ips attractants in Zhuhai, Guangdong, China since 2016. Potential distributions of these two species in Asia, inferred using MaxEnt, is extensive, given the high projected environmental suitability in North America, South America, Mediterranean Europe, Northern Africa, and Eastern Asia. The host plant of I. calligraphus from Zhuhai was identified as slash pine Pinus elliottii using DNA barcoding of gut contents from trapped individuals. CONCLUSION: This is the first report of the establishment of two American pine bark beetles, I. calligraphus and I. grandicollis in continental Asia. The gut content of both species suggests that these pest feeds on a non-native host. Whether the two species present high-risk to Asian forests will become clear with more research on their interactions with native pines.

Ultrastructure of the Sensilla on the Antennae and Mouthparts of Bean Weevils, Megabruchidius dorsalis (Coleoptera: Bruchinae).

Megabruchidius dorsalis (Fåhraeus, 1839) (Coleoptera: Bruchinae) is an important pest that damages the seeds of Gleditsia L. (Fabaceae: Caesalpinioideae). This beetle searches for host plants with its sensory system. To further explore the mechanisms of host location and to understand the ultrastructure of M. dorsalis, we examined the morphology and distribution of its sensilla on the antennae and mouthparts of male and female adults, using scanning electron microscopy (SEM). Both male and female antennae are serrated and can be divided into scape, pedicel, and flagellum. There were seven types and eight subtypes of antennal sensilla, including Bohm bristles (BB), two subtypes of sensilla trichoid (ST1, ST2), two subtypes of sensilla chaetica (SC1, SC2), four subtypes of sensilla basiconic (SB1, SB2, SB3, SB4), sensilla cavity (SCa), sensilla auricillica (SA), and sensilla gemmiformium (SG). Five types of maxillary and labial palp sensilla in the mouthparts were observed: sensilla chaetica (SC), sensilla trichoidea (ST), sensilla styloconica (SSt), sensilla coeloconica (SCo), and sensilla digitiform (SD). No sexual dimorphism in sensilla type was observed, but there were variations between males and females in the numbers and distribution along the antennae. There were more SA in males than in females, while the number of ST sensilla in the maxillary palps were lower in males than in females. ST1 were most abundant in both sexes. We discussed potential function related to structure via comparisons with previous investigations of bruchids and other insects. Our results provide a theoretical basis for further studies on sensory physiological function, using semiochemicals as effective biological controls of M. dorsalis.

Does Larval Rearing Diet Lead to Premating Isolation in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)?

Host plant preference during the larval stage may help shape not only phenotypic plasticity but also behavioral isolation. We assessed the effects of diet on population parameters and mate choice in Spodoptera litura. We raised larvae fed on tobacco, Chinese cabbage, or an artificial diet, and we observed the shortest developmental time and highest fecundity in individuals fed the artificial diet. However, survival rates were higher for larvae on either of the natural diets. Population parameters including intrinsic rate of increase and finite rate of increase were significantly higher with the artificial diet, but this diet led to a lower mean generation time. Copulation duration, copulation time, and number of eggs reared significantly differed between diets. In terms of mate choice, females on the artificial diet rarely mated with males fed on a natural host. Our results support the hypothesis that different diets may promote behavioral isolation, affecting mating outcomes. Thus, findings for populations fed an artificial diet may not reflect findings for populations in the field.

Different Host Plants Distinctly Influence the Feeding Ability of the Brown Citrus Aphid Toxoptera citricida.

Piercing-sucking insects are important crop pests, and an understanding of their feeding behavior and population development plays a crucial role in studying insect population dynamics and crop resistance. In our study, we examined the probing behavior of the brown citrus aphid, Toxoptera citricida, using electropenetrography and assessed its population development after 8 days on seven host plants: Yuzu, Citrus junos Sieb. ex Tanaka; Rough Lemon, C. jambhiri Lush.; 'Luofu' kumquat, Fortunella margarita Swingle; 'Olinda' valencia orange, C. sinensis (L.) Osbeck; 'Yanxiwanlu' Ponkan, C. reticulata Blanco; 'Rohde Red' valencia orange, C. sinensis; and 'Eureka' lemon, C. limon (L.) Osbeck. The results demonstrated that probing by the brown citrus aphid differed significantly according to the target hosts. Toxoptera citricida produced significantly more pathway activities on Eureka than on Rough Lemon and Yuzu. Toxoptera citricida spent more time from the first probe to first salivation into phloem sieve elements on Eureka compared to Yuzu. In addition, the total duration of ingestion from sieve cells of each aphid in the phloem-feeding phase was shortest on Eureka, and this was significantly shorter than that on Yuzu, Rough Lemon, Luofu, and Olinda. The population number of T. citricida on Eureka after 8 days was significantly lower than that on the other hosts. Overall, Eureka was found to have obvious resistance to T. citricida, whereas Yuzu and Rough Lemon were susceptible host plants. These results provide a theoretical basis for exploring aphid-resistant fruit tree resources using resistant varieties.

Delayed Mating with Multiple Partners Decreases Indexes of Mating in Female and Male Spodoptera litura (Lepidoptera: Noctuidae).

Delayed mating is an effective strategy that can decrease the fecundity of a pest by reducing the time that females have to mate. This disruption does not completely inhibit mating and may lead to multiple matings. The effects of delayed mating with multiple partners on different indexes of mating in female and male Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) were determined in this study. The delay in mating significantly decreased the number of matings and the mating success of both sexes and the male contribution to reproduction. Compared with the effect on female fecundity, the delayed mating with multiple partners had a greater effect on the male mate contribution to fecundity. The longevity of females and males increased significantly with a 72 h delay in mating. Linear regression analysis showed negative relationships between delayed mating and fecundity and number of matings in both sexes. Thus, delayed mating with multiple partners can disrupt the mating and reproductive potential of S. litura.

Delay in Mating Reduces Reproductivity but Increases Life Span in Tobacco Cutworm, Spodoptera litura Fabricius (Lepidoptera: Noctuidae).

A pheromone-mediated mating disruption is a vital tool in the management of insect population dynamics that not only prevents mating but also delays mating in the target insect. Here, we examined the effect of delayed mating on the longevity and reproductive performance of the global pest, Spodoptera litura Fabricius (Lepidoptera: Noctuidae). Delayed mating was imposed on both sexes simultaneously, males only, and females only. The results showed that a 30-40% reduction in the successful mating rate of S. litura was caused by 7-d delay in mating. Increased mating ages of both sexes of S. litura resulted in a significant decrease in 3-d-old delayed mating, followed by an increase in mean duration of copulation. Furthermore, delayed mating had a significantly negative influence on the number of S. litura eggs produced. Mating delay imposed on both sexes simultaneously had a significantly greater effect on longevity and the number of eggs than when it was applied to either sex alone, and females were more severely affected by delayed mating than males in terms of longevity. Percentage of mating, fecundity, and female longevity were all significantly correlated with the number of days delayed mating. However, the hatching rate of eggs was not significantly affected by an increased delay in mating. Overall, our results indicated that delayed mating in both females and males drastically reduced the females' reproductive output, which itself was affected more by increasing the age at mating of females than males.