Tissue Localization of the Endosymbiotic Bacterium “Candidatus Blochmannia floridanus” in Adults and Larvae of the Carpenter Ant Camponotus floridanus

The distribution of endosymbiotic bacteria in different tissues of queens, males, and workers of the carpenter ant Camponotus floridanus was investigated by light and electron microscopy and by in situ hybridization. A large number of bacteria could be detected in bacteriocytes within the midguts of workers, young virgin queens, and males. Large amounts of bacteria were also found in the oocytes of workers and queens. In contrast, bacteria were not present in oocyte-associated cells or in the spermathecae of mature queens, although occasionally a small number of bacteria could be detected in the testis follicles of males. Interestingly, the number of bacteriocytes in mature queens was strongly reduced and the bacteriocytes contained only very few or no bacteria at all, although the endosymbionts were present in huge amounts in the ovaries of the same animals. During embryogenesis of the deposited egg, the bacteria were concentrated in a ring of endodermal tissue destined to become the midgut in later developmental stages. However, during larval development, bacteria could also be detected in other tissues although to a lesser extent. Only in the last-instar larvae were bacteria found exclusively in the midgut tissue within typical bacteriocytes. Tetracycline and rifampin efficiently cleansed C. floridanus workers of their symbionts and the bacteriocytes of these animals still remained empty several months after treatment had ceased. Despite the lack of their endosymbionts, these adult animals were able to survive without any obvious negative effect under normal cultivation conditions.     

Population and colony structure of the carpenter ant Camponotus floridanus

The colony and population structure of the carpenter ant, Camponotus floridanus, were investigated by multilocus DNA fingerprinting using simple repeat motifs as probes [e.g. (GATA)₄]. The mating frequency of 15 queens was determined by comparing the fingerprint patterns of the queen and 17–33 of her progeny workers. C. floridanus queens are most probably singly mated, i.e. this species is monandrous and monogynous (one queen per colony). C. floridanus occurs in all counties of mainland Florida and also inhabits most of the Key islands in the southern part of Florida. We tested whether the two mainland populations and the island populations are genetically isolated. Wright's F_ST and Nei's D-value of genetic distance were calculated from intercolonial bandsharing-coefficients. The population of C. floridanus is substructured (F_ST= 0.19 ± 0.09) and the highest degree of genetic distance was found between one of the mainland populations and the island populations (D= 0.35). Our fingerprinting technique could successfully be transferred to 12 other Camponotus species and here also revealed sufficient variability to analyse the genetic structure. In three of these species (C. ligniperdus, C. herculeanus and C. gigas) we could determine the mating frequency of the queen in one or two colonies, respectively.     

Timekeeping Through Social Contacts: Social Synchronization of Circadian Locomotor Activity Rhythm in the Carpenter Ant Camponotus paria

In ant colonies a large proportion of individuals remain inside nests for most of their lives and come out only when necessary. It is not clear how, in a nest of several thousand individuals, information about local time is communicated among members of the colony. Central to this seem to be circadian clocks, which have an intrinsic ability to keep track of local time by entraining to environmental light-dark, temperature, and social cycles. Here, the authors report the results of their study aimed at understanding the role of cyclic social interactions in circadian timekeeping of a day-active species of carpenter ant Camponotus paria. The authors found that daily social interactions with visitors (worker ants) was able to synchronize the circadian locomotor activity rhythm of host worker ants and queens, in one-on-one (pair-wise) and multi-individual (group-wise) interactions. Interestingly, the outcome of cyclic social interactions was context specific; when visitor workers socially interacted with host workers one-on-one, host workers considered the time of interaction as subjective day, but when visitor workers interacted with a group of workers and queens, the hosts considered the time of interaction as subjective night. These results can be taken to suggest that members of the ant species C. paria keep track of local time by socially interacting with workers (foragers) who shuttle in and out of the colony in search of food. (Author correspondence: vsharma@jncasr.ac.in)     

Cuticular body hairs mediate clumping of small Camponotus floridanus larvae

The body surface of insects usually carries cuticular hairs. Commonly, important functions of these structures are to prevent drowning and to defend against predators. Here, we report on our studies on hairs at the surface of larvae of the ant species Camponotus floridanus and Camponotus sericeiventris. First, we present data supporting the hypothesis that anti-drowning properties of the surface might rely on cuticular hairs. Second, we show that especially in young larvae body hairs serve as attachment and interlocking devices mediating clumping of larvae facilitating transport by workers. Based on our observations, we speculate that clumping also enhances larval perceptibility. Taken together, larval cuticular hairs seem to have at least two important functions augmenting chances of larval survival. Obviously, despite their immobility, young Camponotus larvae support childcare in the ant colony providing an arsenal of cuticular hairs on their body surface.     

Systemic gene knockdown in Camponotus floridanus workers by feeding of dsRNA

RNA interference (RNAi) technology enables to study specific gene functions also in social insects, which are otherwise difficult to access for genetic manipulations. The recent sequencing of the genomes from seven ant species made these members of the Formicidae available for knockdown studies. However, for this purpose the RNAi technology first needs to be adapted for application in ants. Studies on other insects show that the effectiveness of RNAi is quite species-specific and can depend on several experimental parameters such as the investigated stage of the insect, the target gene and/or the dsRNA delivery method. RNAi in ants through feeding of dsRNA is a preferable approach, since knockdown can be achieved in individuals without interfering with the animal’s physiology in contrast to injection of dsRNA. Here, we present a protocol for gene knockdown in Formicidae by feeding of dsRNA to worker animals. The expression of a peptidoglycan recognition protein gene, PGRP-LB, was efficiently knocked down in the body of Camponotus floridanus worker ants. Moreover, we describe a relatively cheap method to extract dsRNA from bacteria in order to obtain large quantities needed for feeding experiments.     

Tissue localization of the endosymbiotic bacterium "Candidatus Blochmannia floridanus" in adults and larvae of the carpenter ant Camponotus floridanus

The distribution of endosymbiotic bacteria in different tissues of queens, males, and workers of the carpenter ant Camponotus floridanus was investigated by light and electron microscopy and by in situ hybridization. A large number of bacteria could be detected in bacteriocytes within the midguts of workers, young virgin queens, and males. Large amounts of bacteria were also found in the oocytes of workers and queens. In contrast, bacteria were not present in oocyte-associated cells or in the spermathecae of mature queens, although occasionally a small number of bacteria could be detected in the testis follicles of males. Interestingly, the number of bacteriocytes in mature queens was strongly reduced and the bacteriocytes contained only very few or no bacteria at all, although the endosymbionts were present in huge amounts in the ovaries of the same animals. During embryogenesis of the deposited egg, the bacteria were concentrated in a ring of endodermal tissue destined to become the midgut in later developmental stages. However, during larval development, bacteria could also be detected in other tissues although to a lesser extent. Only in the last-instar larvae were bacteria found exclusively in the midgut tissue within typical bacteriocytes. Tetracycline and rifampin efficiently cleansed C. floridanus workers of their symbionts and the bacteriocytes of these animals still remained empty several months after treatment had ceased. Despite the lack of their endosymbionts, these adult animals were able to survive without any obvious negative effect under normal cultivation conditions.     

Caste- and sex-specific adaptations within the olfactory pathway in the brain of the ant Camponotus floridanus

Olfaction plays a key role in mediating ant behavior, and ant societies are characterized by caste- and sex-specific division of labor. We propose that caste- and sex-specific adaptations in the olfactory pathway promote differences in olfactory behavior. This study compares olfactory centers in the brain of large (major) workers, small (minor) workers, virgin queens, and males of the carpenter ant Camponotus floridanus. The number of glomeruli in the antennal lobe was similar in the female castes, although the glomerular volumes differed. Males had approximately 45% fewer glomeruli compared to females (approximately 258 and approximately 434) and one antennal sensory tract was absent. A dual output pathway to the mushroom bodies was present in males. In contrast to females, however, the number of glomeruli connected to the medial antennocerebral tract was substantially smaller than those associated with the lateral tract. All glomeruli in the male antennal lobe contained serotonergic processes, whereas in the female castes glomeruli in the large tract six cluster lacked serotonergic innervations. We conclude that differences in general glomerular organization are subtle among the female castes, but sex-specific differences in the number, connectivity and neuromodulatory innervation of glomeruli are substantial and likely to underlie differences in olfactory processing and learning.     

Early Learning and the Recognition of Conspecific Cocoons by Carpenter Ants (Camponotus spp.)

Groups of newly-eclosed workers of two carpenter ant species (Camponotus floridanus and C. tortuganus) were reared in the presence of conspecific cocoons, cocoons of the other species, or were kept without cocoons. Groups of older workers (> 20 days), previously exposed to conspecific brood in their natal nest, were familiarized with either conspecific or heterospecific cocoons. After 14 days of exposure, groups were subjected to short-term (5 min) and long-term (10 day) preference tests. Young and older workers retrieved and retained many cocoons of both species, familiar and unfamiliar. However, a pattern of non-exclusive discrimination emerged: (1) Young workers exposed to conspecific cocoons picked up and retrieved conspecifics before unfamiliar heterospecifics, and retained conspecifics longer. (2) Young workers generally required experience with conspecifics to develop this preference, as those exposed to only heterospecifics and those deprived of cocoons were impartial in short-term tests. However, in long-term tests (5–10 days), naive young workers significantly preferred unfamiliar conspecifics. (3) Older workers preferred conspecific cocoons, whether familiar or unfamiliar, in short-term tests, but their tolerance for heterospecifics in the longer term (5 days) could be increased by recent familiarization. In no case did young or older ants significantly prefer familiar heterospecifics to conspecifics. These results confirm a role for early learning in brood recognition by carpenter ants, but suggest that it is less important than in Formica species studied by previous authors.     

Preparation and Characterization of Nanoliposomes Loaded with the Antimicrobial Peptide CGA-N9

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides (AMPs) are considered ideal antibiotic candidates, but their therapeutic ability and application are restricted...     

Characterization and Antilisterial Effect of Phosphatidylcholine Nanovesicles Containing the Antimicrobial Peptide Pediocin

Encapsulation may provide increased stability and antimicrobial efficiency to bacteriocins. In this work, the antilisterial peptide pediocin was encapsulated in nanovesicles prepared from partially purified soybean phosphatidylcholine. The maintenance of antimicrobial activity and properties of free and encapsulated pediocin was observed during 13 days at 4 °C, and after this period, the encapsulated pediocin retained 50 % its initial activity. The maintenance of the bioactive properties of free and encapsulated pediocin was observed against different species of Listeria, inhibiting Listeria monocytogenes, Listeria innocua and Listeria ivanovii. The size of vesicles containing pediocin was determined by dynamic light scattering as an average of 190 nm, with little change throughout the observation period. Polydispersity index values were around 0.201 and are considered satisfactory, indicating an adequate size distribution of liposomes. The efficiency of encapsulation was 80 %. Considering these results, the protocol used was appropriate for the encapsulation of this bacteriocin. Results demonstrate the production of stable nanoparticulate material. The maintenance of the properties of pediocin encapsulated in liposomes is fundamental to prospect the stability in different conditions of the food matrix.     

Molecular Characterization of Barley 3H Semi-Dwarf Genes

The barley chromosome 3H accommodates many semi-dwarfing genes. To characterize these genes, the two-rowed semi-dwarf Chinese barley landrace ‘TX9425’ was crossed with the Australian barley variety ‘Franklin’ to generate a doubled haploid (DH) population, and major QTLs controlling plant height have been identified in our previous study. The major QTL derived from ‘TX9425’ was targeted to investigate the allelism of the semi-dwarf gene uzu in barley. Twelve sets of near-isogenic lines and a large NILF₂ fine mapping population segregating only for the dwarfing gene from ‘TX9425’ were developed. The semi-dwarfing gene in ‘TX9425’ was located within a 2.8 cM region close to the centromere on chromosome 3H by fine mapping. Molecular cloning and sequence analyses showed that the ‘TX9425’-derived allele contained a single nucleotide substitution from A to G at position 2612 of the HvBRI1 gene. This was apparently the same mutation as that reported in six-rowed uzu barley. Markers co-segregating with the QTL were developed from the sequence of the HvBRI1 gene and were validated in the ‘TX9425’/‘Franklin’ DH population. The other major dwarfing QTL derived from the Franklin variety was distally located on chromosome 3HL and co-segregated with the sdw1 diagnostic marker hv20ox2. A third dwarfing gene, expressed only in winter-sown trials, was identified and located on chromosome 3HS. The effects and interactions of these dwarfing genes under different growing conditions are discussed. These results improve our understanding of the genetic mechanisms controlling semi-dwarf stature in barley and provide diagnostic markers for the selection of semi-dwarfness in barley breeding programs.     

Molecular and Structural Characterization of Barley Vernalization Genes

Vernalization, the requirement of a period of low temperature to induce transition from the vegetative to reproductive state, is an evolutionarily and economically important trait in the Triticeae. The genetic basis of vernalization in cultivated barley (Hordeum vulgare subsp. vulgare) can be defined using the two-locus VRN-H1/VRN-H2 model. We analyzed the allelic characteristics of HvBM5A, the candidate gene for VRN-H1, from ten cultivated barley accessions and one wild progenitor accession (subsp. spontaneum), representing the three barley growth habits – winter, facultative, and spring. We present multiple lines of evidence, including sequence, linkage map location, and expression, that support HvBM5A being VRN-H1. While the predicted polypeptides from different growth habits are identical, spring accessions contain a deletion in the first intron of HvBM5A that may be important for regulation. While spring HvBM5A alleles are typified by the intron-localized deletion, in some cases, the promoter may also determine the allele type. The presence/absence of the tightly linked ZCCT-H gene family members on chromosome 4H perfectly correlates with growth habit and we conclude that one of the three ZCCT-H genes is VRN-H2. The VRN-H2 locus is present in winter genotypes and deleted from the facultative and spring genotypes analyzed in this study, suggesting the facultative growth habit (cold tolerant, vernalization unresponsive) is a result of deletion of the VRN-H2 locus and presence of a winter HvBM5A allele. All reported barley vernalization QTLs can be explained by the two-locus VRN-H1/VRN-H2 model based on the presence/absence of VRN-H2 and a winter vs. spring HvBM5A allele. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

直链烷基苯磺酸钠对中国林蛙抗菌肽基因表达的影响

近年来由于生境丧失及环境污染等原因,中国林蛙野生种群急剧减少.为深入研究水体中普遍存在的阴离子表面活性剂直链烷基苯磺酸钠(linear alkylbenzene sulfonate,LAS)对中国林蛙生存的影响,通过急毒实验确定LAS对分别处于发育阶段26、32、42、46期的中国林蛙的半致死浓度.同时利用1、4、8 mg/L LAS处理不同发育阶段中国林蛙,研究LAS对中国林蛙存活率、发育状态的影响,并利用real-time PCR技术检测中国林蛙抗菌肽基因表达水平变化.结果表明,当处理时间为96 h,LAS浓度达到12.33 mg/L以上时会对不同发育阶段中国林蛙产生较为严重的致死效应.1~8 mg/L浓度范围的LAS虽然对中国林蛙存活和发育没有明显影响,但是改变了其抗菌肽基因表达模式.特别是中国林蛙胚后发育后期(32期)和变态期(42期),4~8 mg/L LAS会在不同程度上抑制prepropalustrin-2CE3、preprobrevinin-2CE1和preprotemporin-1CEd1抗菌肽基因表达.总体而言,高浓度LAS对中国林蛙免疫系统造成一定影响,可能使该物种更易受到致病菌的侵袭.研究结果为化学污染物对中国林蛙免疫系统影响积累了实验数据,同时为研究中国林蛙抗菌肽基因表达的调控通路提供一定的理论依据.     

Molecular phylogeny of Camponotus ants in Korea

The molecular phylogeny of 12 species of Camponotus ants in Korea was examined using random amplified polymorphic DNA (RAPD) markers as inputs for an analysis of molecular variance (AMOVA) and cluster analysis to describe the relationships between species. For comparison, morphometric data (based on 10 morphological characters) were also gathered for phylogenetic analysis. Assessments of similarity between species were made, and the results of these assessments are compared for the molecular and morphological data sets. In the morphometric analysis, the following groups were identified: (i) C. atrox, C. kiusuensis, C. japonicus and C. concavus (93% similarity); (ii) C. sp. 1 (be diverging at 80% similarity); (iii) C. jejuensis, C. sp. 3, C. itoi, C. nawai and C. tokioensis (94.5% similarity); and (iv) C. nipponensis and C. quadrinotatus (94.5% similarity). Formica fusca was 73.5% similar to the 12 Camponotus species studied here. The group comprising C. nawai and C. tokioensis had the highest similarity index (97.36%), followed by the group comprising C. jejuensis and C. sp. 3 (95%), then C. atrox and C. kiusuensis (94.5%), and then C. nipponensis and C. quadrinotatus (93.5%). In the molecular analysis the following groups were identified: (i) C. atrox and C. jejuensis (30% similarity); (ii) C. concavus, C. japonicus and C. itoi (25% similarity); (iii) C. kiusuensis, C. nawai, C. sp. 3, C. nipponensis, C. quadrinotatus and C. sp. 1 (24% similarity); and (iv) C. tokioensis (be diverging at 23% similarity). The most closely related group in the molecular analysis was C. nawai and C. sp. 3 (75% similarity), followed by C. concavus and C. japonicus (50.5% similarity), then C. atrox and C. jejuensis (30%), and then C. quadrinotatus and C. sp. 1. Camponotus tokioensis was the least closely related to other species among the 12 species studied. Although C. jejuensis and C. tokioensis were found to be 93.6% similar on the basis of morphometric data, molecular data indicated only 23% similarity, the lowest similarity index between any two species considered here. Camponotus jejuensis has marked morphological similarities to C. tokioensis, but on the basis of molecular data gathered in the present study, we refute the proposal that they are synonyms or sister species.     

Interindividual Variability and Idiosyncrasy in Social Behaviours in the Ant Camponotus vagus Scop

Large intraspecific variability was found in interactive ant behaviours. However, a time study of callow and mature Camponotus vagus workers showed some stability in relationships within pairs of workers or in given individuals. Individual idiosyncrasies were observed as tendencies to simultaneously display a single level of activity for several behaviours. Thus some mature workers reverse the food flow during trophallactic exchanges, spend more time exchanging food and are more aggressive toward intruders. The time spent by callows in their main activity, brood-tending, allows discrimination on two activity levels. Moreover, callows deprived of any relationship with older nestmates show different levels of activity in the duration and intensity of aggressive and non-aggressive behaviours. The causality of these behavioural idiosyncrasies could possibly be explained in part by the social experience of each individual.