Culture-independent identification of gut bacteria in fourth-instar red imported fire ant, Solenopsis invicta Buren, larvae

Red imported fire ants (RIFA), Solenopsis invicta Buren, are medical, urban, and agricultural pests from South America. They are successful invaders due to their preference for disturbed habitats, high reproductive rates, and the ability to feed on a wide variety of food items (omnivorous). Fourth-instar larvae are used by the colony to digest solid food and then regurgitate it for consumption by workers and queens. Larvae are an ideal source of investigations of endosymbiotic bacteria possibly involved in nutrient distributions. Our study utilized 16S rDNA sequencing to describe the composition of the bacterial community in fourth-instar ant larvae in order to identify possible endosymbiotic bacteria present therein. The 16S rRNA gene was directly amplified from mixed-population DNA of whole fire ant larval guts and cloned into Escherichia coli. Bacterial communities from three geographically separated RIFA colonies were examined. Sequenced bacterial clones from guts were determined to be predominantly from the phylum Proteobacteria and the family Enterobacteriaceae. Our results did not detect the presence of endosymbiotic bacteria in the guts of RIFA larvae among the colonies. In addition, minimal species overlap was found when bacterial inventories were compared among colonies. Thus, bacteria coadapted with red imported fire ant larvae were not detected. Identified bacteria were not closely affiliated with endosymbiotic bacteria common in other insect species. Bacteria communities appeared to be unique to each geographical location and were determined by the foods consumed by the ants.     

Fungus Exposed Solenopsis invicta Ants Benefit from Grooming

We investigated aspects of resistance to entomopathogenic fungi in the social insect Solenopsis invicta, the red imported fire ant (RIFA). RIFA reared individually were significantly more susceptible to the entomopathogenic fungi Metarhizium anisopliae var. anisopliae M09 than reared in groups. Fungus exposed ants performed more self-grooming behavior when isolated as individuals and received more allo-grooming when accompanied with four healthy nestmates. Using fluorescence microscopy, we counted the number of fluorescein isothiocyanate (FITC)-labeled conidia on the cuticle of fungus exposed ants reared individually or as groups. The number of conidia on the surface of grouped ants decreased more rapidly than on isolated individuals. Allo-grooming behavior appears to be important in removing the conidia on the surface of RIFA. Individuals help fungus exposed ants by performing intensive grooming behaviors, which either risk infecting themselves or get them immunized as social immunity. We show evidence that contacting with fungus exposed ants would decrease susceptibility of nestmates to the fungus. All these results indicate that RIFA benefit from grooming behavior to fight against the fungal pathogens. Future advances in biological control of RIFA with entomopathogenic fungi are also discussed.     

红火蚁入侵草坪过程中蚂蚁类群变动趋势

观察研究了红火蚁入侵草坪之后蚂蚁群落结构的变动趋势。对各处理区在不同时期的蚂蚁优势种进行了分析,结果表明,与对照区相比红火蚁入侵后各处理区的蚂蚁优势种发生了明显改变,红火蚁替代了原来的优势蚂蚁种类,并在数量上占主导地位,而且各处理区的蚂蚁类群多样性、均匀度降低,优势度明显增大。通过分析红火蚁入侵过程中各处理区的蚂蚁类群相似性,表明轻度发生区与对照区的蚂蚁类群相似性系数q一直处在0.25-0.50之间,为中等不相似;中度发生区与对照区的蚂蚁类群相似性系数q一直处在0.075-0.444之间,为中等不相似或极不相似;重度发生区与对照区的蚂蚁类群相似性系数q一直处在0.0-0.222之间,为极不相似,这也说明了不同的红火蚁危害程度对本地蚂蚁类群结构的影响是不同的,红火蚁种群数量越大,对本地蚂蚁类群的影响也越大,排挤或取代它们所需要的时间也越短。     

The potential suitability of Jiangsu Province, east China for the invasive red imported fire ant, Solenopsis invicta

The red imported fire ant Solenopsis invicta Buren (RIFA), an invasive pest that has diverse detrimental impacts on the communities it invades, was recently discovered in China and has the potential to colonize numerous other regions. Using the model of Korzukhin et al. as modified by Morrison et al. and the biological and ecological characteristics of RIFA, we show that Jiangsu Province is a potentially suitable establishment area of RIFA. An isotherm map made by ArcMap, a Geographic Information System, indicated that several regions of Jiangsu Province (Changzhou, Liyang, Wuxian Dongshan, Nanjing and Lvsi) are at higher risk of S. invicta infestation, especially from late July and early August. Quarantine officials should be vigilant for any accidental introductions of this pest in the susceptible regions and time.     

Infection characteristics of Solenopsis invicta virus 2 in the red imported fire ant, Solenopsis invicta

Solenopsis invicta virus 2 (SINV-2) is the second virus identified from the fire ant, S. invicta Buren. SINV-2 is unique among positive-strand RNA viruses from insects by possessing four cistrons in a monopartite genome. Fire ant colonies testing positive for SINV-2 by RT-PCR did not exhibit any discernable symptoms. RT-PCR-based surveys for SINV-2 among 688 fire ant mounds in Alachua County, Florida, sampled during the period January, 2006 through December, 2007 showed that the prevalence of SINV-2 among nests ranged from 1.6% to 16.4%. Unlike S. invicta virus 1, no seasonal-associated prevalence was observed with regard to SINV-2 infection among fire ant colonies. No social form specificity was evident; SINV-2 was found in both monogyne and polygyne S. invicta ants. Real-time quantitative PCR experiments showed that SINV-2 genome equivalents per individual ant ranged from 1.9 × 10⁷ in. pupae to 4.3 × 10¹¹ in. inseminated queens. The SINV-2 infection was detected in all ant stages examined (eggs, larvae, pupae, workers, and queens). Tissue tropism studies indicated that the alimentary canal (specifically the midgut) is most likely the susceptible tissue. SINV-2 was successfully transmitted to uninfected S. invicta ants by feeding a partially purified homogenate of SINV-2-infected ants. The SINV-2 transmission rate ranged from 30% to 80%, and both positive (genomic) and negative (replicative) SINV-2 RNA strands accumulated in recipient ants over the course of the experiment. These results indicated that SINV-2 replicates within S. invicta.     

Pathogenicity of Beauveria bassiana ANU1 to the red imported fire ant,Solenopsis invicta workers in Korea

The red imported fire ant (RIFA), Solenopsis invicta, is one of the globalized invasive pests. This study focused on pathogenicity and virulence of entomopathogenic fungi as one of the biological control agents to RIFA workers under different temperatures. The fungal pathogen, Beauveria bassiana ANU1 was isolated from Korea in 2015 and showed the pathogenicity to RIFA workers. A conidial suspension (1 × 10⁷ conidia/ml) induced a low mortality from day 2 after treatment and reached to 100% mortality at day 7 and day 8 after treatment for major and minor workers, respectively. The median lethal concentrations of B. bassiana ANU1 were calculated as 3.9 × 10³ for major and 4.6 × 10³ for minor workers at day 7 after treatment. Low temperatures decreased a virulence of B. bassiana ANU1 (1 × 10⁷ conidia/ml) to RIFA and showed mortality of 26.6% for major and 20% for minor workers. Based on bioassay results, this study provides one of possibilities of effective and successful strategy for controlling RIFA by entomopathogenic fungi.     

Analysis on new occurrence records of red imported fire ant (Solenopsis invicta Buren) in China in 2021

Solenopsis invicta Buren, also known as the red imported fire ant (RIFA), has had a very negative effect on biological diversity, public safety, agriculture, and economics. Because of the growth in global trade, the RIFA threat to China will continue to increase. To better understand characteristics of newly observed distributions of RIFA, we studied the effects of three factors including provincial areas, landcover types, and climatic conditions on the 130 new occurrence records in 2021. (1) We found that RIFA preferred to invade Sichuan and Guangxi, and provinces that are large agricultural producers were more vulnerable to invasive RIFA. Guangdong was the most damaged province, and wealthy provinces, especially southeastern coastal provinces, were more at risk and suffered a bigger loss from the further RIFA threat. (2) Compared with other habitats, plantations received more significant damage from invasive RIFA. (3) Areas with an annual precipitation of 516.4 mm, annual average temperature of 18.6°C, or elevation of 569.9 m had a great abundance of invasive RIFA. Our study suggests that stringent inspection and quarantine measures are necessary in new occurrence areas to prevent further RIFA outbreak, and highlights the need for cross-provincial cooperation and national coordination to slow its spread.     

Enzyme polymorphisms in the fire ant,Solenopsis invicta (hymenoptera: formicidae)

A survey of allozyme variability in the fire ant, Solenopsis invicta Buren, was conducted using samples collected throughout the species' range in North America. A substantial amount of variability was found, although heterozygosity estimates were considerably less than estimates for diploid non-hymenopterous species. The presence of this variability raises the possibility of multiple introductions of this ant to North America. Although based on a limited sample, our data indicate no differences in variability between individuals from monogynous and polygynous populations or between individuals from the Mobile, Alabama area (presumed point of introduction) and outside the area. Individuals from polygynous colonies shared all detected alleles with individuals from monogynous colonies indicating the lack of long-term reproductive isolation between the two types of populations.     

Elucidating how the red imported fire ant (Solenopsis invicta) diffused spatiotemporally among different landscapes in north Taiwan, 2008–2015

Solenopsis invicta Buren, also known as the red imported fire ant (RIFA), has had a large negative impact on human and livestock health. However, few studies have further investigated the influence of human land use, which is an important factor affecting the habitats of insects, on the expansion of RIFAs. In addition, there is a lack of knowledge of the empirical associations between RIFA diffusion and land use within countries. Therefore, the objectives of this study were to provide an approach to delineate the areas of RIFA infestations and explore how land use influences the spatiotemporal diffusion of S. invicta. We used RIFA data from 2008 to 2015 from the RIFA surveillance system, which was conducted by the National RIFA Control Center in Taiwan. Two regions in Taiwan with different RIFA infestation levels were investigated. The ordinary kriging method was applied to show the spatial intensity of RIFAs, and the extreme distance estimator method was applied to determine the critical dispersal distances, which showed the distance of the highest probability of RIFAs in two consecutive years. In addition, network analyses were used to identify RIFA invasion routes between land‐use types. Finally, bivariate local indicators of spatial association were used to capture the invasion process in time and space. The results showed, paddy fields, main roads, and warehouses were identified as the top three land‐use types of diffusion sources. On average, the critical RIFA dispersal distances were 600 and 650 m in two consecutive years in high‐ and low‐infestation regions, respectively. Finally, RIFAs were likely to diffuse between main roads and warehouses in the low‐infestation region. Therefore, it is suggested that RIFA control activities be implemented at least 600 m from the observed spot. Additionally, control activities should be conducted on the identified three land‐use types of diffusion sources in the high‐infestation region, and the roadsides between main roads and warehouses in the low‐infestation region to prevent the accidental spread of RIFAs.     

红火蚁病原真菌分离鉴定及致病性研究

红火蚁Solenopsis invicta是世界上最具破坏力和威胁性的入侵检疫害虫之一,对农林业生产安全、公共设施以及人体健康都造成了巨大影响。为筛选对红火蚁有致病性的病原真菌,本研究通过组织分离法对自然罹病的红火蚁进行致病菌分离,结合病原形态学及分子生物学的方法对病原真菌进行鉴定并采用喷雾法测定了病原真菌对红火蚁的致病力。结果表明：从罹病红火蚁上分离出3株病原真菌HHYSFJ01、HHYSFJ02和HHYSFJ05,分别为红绶曲霉Aspergillus nomius、淡紫拟青霉Purpureocillium lilacinum和球孢白僵菌Beauveria bassiana。致病性测定,这3株病原菌对红火蚁均具有较强的致病性,且3株病原菌致病性间差异显著。经1.0×10^8孢子/mL孢子悬浮液喷雾处理红火蚁10 d后,球孢白僵菌菌株HHYSFJ05的致病性最强,红火蚁工蚁的校正死亡率为84.71%,LT50为4.67 d;红绶曲霉菌株HHYSFJ01和淡紫拟青霉菌株HHYSFJ02次之,红火蚁工蚁的校正死亡率分别为78.82%和73.29%,LT50分别为4.99 d和5.20 d。     

Isolation, characterization, and molecular identification of bacteria from the red imported fire ant (Solenopsis invicta) midgut

Bacteria were isolated and cultured from the red imported fire ant (Solenopsis invicta) midgut. The small-subunit ribosomal RNA gene, (16s rRNA gene, approximately 1500 bp) was amplified from bacterial genomic DNA using the polymerase chain reaction and consensus sequence primers. Restriction fragment length polymorphism analysis revealed 10 unique profiles, indicating that at least 10 different bacteria are present in red imported fire ant midguts. The 16s rRNA gene sequence was determined for these isolates and queried against the NCBI genetic database. The results identified all isolates to at least the genus level. Antibiotic resistance profiles and biochemical activities were also determined for these species. This work provides the basis for a wider characterization of bacterial distributions in fire ant colonies and provides strains suitable for genetic manipulation to develop novel methods of fire ant control.     

Aggressive interactions between Solenopsis invicta and Linepithema humile (Hymenoptera: Formicidae) under laboratory conditions

The Argentine ant, Linepithema humile (Mayr), and the red imported fire ant, Solenopsis invicta Buren, are natural agonists in their country of origin. Since the first report of L. humile in California in 1907 its range expanded statewide, displacing native ant species wherever it spread. Since the discovery of established populations of S. invicta in southern California in 1998, it has been restricted to discrete areas of southern California. However, as these discrete populations expand, they are encountering large populations of L. humile, which are effective competitors for available resources and are particularly aggressive in their encounters with other ant species such as S. invicta. Most Dolichoderine ants such as L. humile do not prefer to forage on baits made with defatted corn grit and soybean oil typically used in red imported fire ant control programs. Applications of these baits in areas where distributions of these species overlap might selectively affect populations of S. invicta and give L. humile a competitive advantage. Three laboratory experiments were conducted to determine the competitive outcomes between S. invicta pitted against L. humile: 1) agonistic behavior of workers in small arenas, 2) colony interactions with different population ratios, and 3) the effects of pyriproxyfen on the competitiveness of S. invicta against L. humile. Populations of S. invicta consisting of major workers killed more L. humile than did minors or a mixture of majors and minors. When paired against L. humile colonies consisting of 1,100 workers, colonies consisting of 38 S. invicta workers were easily defeated by L. humile. Colonies consisting of 450 S. invicta workers plugged their nest entrances, but they were ultimately defeated by L. humile after 13 d. The S. invicta colonies consisting of 1,100 workers took control of the bridge connecting the colonies, invaded the L. humile colony, killed the Argentine ant queens, and removed their brood. Pyriproxyfen-treated fire ant workers took significantly longer to chase the Argentine ants from a connecting bridge than did untreated fire ants. Thus, fire ant baits may have long-term effects on intercolonial aggression between S. invicta and L. humile, especially when Argentine ant populations are high in the summer.     

Diversity and geographic variation of endosymbiotic bacteria in natural populations of the pea aphid (Acyrthosiphon pisum) in China

Bacterial symbionts in aphids are known to benefit the insect host and associated with aphid’s ecological adaptation. The pea aphid (Acyrthosiphon pisum), an important legume pest worldwide, carries at least eight endosymbionts, providing a model system to study insect–bacteria interactions. However, species diversity and geographic variations of endosymbionts are unknown in Chinese populations; therefore, we characterized symbiont communities and diversity of 27 pea aphid samples from 13 geographic populations of China. Via amplicon high-throughput sequencing and diagnostic PCR, we found that bacterial communities of Chinese populations were dominated by Proteobacteria and Firmicutes. Among eight known endosymbionts, five (Buchnera, Serratia, Hamiltonella, Regiella, and Rickettsia) were detected by both methods, with a specific geographical distribution. The obligate symbiont, Buchnera, was present in all aphid samples, while the four facultative symbionts showed a significant geographic variation. Each population was randomly infected with distinct endosymbionts, ranging from three to five species. Serratia and Rickettsia showed relatively higher abundance in central regions of China, Regiella was predominant in eastern and western China, whereas Hamiltonella showed an extremely low abundance and was absent in four populations. Samples grouped by altitudes showed a significant diversity difference, whereas there was no significant difference between red and green body colors. Bacterial community structures of the Chinese pea aphid populations were mainly influenced by environmental factors, other than body colors. These data can guide the development of potential biocontrol techniques against this aphid.     

感染球孢白僵菌后红火蚁体内蛋白质含量的变化

为了解球孢白僵菌Beauveria bassiana成功感染红火蚁Solenopsis invicta的生理生化机制, 本研究在室内条件下测定了经球孢白僵菌感染后红火蚁工蚁、蛹和幼蚁体内蛋白质含量的变化。结果表明: 红火蚁各虫态感染球孢白僵菌后体内蛋白质含量变化存在差异。在接种后12-72 h, 工蚁体内蛋白质含量在24 h时最高, 为47.12 mg/g, 显著低于对照的56.40 mg/g(P＜0.05), 此后呈下降趋势, 72 h时蛋白质含量下降至25.16 mg/g。幼蚁在接种36 h时体内蛋白质含量最高, 为24.13 mg/g, 此后呈下降趋势, 72 h时蛋白质含量为8.95 mg/g, 显著低于对照的24.80 mg/g。蛹感染球孢白僵菌后体内蛋白质含量一直呈下降趋势, 从12 h的36.57 mg/g降到72 h的10.42 mg/g, 与对照的38.61 mg/g相比差异显著(P＜0.05)。结果显示球孢白僵菌感染能显著降低红火蚁各虫态体内蛋白质含量。     

严防危险性害虫红火蚁入侵

原产南美洲的红火蚁SolenopsisinvictaBuren,先后入侵美国和澳洲,最近又入侵中国台湾,对人类健康和环境安全构成重大威胁。该文介绍了红火蚁的形态特征、与近源种的形态区别、生物学特性、入侵特性、危害以及预防、控制等方面的知识,最后提出我国大陆需要高度警惕,严防红火蚁入侵。     

Dynamic changes in host–virus interactions associated with colony founding and social environment in fire ant queens (Solenopsis invicta)

The dynamics of host – parasite interactions can change dramatically over the course of a chronic infection as the internal (physiological) and external (environmental) conditions of the host change. When queens of social insects found a colony, they experience changes in both their physiological state (they develop their ovaries and begin laying eggs) and the social environment (they suddenly stop interacting with the other members of the mother colony), making this an excellent model system for examining how these factors interact with chronic infections. We investigated the dynamics of host – viral interactions in queens of Solenopsis invicta (fire ant) as they transition from mating to colony founding/brood rearing to the emergence of the first workers. We examined these dynamics in naturally infected queens in two different social environments, where queens either founded colonies as individuals or as pairs. We hypothesized that stress associated with colony founding plays an important role in the dynamics of host – parasite interactions. We also hypothesized that different viruses have different modalities of interaction with the host that can be quantified by physiological measures and genomic analysis of gene expression in the host. We found that the two most prevalent viruses, SINV‐1 and SINV‐2, are associated with different fitness costs that are mirrored by different patterns of gene expression in the host. In fact SINV‐2, the virus that imposes the significant reduction of a queen's reproductive output is also associated with larger changes of global gene expression in the host. These results show the complexity of interactions between S. invicta and two viral parasites. Our findings also show that chronic infections by viral parasites in insects are dynamic processes that may pose different challenges in the host, laying the groundwork for interesting ecological and evolutionary considerations.     

Multiple virus infections occur in individual polygyne and monogyne Solenopsis invicta ants

Concurrent infections of Solenopsis invicta colonies with S. invicta virus 1 (SINV-1), SINV-2, and SINV-3 has been reported. However, whether individual ants were capable of supporting multiple virus infections simultaneously was not known, nor whether the social form of the colony (polygyne or monogyne) had an influence on the occurrence of multiple infection rates in individual ants. S. invicta field populations were sampled sequentially to establish whether multiple virus infections co-occurred in individual worker ants. In addition, the intra-colony virus infection rates were compared in monogyne and polygyne field colonies to determine whether social form played a role in the viral infection prevalence. All combinations of virus infection (SINV-1, SINV-2, or SINV-3 alone, SINV-1 & SINV-2, SINV-1 & SINV-3, SINV-2 & SINV-3, and SINV-1, SINV-2 & SINV-3) were detected in individual worker ants as well as queens in the field. Thus, individual S. invicta ants can be infected simultaneously with all combinations of the S. invicta viruses. Colony social form did have an influence on the intra-colony prevalence of multiple S. invicta virus infections. Polygyne colonies exhibited significantly greater intra- and inter-colony single and multiple virus infections compared with monogyne colonies.     

Population genetics and history of the introduced fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), in Australia

Abstract  The red imported fire ant, Solenopsis invicta , a damaging invasive pest, was discovered in February 2001 in Brisbane, Australia at two sites, Fisherman Islands and suburban Richlands-Wacol. Using four microsatellite loci and the protein marker Gp-9 , we compared the two infestations with each other, and with potential source populations in North and South America to better understand the history of their introduction to Brisbane. Based on an analysis of molecular variance, as well as a maximum likelihood tree of colonies from the two Australian sites, we found that the two sites were genetically distinct and were almost certainly introduced separately. All of the colonies at Fisherman Islands were monogynous, headed by a single queen, while the Richlands-Wacol site had a mixture of single-queen monogynous and multiple-queen polygynous colonies. However, the monogynous and polygynous colonies at the Richlands-Wacol site were not genetically distinct from each other, and probably constitute a single, mixed introduction. Based on allele frequencies at the microsatellite loci, and Gp-9 , both Australian infestations were more similar to North American populations than to South American, though the Fisherman Islands infestation was intermediate, making it difficult to assign. Thus, there has been one introduction from either a North or South American monogynous population at Fisherman Islands, and one introduction from a mixed monogynous/polygynous North American population at Richlands-Wacol. These findings have implications for the control of the current infestations, as well as for the quarantine regulations necessary to prevent additional introductions to Australia.     

Culture-Independent Characterization of the Microbiota of the Ant Lion Myrmeleon mobilis (Neuroptera: Myrmeleontidae)

Ant lions are insect larvae that feed on the liquefied internal components of insect prey. Prey capture is assisted by the injection of toxins that are reportedly derived from both the insect and bacterial symbionts. These larvae display interesting gut physiology where the midgut is not connected to the hindgut, preventing elimination of solid waste until adulthood. The presence of a discontinuous gut and the potential involvement of bacteria in prey paralyzation suggest an interesting microbial role in ant lion biology; however, the ant lion microbiota has not been described in detail. We therefore performed culture-independent 16S rRNA gene sequence analysis of the bacteria associated with tissues of an ant lion, Myrmeleon mobilis. All 222 sequences were identified as Proteobacteria and could be subdivided into two main groups, the α-Proteobacteria with similarity to Wolbachia spp. (75 clones) and the γ-Proteobacteria with similarity to the family Enterobacteriaceae (144 clones). The Enterobacteriaceae-like 16S rRNA gene sequences were most commonly isolated from gut tissue, and Wolbachia-like sequences were predominant in the head and body tissue. Fluorescence in situ hybridization analyses supported the localization of enterics to gut tissue and Wolbachia to nongut tissue. The diversity of sequences isolated from freshly caught, laboratory-fed, and laboratory-starved ant lions were qualitatively similar, although the libraries from each treatment were significantly different (P = 0.05). These results represent the first culture-independent analysis of the microbiota associated with a discontinuous insect gut and suggest that the ant lion microbial community is relatively simple, which may be a reflection of the diet and gut physiology of these insects.     

Group defense by colony-founding queens in the fire ant Solenopsis invicta

Mutualistic associations among nonkin can form when animalsin groups have a greater chance of overcoming challenges presentedby the environment than do solitary animals. Colony foundingby small groups of unrelated queens, a habit documented in severalspecies of ants, is often interpreted as a mutualistic interactionselected by intense competition among incipient colonies. However,many new colonies in these species are founded in areas wheretheir chief enemies are mature ant colonies, rather than othernewly founded colonies. In this study, we tested whether groupnest-founding in the fire ant Solenopsis invicta improved theability of queens to survive attacks by mature colonies. Inthe laboratory, queens in groups of three were more likely thansolitary queens to survive attacks by workers of the nativefire ant Solenopsis geminata. When newly mated queens were establishedexperimentally in the field, workers from mature S. invictacolonies caused the majority of queen deaths. Queens in groupsof two, but not in groups of four, had higher survival ratesthan did solitary queens during the period between colony establishmentand the appearance of the first workers. The advantage of cooperativedefense approximately counterbalanced the disadvantages causedby competition within foundress associations of two to threequeens. Previous studies have shown that colonies founded bymultiple queens produce larger worker populations than coloniesfounded by solitary queens; however, experimentally increasingworker number in incipient colonies had no effect on colonysurvival in the field.