Co-Occurrence of Wing Deformity and Impaired Mobility of Alates with Deformed Wing Virus in Solenopsis invicta Buren (Hymenoptera: Formicidae).

Deformed wing virus (DWV), a major honey bee pathogen, is a generalist insect virus detected in diverse insect phyla, including numerous ant genera. Its clinical symptoms have only been reported in honey bees, bumble bees, and wasps. DWV is a quasispecies virus with three main variants, which, in association with the ectoparasitic mite, Varroa destructor, causes wing deformity, shortened abdomens, neurological impairments, and colony mortality in honey bees. The red imported fire ant, Solenopsis invicta Buren, is one of the most-invasive and detrimental pests in the world. In this study, we report the co-occurrence of DWV-like symptoms in S. invicta and DWV for the first time and provide molecular evidence of viral replication in S. invicta. Some alates in 17 of 23 (74%) lab colonies and 9 of 14 (64%) field colonies displayed deformed wings (DWs), ranging from a single crumpled wing tip to twisted, shriveled wings. Numerous symptomatic alates also exhibited altered locomotion ranging from an altered gait to the inability to walk. Deformed wings may prevent S. invicta alates from reproducing since mating only occurs during a nuptial flight. The results from conventional RT-PCR and Sanger sequencing confirmed the presence of DWV-A, and viral replication of DWV was confirmed using a modified strand-specific RT-PCR. Our results suggest that S. invicta can potentially be an alternative and reservoir host for DWV. However, further research is needed to determine whether DWV is the infectious agent that causes the DW syndrome in S. invicta.

Complete chromosome-level genome assembly data from the tawny crazy ant, Nylanderia fulva (Mayr) (Hymenoptera: Formicidae).

The tawny crazy ant, Nylanderia fulva (Mayr) (Hymenoptera: Formicidae) has a native range that extends from northern Argentina to southern Brazil. In the U.S.A. this species has often been misidentified as Nylanderia (Paratrechina) pubens or N. cf. pubens and has likely been present in Florida and Texas for several decades [1]. In the early 2000's explosive population growth in Texas and neighboring states drew renewed taxonomic focus. Genetic analyses [2,3] aided in identifying the pest species as N. fulva. This species poses an invasive threat to native flora and fauna and human structures. In its invasive range it has been reported to displace another invasive species, the red imported fire ant. The specimens used for genome sequencing were obtained from the coastal region of Mississippi. DNA was extracted from pupae. The genome data set was deposited to the National Center for Biotechnology Information as submission ID: SUB10775679, Project ID: PRJNA796544, Accession IDs: SAMN24895442 and JAKFQQ000000000. The organism taxid is 613905, locus tag prefixes are L1K79. The assembly, USDA_Nfulva_1.0, was generated in collaboration with Dovetail Genomics (now Cantata Bio) to yield a chromosome-level assembly of 375 Mb with a 15.67 Mb N50 and 78X coverage and revealing 16 putative chromosomes. This high-quality chromosome-level genome assembly was released prior to publication as a public service to the research community.

Gross Morphology of Diseased Tissues in Nezara viridula (Hemiptera: Pentatomidae) and Molecular Characterization of an Associated Microsporidian.

Nezara viridula (L.) (Hemiptera: Pentatomidae), commonly known in the U.S. as the southern green stink bug (SGSB), is a cosmopolitan, highly polyphagous feeder that causes severe damage to a wide range of agronomically important crops such as fruit, vegetable, grain, tobacco, and cotton, throughout much of the United States, and is a global pest of considerable ecological, agricultural, and economical interest. During dissection of female Nz. viridula, conspicuous black and brown spots or lesions were observed on various internal organs. To determine the cause of these spots or lesions, tissues of fat body, spermatheca, ovaries, and ovulated eggs were collected from healthy and infected individuals. The gross morphology of the spots was characterized, and the microorganisms associated with the infection were identified by amplicon sequencing of the V4 region of the small subunit rRNA gene. The presence of a microsporidian pathogen Nosema maddoxi, Becnel, Solter, Hajek, Huang, Sanscrainte, & Estep (Microsporidia: Nosematidae) which has been observed on other species of stink bug, was evidenced for the first time. The characterization of the gross morphology of this associated microsporidian may enable more rapid determination of microsporidia infection in stink bug colonies and field populations.

Trypanosomatids Associated in the Alimentary Canal of Bagrada hilaris (Hemiptera: Pentatomidae).

Bagrada hilaris (Burmeister) is an invasive pest of economically important crops in the United States. During physiological investigations of B. hilaris, a flagellated protozoan was discovered in the alimentary canal of many specimens. This manuscript characterizes the morphology and molecular identification of the trypanosomatid, which appears similar to trypanosomatids identified in other stink bug species. It has been identified as a species in the Blastocrithidia genus based on morphological characteristics and molecular analyses.

Development of a Physiological Age-Grading System for Nezara viridula (Hemiptera: Pentatomidae).

The southern green stink bug (SGSB), Nezara viridula (L.), is an important agricultural pest in the United States. Limited information is available on the morphology of the female's reproductive system in relation to morphological changes associated with the number of eggs produced and egg masses oviposited. The ability to assess reproductive health and reproductive status based on ovarian morphology (i.e., physiological age-grading) can be an important tool for evaluating field populations and laboratory colonies intended for the application of different management strategies and experimental trials. Thus, the goal of this study was to develop a physiological age-grading system for SGSB. Females aged from 0 to 79 d chronologically randomly selected from laboratory colonies and dissected to assess ovarian morphology. Specific morphological differences in ovarian structures including differentiation of the ovarioles, deposition of yolk in the most proximal follicle, quantity and appearance of follicular relics, expansion of the lateral oviducts, and number of developing follicles per ovariole were related to chronological age, the number of eggs produced and number of egg masses. Based on specific combinations of these morphological characteristics, the continuum of ovarian development was divided into three nulliparous (i.e., 'no eggs'; N1, N2, and N3) and three parous stages (i.e., 'with eggs'; P1, P2, and P3). Direct relationships were noted between number of eggs produced and physiological age with over 7-fold higher number of eggs and 14-fold higher number of egg masses associated with the P2 and P3 stages, respectively.

Insecticidal and Enzyme Inhibitory Activities of Isothiocyanates against Red Imported Fire Ants, Solenopsis invicta.

Contact and fumigation toxicity of four isothiocyanates (ITCs), including allyl isothiocyanate (AITC), 3-butenyl isothiocyanate (3BITC), 3-(methylthio) propyl isothiocyanate (3MPITC) and 2-phenylethyl isothiocyanate (2PEITC), were evaluated against the red imported fire ant worker, Solenopsis invicta Buren. 2PEITC and 3MPITC exhibited strong contact toxicity. The median lethal dose (LD50)value of AITC, 2PEITC and 3MPITC were 7.99, 2.36 and 2.09 µg/ant respectively. In addition, AITC and 3MPITC also showed strong fumigation toxicity but not 2PEITC. The median lethal concentration (LC50) values of AITC and 3MPITC were 32.49 and 57.6 µg/L, respectively. In contrast, 3BITC did not exhibit any contact and fumigation toxicity even at 100 µg/µL. Esterase (EST), glutathione S-transferase (GST) and acetylcholinesterase (AChE)-inhibiting activities were assessed for three ITCs in S. invicta workers. All three ITCs inhibited both EST and GST activities but not AChE. The in vitro half maximal inhibitory concentration (IC50)values of AITC, 2PEITC and 3MPITC for GST were 3.32, 0.61 and 0.66 µg/µL, respectively. These results suggested that naturally occurring ITCs might be potentially useful for developing fire ants control products.

Electrophysiological Responses of Eighteen Species of Insects to Fire Ant Alarm Pheromone.

Olfaction plays a dominant role in insect communication. Alarm pheromones, which alert other insects of the same species of impending danger, are a major class of releaser pheromones. The major components of alarm pheromones in red imported fire ants, honeybees and aphids have been identified as 2-ethyl-3,6-dimethylpyrazine (2E-3,6-DP), isopentyl acetate (IPA), and E-ß-farnesene (EßF), respectively. In this study, electroantennography (EAG) responses to EDP (a mixture of 2-ethyl-3,6-dimethylpyrazine and 2-ethyl-3,5-dimethylpyrazine), IPA and EßF were investigated in a wide range of insect species. Beside imported fire ants, the EDP (2-ethyl-3,6(5)-dimethylpyrazine) elicited significant EAG response from all other tested insects, including six ant species and one hybrid ant, honeybee, bagrada bug, lady beetle, housefly, small hive beetle, yellow fever mosquito, termite, bedbug, water hyacinth weevil, southern green stink bug and two aphid species. In contrast, IPA elicited significant EAG response only in the honeybee, red imported fire ant, an Aphaenogaster ant, and the water hyacinth weevil. The EßF only elicited EAG responses in two aphids, small hive beetle and housefly. The results clearly indicate that EDP can be detected by widespread insect species that did not coevolve with S. invicta and further suggested alkylpyrazine may activate multiple generally tuned olfactory receptors (ORs) across a wide number of insect species.

Discovery of Aphis ruborum (Hemiptera: Aphididae) and Aphelinus varipes (Hymenoptera: Aphelinidae) on Cultivated Strawberry in Mississippi, USA.

An adventive aphid and novel host-parasitoid association from cultivated strawberry (Fragaria × ananessa Duch. cv. Chandler; Fragaria × ananessa Duch. cv. Camarosa) in Mississippi, USA are reported herein. The aphid, first detected in high tunnel cultivation, was found predominately on newly emerged, not fully developed leaflets of daughter plants in the Fall of 2016. By 2017, aphids and their associated mummies were observed on fully developed leaflets on mother plants of both cultivars. The aphid was identified as Aphis ruborum (Börner & Schilder) using morphology and DNA barcoding studies. In addition, DNA barcoding identified parasitoid adults emerging from aphid mummies as two cryptic species, Aphelinus varipes (Foerster) and Aphelinus albipodus Hayat and Fatima. Occurrence of A. ruborum in Mississippi represents a new state record and the eastern-most established record in the United States. The A. ruborum - A. varipes or A. albipodus host-parasitoid association is reported for the first time anywhere in the world.

Female Reproductive System Morphology and the Development of a Physiological Age-Grading System for Bagrada hilaris (Hemiptera: Pentatomidae).

In this paper, we describe the morphology of the female Bagrada hilaris (Burmeister) reproductive system and develop a physiological age-grading system related to egg production. The female reproductive system is composed of two meroistic and telotrophic ovaries each containing 5-6 tubular ovarioles. The ovarioles unite into the lateral oviduct which combine to form the common oviduct. The ovarioles are composed of two regions; the distal germarium and the tubular vitellarium which contains maturing follicles. Each follicle is surrounded by a layer of cells; the follicular epithelium. As the follicle passes from the ovariole to the lateral oviducts the follicular epithelial cells slough off and accumulate in the base of the ovarioles and are known as follicular relics. The continuum of ovarian development is divided into two categories: nulliparous ('nonreproducing') and parous ('reproducing'). The nulliparous category is characterized by the absence of follicular relics or eggs in the oviducts as opposed to the parous category where follicular relics and, in many cases, eggs occurred in the oviducts. The nulliparous category is divided further into two stages; N1 and N2 based on ovariole differentiation. The parous category is divided into three stages; P1, P2, and P3, based on the quantity and appearance of follicular relics. Females characterized as P3 produced three times more eggs (79.2 eggs ± 5.7) than females characterized as P1 (27.1 eggs ± 6.0).

Insecticidal Activity of Methyl Benzoate Analogs Against Red Imported Fire Ants, Solenopsis invicta (Hymenoptera: Formicidae).

Although insecticidal properties of certain benzoates have been investigated for pest insects and mites, toxicity of benzoates to the red imported fire ants, Solenopsis invicta Buren, has never been reported. In this study, 15 commercially available benzoates were assessed for their contact and fumigation toxicity to S. invicta workers and their chemical structure-activity relationships. Among tested benzoates, benzylbenzoate, n-pentybenzoate, and n-hexylbenzoate were three most potent contact toxins against S. invicta workers (mean LD50 value = 23.31, 35.26, 35.99 µg per ant, respectively) and methyl-3-methoxybenzoate, methyl-3-methylbenzoate, and methylbenzoate were the three most potent fumigants (mean LC50 value = 0.61, 0.62, 0.75 µg/ml, respectively). For nonsubstituted alkyl benzoates (esters of benzoic acid and C1-C6 linear alcohols), the contact toxicity was positively correlated to the alkyl chain length (r = 0.89), while the fumigation toxicity was negatively correlated (r = 0.90). Presence of a methoxyl group at either the ortho or meta position of methylbenzoate significantly increased its contact toxicity, so did a methyl group at meta position. However, presence of a methyl group at ortho position reduced the contact toxicity. Presence of methyl or methoxyl group at the meta position did not have significant effect on the fumigation toxicity; however, methyl, methoxyl, chloro, or nitro groups at the ortho position significantly reduced fumigation toxicity. Hexylbenzoate has neither known Occupational Safety and Health Administration hazards nor aquatic toxicity, and methyl 3-methoxybenzoate is not considered a hazardous substance, indicating a great potential for their application in fire ant management.

Differential Gene Expression in Red Imported Fire Ant (Solenopsis invicta) (Hymenoptera: Formicidae) Larval and Pupal Stages.

Solenopsis invicta Buren is an invasive ant species that has been introduced to multiple continents. One such area, the southern United States, has a history of multiple control projects using chemical pesticides over varying ranges, often resulting in non-target effects across trophic levels. With the advent of next generation sequencing and RNAi technology, novel investigations and new control methods are possible. A robust genome-guided transcriptome assembly was used to investigate gene expression differences between S. invicta larvae and pupae. These life stages differ in many physiological processes; of special importance is the vital role of S. invicta larvae as the colonies' "communal gut". Differentially expressed transcripts were identified related to many important physiological processes, including digestion, development, cell regulation and hormone signaling. This dataset provides essential developmental knowledge that reveals the dramatic changes in gene expression associated with social insect life stage roles, and can be leveraged using RNAi to develop effective control methods.

Oviposition Strategies in Beneficial Insects.

AIMS AND SCOPE: The aim of this special collection is to highlight the importance of reproduction (ie, oviposition) in the life history, population dynamics, survival, and fitness of beneficial insects, broadly defined. Specific areas of interest include the (1) identification of natural products to boost oviposition; (2) importance of chemical cues in oviposition site selection; (3) influence of host plant defenses on oviposition success; (4) reproductive physiology and the frequency of egg laying; (5) trade-offs between maternal size and egg size; (6) foraging behavior, host selection, and oviposition in parasitoids; and (7) oviposition decisions in the face of intraguild predators.

The Reproductive Morphology and Physiological Age Grading of the Female Salvinia Weevil, Cyrtobagous salviniae Calder and Sands.

The morphology of the female Cyrtobagous salviniae Calder and Sands reproductive system is similar to other weevil species being meroistic and telotrophic. The reproductive system is composed of 2 ovaries each containing 2 ovarioles where the follicles mature. A physiological age grading system was developed where the continuum of ovarium development was divided into 2 nulliparous and 3 parous classes. This was based on the differentiation of the ovarioles, presence, and appearance of follicular relics, cuticle hardness/coloration, and fat body quantity/appearance. High correlation occurred between the parous classes and number of eggs produced where the P3 class had over 9-fold higher number of eggs in comparison with the P1 class. Mean number of eggs produced for each parous class was significantly different, however, overlap occurred. Such a system enables a determination of the past, present, and future reproductive status of field populations and mass-rearing colonies.

Genome Sequences of Three Strains of Aspergillus flavus for the Biological Control of Aflatoxin.

Aflatoxin is a carcinogenic contaminant of many commodities that are infected by Aspergillus flavus Nonaflatoxigenic strains of A. flavus have been utilized as biological control agents. Here, we report the genome sequences from three biocontrol strains. This information will be useful in developing markers for postrelease monitoring of these fungi.

Allantoin Crystal Formation in Bagrada hilaris (Burmeister) (Heteroptera: Pentatomidae) Females.

Bagrada hilaris is a polyphagous herbivore reported as an invasive pest in the United States. During the course of dissecting Burmeister hilaris unique crystals were observed in both the midgut and oviducts. Crystals were identified using X-ray diffraction techniques. Both acicular (i.e., needle-like, slender, and/or tapered) and cubic (i.e., cube shaped) crystals were observed in six of 75 individuals examined (8.0%). The crystals were mainly observed in females (6.7%), followed by males (1.3%) with no crystals observed in the minimal number of nymphs examined (0%). Crystals of both types were detected in the midgut and lateral oviducts of the females and midgut in males. The acicular crystals often appeared as distinct bundles when present in the midgut and oviducts. Crystals varied in size with the acicular crystals ranging from 0.12 mm to 0.5 mm in length although the cubic crystals ranged in length from 0.25 mm to over 1.0 mm with widths of ∼0.25 mm. The cubic crystals were identified as allantoin although the acicular crystals were most likely dl-allantoin in combination with halite. While allantoin in a soluble form is often found in insect tissues and excreta; being present as a crystal, especially in such a large form, is curious and raises some interesting questions. More research is warranted to further understand mechanisms associated with such crystal formation in B. hilaris and can lead to a better understanding of the excretory process in this species and the role allantoin plays in the elimination of excess nitrogen.

Update on the defensive chemicals of the little black ant, Monomorium minimum (Hymenoptera: Formicidae).

Alkaloids, including 2,5-dialkylpyrrolidines and 2,5-dialkylpyrrolines, have been reported to be components in the venom of little black ants, Monomorium minimum (Buckley). Two fatty amines were recently reported as minor compounds. By analyzing the discharge collected from the stinger apparatus (milking), this study revealed the presence of an additional seven compounds in the defensive secretion of this ant species. Compounds identified were 9-decenyl-1-amine, N-methylenedecan-1-amine, N-methylenedodecan-1-amine, 2-(1-non-8-enyl)-5-(1-hex-5-enyl)-1-pyrroline, N-methyl-2-(hex-5-enyl)-5-nonanyl-1-pyrrolidine, ß-springene ((E,E)-7,11,15-trimethyl-3-methylene-1,6,10,14-hexadecatetraene) and neocembrene ((E,E,E)-1-isopropenyl-4,8,12-trimethylcyclotetradeca-3,7,11-triene). ß-springene and neocembrene were found only in the defensive secretion of queens. Analyses of the contents of isolated poison and Dufour's glands of the queen indicated that all amines and alkaloids were from the poison gland and that ß-springene and neocembrene were from the Dufour's gland. This demonstrated that the defensive secretion in M. minimum queens consists of components from both glands. This is also the first report on the natural occurrence of 9-decenyl-1-amine, N-methylenedecan-1-amine, and N-methyllenedodecan-1-amine.