The Effect of the Hypertrophy Virus (MdSGHV) on the Ultrastructure of the Salivary Glands of Musca domestica (Diptera: Muscidae)

The salivary glands of insects play a key role in the replication cycle and vectoring of viral pathogens. Consequently, Musca domestica (L.) (Diptera: Muscidae) and the Salivary Gland Hypertrophy Virus (MdSGHV) serve as a model to study insect vectoring of viruses. A better understanding of the structural changes of the salivary glands by the virus will help obtain a better picture of the pathological impact the virus has on adult flies. The salivary glands are a primary route for viruses to enter a new host. As such, studying the viral effect on the salivary glands is particularly important and can provide insights for the development of strategies to control the transmission of vector-borne diseases, such as dengue, malaria, Zika, and chikungunya virus. Using scanning and transmission electron microscopic techniques, researchers have shown the effects of infection by MdSGHV on the salivary glands; however, the exact location where the infection was found is unclear. For this reason, this study did a close examination of the effects of the hypertrophy virus on the salivary glands to locate the specific sites of infection. Here, we report that hypertrophy is present mainly in the secretory region, while other regions appeared unaffected. Moreover, there is a disruption of the cuticular, chitinous lining that separates the secretory cells from the lumen of the internal duct, and the disturbance of this lining makes it possible for the virus to enter the lumen. Thus, we report that the chitinous lining acts as an exit barrier of the salivary gland.     

Localization and Bioreactivity of Cysteine-Rich Secretions in the Marine Gastropod Nucella lapillus

Marine biodiversity has been yielding promising novel bioproducts from venomous animals. Despite the auspices of conotoxins, which originated the paradigmatic painkiller Prialt, the biotechnological potential of gastropod venoms remains to be explored. Marine bioprospecting is expanding towards temperate species like the dogwhelk Nucella lapillus, which is suspected to secrete immobilizing agents through its salivary glands with a relaxing effect on the musculature of its preferential prey, Mytilus sp. This work focused on detecting, localizing, and testing the bioreactivity of cysteine-rich proteins and peptides, whose presence is a signature of animal venoms and poisons. The highest content of thiols was found in crude protein extracts from the digestive gland, which is associated with digestion, followed by the peribuccal mass, where the salivary glands are located. Conversely, the foot and siphon (which the gastropod uses for feeding) are not the main organs involved in toxin secretion. Ex vivo bioassays with Mytilus gill tissue disclosed the differential bioreactivity of crude protein extracts. Secretions from the digestive gland and peribuccal mass caused the most significant molecular damage, with evidence for the induction of apoptosis. These early findings indicate that salivary glands are a promising target for the extraction and characterization of bioactive cysteine-rich proteinaceous toxins from the species.     

Biology and Trapping of Stable Flies (Diptera: Muscidae) Developing in Pineapple Residues (Ananas comosus) in Costa Rica

Pineapple production in Costa Rica increased nearly 300-fold during the last 30 yr, and >40,000 hectares of land are currently dedicated to this crop. At the end of the pineapple cropping cycle, plants are chopped and residues incorporated into the soil in preparation for replanting. Associated with increased pineapple production has been a large increase in stable fly, Stomoxys calcitrans (L.), populations. Stable flies are attracted to, and oviposit in, the decomposing, chopped pineapple residues. In conjunction with chemical control of developing larvae, adult trapping is an important control strategy. In this study, four blue-black fabric traps, Nzi, Vavoua, Model H, and Ngu, were compared with a white sticky trap currently used for stable fly control in Costa Rica. Overall, the white sticky trap caught the highest number of stable flies, followed by the Nzi, Vavoua, Model H, and Ngu. Collections on the white sticky trap increased 16 d after residues were chopped; coinciding with the expected emergence of flies developing in the pineapple residues. During this same time period, collections in the blue-black fabric traps decreased. Sex ratio decreased from >7:1 (females:males) 3–7 d after chopping to 1:1 at 24–28 d. White sticky, Nzi and Vavoua traps collected similar numbers of colonizing flies 3–7 d after residues were chopped. However, white sticky traps collected more flies once emergence from the pineapple residues began. Although white sticky traps collected more flies than fabric traps, they remain labor intensive and environmentally unsound because of their disposable and nonbiodegradable nature.     

The Complete Mitochondrial Genome of Brachmia macroscopa (Lepidoptera: Gelechiidae) and Its Related Phylogenetic Analysis

The sweet potato leaf folder, Brachmia macroscopa, is an important pest in China. The complete mitogenome, which consists of 13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and an A + T-rich region, was sequenced and found to be 15,394 bp in length (GeneBank no. KT354968). The gene order and orientation of the B. macroscopa mitogenome were similar to those of other sequenced lepidopteran species. All of the PCGs started with ATN as the canonical start codon except for cox1, which started with CGA. In regard to stop codons, most PCGs stopped at TAA except for cox2, which stopped at TA, and nad4, which stopped at a single T. Thirteen PCGs of the available species (33 taxa) were used to demonstrate phylogenetic relationships. The ditrysian cluster was supported as a monophyletic clade at high levels by using maximum likelihood and Bayesian methods. The apoditrysian group, covering the Gelechioidea, formed a monophyletic clade with a bootstrap value of 88% and a posterior probability of 1.00. The superfamily Gelechioidea was supported as a monophyletic lineage by a posterior probability of 1.00.     

Status of Bactrocera invadens (Diptera: Tephritidae) in Mango-Producing Areas of Arba Minch,Southwestern Ethiopia

Bactrocera invadens, the Asian fruit fly, was first reported in Kenya in 2003, and it spread fast to most tropical countries in Africa. To our knowledge, there is no detailed data on the fruit damage and status of fruit flies in Arba Minch and elsewhere in Ethiopia. Hence, information on the species composition and pest status of the fruit fly species is urgent to plan management strategies in the area. Fruit flies were captured using male parapheromone-baited traps. Matured mango (Mangifera indica) fruits were collected from randomly selected mango trees and incubated individually in cages (15 by 15 by 15 cm) with sandy soil. B. invadens was the predominant (96%; 952 of 992) captured species and the only fruit fly species emerging from mango fruits incubated in the laboratory. The mean number of adult B. invadens emerging per mango fruit was 35.25, indicating that the species is the most devastating mango fruit fly in the area. The loss due to this species would be serious if no management strategies are implemented.     

Cephalopods as Vectors of Harmful Algal Bloom Toxins in Marine Food Webs

Here we summarize the current knowledge on the transfer and accumulation of harmful algal bloom (HAB)-related toxins in cephalopods (octopods, cuttlefishes and squids). These mollusks have been reported to accumulate several HAB-toxins, namely domoic acid (DA, and its isomers), saxitoxin (and its derivatives) and palytoxin (and palytoxin-like compounds) and, therefore, act as HAB-toxin vectors in marine food webs. Coastal octopods and cuttlefishes store considerably high levels of DA (amnesic shellfish toxin) in several tissues, but mainly in the digestive gland (DG)—the primary site of digestive absorption and intracellular digestion. Studies on the sub-cellular partitioning of DA in the soluble and insoluble fractions showed that nearly all DA (92.6%) is found in the cytosol. This favors the trophic transfer of the toxins since cytosolic substances can be absorbed by predators with greater efficiency. The available information on the accumulation and tissue distribution of DA in squids (e.g., in stranded Humboldt squids, Dosidicus gigas) is scarcer than in other cephalopod groups. Regarding paralytic shellfish toxins (PSTs), these organisms accumulate them at the greatest extent in DG >> kidneys > stomach > branchial hearts > posterior salivary glands > gills. Palytoxins are among the most toxic molecules identified and stranded octopods revealed high contamination levels, with ovatoxin (a palytoxin analogue) reaching 971 μg kg⁻¹ and palytoxin reaching 115 μg kg⁻¹ (the regulatory limit for PlTXs is 30 μg kg⁻¹ in shellfish). Although the impacts of HAB-toxins in cephalopod physiology are not as well understood as in fish species, similar effects are expected since they possess a complex nervous system and highly developed brain comparable to that of the vertebrates. Compared to bivalves, cephalopods represent a lower risk of shellfish poisoning in humans, since they are usually consumed eviscerated, with exception of traditional dishes from the Mediterranean area.     

A comparison of monitoring systems used for Ceratitis species (Diptera: Tephritidae) in South Africa

For 16 years a bucket-type trap known as the Sensus fruit fly trap has been used to monitor three fruit fly pest species Ceratitis capitata, Ceratitis rosa and Ceratitis cosyra in South African fruit industries. The relative efficiencies of lures sold for monitoring fruit flies with the Sensus trap in South Africa were determined in field experiments where laboratory-reared C. capitata, C. rosa and C. cosyra were released in a mango orchard within a few metres of Sensus traps containing either Capilure (trimedlure or tert-butyl 4 (or 5) -chloro-2-methylcyclohexane carboxylate), Ceratitislure (protein hydrolysate plus β-caryophyllene) or Questlure (protein hydrolysate plus plant extracts). When using 12-day-old flies, Capilure caught 3 times more C. capitata males than C. rosa males and this difference was more extreme when 3-day-old flies were released. Ceratitislure caught significantly more 12-day-old C. cosyra males than 12-day-old C. capitata males, but the difference was reversed when 3-day-old flies were compared. Questlure showed the least differences between species and age but recovered the lowest proportion of released species. Further comparisons were conducted in an orchard with wild flies using other known attractants in larger yellow Probodelt bucket traps. Capilure caught more male C. capitata than BioLure Fruit Fly 3-component, but BioLure 2-component (trimethylamine and ammonium acetate) was more effective than Questlure for C. capitata females. The 3-component lure was also more effective than both Capilure and Questlure for male and female C. rosa, respectively. Ceratitislure was the most effective lure for male C. cosyra flies and BioLure 3-component was more effective than Ceratitislure and Questlure for female C. cosyra flies. The intervention threshold of 4 flies/trap/week previously used in citrus with Capilure for C. rosa was lowered to 2 C. rosa/trap/week when using the Sensus trap due to the lower sensitivity of this trap-lure combination found for C. rosa in this study. The 3-component lure, or the 2-component combination of trimethylamine and ammonium acetate in a ratio of 1:8 in the Sensus trap capsule, would be more effective for both sexes of all three Ceratitis species than the Questlure that is currently being used.     

Histopathological Changes in Third-Instar and Adult Anastrepha ludens (Diptera: Tephritidae) After in vitro Heat Treatment

The Mexican fruit fly, Anastrepha ludens Loew (Diptera: Tephritidae), is one of the most harmful pests of mango causing direct damage by oviposition on the fruit pulp. Mango for export is subjected to hydrothermal treatment as a quarantine method for the control of this pest, but exposure to heat for long periods of time reduces considerably the quality and shelf-life of treated fruit. The aim of this work was to study morphological changes of third-instar larvae and adults of A. ludens after in vitro exposure to high temperature at sublethal times. A heating block system was used to expose larvae at 46.1°C for 19.6 and 12.9 min, producing 94.6 and 70% mortality, respectively. Treated larvae were processed for optical microscopy. A fraction of surviving treated larvae was separated into containers with artificial diet to allow development into adults. Adult sexual organs were dissected and processed for transmission electron microscopy analysis. Results showed that 94.6% of the treated larvae died at 46.1°C for 19.6 min and none of the surviving larvae eclosed to adulthood, as they developed as malformed puparia. For the in vitro treatment at 46.1°C during 12.9 min, 70% of the treated larvae died and only 3.75% reached the adult stage, but ultrastructural damage in the male testes and in the female ovaries was observed. Additionally, 11.1% of the adult flies from the in vitro treatment also showed wing malformation and were incapable of flying. The analysis showed that surviving flies were unable to reproduce.     

Identification and Function Analysis of enolase Gene NlEno1 from Nilaparvata lugens (St?l) (Hemiptera:Delphacidae)

The enolase [EC 4.2.1.11] is an essential enzyme in the glycolytic pathway catalyzing the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP). In this study, a full-length cDNA encoding α-enolase was cloned from rice brown planthopper (Nilaparvata lugens) and is provisionally designated as NlEno1. The cDNA sequence of NlEno1 was 1,851 bp with an open reading frame (ORF) of 1,305 bp and encoding 434 amino acids. The deduced protein shares high identity of 80–87% with ENO1-like protein from Hemiptera, Diptera, and Lepidoptera speices. The NlEno1 showed the highest mRNA expression level in hemolymph, followed by fat body, salivary gland, ovaries and egg, and showed trace mRNA levels in testis. The mRNA of NlEno1 showed up-regulated level in virulent N. lugens population Mudgo, IR56 and IR42 when compared with TN1 population. Injection of double-stranded RNA (dsRNA) of NlEno1 into the adults significantly down-regulated the NlEno1 mRNA level along with decreased eggs and offspring. Moreover, injection of NlEno1-dsRNA decreased mRNA level of Vitellogenin (Vg) gene. These results showed that the NlEno1, as a key glycolytic enzyme, may play roles in regulation of fecundity and adaptation of N. lugens to resistant rice varieties.     

Disruption of Darna pallivitta (Lepidoptera: Limacodidae) by Conventional and Mobile Pheromone Deployment

Identification of the Darna pallivitta (Moore) pheromone component n-butyl (E)-7,9-decadienoate (E7,9-10:COOn-Bu) has made it possible to investigate communication disruption to control this lepidopteran pest. Conventional communication disruption trials showed marked decreases in the mean number of male moths captured in E7,9-10:COOnBu-treated fields compared with control fields. For traps baited with E7,9-10:COOnBu, percent disruptions were 94.4% and 92.1% for septa (1 g pheromone/ha, 1-wk trial duration) and spirals (6 g pheromone/ha, 8-wk trial duration) respectively. For traps baited with virgin female moths, percent disruption was 73.3% using septa disruptors (1 g pheromone/ha, 1-wk trial duration). Mobile communication disruption using Bactrocera cucurbitae (Coquillett) as carriers for E7,9-10:COOn-Bu was evaluated in the following three areas: fly survivorship, attraction of male moths to treated flies, and moth disruption in a small-scale field trial. Topical application of E7,9-10:COOnBu showed no significant decrease in survivorship at 50 and 80 µg/fly. However, decreased survivorship was observed at 100 µg/fly and linear regression showed E7,9-10:COOnBu dose was significantly correlated with B. cucurbitae survivorship. Traps containing honey–pheromone-fed flies attracted and caught D. pallivitta over a 1-wk period, demonstrating the attractiveness of the carrier. Releasing E7,9-10:COOnBu-fed B. cucurbitae (∼2 g pheromone/ha, 1-wk trial duration) resulted in significantly reduced trap catches in treatment fields compared with control fields on the first 2 d of the field trial. Percent disruptions were 84.7% (day 1) and 56.0% (day 2). These results suggest that both conventional communication disruption and mobile communication disruption have potential to control D. pallivitta.     

Flight Capacity of Bactrocera dorsalis (Diptera: Tephritidae) Adult Females Based on Flight Mill Studies and Flight Muscle Ultrastructure

The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is considered a major economic threat in many regions worldwide. To better comprehend flight capacity of B. dorsalis and its physiological basis, a computer-monitored flight mill was used to study flight capacity of B. dorsalis adult females of various ages, and the changes of its flight muscle ultrastructures were studied by transmission electron microscopy. The flight capacity (both speed and distance) changed significantly with age of B. dorsalis female adults, peaking at about 15 d; the myofibril diameter of the flight muscle of test insects at 15-d old was the longest, up to 1.56 µm, the sarcomere length at 15-d old was the shortest, averaging at 1.37 µm, volume content of mitochondria of flight muscle at 15-d old reached the peak, it was 32.64%. This study provides the important scientific data for better revealing long-distance movement mechanism of B. dorsalis.     

Intrabody Tetrodotoxin Distribution and Possible Hypothesis for Its Migration in Ribbon Worms Cephalothrix cf. simula (Palaeonemertea,Nemertea)

Tetrodotoxin (TTX) is a potent neurotoxin found in many marine and terrestrial animals, but only a few species, such as the ribbon worms of the genus Cephalothrix, accumulate it in extremely high concentrations. The intrabody distribution of TTX in highly toxic organisms is of great interest because it helps researchers to understand the pathways by which the toxin migrates, accumulates, and functions in tissues. Using immunohistochemistry with anti-TTX antibodies, the authors of this study investigated the toxin’s distribution inside the organs, tissues, and cells of Cephalothrix cf. simula. The cell types of TTX-positive tissues were identified by light microscopy. The main sites of TTX accumulation occurred in the secretory cells of the integuments, the microvilli of the epidermal ciliary cells, cephalic glands, the glandular epithelia of the proboscises, the enterocytes of the digestive systems, and nephridia. Obtained data suggest the toxin migrates from the digestive system through blood vessels to target organs. TTX is excreted from the body through the nephridia and mucus of epidermal cells.     

Spatial and temporal abundance of the solanum fruit fly, Bactrocera latifrons (Hendel), in Morogoro,Tanzania

Bactrocera latifrons is a newly invasive pest species in eastern Africa. Surveys to determine the spatial and temporal abundance of this fruit fly in the Morogoro Region, Eastern Central Tanzania, were conducted from April 2007 to March 2008. The surveys involved trapping of adult flies and collection of host fruits to determine the emergence of adults. The results indicated that the population of B. latifrons was generally low but that adult flies were present throughout the survey period. The population of B. latifrons based on infestation rates and incidence in host fruits, seems to be relatively high during the wet seasons, probably because of availability of many hosts. However, the population of the pest as determined by trapping, indicated a low population but without a specific pattern. The pest shows minimal response to the used attractants. The surveys further indicate that B. latifrons is more abundant in low to medium altitude areas compared to high altitude areas. The results indicate that, because of its low population, B. latifrons might be a minor pest. Results further suggest the possibility of establishing B. latifrons free areas where export valued solanaceous crops can be grown, but further research over longer periods would be needed to establish this.     

Identification and Physicochemical Properties of the Novel Hemolysin(s) From Oral Secretions of Helicoverpa armigera (Lepidoptera: Noctuidae)

Hemolysins cause the lysis of invading organisms, representing major humoral immunity used by invertebrates. Hemolysins have been discovered in hemolymph of Helicoverpa armigera larvae as immune factors. As oral immunity is great important to clear general pathogens, we presumed that hemolysins may be present in oral secretions (OS). To confirm this hypothesis, we conducted four testing methods to identify hemolysin(s) in larval OS of H. armigera, and analyzed physicochemical properties of the hemolysin in comparison with hemolytic melittin of Apis mellifera (L.) (Hymenoptera: Apidae) venom. We found hemolysin(s) from OS of H. armigera for the first time, and further identified in other lepidopteran herbivores. It could be precipitated by ammonium sulfate, which demonstrates that the hemolytic factor is proteinaceous. Labial gland showed significantly higher hemolytic activity than gut tissues, suggesting that hemolysin of OS is mainly derived from saliva secreted by labial glands. Physicochemical properties of hemolysin in caterpillar’s OS were different from bee venom. It was noteworthy that hemolytic activity of OS was only partially inhibited even at 100°C. Hemolytic activity of OS was not inhibited by nine tested carbohydrates contrary to bee venom melittin. Moreover, effects of metal ions on hemolytic activity were different between OS and bee venom. We conclude that there is at least a novel hemolysin in OS of herbivorous insects with proposed antibacterial function, and its hemolytic mechanism may be different from melittin. Our study enriches understanding of the potential role of hemolysins in insect immunity and provides useful data to the field of herbivorous insect-pathogen research.     

Insecticide-induced increase in the protein content of male accessory glands and its effect on the fecundity of females in the brown planthopper Nilaparvata lugens Stål (Hemiptera: Delphacidae)

The brown planthopper Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is a classical resurgent rice pest induced by insecticides. The past focus on resurgence mechanisms has been on the stimulation of the reproduction of adult females induced by insecticides. To date, the role that males play as a resurgence of N. lugens has not been investigated. The present study examined changes in protein levels in both male accessory glands and female ovaries induced by the insecticides triazophos and deltamethrin as well as the stimulating effect of treated males on the fecundity of adult females via mating following foliar sprays of the insecticides. For adults that had been exposed as nymphs to treated rice plants, the protein content in both the male accessory glands and in the female ovaries of N. lugens were significantly affected by male mating status, insecticide and insecticide concentration. There was a higher protein content in male accessory glands when males were exposed to triazophos as third instars compared to fifth instars, and there was a higher protein content before mating compared to after mating. In addition, the protein levels in male accessory glands after mating for individuals exposed to high doses of the two insecticides as 3rd and 5th instars were significantly lower than untreated control except for exposed to triazophos as 3rd instar, indicating that treated males transferred more male accessory gland protein to adult females via mating. The protein content was also affected by different combinations of treated mating pairs. Adult males (♂_t) developed from third instar nymphs treated with triazophos stimulated the fecundity of the female significantly via mating (♂_t × ♀_ck) with untreated females (♀_ck) (control females), increasing the reproductive rate by 43.5% as compared to the mating (♂_ck × ♀_ck) of untreated males and females. Also, the fecundity of the females after the mating (♂_t × ♀_t) of treated males and females was significantly higher than that after the mating (♂_ck × ♀_t) of untreated males with treated females. These findings indicated that the reproductive effects of insecticide on males can be transferred to females via mating. The present findings provide valuable information for understanding the potential role that males play in the pesticide-induced resurgence of N. lugens.     

Apoptogenic Metabolites in Fractions of the Benthic Diatom Cocconeis scutellum parva

Benthic diatoms of the genus Cocconeis contain a specific apoptogenic activity. It triggers a fast destruction of the androgenic gland in the early post-larval life of the marine shrimp Hippolyte inermis, leading to the generation of small females. Previous in vitro investigations demonstrated that crude extracts of these diatoms specifically activate a dose-dependent apoptotic process in human cancer cells (BT20 breast carcinoma) but not in human normal lymphocytes. Here, a bioassay-guided fractionation has been performed to detect the apoptogenic compound(s). Various HPLC separation systems were needed to isolate the active fractions, since the apoptogenic metabolite is highly active, present in low amounts and is masked by abundant but non-active cellular compounds. The activity is due to at least two compounds characterized by different polarities, a hydrophilic and a lipophilic fraction. We purified the lipophilic fraction, which led to the characterization of an active sub-fraction containing a highly lipophilic compound, whose molecular structure has not yet been identified, but is under investigation. The results point to the possible medical uses of the active compound. Once the molecular structure has been identified, the study and modulation of apoptotic processes in various types of cells will be possible.     

Developmental Biology of Zeugodacus cucurbitae (Diptera: Tephritidae) in Three Cucurbitaceous Hosts at Different Temperature Regimes

Fruit flies are key pests of cucurbits in many parts of the world, including Tanzania. Developmental biology of Zeugodacus cucurbitae (Coquillett) has been determined across temperature regimes in some cucurbitaceous hosts, in limited geographies. This study was conducted to determine duration and survival rates of immature stages of Z. cucurbitae in three cucurbitaceous hosts, at different temperature regimes. It was hypothesized that temperature and cucurbitaceous hosts influence duration and survival of immature stages of Z. cucurbitae. We conducted experiments in the environmental chamber set at 75 ± 10% RH and a photoperiod of 12:12 (L:D) h, at temperatures of 20, 25, and 30°. Our results showed that duration and survival of immature stages of Z. cucurbitae differed significantly among the temperature regimes but not among the hosts. Egg incubation period as well as larval and pupal stages were significantly longer (P < 0.0001) at low temperature in all three hosts Likewise, survival rate of all immature stages were significantly higher (P < 0.0001) at higher than lower temperatures. The three hosts, cucumber (Cucumis sativus), watermelon (Citrullus lanatus (Thunb.) Matsum. and Nakai), and pumpkin (Cucurbita pepo) did not significantly affect duration or survival rates of immature stages of Z. cucurbitae. The low developmental thresholds were estimated at 15.88, 13.44, and 12.62 for egg, larva and pupa, respectively. These results further confirm that Z. cucurbitae is well adapted to warm climate, which dominates many areas of Tanzania.