A novel technique for the precise measurement of CO2 production rate in small aquatic organisms as validated on aeshnid dragonfly nymphs.

The present study describes and validates a novel yet simple system for simultaneous in vivo measurements of rates of aquatic CO2 production (MCO2 ) and oxygen consumption (MO2 ), thus allowing the calculation of respiratory exchange ratios (RER). Diffusion of CO2 from the aquatic phase into a gas phase, across a hollow fibre membrane, enabled aquatic MCO2  measurements with a high-precision infrared gas CO2 analyser. MO2  was measured with a PO2  optode using a stop-flow approach. Injections of known amounts of CO2 into the apparatus yielded accurate and highly reproducible measurements of CO2 content (R2=0.997, P<0.001). The viability of in vivo measurements was demonstrated on aquatic dragonfly nymphs (Aeshnidae; wet mass 2.17 mg-1.46 g, n=15) and the apparatus produced precise MCO2  (R2=0.967, P<0.001) and MO2  (R2=0.957, P<0.001) measurements; average RER was 0.73±0.06. The described system is scalable, offering great potential for the study of a wide range of aquatic species, including fish.

Autecology, feeding preferences and reproductive biology of Chorthippus (Glyptobothrus) vagans (Eversmann, 1848) (Orthoptera: Gomphocerinae) in Mediterranean ecosystems.

Chorthippus vagans is a common species of Gomphocerinae (Orthoptera) on the Iberian Peninsula. It is endangered in Central Europe where information about its ecological requirements is available; however, aspects of its biology are almost unknown in Mediterranean ecosystems, where it is widespread and common. The focus of this study was to determine how C. vagans adjusts its biology to environmental features of this ecosystem and to interpret how it may be affected by the ecological changes related to the re-vegetation programme linked to the construction of the Breña dam (SW Spain). The research parameters included the autoecology, feeding response and some aspects of reproduction of this species in the Southern Iberian Peninsula. To determine the local distribution and phenology of C. vagans, monthly samplings were conducted (2007-2010) in 12 sampling plots. For the food selection tests, ten nymphs and ten adults were placed individually in insectaries under controlled conditions. Grasses (Lolium sp.) and shrubs (Cistus sp.) were supplied ad libitum in two types of tests, monospecific and mixed diet. The reproductive biology was analysed by both observations of anatomical structures (integument, bristles, tibial spines, tarsal claws and mandibles) and ovarian dissections of 29 females and in laboratory rearing experiments with 15 pairs of adults. The results indicate that C. vagans shows an extended activity period which peaks at the end of summer. It is a polyphagous grasshopper, although adults show a slight preference for grasses. In addition, it is a univoltine species with spring-summer breeding activity.

Plant resistance reduces the strength of consumptive and non-consumptive effects of predators on aphids.

1. The impact of predators on prey has traditionally been attributed to the act of consumption. Prey responses to the presence of the predator (non-consumptive effects), however, can be as important as predation itself. While plant defences are known to influence predator-prey interactions, their relative effects on consumptive vs. non-consumptive effects are not well understood. 2. We evaluated the consequences of plant resistance and predators (Hippodamia convergens) on the mass, number of nymphs, population growth, density and dispersal of aphids (Macrosiphum euphorbiae). We tested for the effects of plant resistance on non-consumptive and consumptive effects of predators on aphid performance and dispersal using a combination of path analysis and experimental manipulation of predation risk. 3. We manipulated plant resistance using genetically modified lines of tomato (Solanum lycopersicum) that vary incrementally in the expression of the jasmonate pathway, which mediates induced resistance to insects and manipulated aphid exposure to lethal and risk predators. Predation risk predators had mandibles impaired to prevent killing. 4. Plant resistance reduced predation rate (consumptive effect) on high resistance plants. As a consequence, predators had no impact on the number of nymphs, aphid density or population growth on high resistance plants, whereas on low resistance plants, predators reduced aphid density by 35% and population growth by 86%. Path analysis and direct manipulation of predation risk showed that predation risk rather than predation rate promoted aphid dispersal and varied with host plant resistance. Aphid dispersal in response to predation risk was greater on low compared to high resistance plants. The predation risk experiment also showed that the number of aphid nymphs increased in the presence of risk predators but did not translate into increased population growth. 5. In conclusion, the consumptive and non-consumptive components of predators affect different aspects of prey demography, acting together to shape prey population dynamics. While predation risk accounts for most of the total effect of the predator on aphid dispersal and number of nymphs, the suppressive effect of predators on aphid population occurred largely through consumption. These effects are strongly influenced by plant resistance levels, suggesting that they are context dependent.

The role of Rhipicephalus sanguineus sensu lato saliva in the dissemination of Rickettsia conorii in C3H/HeJ mice.

Animal models have been developed for the study of rickettsial pathogenesis. However, to understand what occurs during the natural route of rickettsial transmission via the tick bite, the role of tick saliva should be considered in these models. To address this, we analysed the role of tick saliva in the transmission of Rickettsia conorii (Rickettsiales: Rickettsiaceae) in a murine host by intradermally (i.d.) inoculating two groups of susceptible C3H/HeJ mice with this Rickettsia, and infesting one group with nymphal Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks. Quantification of bacterial loads and mRNA levels of interleukin-1ß (IL-1ß), IL-10 and NF-κB was performed in C3H/HeJ lung samples by real-time quantitative polymerase chain reaction (PCR) and real-time reverse transcriptase PCR, respectively. Lung histology was examined to evaluate the pathological manifestations of infection. No statistically significant difference in bacterial load in the lungs of mice was observed between these two groups; however, a statistically significant difference was observed in levels of IL-1ß and NF-κB, both of which were higher in the group inoculated with rickettsiae but not infected with ticks. Lung histology in both groups of animals revealed infiltration of inflammatory cells. Overall, this study showed that i.d. inoculation of R. conorii caused infection in the lungs of C3H/HeJ mice and tick saliva inhibited proinflammatory effects.

Description of the immature stages of the planthopper Lacertinella australis (Hemiptera: Delphacidae).

The five immature stages of the planthopper Lacertinella australis (Remes Lenicov and Rossi Batiz) (Hemiptera: Delphacidae: Saccharosydnini) are described and illustrated. The main characters that allowed us to distinguish the various stages were body size, number of tarsomeres and metatibial spines, and number of teeth on the spur. New biological data based on laboratory rearing and field observations showed that L. australis can carry out its biological cycle successfully on the graminaceous pampas grass (Cortaderia spp. Stapf (Poales: Poaceae)). In addition, the efficient rearing in captivity, the high survivorship registered, and overwintering only on this host plant suggests that L. australis is a potential biocontrol agent of this invasive graminaceous weed. This study provides information about the immature stages, including a key for their identification, based on laboratory reared specimens and field observations.

First description of the nymph and larva of Dermacentor raskemensis (Acari: Ixodidae), parasites of pikas and other small mammals in Central Asia.

Dermacentor raskemensis Pomerantzev, 1946 is one of the rare Asian species in this genus. The immature stages of this species have never been described. Reexamination of D. raskemensis holdings stored in the United States National Tick Collection revealed a collection lot containing reared nymphs and larvae of this species. This collection made it possible for us to find numerous nymphs and larvae of D. raskemensis among previously unidentified material collected in the field. Both immature stages of D. raskemensis are described here for the first time. Nymphs of D. raskemensis can be distinguished from those of other Dermacentor species in the region by small spiracular plate, relatively short and obtuse lateral projections of basis capituli dorsally, relatively short spurs on coxa I and the internal spur is characteristically very broadly rounded at its apex, and very small spur on coxa IV, whereas larvae of D. raskemensis can be distinguished from other Dermacentor by relatively short and obtuse lateral projections of basis capituli, approximately 6 denticles in the median files on hypostome, and relatively short, broad, and rounded spur on coxa I. The nymphs and larvae of D. raskemensis studied originate from Afghanistan, India, Iran, and Pakistan, where they were collected from pikas and other small mammals.

Importance and physiological effects of hemolymphagy in triatomines (Hemiptera: Reduviidae).

Triatomines are hematophagous insects and the vectors for Trypanosoma cruzi in the Americas. Although their preferred meal is blood from vertebrate hosts, unfed triatomine nymphs are often seen feeding on different arthropod species. Triatomine saliva has a wide range of activities that aid the hematophagic process. However, nothing is known about its role during hemolymphagy. In the current study, we reproduced hemolymphagy under laboratory conditions and evaluated the influence of hemolymphagy on the survival of Triatoma infestans nymphs. The effects of saliva on the activation of the prophenoloxidase cascade in the invertebrate host and the influence of the saliva on the motility of the bugs and contractions of the dorsal vessels were assessed. Hemolymphagy prolonged the survival rate of T. infestans first instars from 60 to >120 d compared with unfed nymphs. The saliva from T. infestans caused a 50% reduction in the amplitude and frequency of the dorsal vessel contractions of adult Rhodnius prolixus and induced paralysis for >10 min when the saliva was injected into second instars. T. infestans saliva was able to inhibit the activation of the prophenoloxidase cascade from a R. prolixus hemolymph, but had no effect on the phenoloxidase activity after the cascade was activated. The paralyzing molecule in the saliva was <5 kDa and probably had no proteic or lipidic characteristics. These results suggest that triatomine saliva may play an important role during hemolymphagy by inducing paralysis and suppressing immune responses in the invertebrate host. The importance of hemolymphagy for the survival of nymphs in periods when vertebrate blood is scarce is also discussed.

A transwell assay method to evaluate Borrelia burgdorferi sensu stricto migratory chemoattraction toward tick saliva proteins.

We developed a transwell assay to quantify migration of the Lyme disease agent, Borrelia burgdorferi sensu stricto (s.s.), toward Ixodes scapularis salivary gland proteins. The assay was designed to assess B. burgdorferi s.s. migration upward against gravity through a transwell polycarbonate membrane overlaid with 6% gelatin. Borreliae that channeled into the upper transwell chamber in response to test proteins were enumerated by flow cytometry. The transwell assay measured chemoattractant activity for B. burgdorferi s.s. from salivary gland extract (SGE) harvested from nymphal ticks during bloodmeal engorgement on mice 42 h post-attachment and saliva collected from adult ticks. Additionally, SGE protein fractions separated by size exclusion chromatography demonstrated various levels of chemoattractant activity in the transwell assay. Sialostatin L, and Salp-like proteins 9 and 11 were identified by mass spectrometry in SGE fractions that exhibited elevated activity. Recombinant forms of these proteins were tested in the transwell assay and showed positive chemoattractant properties compared to controls and another tick protein, S15A. These results were reproducible providing evidence that the transwell assay is a useful method for continuing investigations to find tick saliva components instrumental in driving B. burgdorferi s.s. chemotaxis.

Structure and function of the stylets of hematophagous Triatominae (Hemiptera: Reduviidae), with special reference to Dipetalogaster maxima.

Kissing bugs (Hemiptera: Reduviidae: Triatominae) are able to bend their rod-like maxillae while searching for blood vessels in the tissue of their vertebrate hosts. Little is known about the working mechanisms of these bending movements and the distal opening of the food channel. We compared the morphological structure of the stylets (mandibles and maxillae) of four triatomine species and analyzed the feeding process of Dipetalogaster maxima (Uhler, 1894). The maxillae of triatomine bugs are interlocked by a tongue-and-groove system, allowing longitudinal sliding. While penetrating the host tissue, the animals perform rapid alternate back and forth movements of the maxillae. The resistance of the surrounding tissue pushes the asymmetric apex of the maxillae away from its straight path, i.e., if one individual maxilla is protracted alone, its tip curves inwards, and the other maxilla follows. Once a blood vessel is tapped, the spine-like tip of the left maxilla splays outwards. Apically, each of the maxillae features an abutment, the left one exhibiting a notch that presumably facilitates splaying. The mechanical interaction of the two maxillary abutments enables the distal opening of the food channel but might also support the movements of the maxillary bundle attributable to different bending moment distributions.

Host-specific expression of Ixodes scapularis salivary genes.

Ixodes scapularis vectors several pathogens including Borrelia burgdorferi, the agent of Lyme disease. Nymphal and larval stages, and the pathogens transmitted by I. scapularis are maintained in a zoonotic cycle involving rodent reservoir hosts, predominantly Peromyscus leucopus. Humans are not reservoir hosts, however, accidental encounters of infected ticks with humans, results in pathogen transmission to the human host. Laboratory models of non-reservoir hosts such as guinea pigs develop a strong immune response to tick salivary proteins and reject ticks upon repeated tick infestations. Anecdotal and scientific evidence suggests that humans that get frequent tick bites might also develop resistance to ticks. Mus musculus, the laboratory model of natural host, does not develop resistance to I. scapularis upon repeated tick infestations. Addressing this dichotomy in vector-host interaction, we present data that suggest that the salivary transcriptome and proteome composition is different in mouse and guinea pig-fed I. scapularis, and that these differences might contribute to differences in host immune responses. These findings reveal a new insight into vector-host interactions and offer a functional paradigm to better understand the phenomenon of acquired tick-resistance.

Liposome encapsulation and EDTA formulation of dsRNA targeting essential genes increase oral RNAi-caused mortality in the Neotropical stink bug Euschistus heros.

BACKGROUND: The Neotropical stink bug Euschistus heros is a major pest in soybean fields. Development of highly species-specific pesticides based on RNA interference (RNAi) could provide a new sustainable and environmentally friendly control strategy. RESULTS: Here, the potential of RNAi as a pest control tool against E. heros was assessed. First, target gene selection using a microinjection approach was performed. Seven of the 15 candidate genes tested exhibited > 95% mortality after hemolymph injection of 27.5 ng dsRNA. Subsequently, dsRNA was administered orally using different formulations: naked dsRNA, liposome-encapsulated-dsRNA and dsRNA formulated with EDTA. Liposome-encapsulated dsRNA targeting vATPase A and muscle actin led to significant mortality after 14 days (45% and 42%, respectively), whereas EDTA-formulated dsRNA did so for only one of the target genes. Ex vivo analysis of the dsRNA stability in collected saliva indicated a strong dsRNA-degrading capacity by E. heros saliva, which could explain the need for dsRNA formulations. CONCLUSION: The results demonstrate that continuous ingestion of dsRNA with EDTA or liposome-encapsulated dsRNA can prevent dsRNA from being degraded enzymatically and suggest great potential for using these formulations in dsRNA delivery to use RNAi as a functional genomics tool or for pest management of stink bugs. © 2018 Society of Chemical Industry.

A behavioral evaluation of the effects of ingestion of Linguatula serrata nymphs in rats.

Nasopharyngeal linguatuliasis known as Halzoun or Marrara syndrome occurs following ingestion of raw or undercooked viscera, including lymph nodes, liver and lung of infected animals. The present study was aimed to investigate the behavioral changes induced by ingestion of Linguatula serrata nymphs in rats. For this purpose, 24 rats were divided into four groups and orally ingested with 0 (control), 15, 30 and 60 L. serrata nymphs, respectively. Sneezing, mouth and tongue movements and mouth opening numbers were counted and the duration of mouth and nose grooming was measured at 10-min blocks for 30 min. Ingestion of 0 (normal saline) number of nymph produced negligible behaviors, whereas 15, 30 and 60 numbers of nymphs increased the above-mentioned behaviors when compared to normal saline (0 nymph) group. In this context, 60 number of nymphs produced more behavioral changes than 15 nymphs. We concluded that ingestion of L. serrata nymphs can produce behavioral changes in orofacial area in rats.

Identification of Lyme borreliae proteins promoting vertebrate host blood-specific spirochete survival in Ixodes scapularis nymphs using artificial feeding chambers.

Lyme borreliosis, the most common vector-borne illness in Europe and the United States, is caused by spirochetes of the Borrelia burgdorferi sensu lato complex and transmitted by Ixodes ticks. In humans, the spirochetes disseminate from the tick bite site to multiple tissues, leading to serious clinical manifestations. The ability of spirochetes to survive in ticks during blood feeding is thought to be essential for Lyme borreliae to be transmitted to different vertebrate hosts. This ability is partly attributed to several B. burgdorferi proteins, including BBA52 and Lp6.6, which promote spirochete survival in nymphal ticks feeding on mice. One of the strategies to identify such proteins without using live animals is to feed B. burgdorferi-infected ticks on blood via artificial feeding chambers. In previous studies, ticks were only fed on bovine blood in the feeding chambers. In this study, we used this chamber model and showed that I. scapularis ticks will not only acquire bovine blood but human and quail blood as well. The latter two are the incidental host and an avian host of Lyme borreliae, respectively. We also investigated the roles that BBA52 and Lp6.6 play in promoting spirochete survival in nymphal ticks fed on human or quail blood. After feeding on human blood, spirochete burdens in ticks infected with an lp6.6-deficient B. burgdorferi were significantly reduced, while bba52-deficient spirochete burdens in ticks remained unchanged, similar to the wild-type strain. No strain showed a change in spirochete burdens in ticks fed on quail blood. These results indicate that Lp6.6 plays a role for B. burgdorferi in nymphs fed on human but not quail blood. Such information also demonstrates that the artificial feeding chamber is a powerful tool to identify B. burgdorferi proteins that promote vertebrate host blood-specific spirochete survival in I. scapularis ticks.

Genomics of interaction between the brown planthopper and rice.

Rice (Oryza sativa L.) and the brown planthopper (Nilaparvata lugens (Stål)) form a model system for dissection of the mechanism of interaction between insect pest and crop. In this review, we focus on the genomics of BPH-rice interaction. On the side of rice, a number of BPH-resistance genes have been identified genetically. Thirteen of these genes have been cloned which shed a light on the molecular basis of the interaction. On the aspect of BPH, a lot of salivary proteins have been identified using transcriptome and proteome techniques. The genetic loci of virulence were mapped in BPH genome based on the linkage map. The understanding of interaction between BPH and rice will provide novel insights into efficient control of this pest.

Description of a new soft tick species (Acari: Argasidae: Ornithodoros) associated with stream-breeding frogs (Anura: Cycloramphidae: Cycloramphus) in Brazil.

In this study, we present a morphological description of immature and adult specimens of Ornithodoros saraivai n. sp., a tick associated with the frog Cycloramphus boraceiensis (Cycloramphidae) at São Sebastião island, located in the São Paulo state seaboard, Brazil. While larvae of O. saraivai are ecologically related to Ornithodoros faccinii, another soft tick associated with cycloramphids, the combination of 7 sternal pairs, 16 dorsal pairs, a pyriform dorsal plate and a partially toothed hypostome constitute unique characters of the O. saraivai larvae. One undetermined nymphal instar and adults of O. saraivai are similar to mature specimens of the Ornithodoros talaje species group; however, the O. saraivai specimens can be recognized by the presence of a robust bean-shaped spiracle with a large spiracular plate and more than two long seta in palpal article I. Identical partial sequences of the mitochondrial 16S rDNA gene confirmed the identity for all collected stages and for two cohorts of laboratory-reared larvae of O. saraivai. A Bayesian and Maximum Parsimony inferred phylogenetic trees support the position of O. saraivai in a clade with O. faccinii, suggesting the existence of an Ornithodoros lineage that evolved in association with amphibians.

Gene expression changes in the tyrosine metabolic pathway regulate caste-specific cuticular pigmentation of termites.

In social insects, all castes have characteristic phenotypes suitable for their own tasks and to engage in social behavior. The acquisition of caste-specific phenotypes was a key event in the course of social insect evolution. However, understanding of the genetic basis and the developmental mechanisms that produce these phenotypes is still very limited. In particular, termites normally possess more than two castes with specific phenotypes (i.e. workers, soldiers, and reproductives), but proximate developmental mechanisms are far from being fully understood. In this study, we focused on the pigmentation of the cuticle as a model trait for caste-specific phenotypes, during the molts of each caste; workers, soldiers, presoldiers (intermediate stage of soldiers), and alates (primary reproductives) in Zootermopsis nevadensis. Expression patterns of cuticular tanning genes (members of the tyrosine metabolic pathway) were different among each molt, and high expression levels of several "key genes" were observed during each caste differentiation. For the differentiation of castes with well-tanned cuticles (i.e. soldiers and alates), all focal genes except DDC in the former were highly expressed. On the other hand, high expression levels of yellow and aaNAT were observed during worker and presoldier molts, respectively, but most other genes in the pathway were expressed at low levels. RNA interference (RNAi) of these key genes affected caste-specific cuticular pigmentation, leading to soldiers with yellowish-white heads and pigmented mandibular tips, presoldiers with partly pigmented head cuticles, and alates with the yellow head capsules. These results suggest that the pigmentation of caste-specific cuticles is achieved by the regulation of gene expression in the tyrosine metabolic pathway.

Stylet morphometrics and citrus leaf vein structure in relation to feeding behavior of the Asian citrus psyllid Diaphorina citri, vector of citrus huanglongbing bacterium.

The Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae), is the primary vector of the phloem-limited bacterium Candidatus Liberibacter asiaticus (LAS) associated with huanglongbing (HLB, citrus greening), considered the world's most serious disease of citrus. Stylet morphometrics of ACP nymphs and adults were studied in relation to citrus vein structure and to their putative (histologically verified) feeding sites on Valencia orange leaves. ACP nymphs preferred to settle and feed on the lower (abaxial) side of young leaves either on secondary veins or on the sides of the midrib, whereas adults preferred to settle and feed on the upper (adaxial) or lower secondary veins of young or old leaves. Early instar nymphs can reach and probe the phloem probably because the distance to the phloem is considerably shorter in younger than in mature leaves, and is shorter from the sides of the midrib compared to that from the center. Additionally, the thick-walled 'fibrous ring' (sclerenchyma) around the phloem, which may act as a barrier to ACP stylet penetration into the phloem, is more prominent in older than in younger leaves and in the center than on the sides of the midrib. The majority (80-90%) of the salivary sheath termini produced by ACP nymphs and adults that reached a vascular bundle were associated with the phloem, whereas only 10-20% were associated with xylem vessels. Ultrastructural studies on ACP stylets and LAS-infected leaves suggested that the width of the maxillary food canal in first instar nymphs is wide enough for LAS bacteria to traverse during food ingestion (and LAS acquisition). However, the width of the maxillary salivary canal in these nymphs may not be wide enough to accommodate LAS bacteria during salivation (and LAS inoculation) into host plants. This may explain the inability of early instar nymphs to transmit LAS/HLB in earlier reports.

Mouthparts and stylet penetration of the lac insect Kerria lacca (Kerr) (Hemiptera:Tachardiidae).

Hitherto less known aspects on mouthpart morphology and penetration mechanism of the lac insect Kerria lacca have been explored. Unique details of the mouthparts, i.e. morphology of labium and stylets and salivary sheath have been brought out. The gross morphology of the mouthparts though resembled other plant sucking homopterans; a two-segmented labium with symmetrically distributed six pairs of contact-chemoreceptors on its surface was distinct; the mandibular stylets had serrations on its extreme apical region, while the maxillary stylets had their external surface smooth with parallel longitudinal grooves on their inner surface. Formation of flanges, salivary sheath and penetration pathway observed along with probing and penetration of the stylets intracellularly up to the phloem cells, as illustrated herein, are the addition to the existing knowledge on the structural details of the mouthparts and the feeding behavior thereupon.

Feeding of Boophilus microplus larvae on a partially defined medium through thin slices of cattle skin.

Larvae of the cattle tick Boophilus microplus will attach to thin (0-3--0-5 mm) slices of cattle skin and engorge on a partially defined medium at 35 degrees C. Forty-seven to 83 per cent of the larvae had engorged by 8 days, and 51--71 per cent of these moulted to nymphs. Tissue culture medium alone allowed little growth unless supplemented with dialysed, freeze dried bovine serum (7 per cent, w/v). This medium could be further difined by substituting purified bovine serum albumin (Cohn fraction V) for the dialysed bovine serum. In one experiment, nymphs of Haemaphysalis longicornis engorged and later moulted to adults. Skin slices were used fresh or after freeze-drying and storing at --25 degrees C. The possible uses of the culture method are discussed.