Glyphodes pyloalis Walker (Lepidoptera: Pyralididae) is a common pest in sericulture and has developed resistance to different insecticides. However, the mechanisms involved in insecticide resistance of G. pyloalis are poorly understood. Here, we present the first whole-transcriptome analysis of differential expression genes in insecticide-resistant and susceptible G. pyloalis. Clustering and enrichment analysis of DEGs revealed several biological pathways and enriched Gene Ontology terms were related to detoxification or insecticide resistance. Genes involved in insecticide metabolic processes, including cytochrome P450, glutathione S-transferases and carboxylesterase, were identified in the larval midgut of G. pyloalis. Among them, CYP324A19, CYP304F17, CYP6AW1, CYP6AB10, GSTs5, and AChE-like were significantly increased after propoxur treatment, while CYP324A19, CCE001c, and AChE-like were significantly induced by phoxim, suggesting that these genes were involved in insecticide metabolism. Furthermore, the sequence variation analysis identified 21 single nucleotide polymorphisms within CYP9A20, CYP6AB47, and CYP6AW1. Our findings reveal many candidate genes related to insecticide resistance of G. pyloalis. These results provide novel insights into insecticide resistance and facilitate the development of insecticides with greater specificity to G. pyloalis.

The process of autophagy has been detected in the midgut epithelium of four millipede species: Julus scandinavius, Polyxenus lagurus, Archispirostreptus gigas, and Telodeinopus aoutii. It has been examined using transmission electron microscopy (TEM), which enabled differentiation of cells in the midgut epithelium, and some histochemical methods (light microscope and fluorescence microscope). While autophagy appeared in the cytoplasm of digestive, secretory, and regenerative cells in J. scandinavius and A. gigas, in the two other species, T. aoutii and P. lagurus, it was only detected in the digestive cells. Both types of macroautophagy, the selective and nonselective processes, are described using TEM. Phagophore formation appeared as the first step of autophagy. After its blind ends fusion, the autophagosomes were formed. The autophagosomes fused with lysosomes and were transformed into autolysosomes. As the final step of autophagy, the residual bodies were detected. Autophagic structures can be removed from the midgut epithelium via, e.g., atypical exocytosis. Additionally, in P. lagurus and J. scandinavius, it was observed as the neutralization of pathogens such as Rickettsia-like microorganisms. Autophagy and apoptosis ca be analyzed using TEM, while specific histochemical methods may confirm it.

Polistes paper wasps can be used to monitor trace metal contaminants, but the effects of pollution on the health of these insects are still unknown. We evaluated, in a south-eastern area of Spain, whether workers of Polistes dominula collected at urban and rural sites differ in health of midgut tissue and in fluctuating asymmetry, an estimate of developmental noise. We found that wasps collected at the urban sites had abundant lead (Pb)-containing spherites, which were less visible in wasps from the rural sites. Evident ultrastructural alterations in the epithelium of the midgut of the wasps collected at the urban sites included broken and disorganized microvilli, a high amount and density of heterochromatin in the nucleus of epithelial cells, cytoplasmic vacuolization and mitochondrial disruptions. Altogether, these findings suggest a negative effect on the transmembrane transport and a less efficient transcription. On the contrary, a healthy epithelium was observed in wasps from the rural sites. These differences may be preliminarily linked with levels of lead pollution, given that wasps from urban sites had double the Pb concentrations of wasps from rural sites. Level of fluctuating asymmetry was unrelated to wasp origin, thus suggesting no link between developmental noise and Pb-driven pollution.

The digestive system of sea spiders (Pycnogonida) presents peculiarities that have not been discussed in the context of their ecology or feeding behaviour. We investigated the digestive system of two Mediterranean species, a carnivorous species Ammothella longipes and a detritivorous Endeis spinosa, with special focus on its correlation with behavioural feeding habits. The midgut and hindgut sections did not present significant differences between the two species, but major differences were observed in the foregut, reflecting concordance to their diet and their feeding behaviour. Jaws, setose lips, the structure of the pharyngeal filter and musculature of the proboscis are the main differential elements when comparing feeding habits of A. longipes and E. spinosa. These elements are responsible for the reduction of the food pulp down to subcellular size. The digestion process observed in the species studied agrees with that observed in other pycnogonid lineages, but differs from most marine arthropods mainly because of the absence of midgut gland cells and the presence of a unique multifunctional type of midgut epithelial cell. Epithelial digestive cells are present in a small ‘resting’ form during starvation periods. During digestion, secretion granules possibly containing zymogen move to their apical border to be secreted to the midgut lumen, secondary lysosomes are formed and intracellular digestion occurs within them. Residual bodies are formed within the epithelial cell and released to the midgut lumen to be transported towards the hindgut. The characteristics of the digestive process of the pycnogonids studied seem to reflect a plesiomorphic state in arthropods.

A midgut trypanolysin and an agglutinin from Glossina palpalis subspecies were isolated and partially characterized using anion-exchange chromatography and polyacrylamide gel electrophoresis. FPLC fractions of midgut extracts of Glossina palpalis palpalis caused agglutination and lysis of two trypanosome species (Trypanosoma congolense and Trypanosoma brucei brucei). although Glossina palpalis gambiensis caused only agglutination. The trypanolysin and agglutinin were active only in the posterior midguts, were heat labile above 50%C, had a periodic cycle of ‘activity’ in response to bloodmeal intake and were not affected by protease inhibitors or trypsin but were inactivated by pronase. The lytic substance contained two proteins with approximate molecular weights (Mr) of 12000 and 10000 Da respectively. The agglutinin had an approximate Mr of 67000 Da. Gamma-irradiation of the two subspecies caused a temporary inhibition of trypanolytic and agglutinin activities in midgut extracts.

Bacillus sphaericus (Bs) is a potential mosquito larvicide especially against Culex quinquefasciatus (Say), the vector of bancroftian filariasis. It is a gram-positive, aerobic and endospore-forming bacterium. During sporulation, it synthesizes binary endotoxins (molecular weight 42 and 51 kDa proteins) that are highly toxic to mosquito species. The mode of action of the binary toxin of this biolarvicide in the alimentary canal and the ultrastructural events in the tissues of the affected larvae have been demonstrated. Lysosomal components are the most important organelles involved in the metabolism of the foreign molecules encountered by the cells. In this paper, we report the ultrastructural changes in the midgut epithelial cells, with special emphasis on lysosomes (L), in untreated Cx. quinquefasciatus larvae and those treated with the Bs-IAB59 bacterial strain. In the Bs-treated larvae, the size of the L gradually increased, and within 16 h, fine filaments appeared and compartmentalized into small lamellar bodies, within the L. After 36 h, these lamellar bodies appeared to be collapsed, and later disintegrated in the 48th hour. In the untreated larvae, such ultrastructural changes in the L were not seen. These observations suggested that, though the target site of action of the Bs toxin is the midgut epithelial cells, ultrastructural changes in the L compartments play a vital role in the mortality of the mosquito larvae.

Differentiation of bloodstream-form trypanosomes into procyclics in tsetse flies (Diptera: Glossinidae) is a crucial step in the establishment of midgut infections. A number of factors have been implicated in the transformation process, including enzymes and lectins or lectin-like molecules. Recently, Glossina proteolytic lectin (Gpl) gene, which encodes a protein with both lectin and trypsin activities has been shown to stimulate transformation of bloodstream-form trypanosomes into procyclics in vitro. Using RT-PCR, we show that the induction of Gpl gene expression by blood meal occurs only in Glossina fuscipes fuscipes Newstead, Glossina austeni Newstead, Glossina pallidipes Austen, and not in the Anopheles gambiae Giles sensu stricto, Phlebotomus duboscqi Neveu-Lemaire, Rhipicephalus appendiculatus Neumann and Stomoxys calcitrans (Linnaeus). The expression means of Gpl mRNA in G. f. fuscipes following a blood meal were significant (P < 0.05) with low expression in teneral flies and reaching a maximum between 48 and 72 h (P < 0.05), suggesting time-dependent regulation of the transcription. The expression of the Gpl gene was significantly lower (P < 0.05) in G. f. fuscipes fed on blood meal infected with Trypanosoma brucei brucei as compared with G. f. fuscipes fed on uninfected blood meal. This suggests some form of interaction of T. b. brucei or the parasite products with Gpl within the tsetse midgut leading to down-regulation of the Gpl gene. Additionally, refractory G. f. fuscipes expressed higher (P < 0.05) transcript abundance than the susceptible G. pallidipes.

Malarial gametocytes circulate in the peripheral blood of the vertebrate host as developmentally arrested intra-erythrocytic cells, which only resume development into gametes when ingested into the bloodmeal of the female mosquito vector. The ensuing development encompasses sexual reproduction and mediates parasite transmission to the insect. In vitro the induction of gametogenesis requires a drop in temperature and either a pH increase from physiological blood pH (ca pH 7·4) to about pH 8·0, or the presence of a gametocyte-activating factor recently identified as xanthurenic acid (XA). However, it is unclear whether either the pH increase or XA act as natural triggers in the mosquito bloodmeal. We here use pH-sensitive microelectrodes to determine bloodmeal pH in intact mosquitoes. Measurements taken in the first 30 min after ingestion, when malarial gametogenesis is induced in vivo, revealed small pH increases from 7·40 (mouse blood) to 7·52 in Aedes aegypti and to 7·58 in Anophěles stephensi. However, bloodmeal pH was clearly suboptimal if compared to values required to induce gametogenesis in vitro. Xanthurenic acid is shown to extend the pH-range of exflagellation in vitro in a dose-dependent manner to values that we have observed in the bloodmeal, suggesting that in vivo malarial gametogenesis could be further regulated by both these factors.

Binding to the midgut microvillar surface in the sandfly Phlebotomus papatasi is a prerequisite for successful development of Leishmania major within the gut of the vector. This paper describes a method for detecting microvillar-associated proteins which act as ligands for the parasite surface glycoconjugate lipophosphoglycan (LPG). Adhesion of LPG to midgut proteins was visualized by probing midgut extracts with LPG using a Western ligand blotting technique. Procyclic L. major LPG bound to a microvillar polypeptide band of 65 kDa (estimated in the non-reduced state) and bound variably to several lower molecular weight bands, probably degradation products or subunits of the primary binding polypeptides. Specificity of binding was confirmed by co-incubating biotinylated LPG with an LPG-specific mAb which resulted in a great reduction in binding.

Ultrastructural studies were carried out on wild Kenyan sandflies (Phlebotomus pedifer) naturally infected with Leishmania aethiopica. The promastigotes were attached to structures resembling pieces of a peritrophic membrane. They were located amongst long microvilli and also deep in the cytoplasm of the gut cells. The factors which influence the invasion of the parasites into cells are not known and need to be investigated.