Effects of chronic oral exposure to insecticide teflubenzuron on the midgut of the honey bee Apis mellifera workers: histopathological insights into pesticide toxicity.

The honey bee Apis mellifera plays a significant role as a pollinator of native and cultivated plants, by increasing the productivity of several cultures, preserving the flora, and producing forest seeds. However, bee populations are declining worldwide, including A. mellifera, due to Colony Collapse Disorder, mainly resulting from the constant use of pesticides in the crops. Teflubenzuron is a physiological insecticide that belongs to the benzoylurea group, which inhibits chitin synthesis, the main component of the insect integument classified as safe for non-target insects, including bees. However, its effect on non-target organs of insects remains unknown. The midgut is the main organ of the digestive tract, which works in digestion and absorption and may be exposed to pesticides that contaminate food resources. The present work aimed to verify if the insecticide teflubenzuron is toxic and has histopathological effects on the midgut of A. mellifera adult workers. Workers exposed orally and chronically to the field-realistic concentration of teflubenzuron present 81.54% mortality. The epithelium of the midgut of these bees presents high vacuolization, spherocrystals, cell fragments released to the organ lumen, apocrine secretion, nuclear pyknosis, loss of cell-cell contact, and damage to regenerative cell nests and to the peritrophic matrix. These results indicate that the chitin synthesis-inhibiting insecticide teflubenzuron is toxic to A. mellifera after chronic oral exposure, at realistic field concentration, although it is classified as non-toxic to adult and non-target insects.

Midgut morphology of the predator mosquito Lutzia bigoti (Diptera: Culicidae) and its implications for feeding behavior.

Lutzia mosquitoes (Theobald, 1903) are predaceous during their larval stages, but the adult feeding is not clearly understood, especially in relation to blood feeding. In case these mosquitoes are harmless to humans and related animals, they can be useful in biological control of mosquito vectors of pathogens. Investigating the midgut morphology is a good strategy to understand the feeding behavior of this species. The midgut in Lutzia bigoti Bellardi, 1862 displays two distinct portions, a thin anterior midgut and a more dilated posterior midgut. Digestive cells form a single epithelium in the midgut. These cells have long and packed microvilli at their apex and membrane infoldings at their basal portion, the basal labyrinth. The epithelium is supported by a basal lamina. Regarding their cytoplasm, it is noteworthy the abundance of mitochondria, distributed in an apical-basal fashion, and also a whirl-shaped endoplasmic reticulum in the posterior midgut. Basal cells are also found in the midgut of L. bigoti, resembling regenerative cells. The general organization of L. bigoti's midgut closely resembles that of numerous hematophagous mosquitoes previously documented. However, it diverges due to the presence of a peritrophic matrix even when exclusively fed on sugary solutions. Peculiar aspects of L. bigoti's midgut are discussed and compared to those of other mosquito species.

Cuticle melanization and the expression of immune-related genes in the honeybee Apis mellifera (Hymenoptera: Apidae) adult workers.

The global decline of bee populations has several factors, including pathogens, which need overcome the insect defenses such as the physical barriers, the body cuticle and peritrophic matrix (primary defenses), as well as the secondary defenses with antimicrobial peptides (AMPs) and the enzyme lysozyme. The regulation of immune defenses according to the infection risks raises questions about the immunity of social bees due to their exposition to different pathogens pressures during the adult lifespan and tasks performed. This study evaluated the primary (body cuticle melanization, peritrophic matrix and cpr14 expression) and secondary (AMPs and lysozyme expression) defenses of the honeybee Apis mellifera workers according to the age and tasks. The expression of malvolio was used to detect precocious forage tasks outside the colony. Forager workers have higher amount of cuticular melanization in the body cuticle than nurse, but not when the age effect is retired, indicating the gradual acquisition of this compound in the integument of adult bees. The relative value of chitin in the peritrophic matrix and cpr14 mRNA are similar in all bees evaluated, suggesting that these components of primary defenses do not change according to the task and age. Differential expression of genes for AMPs in workers performing different tasks, within the same age group, indicates that the behavior stimulates expression of genes related to secondary immune defense. The expression of malvolio gene, accelerating the change in workers behavior, and those related to immune defense suggest the investment in secondary defense mechanisms when the primary defense of the body cuticle is not yet completed.

Do calcium oxalate crystals protect against herbivory?

Calcium oxalate (CaOx) crystals have challenged human curiosity since the advent of microscopy. These crystals are linked to the control of calcium levels in plant cells, but they have also been attributed several other functions, including protection against herbivory. However, the protection offered by CaOx crystals against herbivory may be overstated, as claims have been mainly based on their shapes and hard and indigestible nature rather than on experimental evidence. I contend that it is improbable that a constitutive defense, present since very early in the evolution of plants, has not been superseded by herbivores, especially insects. Here, I present arguments and evidence that suggest that these crystals have low efficiency in protecting plants against herbivores. First, I argue that insects with chewing mouthparts possess a semipermeable structure that protects their midgut, minimizing damage from crystals. Second, the action of CaOx crystals is purely mechanical and similar to other inert materials such as sand. Therefore, CaOx crystals only provide effective protection from herbivory in very particular cases and should not be considered an effective defense without supporting experimental evidence.

The role of the peritrophic matrix and red blood cell concentration in Plasmodium vivax infection of Anopheles aquasalis.

BACKGROUND: Plasmodium vivax is predominant in the Amazon region, and enhanced knowledge of its development inside a natural vector, Anopheles aquasalis, is critical for future strategies aimed at blocking parasite development. The peritrophic matrix (PM), a chitinous layer produced by the mosquito midgut in response to blood ingestion, is a protective barrier against pathogens. Plasmodium can only complete its life-cycle, and consequently be transmitted to a new host, after successfully passing this barrier. Interestingly, fully engorged mosquitoes that had a complete blood meal form a thicker, well-developed PM than ones that feed in small amounts. The amount of red blood cells (RBC) in the blood meal directly influences the production of digestive enzymes and can protect parasites from being killed during the meal digestion. A specific study interrupting the development of the PM associated with the proteolytic activity inhibition, and distinct RBC concentrations, during the P. vivax infection of the New World malaria vector An. aquasalis is expected to clarify whether these factors affect the parasite development. RESULTS: Absence of PM in the vector caused a significant reduction in P. vivax infection. However, the association of chitinase with trypsin inhibitor restored infection rates to those of mosquitoes with a structured PM. Also, only the ingestion of trypsin inhibitor by non-chitinase treated mosquitoes increased the infection intensity. Moreover, the RBC concentration in the infected P. vivax blood meal directly influenced the infection rate and its intensity. A straight correlation was observed between RBC concentrations and infection intensity. CONCLUSIONS: This study established that there is a balance between the PM role, RBC concentration and digestive enzyme activity influencing the establishment and development of P. vivax infection inside An. aquasalis. Our results indicate that the absence of PM in the midgut facilitates digestive enzyme dispersion throughout the blood meal, causing direct damage to P. vivax. On the other hand, high RBC concentrations support a better and thick, well-developed PM and protect P. vivax from being killed. Further studies of this complex system may provide insights into other details of the malaria vector response to P. vivax infection.

Spinosad-mediated effects on the walking ability, midgut, and Malpighian tubules of Africanized honey bee workers.

BACKGROUND: The global decline in Apis mellifera colonies is attributed to multiple factors, including pesticides. The bioinsecticide spinosad was initially recognized as safe for non-target organisms; however, its toxicity has been changing this view. Here, we investigated the survival, behavioral changes, and structural changes in the midgut and Malpighian tubules of A. mellifera treated orally with a spinosad formulation. RESULTS: The field-recommended concentration of spinosad killed 100% of the bees. The 5% and 50% lethal concentrations (LC5 and LC50 , respectively) of spinosad altered the behavioral activity, reducing the walking distance and velocity, and increased the resting time in comparison to the control. The LC50 caused disorganization of the epithelia of tested organs and induced oxidative stress and cell death. CONCLUSIONS: The present work provides new insights into the debate about the role of bioinsecticides in the mortality of Africanized honey bees. Even at very low concentrations, the spinosad formulation was toxic to the vital organs midgut and Malpighian tubules and adversely affected walking behavior. This detailed evaluation of the impact of the bioinsecticide on A. mellifera will contribute to the clarification of disturbances probably caused by spinosad formulations, which can be used to develop more sustainable protocols in agriculture. © 2017 Society of Chemical Industry.

Sublethal dose of deltamethrin damage the midgut cells of the mayfly Callibaetis radiatus (Ephemeroptera: Baetidae).

In insects, the midgut performs multiple physiologic functions (e.g., digestion and nutrients absorption) and serves as a physical/chemical barrier against pathogens and chemical stressors such as deltamethrin, a pyrethroid insecticide, commonly used in insect control that are agricultural pests and human disease vectors. Here, we described the midgut cell ultrastructure of Callibaetis radiatus nymphs, which are bioindicators of water quality and the ultrastructural alterations in midgut under sublethal exposure to deltamethrin at three different periods (1, 12, 24 h). The digestive cells of deltamethrin-unexposed nymphs had long microvilli, many mitochondria in the apical cytoplasm, a rough endoplasmic reticulum, a basal labyrinth with openings for hemocele, and the midgut peritrophic matrix which is classified as type I. Nymphs exposed to deltamethrin exhibited digestive cells rich in autophagic vacuoles, basal labyrinth loss, and microvilli disorganization since the first hour of contact with deltamethrin. However, these midgut tissues underwent to autophagic cellular recovery along the 24 h of exposure to deltamethrin. Thus, the sublethal exposure to deltamethrin is sufficient to disturb the ultrastructure of C. radiatus midgut, which might reduce the abilities of these insects to survive in aquatic environments contaminated by pyrethroids.

Termiticidal lectins from Myracrodruon urundeuva (Anacardiaceae) cause midgut damage when ingested by Nasutitermes corniger (Isoptera: Termitidae) workers.

BACKGROUND: Myracrodruon urundeuva is a hardwood tree, and its bark, heartwood and leaf contain lectins (MuBL, MuHL and MuLL respectively) with termiticidal activity against Nasutitermes corniger. In this work, the effects of these lectins on the midgut of N. corniger workers were evaluated. RESULTS: The insects were supplied with an artificial diet containing the lectins at their respective LC50 (previously determined). At 48 h after treatment, the midguts were dissected and fixed for histopathology analyses. Toluidine-blue-stained midguts from lectin-treated workers showed disorganisation, with the presence of debris in the lumen and the absence of brush border. Fluorescence microscopy revealed that the numbers of digestive and proliferating cells were lower in lectin-treated individuals than in the control, and caspase-3 staining confirmed the occurrence of cell apoptosis. Enteroendocrine cells were not seen in the treated individuals. The midguts from treated insects showed greater staining for peroxidase than the control, suggesting that the lectins caused oxidative stress. Staining with wheat germ agglutinin conjugated to FITC revealed that the lectins interfered with the integrity of the peritrophic matrix. CONCLUSION: This study showed that termiticidal lectins from M. urundeuva cause severe injuries, oxidative stress and cell death in the midgut of N. corniger workers. © 2016 Society of Chemical Industry.

Effects of specific antisera targeting peritrophic matrix-associated proteins in the sand fly vector Phlebotomus papatasi.

In many hematophagous insects, the peritrophic matrix (PM) is formed soon after a blood meal (PBM) to compartmentalize the food bolus. The PM is an important component of vector competence, functioning as a barrier to the development of many pathogens including parasites of the genus Leishmania transmitted by sand flies. PM morphology and permeability are associated with the proteins that are part of the PM scaffolding, including several peritrophins, and chitin fibers. Here, we assessed the effects of specific antisera targeting proteins thought to be an integral part of the PM scaffolding and its process of maturation and degradation. Phlebotomus papatasi sand flies were fed with red blood cells reconstituted with antisera targeting the chitinase PpChit1, and the peritrophin PpPer2. Sand fly midguts were dissected at different time points and processed for light microscopy (LM), confocal and transmission electron (TEM) microscopies (24, 42-46, 48 and 72h PBM), scanning electron (SEM) (48h PBM) and atomic force (AFM) (30h PBM) microscopies. TEM and WGA-FITC staining indicate PM degradation was significantly delayed following feeding of flies on anti-PpChit1. AFM analysis at 30h PBM point to an increase in roughness' amplitude of the PM of flies that fed on either anti-PpChit1 or anti-PpPer2. Collective, our data suggest that antibodies targeting PM-associated proteins affects the kinetics of PM maturation, delaying its degradation and disruption and are potential targets on transmission-blocking vaccines strategies.

Effect of mouse antisera targeting the Phlebotomus papatasi midgut chitinase PpChit1 on sandfly physiology and fitness

In sandflies, the absence of the peritrophic matrix (PM) affects the rate of blood digestion. Also, the kinetics of PM secretion varies according to species. We previously characterised PpChit1, a midgut-specific chitinase secreted in Phlebotomus papatasi (PPIS) that is involved in the maturation of the PM and showed that antibodies against PpChit1 reduce the chitinolytic activity in the midgut of several sandfly species. Here, sandflies were fed on red blood cells reconstituted with naïve or anti-PpChit1 sera and assessed for fitness parameters that included blood digestion, oviposition onset, number of eggs laid, egg bouts, average number of eggs per bout and survival. In PPIS, anti-PpChit1 led to a one-day delay in the onset of egg laying, with flies surviving three days longer compared to the control group. Anti-PpChit1 also had a negative effect on overall ability of flies to lay eggs, as several gravid females from all three species were unable to lay any eggs despite having lived longer than control flies. Whereas the longer survival might be associated with improved haeme scavenging ability by the PM, the inability of females to lay eggs is possibly linked to changes in PM permeability affecting nutrient absorption.

Disruption of the peritrophic matrix by exogenous chitinase feeding reduces fecundity in Lutzomyia longipalpis females

Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.