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.     

Oviposition behaviour of Phlebotomus argentipes - A laboratory-based study

The breeding habitat of sandflies is a little studied and poorly understood phenomenon. More importantly, oviposition behaviour is a largely neglected aspect of sandfly biology and this knowledge gap further undermines our understanding of the biology of sandflies. Pheromones released by the eggs play an important role in identifying good sites for oviposition by female insects. Several recent studies have examined the oviposition pheromone. The present study provides a preliminary report on the oviposition behaviour of Phlebotomus argentipes, the only vector of kala-azar (or visceral leishmaniasis) on the Indian sub-continent. Sandflies prefer to oviposit their eggs on surfaces that contain organic substances, especially substances with an odour of decaying animal products and the remains of conspecific eggs. The results presented here suggest that the odour released by the organic substances of old sandfly colony remains that contain dead flies, old unhatched eggs, larval food containing vertebrate faeces, frass and other organic matter serves as an attractant for the ovipositing females of P. argentipes and hence greatly increases the number of oviposited eggs compared to eggs deposited in controlled oviposition pots. This result will be helpful in maintaining an efficient colony of P. argentipes and may be a promising tool for monitoring and controlling the target insect as part of a synergistic approach.     

On oviposition in weevils of the genus <Emphasis Type="Italic">Larinus</Emphasis> Dej. (Coleoptera,Curculionidae)

The following actions performed by females of several Larinus Dej. species during egg laying are described: search of an appropriate place on the plant, making the hole for the egg, oviposition proper, and sealing the hole. The hole preparation takes the longest time and the greatest effort. Only one individual usually completes development in one flower head. Females of Larinus vulpes Ol. prefer larger flower heads for oviposition and occasionally lay eggs into stems. The ability of females to distinguish the flower heads with already laid eggs is discussed. Species of Larinus may be divided into two groups with “early” and “late” oviposition. The evolution of egg laying in the genus Larinus is discussed.     

Resource-Dependent Clutch Size Decisions and Size-Fitness Relationships in a Gregarious Ectoparasitoid Wasp, <Emphasis Type="Italic">Bracon brevicornis</Emphasis>

Gregarious parasitic wasps, which lay more than one egg into or onto a host arthropod’s body, are usually assumed to lay an optimal number of eggs per host. If females would lay too few eggs, some resources may be wasted, but if females lay too many eggs, offspring may develop into substantially smaller-sized adults or may not develop successfully and die. The availability of hosts can further influence a female’s clutch size decision, as more eggs should be laid when hosts are scarce. Here, we analyzed clutch size decisions and the fitness consequences thereof in the ectoparasitic wasp Bracon brevicornis (Hymenoptera: Braconidae), a potential biocontrol agent against pest moth species. For experiments, larvae of the Mediterranean flower moth, Ephestia kuehniella (Lepidoptera: Pyralidae) were used. Using artificially created as well as naturally laid clutches of eggs, the effects of clutch size on fitness of first (F1) and second (F2) generation offspring were investigated. Our results revealed that the fitness consequences of large clutches included both increased mortality and smaller adult sizes of the emerging offspring (F1). Smaller F1 females matured fewer eggs during their lifetime and their offspring (F2) had reduced egg-to-adult survival probability. Naturally laid clutches varied with host size up to a maximum, which probably reflects egg limitation. Clutches remained smaller than the calculated optimal (Lack) clutch size and females responded to high host availability with a decreased number of eggs laid. We thus conclude that large clutches may result in significantly smaller offspring with reduced fitness, and that host size as well as host availability influence the clutch size decision made by B. brevicornis females.     

Comparison of Bloodmeal Digestion and the Peritrophic Matrix in Four Sand Fly Species Differing in Susceptibility to Leishmania donovani

The early stage of Leishmania development in sand flies is closely connected with bloodmeal digestion. Here we compared various parameters of bloodmeal digestion in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to Leishmania donovani, to study the effects on vector competence. The volume of the bloodmeal ingested, time of defecation of bloodmeal remnants, timing of formation and degradation of the peritrophic matrix (PM) and dynamics of proteolytic activities were compared in four sand fly species. Both proven vectors of L. donovani showed lower trypsin activity and slower PM formation than refractory species. Interestingly, the two natural L. donovani vectors strikingly differed from each other in secretion of the PM and midgut proteases, with P. argentipes possessing fast bloodmeal digestion with a very high peak of chymotrypsin activity and rapid degradation of the PM. Experimental infections of P. argentipes did not reveal any differences in vector competence in comparison with previously studied P. orientalis; even the very low initial dose (2×103 promastigotes/ml) led to fully developed late-stage infections with colonization of the stomodeal valve in about 40% of females. We hypothesise that the period between the breakdown of the PM and defecation of the bloodmeal remnants, i.e. the time frame when Leishmania attach to the midgut in order to prevent defecation, could be one of crucial parameters responsible for the establishment of Leishmania in the sand fly midgut. In both natural L. donovani vectors this period was significantly longer than in S. schwetzi. Both vectors are equally susceptible to L. donovani; as average bloodmeal volumes taken by females of P. argentipes and P. orientalis were 0.63 μl and 0.59 μl, respectively, an infective dose corresponding to 1–2 parasites was enough to initiate mature infections.     

Egg Deposition Behavior in the Haplodiploid Sawfly Athalia rosae ruficornis Jakovlev (Hymenoptera: Symphyta: Tenthredinidae)

The egg deposition behavior of the turnip sawfly, Athalia rosae (Hymenoptera: Symphyta), is described. Both unmated and mated females lay eggs individually inside of fresh young leaves of cruciferous plants. During an oviposition event, females exhibit a distinct pause in abdominal contractions just before the actual egg deposition act. Unmated females show a longer pause (11.31 s on average) than mated females (4.38 s on overall average). By employing an eye color mutation, the sex of the eggs laid by females was ascertained. Females mated once lay mostly fertilized (diploid female) eggs initially but begin to lay a considerable number of unfertilized (haploid male) eggs later in life. The laying of an unfertilized egg is associated with a longer pause (6.98 s on average) than the laying of a fertilized egg (3.76 s on average). These results are in contrast to previous reports on apocritan Hymenoptera, where the presence of a pause or a longer pause during oviposition was associated with the deposition of fertilized eggs rather than unfertilized eggs. The possibility that mated Athalia rosae females control fertilization and its implications for sex allocation strategies are discussed.     

Reassociation of cortical secretory vesicles with sea urchin egg plasma membrane: Assessment of binding specificity

Summary An assay has been developed for quantitating the reassociation of cortical secretory vesicles (CVs) with fragments of sea urchin egg plasma membrane attached to glass slides (PM lawns). Binding ofS. pupuratus CVs to homologous PM lawns increased with time and CV concentration. The observation that CV binding was blocked by chymotrypsin digestion of the PM fragments suggested that a PM protein(s) is required for reassociation. The possibility that the extent of CV lysis that occurred during CV preparation (15.4±3.8% as assessed by ovoperoxidase assay) influenced reassociation was investigated by determining the effect of CV content proteins (isolated as fertilization product) on binding. Various concentrations of fertilization product (up to equivalent amounts of fertilization product and CV protein) had no effect on CV binding. The specificity of binding was investigated by assessing the ability of CVs to bind to PM lawns prepared from human red blood cells, and by determining the ability of heterologous vesicles to bind to egg PM fragments. PM lawns from HRBCs did not support CV binding; however, PM lawns prepared from the eggs of several species of sea urchin did bindS. pupuratus CVs. Vesicles from a partially purified preparation of yolk platelets bound to egg PM lawns with low efficiency (1/7 that of CVs), but immunofluorescence analysis with an anti-hyalin monoclonal antibody demonstrated that 74±9% of the bound vesicles were CVs that contaminated the yolk platelet preparation. Dioleoylphosphatidyl choline liposomes were also unable to bind to egg PM lawns. These results are consistent with hypothesis that CV binding to egg PM lawns is a specific, protein-mediated event.     

Virgin wasps develop ovaries on par with mated females, but lay fewer eggs

In the primitively eusocial wasp Ropalidia marginata, mating is not necessary for a female wasp to develop her ovaries, lay eggs, and even to become the sole egg layer of her colony despite the presence of other mated nestmates. Here, we show that virgin wasps do not differ from their mated counterparts in the extent and rapidity of their ovarian development, in the proportion of individuals that build a nest and laid eggs, and in the time taken to do so. However, a significantly larger proportion of virgin females showed resorbing oocytes, and laid fewer eggs as compared to mated individuals. Thus, virgin females have the ability to develop ovaries and lay eggs but also to refrain from necessarily laying all mature eggs produced, before mating opportunities arise. This dual ability would be adaptive in haplodiploid, tropical species with perennial nesting cycles and frequent opportunities for workers to become replacement queens or solitary nest foundresses throughout the year.     

Comparative fecundity and survival rates of Phlebotomus papatasi sandflies membrane fed on blood from eight mammal species

Female sandflies, Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae), were fed via chicken membrane on heparinized blood from eight species of mammal (human, horse, cow, pig, dog, rabbit, guinea-pig, hamster) and their reproductive success rates were compared. No appreciable differences between those fed on human and animal blood were detected with respect to the proportion of flies that fed successfully, mortality-rate within 24h, number of eggs laid per blood-fed female or egg viability. When mass-rearing sandflies for research purposes, membrane-feeding avoids practical difficulties encountered if sandflies are allowed to feed on live hosts (i.e. anaesthesia, distress from handling and postfeeding inflammation) and reduction of sandfly fecundity due to host antibody interference. Use of animal blood also eliminates risks of accidental transmission of human blood-borne pathogens, e.g. hepatitis B and human immunodeficiency virus (HIV), and is less expensive than maintenance of animals and their preparation for sandfly feeding.     

The Insect Growth Regulator Pyriproxyfen Terminates Egg Diapause in the Asian Tiger Mosquito,Aedes albopictus

The Asian tiger mosquito, Aedes albopictus, is a highly invasive mosquito species that transmits chikungunya and dengue. This species overwinters as diapausing eggs in temperate climates. Early diapause termination may be a beneficial strategy for winter mosquito control; however, a mechanism to terminate the diapause process using chemicals is not known. We tested the hypothesis that a hormonal imbalance caused by the administration of juvenile hormone analog would terminate egg diapause in A. albopictus. We tested the insect growth regulator pyriproxyfen on all developmental stages to identify a susceptible stage for diapause termination. We found that pyriproxyfen treatment of mosquito eggs terminated embryonic diapause. The highest rates of diapause termination were recorded in newly deposited (78.9%) and fully embryonated (74.7%) eggs at 0.1 and 1 ppm, respectively. Hatching was completed earlier in newly deposited eggs (25–30 days) compared to fully embryonated eggs (71–80 days). The combined mortality from premature diapause termination and ovicidal activity was 98.2% in newly deposited and >98.9% in fully embryonated eggs at 1 ppm. The control diapause eggs did not hatch under diapausing conditions. Pyriproxyfen exposure to larvae, pupae and adults did not prevent the females from ovipositing diapausing eggs. There was no effect of pyriproxyfen on diapausing egg embryonic developmental time. We also observed mortality in diapausing eggs laid by females exposed to pyriproxyfen immediately after blood feeding. There was no mortality in eggs laid by females that survived larval and pupal exposures. In conclusion, diapausing eggs were the more susceptible to pyriproxyfen diapause termination compared to other life stages. This is the first report of diapause termination in A. albopictus with a juvenile hormone analog. We believe our findings will be useful in developing a new control strategy against overwintering mosquito populations.     

A Theoretical Model of the Winter Survival Dynamics of Meloidogyne spp. Eggs and Juveniles

A theoretical model of the winter survival dynamics of Meloidagyne spp. was developed by considering the roles of egg hatching and juvenile mortality and the initial populations of eggs and juveniles at the onset of winter. Two distinct patterns of juvenile dynamics appear which depend upon the numerical values of the model parameters. The model predicts whether eggs or juveniles are the major component of overwintering nematode populations at any time prior to planting. The model could be elaborated to include egg viability and differential mortality of eggs but at some cost in ease of mathematical analysis. A general procedure for fitting the proposed model to experimental or observational data is outlined.     

Aggregated oviposition in the Simulium damnosum complex is mediated by eggs in a laboratory bioassay

Abstract. . The phenomenon of aggregated oviposition in blackflies (Diptera: Simuliidae) was investigated under controlled laboratory conditions, using wild-caught females of the Simulium damnosum complex in Sierra Leone. A method was developed for inducing Simulium females to lay eggs, and used as a bioassay to measure the responses of gravid S. damnosum s.l. to freshly laid eggs of the same species complex. In a series of two-choice tests, significantly more ovipositing flies chose substrates already containing eggs over control substrates ( P = 0.004). The time from introduction of flies into the oviposition system to the onset of egg-laying was significantly less when eggs were already present ( P = 0.049). Flies responded more quickly when more eggs were present and the relationship between egg-batch number and the time of this response was curvilinear ( P = 0.012). Ecological advantages and disadvantages of such aggregation behaviour and the possible role of semiochemicals in its mediation are discussed.     

Lifetime ovipositional patterns of mated and virgin onion flies,Delia antiqua (Diptera: Anthomyiidae)

Over their 47.2±1.9 (mean±SEM) day lifespan, mated onion flies, Delia antiqua, oviposited more uniformly than did virgins. Mated females began ovipositing at 6.4±0.2 days old and regularly deposited 14.2±0.6 eggs/day for 3-4 weeks. Thereafter, oviposition slowed and stopped at about 1 week before death. Virgin flies began ovipositing 24.7±1.5 days into their 59.0±3.8 day lifespan, and deposited eggs at an increasing rate for 3-4 weeks, generating a mean overall ovipositional rate of 5.8±0.5 eggs/day. The later onset of virgin oviposition (ovipositional activation) and not a shorter ovipositional period largely accounted for the disparity between virgin and mated female lifespans. Mean lifetime egg production of mated females was 475±27 eggs versus 179±30 eggs for virgins. Ovipositional and post-ovipositional periods (34.4±1.8 and 7.2±1.0 days) for mated females were not significantly different from those of virgin females (30.9±3.6 and 6.7±1.2 days). Over 90% of virgin females laid eggs. The distinction between mated and virgin ovipositional patterns may be specifically attributed to differences in: (1) egg maturation, (2) age at ovipositional activation, and (3) egg-laying rate.     

Age-Dependent Effects of Oral Infection with Dengue Virus on Aedes aegypti (Diptera: Culicidae) Feeding Behavior,Survival, Oviposition Success and Fecundity

Background

Aedes aegypti is the main vector of dengue, a disease that is increasing its geographical range as well as incidence rates. Despite its public health importance, the effect of dengue virus (DENV) on some mosquito traits remains unknown. Here, we investigated the impact of DENV-2 infection on the feeding behavior, survival, oviposition success and fecundity of Ae. aegypti females.

Methods/Principal Findings

After orally-challenging Ae. aegypti females with a DENV-2 strain using a membrane feeder, we monitored the feeding behavior, survival, oviposition success and fecundity throughout the mosquito lifespan. We observed an age-dependent cost of DENV infection on mosquito feeding behavior and fecundity. Infected individuals took more time to ingest blood from anesthetized mice in the 2^nd and 3^rd weeks post-infection, and also longer overall blood-feeding times in the 3^rd week post-infection, when females were around 20 days old. Often, infected Ae. aegypti females did not lay eggs and when they were laid, smaller number of eggs were laid compared to uninfected controls. A reduction in the number of eggs laid per female was evident starting on the 3^rd week post-infection. DENV-2 negatively affected mosquito lifespan, since overall the longevity of infected females was halved compared to that of the uninfected control group.

Conclusions

The DENV-2 strain tested significantly affected Ae. aegypti traits directly correlated with vectorial capacity or mosquito population density, such as feeding behavior, survival, fecundity and oviposition success. Infected mosquitoes spent more time ingesting blood, had reduced lifespan, laid eggs less frequently, and when they did lay eggs, the clutches were smaller than uninfected mosquitoes.     

Interactions of mating,egg production and death rates in females of the Mediterranean fruit fly,Ceratitis capitata.

Costs of reproduction include costs of producing eggs and of mating itself. In the present study, we made an experimental investigation of costs of reproduction in the Mediterranean fruit fly (medfly, Ceratitis capitata). We demonstrated that virgins live longer than non-virgin females. However, in strong contrast to most findings within the Diptera, non-virginity had no detectable effect on egg production. Therefore the increased longevity of the virgin females cannot be attributed to an increase in egg production in non-virgin females, and instead indicates a cost of mating. A comparison of the life spans of normal females and those sterilized by low doses of X-irradiation, revealed an additional cost of egg production. There were no significant differences in remating levels between females that did and did not lay eggs, showing that the cost of producing eggs is independent of mating frequency. Medfly females therefore suffer a decrease in survival as a result of egg production and of mating, and these costs are independent of one another. To put our results into context, we reviewed the existing literature on the effects of mating on longevity, egg production and sexual receptivity for 64 species of Diptera, and examined the pattern of mating effects that emerged.     

Do shiny cowbird females adjust egg pecking behavior according to the level of competition their chicks face in host nests?

Interspecific brood parasites, like the shiny cowbird (Molothrus bonariensis), lay eggs in nests of other species. Shiny cowbird females peck and puncture eggs when they parasitize host nests. This behavior increases the survival of cowbird chicks when they have to compete for food with larger nestmates. However, cowbird chicks may benefit from smaller nestmates as they increase food provisioning by parents and the cowbird chicks secure most extra provisioning. We investigated whether egg-pecking behavior by female shiny cowbirds might be adjusted to the competition that their chicks face in host nests. We found that more host eggs are destroyed per cowbird egg laid in a larger-bodied host (chalk-browed mockingbird, Mimus saturninus, 70-75 g) than a smaller-bodied host (house wrens, Troglodytes aedon, 12-13 g). We also tested egg-pecking preferences in choice experiments with female cowbirds in captivity and found cowbirds presented with eggs in artificial nests pecked first and more frequently, and punctured more frequently the larger egg when this was a host egg, but not when this was a cowbird egg. Our results are partially consistent with the hypothesis that shiny cowbird females adaptively adjust their egg pecking behavior according to the competition that their chicks face in host nests.     

A simple method to disperse eggs from lepidopteran scalelike egg masses and to observe embryogenesis

Various insect species lay tiny, thin- and soft-shelled eggs in a connected “egg mass”. Especially in several lepidopteran species, the structure of such clustered eggs is covered with complicated scale-like secretion, which has so far prevented analysis of individual embryos. However, few studies on methods to disperse egg clusters of such insects and to compare different methods have been carried out. To overcome these problems, we developed methods to separate egg masses into individual eggs, using two Tortricidae pests, Homona magnanima Diakonoff and Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae). The eggs were successfully separated from each other using potassium hydroxide and sodium hypochlorite. Although the separated eggs no longer continued their embryogenesis, fixation with heptane–paraformaldehyde, permeabilization with heptane–methanol, and staining with several dyes enabled easy observation of embryogenesis. This protocol is expected to be applicable to other insect taxa and will facilitate further morphological and genetic studies in insects that lay egg masses.     

Oviposition Site Choice in the Yellow Dung Fly Scathophaga stercoraria

Female yellow dung flies Scathophaga stercoraria oviposit on the dung surface, which is extremely uneven and other eggs are often already present. We examined the effects of these features on egg placement. We found that: 1. females preferred to lay on small hills on the dung surface and avoided depressions and points; and 2. they ignored the presence of other eggs. These results were confirmed in a field experiment, where the pattern of egg distribution on artificially formed dung pats was consistent with the predictions from the laboratory results. We further showed that survival to adult emergence was best when laying sites were experimentally manipulated so that the eggs were apparently laid on small hills, avoiding the drying effects of small points in the dung and possible drowning by (simulated) rain when laid in depressions in the dung surface. A female dung fly is clearly capable of making subtle decisions about the placement of her eggs, thus increasing her reproductive success.     

Effect of food and temperature on the reproduction of the blowfly, <Emphasis Type="Italic">Calliphora vicina</Emphasis> R.-D. (Diptera,Calliphoridae), a popular model object in biological research

Egg maturation in Calliphora vicina is known to occur within a wide range of temperatures, from 12°C to nearly 30°C (Vinogradova, 1991). Photoperiodism has no effect on this process. Some females enter diapause already at 20°C; their fraction increases at lower temperatures and reaches 100% at 6°C. Reproducing females with eggs can survive for a long time and even lay eggs at low temperatures (4–5°C). Experiments with C. vicina from Leningrad Province revealed some effects of the diet (liver or fish) and temperature on the fly reproduction. At 20 and 25°C, 7–10-day old females begin to oviposit, but at 20°C egg maturation is observed in 98% of females feeding on liver and in only 5% of females feeding on fish. On the liver diet, the mean daily fecundity is significantly correlated with the day of oviposition but not with the temperature. At 20°C a significant correlation is observed between the mean daily fecundity and both the day of oviposition and food. The total number of eggs laid by flies after feeding on fish is half that produced after feeding on liver. The optimal conditions for Calliphora vicina cultivation are a 16-h light day, temperatures within the range from 20 to 25°C, and liver as food.     

Genetic variation in male effects on female reproduction and the genetic covariance between the sexes

Abstract.— Males of many insect species increase the fecundity and/or egg size of their mates through the amount or composition of their nuptial gifts or ejaculate. The genetic bases of such male effects on fecundity or egg size are generally unknown, and thus their ability to evolve remains speculative. Likewise, the genetic relationship between male and female investment into reproduction in dioecious species, which is expected to be positive if effects on fecundity are controlled by at least some of the same genes in males and females, is also unknown. Males of the seed beetle Stator limbatus contribute large ejaculates to females during mating, and the amount of donated ejaculate is positively correlated with male body mass. Females mated to large males lay more eggs in their lifetime than females mated to small males. We describe an experiment in which we quantify genetic variation in the number of eggs sired by males (mated to a single female) and found that a significant proportion of the phenotypic variance in the number of eggs sired by males was explained by their genotype. Additionally, the number of eggs sired by a male was highly positively genetically correlated with his body mass. The between-sex genetic correlation, that is, the genetic correlation between the number of eggs sired by males and the number of eggs laid by females, was highly positive when eggs were laid on Acacia greggii seeds. This indicates that males that sire many eggs have sisters that lay many eggs. Thus, some of the genes that control male ejaculate size (or some other fecundity-enhancing factor) when expressed in males appear to control fecundity when expressed in females. We found no significant interaction between male and female genotype on fecundity.