NC State University Insect Museum

(written by Ann Carr)

Today we had our last seminar session, and I have to say that most of us were sad to see it end. This has been an amazing class and one of the better seminars offered. We sure hope all of you bug enthusiasts have enjoyed following our discussions! We have learned some much, and hope that you learned a little too. Last weeks seminar topic was insect products with a focus on galls. We had a full house this week, so lucky for you there are quite a few papers to go over. So let’s get started! PS need to give a big “Thank You!” to Trish and Steve for making some amazing curry and banana pudding, which might I add pair together very well.

Insect morphology now infiltrates every aspect of our lives. Here, a wasp-inspired cookie crumb garnish adorns a delicious banana pudding. Yummy and educational!

Keith got us started with a paper discussing the phylogeny of the galling aphid species Eriosomatini. This paper suggests that the evolution of galling aphid species is largely affected by the presence of host plant species. Species divergence is closely associated with host plant extinction and subsequent diversification of host plant types. This theory makes sense because of the critical association galling aphids have with their host plants. By comparing insect and gall morphological features the authors are able to construct a very nice evolutionary tree and identify Eriosomatini’s closest sister species.

I followed with a paper examining survival strategies of Contarinia galling midges. Because the midges are most susceptible to mortality when they are in gall-forming phase, they have evolved long diapause periods in the soil. Contarinia midges have 99% of their population laying dormant in the soil. Their diapause periods last 10-17 years depending on emergence. This allows the species to survive extinction if all of their galling populations are exterminated. The authors also believe that because of the limited availability of their host plant, that the Contarinia midges must establish a long-lived population to evade extinction.

Matt talked about a paper that examined what happens when galls are on leaves that are aborted by the host tree. The authors compared two gall structures, simple and complex. They were able to conclude that the more complicated gall structures were able to survive on the forest floor once aborted by the host tree, unlike the simple gall structures. This was described as the “Green Island Effect” where the exterior tissues of the leaf died, though the tissue surrounding the complex gall remained green and viable for a period of time after falling from the host tree.

Trish found a paper that studied sexually dimorphic galls of scale insects. In the genus Apiomorpha females produce round circular galls and males produce long tubular galls. They were also interested in why two populations of the same species produced different galls. The authors did studies to see if the two populations were making different galls because of the host plants they were using. They concluded that the insect determines the gall shape, not the host plant, and during their research they discovered genetic variances between these two populations and re-established them as two separate species.

Andrew discussed a paper that examined why caddisfly larvae line their cases with pine needles. The caddisfly were not using the pine needles for camouflage or defense, but were actually using the pine needles to help anchor and stabilize themselves when confronted with strong or turbulent water currents. The authors note that caddisfly without pine needles would spin and rotate more than caddisfly with pine needles attached to their case.

Steve followed with a paper that talked about silk production in male dance flies. Dance flies are the first type of fly found to produce silk. Structurally their silk looks very similar to silk produced by Lepidopterans. Though, genetic analysis showed there were no similarities in composition between the dance fly silk and other insect silk products. This means that this is an entirely different silk from what is typically found. What was really neat was the purpose of the silk. Male dance flies would skim the surface or water sources and use the silk as a net to collect diatoms, and present the package as a nuptial gift to females.

Colin presented our last paper of the semester, which actually was a review on lac insects and the harvesting of shellac. Lac insects feed on the sap of trees and produce shellac while laying eggs. The shellec is scraped of the trees, harvested and finished all by hand. Variations in shellac are dependent on lac insect species. The paper also discussed the lac agriculture in India and Thailand, cultural demands, and threats to shellac harvesting.

Well that about sums it up for today. Thanks for following along. We hoped you enjoyed it as much as we did!

This week in Insect Morphology Seminar, we discussed several interesting papers about female genitalia and the insect ovipositor!

Steve started by telling us about germline stem cells (GSCs) in the ovaries of an earwig, Opisthocosmia silvestris. Studies in various animal species have shown that stem cells function in specialized microenvironments known as niches. Insect ovaries consist of ovarioles, which consist of the terminal filament, germarium and vitellarium. The morphology of the Drosophila GSC niche in females is known to house three types of somatic cells: terminal filament cells, cap cells, and escort stem cells. In C. elegans, the GSC niche only contains a distal tip cell equipped with long cytoplasmic structures. The female earwig GSCs are morphologically simple and consist of the terminal filament cells and escort cells; cap cells are absent and escort stem cells are not recognizable (see image below for a comparison of the three organisms).

Structure of the GSC niches in Caenorhabditis elegans, Opisthocosmia silvestris, and Drosophila melanogaster

Heather talked about how female cockroaches exchange water with their oothecae. German cockroaches usually carry their oothecae until the eggs are ready to hatch, and the success of embryogenesis may depend on water-balance between the adult female and the developing ootheca. Since oothecae that are detached from the female before embryogenesis is complete often cannot develop (especially in dry environments!), this led scientists to wonder how water is transferred to the ootheca during development. There is an area located on the proximal end of the ootheca that contains small pores that penetrate the escutcheon region of the covering of the ootheca to access the chorion! This may help maintain water balance between female and ootheca, and the German cockroach may represent an important evolutionary link in the transition from oviparity to ovoviviparity!

Colin discussed a very interesting paper on cryptic female choice in spiders and complex genital structures. Female haplogyne spiders of the species Opopaea fosuma have evolved some neat ways to get rid of or block sperm from males they have mated with. Female spiders have the anterior wall of the spermatheca, where sperm is stored before fertilization, heavily sclerotized with a cone-shaped hole in the upper part. Muscles attach to a transverse sclerite that bears a nail-like structure, and when the muscles contract, they press the nail into the hole of the spermatheca. When this happens, the copulatory orifice is enlarged and the resulting suction probably allows deposited sperm to be emptied from the spermatheca (called “sperm dumping”). This mechanism is commonly used among females to influence a male’s chance of fathering their offspring; this is known as cryptic female choice. So what makes the females decide they don’t like a particular male’s sperm? Is he just not up to her standards?

Keith and Andrew both talked about genetic patterning in genitalia of the milkweed bug Oncopeltus fasciatus and the red flour beetle Tribolium castaneum. These two species differ in the anatomical complexity of their genitalia. Researchers found that the posterior Hox genes (abdominal-A and Abdominal-B) were required for proper genital development in O. fasciatus and they regulated Distal-less and homothorax in a similar way in both sexes. They did RNAi knockdown experiments to look at genitalia development and found that the genitalia are not homologous to appendages in Tribolium but they are Oncopeltus. Andrew found a paper that discovered that the same genes that regulate genitalia development also regulate the development of beetle horns. The results provided developmental genetic support for specific anatomical hypotheses of serial homology. The gene functions and interactions describe the developmental patterning of sexually dimorphic structures such as genitalia that have been critical to the diversification of species-rich insect groups.

Matt finished up with a paper talking about mating plugs in scorpions. There are two kinds of plugs in scorpions, sclerotized and unsclerotized (gel-like), which usually harden in the female genital tract. The researchers found a gelatinous mating plug in Euscorpius italicus that is composed largely of sperm – this was previously unknown in arachnids! It was discovered that fluid from the female genital tract causes sperm activation.

Heather gave a great mini-lecture on wax production and insect products. The lecture began with galls – there are over 13,000 species of insects that produce galls, from sawflies to cynipid wasps to adelgids and many flies and true bugs. There is extreme variation in gall morphology, as seen in this images below:

Sawfly gall

Eurosta fly gall

Ocellate gall midge

Gall hat made by George Melika at the International Congress of Hymenopterists in Hungary, 2010

Galls provide nutrition to developing larvae and offer microclimate protection. Some gall inducing stimuli are saliva, maternal secretions, and larval secretions. They have complex external structures, and the mechanisms of how insects make galls and influence the plant’s response is still lagely unknown! Frederik Ronquist calls this the Holy Grail of science! We are hoping someone discovers how the fascinating structures are formed soon.

The next insect products discussed were wasp nests. Their external surfaces can range from smooth to spiky, and they can be constructed from mud, paper, etc. Mud wasps (sphecids, crabronids, etc.) and potter wasps (Vespidae) construct nests from mud and regurgitated water. It is thought that native Americans modeled their pottery after potter wasps nests!! Seeing this image, this isn’t too hard to imagine.

Mud wasp nest

Oothecae are another evolutionary marvel produced by a few types in insect, including cockroaches, which construct their ootheca from calcium oxalate, proteins, uric acid and water, and mantids, which construct theirs from calcium citrate. The ootheca helps protect eggs from predators, microclimate, etc.

We then discussed bees wax, honeybee nests, and some crafty megachilids (leaf cutter bees) that use mud and pebbles to help camouflage their nests into rocksides (see image below). Some megachilids roll cut sections of leaves together and cut a small circle out of a leaf to seal the opening. Some Osmia bees use flower petals instead of leaves to construct the nest!

Osmia nests constructed of flower petals

These amazing insect products made us wonder… could the product itself (like a nest, gall, or ootheca) be considered a part of an insect’s morphology? In ENT 502, we defined morphology as something that has a “form” and a “function”, and morphology is NOT the same as anatomy. Since some insect products can be diagnostic characters (and morphological characters can be diagnostic), can they be considered morphological characters? Or are they ecological characters? A tool has a form and a function though, so does that mean that a stick held by a monkey and used as a hammer could be consider part of the morphology of the monkey? Or a more realistic example – could the nest of a paper wasp be considered part of the wasp’s morphology? What do you think?

(Written by Steve Turner)

This week’s seminar began with another great overview of the topic of the previous week which was the topic of insect male genitalia. We covered a diverse array of topics, which demonstrated once again how broad and far reaching a single morphological structure can be.

Ann got started with a paper on the first transcriptome of the testis-vas deferens-male accessory gland and proteome of the spermatophore from Dermacentor variabilis, commonly known as the infamous dog tick. The paper basically covered the fact that little is known about tick mating, and the knowledge of sperm transfer in ticks is virtually non-existent. The current understanding is that one of the structures of the head of the male tick transfers sperm into the female recepticle. The transcriptome sequencing (which can be used to detect which proteins are being coded for by RNA expression levels) detected that over 4,000 proteins were found in the vas deferens and the male accessory glands. These are passed to the female as part of the spermataphore and is thought to initiate the maturation of the female’s eggs. This mating interaction has only been noted to occur on the tick’s host and may be an important cue for the initiation of blood feeding by the female.

I (Steve) followed with a summary of the male accessory glands of the tettigoniid B. siculus and the proteins produced by them. The paper covered the fact that in Tettigoniidae, male accessory glands produce a spermatophore with 2 components, the spermatophylax and the ampulla. Both are composed of proteins from the accessory glands. The study of the ultrastructure of the accessory glands of B. siculus reveal 2 previously known groups of tubules that open directly into the accessory glands – 1st and 2nd order tubules. The researchers also discovered a third type, which they named intermediate tubules. The 1st and 2nd order tubules enter directly into the ejaculatory ducts whilst the intermediate tubules open into the other two types. It was found that only one protein was commonly produced by all tubules and that the majority of proteins produced in each tubule type are specific to that tubule type. The first order tubules are larger than the other two types and produce larger proteins. It was found that the spermatophylax, which functions as a nuptial gift, comprises most of the spermatophore. This is fed upon by the female whilst the ampulla transfers sperm. The larger the spermatophore, the better, as it allows more sperm to be transferred from the ampulla to the female before she begins to feed on it in turn.

Heather summarized a very nice paper on the effect of sexual selection and how this is a driving force of the evolution of male genitalia in the earwig Euborellia brunneri. The paper describes a system where female choice influences the length of the male genitalia, which are otherwise relatively simple in structure. Co-evolution driven by female sperm choice, such as access to fertilization sites and copulation duration, are thought to have resulted in polymorphism in the length of male genitalia. Males respond to female choice with two strategies of removing sperm from previous males -antagonistic and defensive, such as transferring a larger seminal vesicle which stimulates the female to oviposit sooner. Males with longer genitalia had greater reproductive success and protection from sperm removal as they can deposit sperm deeper within the female. Males with longer genitalia also had a curved tip used to remove rival males’ sperm.

Trish summarized a paper on mating interactions in seed beetles. The males of these beetles have long spines on their genitalia which are capable of inflicting damage on the female reproductive tract. Females have actively been noticed trying to kick males off during mating interactions. One hypothesis is that males gain from directly harming the female as hooking on with the spines allows for a longer copulatory period. This is known as adaptive harm. A second hypothesis is that males benefit from this strategy but females don’t, and that spines only become functional when the benefit to the male outweighs the cost of the damage to the female. The researchers in this paper found that longer spines do not result in a longer copulation period so do not function as an anchor. The researchers hypothesized that they might be used for sperm manipulation.

The lecture this week presented by Trish was on the insect female genitalia and ovipositor, during which a broad range of characteristics and functions were covered. These characters included the ovarioles, of which there are two main kinds dependent upon insect order, panoistic and meroistic. We also heard about how females in some taxa, notably in Hymenoptera, can have highly modified ovipositors which can be used for hunting, defense or injecting host species with eggs, such as is the case with many parasitoid wasp species. It can also be modified into a cutting/drilling tool for oviposition into a wooden substrate.

We also learned about some of the products made by the accessory glands of female insects. These include the complex egg case of cockroaches and mantids, called the ootheca. The accessory glands of parasitoid wasps which inject their eggs into their host also produce substances which protect their eggs from encapsulation by the host organism’s immune system.

(contributed by Ann Carr)

Hello fellow bug enthusiasts! Last week we had an awesome presentation on insect fat bodies, and I’m still amazed at the number of functions that fat bodies are actually involved in. From nutrient storage, hormone synthesis, molecule transport, excretion and reproduction it seems that fat bodies play an essential role in the lives of insects. Its almost like these fat bodies have superpowers! Following the seminar we found some pretty cool papers that each discussed a different facet of these “all-powerful” fat bodies. Unfortunately attendance today was a little low due to the craziness of the oak pollen in Raleigh, but we’d still love to share with you a brief description of these relevant and recent papers investigating fat bodies.

Our first paper describes the importance of fat bodies in honeybees and caste determination of their offspring. For those of you who are not apiary experts (don’t worry most of us aren’t) honeybee colonies are divided into different castes, or working groups. There are the female reproductives or queens, the non-reproductive female workers, and the reproductive male drones. Caste determination for honeybees is done when they are still immatures and this is based mainly on nutritional factors, primarily on a compound called royal jelly. This paper describes how the ingestion of royal jelly by immature female honeybees interacts with fat bodies and promotes the growth and development of ovaries. These means that female immature honeybees exposed to royal jelly become reproductive queens and those not become non-reproductive female workers. This is a perfect example of how fat bodies affect both reproduction and overall development.

Our second paper discusses how the presence of parasitoid larvae affects fat bodies and their nutrient content. Parasitoids depend on hosts to obtain all their nutritional requirements. This places a huge strain on the host, who must balance supporting the parasitoid while still maintaining enough nutrients for themselves. This paper measured inorganic nutrient content in fat bodies after being infested with paraitoids and through to the end of their development time (10 days). They found that after infestation with the parasitoids, fat bodies lost almost half of their nutrient content during parasitoid development. This is a huge amount! The researches believed that the host may be altering their fat body metabolism to accommodate the developing parasitoid. They’re not sure how the insects was able to do this, but it sure is a cool strategy.

The last paper looks at important fat body regulation of hormones and proteins involved in programmed cell death (PCD). PCD is important in maintaining consistent cell numbers and is regulated by specific proteins call apoptosis inhibitors. These apoptosis inhibitors are regulated by the developmental hormone ecdysone, and both are secreted by fat bodies. This paper used different genetic tests to locate apoptosis inhibitor proteins in the body of developing caterpillar larvae and examine what would happen if this protein weren’t produced. They found that apoptosis inhibitors are super important during larval stages because they essentially determine which cells live and which ones die, and are slothed off during molts. The apoptosis proteins were found in all epithelial cells. Without these proteins, all larvae perished.

Well that about sums up our seminar today. Looking forward to next weeks!!

(Written by Steve Turner)

This week began with a discussion of chosen papers based on the previous week’s topic of muscles and flight muscles in insects.

The round table discussion began with Matt Bickerton summarizing a paper based on wing polymorphisms in Pyrrhocoris apterus, a species of hemipteran which has polymorphisim in wing muscles which can lead to the loss of flight. It appears that in the flightless representatives of this species of true bug, both females and males are brachypterous – therefore they do have wings, albeit reduced. As a result of this stunted development, wing muscles are not needed, so 10 days (for males) and 14 days (for females) after the adult population emerges, they are histolyzed (broken down). Eighteen bands of proteins are lost in individuals where flight muscles are lost or reduced. Juvenile hormone is thought to regulate this process. Below is a nice figure showing the histolysis of the muscle.

Following this, Heather Campbell talked us through a nice paper about wing muscles in two species of bees, Apis mellifera and Scaptotrigona postica. The paper focused on differences in wing muscles between virgin queens (Elizabeth I?!) and mated queens, and on changes in muscle fibre densities between castes (age based) of worker bees. The study found that A. mellifera had larger and more numerous flight muscles as it engages in nuptial flight and S. postica does not leave the ground to mate. The paper also found that the nursing stage of workers had thicker muscles than older caste stages, which is surprising as the oldest stage, foraging workers fly far more than this younger caste. The differences may be due to age related muscle deterioration.

I (Steven Turner) decided to talk about how the contraction of insect flight muscle is regulated. Indirect flight muscles power the rapid movement of insect wings contracting up to 1000Hz. This type of flight is seen in many small flying insects and is effective enough to be used in larger insects such as the waterbug, Lethocerus. The oscillation of muscles occurs against a constant low concentration of Ca(2+) as the muscles move. Stretch activation is controlled by tropomyosin and the troponin complex on the thin filament which form cross bridges to allow for stretching of the muscle fibres. This appears to have a similar function in Drosophila and Lethocerus to the skeletal muscle of vertebrates, but is not homologous to it.

Keith Bayless brought some taxonomic background to the round table discussion, informing us of the use of muscles for the identification of medically important blow fly larvae. In this study, the investigators found on each sternite a lattice of muscles that connects to the end of each thoracic segment, which can be seen on insects as a pattern of dots. By taking an average of the arrangement of these dots, a system for classifying the larvae can be achieved. The dot patterns were described in fanciful ways, e.g. one was “snakelike”, or “broken arrow like” etc., however it proved to be a good system for identifying blowfly larvae. It was tested using inexperienced individuals with no previous knowledge of this system, who had high success rates in correctly identifying the maggots. From a personal point of view, it would be very cool to know whether classifications for larvae of other groups of Diptera and Holometabola could be achieved using this method.

Finally Colin informed us all of titin, a protein found in muscles which is responsible for elastic force – so it effectively controls stretching. Deviating slightly from the topic, he talked about an equally intriguing use of titin and its stretching properties for dividing cells in tipulid flies and acridid Orthoptera. When cell division begins, titin spreads out evenly across the spindle, but towards end of division the protein is found on the ends of the spindle. This is hypothesized to be using elastic forces in order to pull apart the daughter cells and completing the cell division.

Keith Bayless completed the week with a very good lecture on the insect fat body and oenocytes. We learned that the primary function of the fat body is to store and release metabolites for energy. The fat body appears to be a “very insect” thing – it is found in a few crustaceans, and myriopods appear to have evolved a similar structure independently. The ultra structure of the fat body is variable dependent on where it is found (periferal areas – near the body wall or alimentary canal). The structure can also be different across different insect orders. In the hemimetabola, for example, the structure is similar throughout. but in holometabola, the fat body can be completely broken down and rearranged during metamorphosis.

The fat body is also important for other functions including vitellogenesis, the production of diapuase proteins and transanimation (converting one amino acid into another). Glyocogen is the primary sugar stored in the fat body. 70% of the weight of the fat body is for triceroglycerol storage, and lipogenesis occurs here (the building up of larger lipids from smaller ones).

Oenocytes are formed as part of the epidermis. In most insect orders, oenocytes migrate and become associated with fat body. There are some exceptions to this, however, as oenocytes remain in the epidermis in Ephemeroptera, Odonata and Hemiptera. The main function of the oenocytes is to make hydrocarbons and lipids for the epicuticle.

Last week I gave a presentation on pulsatory organs and hemolymph, which pretty much turned into a presentation about the insect circulatory system. Insects have an open circulatory system. Their hemolymph (insect blood) is not channeled through veins and is instead free-flowing through the body cavity. Because they lack the veins and arteries needed to get hemolymph to their extremities they have pulsatory organs instead that pump the hemolymph to where it is needed. They are also known as accessory hearts because they pump hemolymph independent of the dorsal vessel (insect heart and aorta).
The insect circulatory system is very interesting and after I put together my presentation I wanted to know more. So this weeks discussion was done differently. Instead of the usual “paper report” format, I asked each of the pundits to elaborate on one of the broad topics covered in my mini-lecture. What they came up with was pretty awesome!

Steve gave a micro-lecture about cercal vessels. The cercal vessels are pulsatory organs that are unique to insects that have long cerci at the base of their abdomen. The kind of pulsatory organs an insect has is customized to what the insect needs. If the insect has no cerci than it lacks the cercal vessel and if is have very short cerci it may have a membrane that helps channel hemolymph into the cerci but it will lack any pumping ability.

After last weeks mini-lecture, Colin was curious if there were any pulsatory organs in insects that are synchronized with the dorsal vessel. After combing the literature, it appears that most pulsatory organs are not in sync with the dorsal vessel or with one another. In fact, cockroaches have six leg hearts and they are never in perfect phase with one another. This is like due to the fact that the pulses are neural but myogenic in origin. Another point Colin made that I thought was really interesting is that there is no link between the heart beat and the squeezing of the abdomen and the insect abdomen is more effective at circulating the hemolymph than the dorsal vessel. Insects have developed so many way to get hemolymph to where it needs to be! It is quite impressive.

Ann talked about some of the immune functions of the hemolymph. We learned that the coagulation of insect hemolymph occurs by two different processes working together; clotting and inflammation. This is unique because in most other animal these two processes occur as one system. She also brought us delicious treats! A flan to represent coagulation and a roll cake to represent a muscle fiber (relevant to the presentation she would be giving that day). They were delicious!!

Delicious examples of coagulation and muscle fiber.

Trish shed some light on how insects tolerate cold and escape death by freezing. Insects mostly fall into two categories; freeze tolerant insects that can survive when the hemolymph freezes and freeze avoiding insects that prevent the hemolymph from freezing. The freeze tolerant insects use protein ice nucleators to limit supercooling and induce freezing at high subzero temperatures which inhibits lethal intracellular freezing. Antifreeze proteins (AFPs) are used by both freeze tolerant and freeze avoidance insects to protect from damage. The mechanism for this type of cryoprotection is not known but it may prevent re-crystallization of the hemolymph and stabilization of the membrane. Think about a frozen blueberry. When it is frozen it looks like a fresh blueberry but as it thaws it begins to collapse in on itself and its juices ooze out. The juices ooze out of holes that were created by crystallized shards that punctured the membrane. A similar thing can happen to cells when they are frozen and the damage that is caused is irreversible in most animals with the exception of the unsinkable Nematode!!