Reading Assignment: Abiotic and Biotic Factors Regulating Inter-Kingdom Engagement between Insects and Microbe Activity on Vertebrate Remains

Overview of Inter-Kingdom Engagement on Vertebrate Remains

• Authors: Heather R. Jordan (Department of Biological Sciences, Mississippi State University) and Jeffery K. Tomberlin (Department of Entomology, Texas A&M University).

• Objective: To review the abiotic and biotic factors regulating arthropod attraction, colonization, and utilization of decomposing remains, specifically focusing on the role of microorganisms (bacteria/fungi) as a primary mechanism regulating arthropod behavior.

• Core Assertion: Microbes serve as the "curtain" pulled back by high-throughput sequencing, revealing a microscopic world that dictates carrion decomposition patterns and insect inter-kingdom engagement.

Forensic Entomology: Foundations and Modern Revisions

• Historical Definition: The application of insect science to legal investigations involving living or deceased people, pets, wildlife, or livestock.

• Postmortem Interval ($PMI$) vs. Time of Colonization ($TOC$):

  • Historically, entomologists were asked to determine $PMI$.

  • Recently, the focus has shifted to determining the age of insects to estimate $TOC$. This differs from $PMI$ due to circumstances like myiasis (colonization before death) or delayed colonization (after death, resulting in a minimum $PMI$).

• Presuppositions impacting Forensic Inferences:

  • Assumption that colonization always occurs after death.

  • Assumption that insect material was originally from the remains in question.

  • Applied datasets from one region to another (lacks geographic specificity).

  • Assuming abiotic conditions at the time of death are the sole influencers of activity.

The Ecological Perspective of Nutrient Recycling

• Vertebrate Remains as Resources: They are unpredictable, nutrient-rich, but limited. This creates intense evolutionary pressure for animals to locate and consume them quickly to avoid competition, starvation, or mating failure.

• The Janzen Paradigm (1977): Challenged the view of microbes as simple "recyclers." Janzen proposed microbes occupy various ecological categories:

  • Competitor: Actively vying for the same resource as animals.

  • Mutualist: Working in tandem with arthropods.

  • Predator: Microbes that may consume or infect other organisms on the remains.

• Inter-Kingdom Communication: Microbes produce volatile organic compounds ($VOCs$) and other semiochemicals that regulate arthropod detection and attraction.

Abiotic Factors: Temperature

• Cellular Impacts of Temperature:

  • Cold Shock: Leads to membrane rigidity, decreased energy, decreased fluidity, ice crystal formation causing cell lysis, and slowing/ceasing of enzyme activity.

  • Optimal Temperature: Increases membrane fluidity, metabolic activity, and enzyme rates, leading to increased cell growth.

  • Heat Shock: Causes protein denaturation and the cessation of cell growth.

  • Extreme Stress (Heat or Cold): Results in increased membrane permeability, loss of $ATP$ synthesis, increased $pH$, increased reactive oxygen species, and eventual cell death.

• Metabolic Response:

  • Intrinsic metabolic rates and energy demands increase with temperature.

  • If resources are limited, higher temperatures can lead to negative growth due to energy deficits.

  • If resources are abundant, energy is invested in biomass growth.

• Cold Adaptation: Microbes increase the proportion of unsaturated fatty acids in their membranes as temperatures decrease to maintain fluidity and prevent leakage.

• Decomposition Rates:

  • Warmer temperatures generally correlate with higher microbial biomass, enzymatic activity, and higher abundance of $VOCs$.

  • Some studies indicate microbially-mediated decomposition can be more efficient at lower temperatures due to lower catabolic requirements and a lack of competition from scavengers.

Abiotic Factors: Water Activity ($a_w$)

• Definition of Water Activity ($a_w$): The partial vapor pressure of water in a substance divided by the standard state partial vapor pressure of water ($a_w = \frac{P}{P_0}$).

• Microbial Requirements:

  • Most microorganisms require RH > 60\,%.

  • Some can survive in > 20\,% \, RH.

  • Gram-negative bacteria are generally more sensitive to low $a_w$ than Gram-positive bacteria.

  • $Staphylococcus \, aureus$ can grow and produce toxin at $a_w$ as low as $0.86$.

• Insect Hygroreception: Insects use hygroreceptors to estimate water loss risks. The "Psychrometer Model" is the best fit for sensing humidity in species like Periplaneta americana, where the cooling effect of water evaporation measures moisture.

• Physical Phenology: High humidity and low temperatures prolong decomposition and keep remains wet. Conversely, hot, dry conditions accelerate moisture evaporation from skin, leading to mummification while internal organs undergo limited internal decay.

Abiotic Factors: Resource Quality and Chemistry

• Heterotrophic Communities: Changes in resource supply quality impact enzyme activity, community composition, and stoichiometry.

• Nutrient Pulse and Putrefaction:

  • Post-death, host immune function ceases, and commensal microbes translocate via upregulation of motility genes and exoenzymes.

  • The environment shifts from aerobic to anaerobic, facilitating putrefaction.

  • Putrefaction involves the fermentation of carbohydrates, lipids, and proteins, which releases gases.

• Stoichiometry (Carbon to Nitrogen Ratios):

  • Mammal carcasses have a narrow $C:N$ ratio ($5:1$ to $8:1$).

  • Fungal biomass $C:N$ ratio: $10:1$ to $15:1$.

  • Bacterial biomass $C:N$ ratio: $3:1$ to $12:1$.

  • Overall microbial biomass ratio: $4:1$ to $8:1$.

  • Low-quality resources (High $C:N$) favor fungi; high-quality resources (Low $C:N$) favor bacteria.

• Adaptation to "Feast or Famine": Microbes utilize rRNA operon copies, extracellular enzyme activity, shifts to a "viable but non-culturable" ($VBNC$) state, and storage of carbon/lipid reserves to survive nutrient variability.

Abiotic Factors: Impact of Narcotics and Drugs

• Entomotoxicology: Insect larvae accumulate drugs from the deceased, which delays colonization or development.

• Microbiome Perturbation:

  • Phencyclidine ($PCP$): In mouse models, $PCP$ treatment altered the gut microbiome, which correlated with behavior changes. Ampicillin administration could abolish $PCP$-induced memory deficits.

  • Antibiotics: Significantly decrease species from the genus Bifidobacterium.

  • Metformin: Increases the abundance of Escherichia coli and uregulates fermentation pathways (e.g., pyruvate fermentation).

  • Other influential drugs: Proton-pump inhibitors ($PPI$), statins, and laxatives.

Biotic Factors: Microbial and Inter-Kingdom Interactions

• Social Interactions:

  • Syntrophy: Metabolic activities where species are mutually dependent; combinations of organisms make reactions thermodynamically favorable.

  • Competition: Intense competition involves antimicrobial secretion, sequestration of communication signals (quorum quenching), and contact-dependent killing.

• Symbiosis Example: Lucilia sericata and Proteus mirabilis.

  • P. mirabilis resides in the gut of the larvae.

  • The bacteria produce "miribilicides" (antibacterial agents) that protect the larvae from pathogenic bacteria.

• Pathogen Dispersal Mechanisms:

  • Host death ceases immune function within specific hours, allowing opportunistic pathogens to proliferate.

  • Dispersal occurs via vertebrate scavengers, insects, soil, and groundwater.

  • Antibiotic resistance ($AbxR$) and virulence genes are transferred between the remains and the environment.

Pathogen Proliferation and Dispersal in the Environment

• Survival Strategies: Bacteria may form endospores, enter $VBNC$ states, or survive inside protists.

• Duration of Survival ($E. coli$ and $Salmonella$):

  • Fresh water: $1 \, \text{day}$.

  • Sediments: $1.5 \, \text{days}$.

  • Soil: $3 \, \text{days}$.

  • Host-adapted E. coli typically cannot compete for nutrients under starvation conditions in non-host environments without added carbon.

• Environmental Factors for Dispersal:

  • Clay particles: Surface charges retard microbial diffusion but increase survival through adsorption.

  • Soil pH: High soil pH is favorable for microbial transport; low pH retains more bacteria in the soil matrix.

  • Topography: Sloped surfaces increase risk of dispersal via runoff.

• Fungal-Specific Dispersal: Active discharge of spores into air/water; use of bacteria or insects for "hitchhiking."

Insects as Pathogen Vectors

• Common Pest Vectors: House flies, blowflies, and cockroaches.

• Isolates Recovered:

  • Salmonella: Survives in house flies for up to a month; survives in blowfly pupae for $29 \, \text{days}$.

  • Other pathogens: Enterobacteriaceae, Staphylococcus, Campylobacter, Claviceps purpurea, and Candida ssp.

• Mechanism: Insects are attracted to $VOCs$ from carrion, which are often contaminated with pathogens. They transmit these pathogens via their excreta or mechanical transfer.

Conclusions

• Decomposition is a complex inter-kingdom process where microbes (bacteria and fungi) regulate arthropod behavior through physiology and chemical signaling.

• Integrating forensic microbiology with forensic entomology is essential for refining estimates of the time of death and colonization.

• The Future: There is a critical need for multidisciplinary investigations to obtain robust forensic evidence from vertebrate remains.